Chimeric ILT Receptor Compositions and Methods

Abstract

Chimeric ILT receptors (CIRs) are provided that include a targeting region, a transmembrane domain, and an intracellular domain (ICD) from ILT2 or ILT4. The ICD includes a signaling region (e.g., CD3 zeta (CD3z)) and optionally a costimulatory region (e.g., CD28, 4-1BB, OX40, etc.). Nucleic acids (e.g., expression vectors) encoding the subject CIRs are also provided, as well as genetically modified cells (e.g., immune cells such as NK cells, T cells, iNKT cells, macrophages, etc.) that express the subject CIRs. For example, genetically modified immune cells such as NK cells that include nucleic acids encoding ILT2 or ILT4 CIRs are provided. The subject CIRs are designed to activate cytotoxicity by immune cells such as NK cells, T cells, iNKT cells, and macrophages against cancers that express HLA-G.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 306,514, filed February 4, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporation by reference of sequence listings provided as XML files The Sequence Listing is provided herewith as Sequence Listing XML "NKLT-001WO_SEQ_LIST.xml" created on February 3, 2023 and having a size of 142,627 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

[0003] I. Introduction Immune cell therapy helps to specifically target diseased cells. This treatment may be curative for both malignant and non-malignant conditions. For example, donor lymphocyte infusion, allogeneic T cells, and allogeneic natural killer (NK) cells can be used to control the growth of leukemia. Furthermore, genetic modification can direct the specificity of immune cells, including T cells, natural killer (NK) cells, γδ T cells, inducible NKT cells, and macrophages, to a given 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). Clinical trials have shown the efficacy of CAR-T therapy, particularly in hematological malignancies such as B-cell leukemia and lymphoma, by targeting antigens such as CD19 or B-cell maturation antigen (BCMA) for multiple myeloma. These targets have distinct expression in B cells and plasma cells and their respective tumor types, and aplasia resulting from extratumoral targeting by CD19 and BCMA CAR-T cells can be replaced by immunoglobulin replacement therapy. Three autologous CD19 CAR-T cell therapy products have been approved by the US FDA. More recently, NK (CAR-NK) cells expressing CARs directed against CD19 have shown therapeutic efficacy.

[0004] Acute myeloid leukemia (AML), also called acute myelogenous leukemia, is a disease of transformed myeloid progenitors and cells more differentiated towards the monocytic lineage. CAR-T therapy against targets such as CD33 and CD123 expressed on AML blasts has challenges with expression of targets on normal myeloid progenitors, and therapeutic ablation leads to highly deleterious cytopenias. CAR or other directed cell therapy against more distinctly expressed targets in AML is needed. One such target is HLA-G, a class 1B (or non-classical) MHC-1 protein.

[0005] HLA-G is abundantly expressed on the surface of trophoblast cells in the fetal placenta and provides a barrier to the fetus from immune attack by the mother through potent inhibition of most classes of lymphoid and myeloid cells. As a mechanism to evade immune attack and thereby promote tumor growth, HLA-G is overexpressed in a variety of tumor types, including AML, whereas hematopoietic progenitor cells do not express HLA-G due to innate tolerance from immune attack.

[0006] The HLA-G gene produces multiple different RNA transcripts through alternative mRNA splicing, which then produce at least seven different protein products. These protein products can exist as secreted forms on the plasma membrane of tumor cells or in the tumor microenvironment, with or without complexation with β2-microglobulin (β2-M). Furthermore, HLA-G can exist as a monomer or disulfide-linked homodimer with an altered tertiary structure. Conventional CAR-T products target antigens using single-chain fragments (scFv) derived from antibodies or camelid-derived VhH domains in chimeric receptors. The epitope targeted by a given scFv on HLA-G is likely to be removed or eliminated in some HLA-G isoforms, eliminating the expression of the isoform containing this epitope but selecting tumor cells expressing an isoform that retains the immunosuppressive activity of HLA-G. There is a need for targeting agents for cell therapy against HLA-G, including all isoforms capable of immunosuppression. Summary of the Invention

[0007] II. Overview The present disclosure provides genetically modified cells engineered to express chimeric receptor proteins, which have affinity and specificity such that the modified cells can stimulate an immune response in a subject. For example, the chimeric receptor protein can target proteins expressed at higher levels in tumor tissues compared to non-transformed normal tissues, generating a cytotoxic or inflammatory response against the tumor. CARs are frequently used to generate antigen-specific recognition of tumor tissues based on the affinity and specificity enabled by antibody-antigen interactions. For the compositions and methods of the present disclosure, affinity and specificity are not maintained by the use of antibodies or VhH relationships, or by the binding of randomly generated peptides to target antigens, but rather by the use of ligand:receptor interactions, where affinity and specificity are maintained by evolution.

[0008] For example, in preferred embodiments, genetically modified cells express chimeric receptors with high affinity for HLA-G. HLA-G is a target protein that can exist in tumor tissue in one or more of seven known forms generated by alternative mRNA splicing and post-translational modifications. HLA-G naturally functions as an agent that suppresses immune responses through engagement with the negative signaling immunoglulin-like transcript 2 (ILT2) and ILT4 receptors on the surface of immune cells. In these preferred embodiments, T cells, NK cells, iNKT cells, or macrophages are engineered such that recognition of active HLA-G by ILT2 or ILT4 instead generates an activation signal. In these preferred embodiments, the intracellular signaling elements of ILT2 or ILT4 are removed and replaced with the ITAM-containing signaling domain of the CD3 zeta chain that drives immune cell activation and cytotoxicity. Such proteins are referred to as "chimeric ILT receptors" or "CIRs."

[0009] Thus, chimeric ILT receptors (CIRs) are provided that comprise a targeting region, a transmembrane domain, and an intracellular domain (ICD) from ILT2 or ILT4, including a signaling region (e.g., CD3 zeta (CD3ζ)) and optionally a costimulatory region (e.g., CD28, 4-1BB, OX40, etc.). The inventors recognize that this approach is advantageous over using antibody-based targeting regions (e.g., scFvs). Antibody-based targeting approaches may lead to the selection of tumor cells expressing HLA-G isoforms that lack the targeted epitope, thus allowing cancer to evade treatment. In contrast, the subject ILT2 or ILT4-based chimeric ILT receptors should target more, and possibly all, HLA-G isoforms, since ILT2 and ILT4 naturally bind to those isoforms. This would greatly reduce, and perhaps eliminate, the ability of cancer cells to evade therapy by selecting for specific HLA-G isoforms.

[0010] ILT2 and ILT4 are structurally similar in the extracellular region, composed of four folded domains (D1, D2, D3, and D4) arranged distal to proximal 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 protein while maintaining their interaction with HLA-G. Cellular proteins other than HLA-G can interact with the D3 and D4 domains of ILT4. To prevent off-target interactions with these proteins that may generate toxicity in a therapeutic setting, it is desirable to eliminate such off-HLA-G interactions. Thus, in some embodiments, ILT2 or ILT4 D3-D4 are replaced with another extracellular domain that functions as the stalk and transmembrane domain in a chimeric receptor fusion to present ILT2 or ILT4 D1-D2 to HLA-G expressing target cells. Examples of stalk proteins that display 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. Thus, in some embodiments, a subject chimeric ILT receptor comprises D1-D2 of ILT2, but lacks D3-D4 of ILT2. In some embodiments, a subject chimeric ILT receptor comprises D1-D2 of ILT4, but lacks D3-D4 of ILT4.

[0011] Some proteins can perform binding interactions with the D1 and D4 regions of ILT4. In the context of D4 ablation, such interactions with D1 alone are likely to be weaker. To further reduce undesired interactions with other cellular proteins other than HLA-G, in some embodiments, mutations are made that code for amino acid replacements in D1 of ILT4. For example, tyrosine 96 can be replaced with any amino acid, including alanine.

[0012] In yet another embodiment, costimulatory elements are included as part of the intracellular portion of the chimeric receptor to improve survival, persistence, cytotoxicity, and ability to secrete cytokines as part of an immune response, while in other embodiments, costimulatory elements are engineered to be expressed separately from the chimeric receptor. These costimulatory elements can be derived from any of the following proteins or combinations thereof: 4-1BB, OX40, ICOS, CD28, CD27, MyD88, IL-1Rα, HVEM, TRANCE, IL-1Rβ, IL-18Rα, CD40, IL-18Rβ, CD30, IL-33Rα, BCMA, or IL-33β.

[0013] In some embodiments, the safety switch can be co-expressed with the chimeric receptor protein in the same cell. The purpose of this switch is to reduce toxicity that may be generated by genetically modified cells containing CIR. Such toxicity may result from hyperactivity causing cytokine release syndrome (CRS), immune cell activation neurotoxicity syndrome (ICANS), off-target extratumoral interactions, or targeting to normal tissues that may express HLA-G. In one such embodiment, the safety switch is inducible caspase 9 (iC9), a fusion of a variant FKBP12 with a truncated form of caspase 9 such that the proapoptotic activity of the caspase is under the control of a synthetic dimerizer ligand, rimiducid.

[0014] Reagents, compositions, kits / systems, and methods relating to chimeric ILT receptors are provided, including methods for making genetically modified cells and methods of treatment (e.g., administering to an individual immune cells such as NK cells, T cells, or macrophages that express a subject CIR). [Brief description of the drawings]

[0015] III. BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of the embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the present invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. The drawings show certain embodiments of the technology, but are not limiting. For clarity and ease of explanation, the drawings are not made to scale, and in some cases, various aspects may be shown exaggerated or enlarged to facilitate understanding of certain embodiments. [Figure 1] 1A-1B: HLA-G and inhibitory receptors of HLA-G. (FIG. 1A) Schematic of HLA-G1 tethered to the plasma membrane of tumor cells complexed with β2-M. Also shown are the receptors of HLA-G (ILT2 protein expressed mainly on lymphocytes and ILT4 expressed mainly on cells derived from myeloid lineage). The four separately folded extracellular domains on ILT2 and ILT4 proteins are labeled D1-D4 according to their proximity to the plasma membrane. The intracellular signaling domains (ICDs) are also shown. (FIG. 1B) Diagram of ILT4 (left) or ILT2 complexed with HLA-G. Unlike classical MHC-I interactions through peptide-binding clefts, ILT proteins interact with the membrane proximal α3 domain of HLA-G and β2-M. The formation of the complex initiates signaling from the ICD of ILT2 or ILT4 and suppression of immune cell activation. [Diagram 2] Formation of ILT4 complexes with dimeric HLA-G. A subset of HLA-G proteins form disulfide-linked dimers in the α1 or α3 domains. Dimeric HLA-G has a higher affinity for ILT2 / 4, likely due to an avidity effect that leads to enhanced ILT signaling and suppression of immune cells. [Diagram 3]Figures 3A-3B: Chimeric ILT proteins for immune cell activation. (Figure 3A) Schematic of chimeric ILT4 receptor (CIR) in which the native ICD of ILT4 is replaced with a signaling domain derived from an immune cell activating receptor or activating adaptor protein. Engagement of CIR with dimeric HLA-G initiates signaling that activates immune cells. (Figure 3B) Diagram of chimeric ILT4 receptor complexed with monomeric HLA-G. [Figure 4] Figures 4A-4B: Interaction of the D1 and D2 domains of ILT2 and ILT4 with HLA-G isoforms. (Figure 4A) Schematic of isolated ILT4 (left) and ILT2 (right) in complex with the HLA-G1 isoform. The D3 and D4 domains of the ILT proteins are not essential for interaction with HLA-G (Figure 4B). Schematic of isolated ILT4 (left) and ILT2 (right) in complex with the HLA-G1 isoform. [Figure 5A] Figures 5A-5B: Interaction of ILT4 D1D2 CIR with HLA-G. (Figure 5A) Schematic of the D1 and D2 domains of ILT4 fused to stalk and transmembrane domains from separate proteins as a chimeric hybrid, and further fused to an activating intracellular signaling moiety. Interaction of D1D2 with HLA-G initiates signaling to activate immune cells. [Figure 5B] Figures 5A-5B: Interaction of ILT4 D1D2 CIR with HLA-G. (Figure 5B) Interaction of a stalk containing ILT4 D1D2 CIR with dimeric HLA-G. [Figure 6A]Figures 6A-6E: Transduction and expression of CIR protein from constructed γ-retroviral vectors. (Figure 6A) Schematic of retroviral construct expressing ILT2 and ILT4 fusion protein, termed chimeric ILT receptor (CIR). D1-D4 indicate coding of extracellular domains D1-D4 derived from native ILT2 or ILT4. STM refers to stem (S), a linker domain that connects the extracellular D domain with the transmembrane domain (TM), which are derived from ILT2, ILT4, or CD8α (CD8a). FL refers to full-length versions of native ILT2 and ILT4. ICD of ILT2 and ILT4 refer to native intracellular domains of ILT2 and ILT4 that promote inhibitory signals to immune cells. BB.ζ refers to intracellular domains derived from fusion with the co-stimulatory domain of 4-1BB and the signaling domain of CD3-ζ, each promoting activating signals to immune cells. Each of these gamma-retroviral constructs also encodes a separate marker protein, ΔCD19, to determine the efficacy of transduction. See also Tables 13B, 15B, 16B, 20B, 21B, 23B, 24B, 28B, 36, and 37 (Figure 6B), transduction of retroviral constructs into primary human T cells. Flow cytometry plots show expression of ΔCD19 marker protein coupled with expression of CIR constructs along with expression of a separate co-transduced marker virus encoding Orange nanolantern rennila luciferase (ONL). Plots shown represent T cells derived from one of two healthy blood donors. Negative control (Neg ctl) shows transduction of ILT2 and ILT4 full-length constructs expressing ILT2 and ILT4 full-length (FL) proteins to inhibit T cell function and serve as a negative control (Neg.Ctl.). (FIG. 6C) Quantification of the efficiency of retroviral transduction into primary human T cell (N=2) donors. (FIG. 6D) Expression of ILT4 ectodomain in transduced human T cells 7 days post-transduction as determined by mean fluorescent intensity of antibody staining as determined by flow cytometry.(FIG. 6E) Expression of the ILT2 extracellular domain in transduced human T cells 7 days post-transduction as determined by mean fluorescent intensity of antibody staining determined by flow cytometry. [Figure 6B-1] See legend to Figure 6A. [Figure 6B-2] See legend to Figure 6A. [Figure 6B-3] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 7A-1] Figures 7A-7B: Transgenic expression of HLA-G isoforms in THP1 acute myeloid leukemia cells. (Figure 7A) Stable expression of γ-retroviral constructs expressing HLA-G1, HLA-G2, and HLA-G5 in THP1 cells lacking endogenous HLA-G protein expression. Transduction efficiency is characterized by the expression of ΔEGFR coexpressed by the retroviral constructs. HLA-G1 expression is easily detected by flow cytometry using MEM-G / 9 antibody specific for HLA-G1. (Figure 7B) Stable expression of GFP-ff luciferase in THP1 cells stably expressing HLA-G1. [Figure 7A-2] See legend to Figure 7A-1. [Figure 7B] See legend to Figure 7A-1. [Figure 8]Control for THP1-HLA-G1 cell proliferation by CIR-T cells. Primary human T cells (N=2 donors) transduced with the indicated ILT2 and ILT4 CIR constructs or full length ILT2 or ILT4 (Neg Ctl) were co-cultured with THP1 cells stably transduced with HLA-G1 isoforms and GFP-ffluc marker. THP1 proliferation or killing was measured periodically by GFP fluorescence in an Incucyte over 48 hours. T cells expressing and HLAG1-specific chimeric antigen receptor (HLA-G CAR), or CD33-specific CAR targeting AML (CD33 CAR) served as positive controls. NT=untransduced. [Figure 9] Short-term cytotoxicity of CIR-T cells. Primary human T cells (N=2 donors) transduced with the indicated ILT2 and ILT4 CIR constructs or full length ILT2 or ILT4 (Neg Ctl) were co-cultured for 24 hours with THP1 cells stably transduced with HLA-G1 isoforms and GFP-ffluc markers. THP1 killing was measured by loss of luciferase activity from tumor targets. T cells expressing and HLAG1-specific chimeric antigen receptor (HLA-G CAR), or CD33-specific CAR targeting AML (CD33 CAR) served as positive controls. NT=untransduced. [Figure 10] Stalk and transmembrane domain modifications of CIR retroviral constructs. A schematic table showing derivatives of CIR4 encoding the HLA-G binding domains D1 and D2 of ILT4 linked to the hinge / stalk and transmembrane domain of CD8α, along with intracellular domains from 4-1BB and CD3ζ. CIR constructs 6-10 encode replacements of the stalk from human immunoglobulin IgG4, the CH3 domain of IgG4, or CD28. These were fused to the transmembrane domains of CD8α or CD28 as indicated. See also Tables 28B and 38-42. [Figure 11A]Figures 11A-11C: Expression of γ-retroviral constructs encoding CIR proteins with derivative stalk and transmembrane domains. (Figure 11A) Transduction efficiency at 7 days (black) and 14 days (grey) after transduction of retroviral constructs encoding the indicated stalk and transmembrane derivatives characterized by the ΔCD19 marker expressed by the same retroviral construct. (Figure 11B) Expression of CIR constructs with alternative stalk and transmembrane domains determined by flow cytometry using an ILT4 antibody, gated by baseline of isotype control. (Figure 11C) Expression of CIR constructs with alternative stalk and transmembrane domains determined by mean fluorescence intensity (MFI) of an ILT4-specific antibody by flow cytometry. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 12] Expression of HLA-G in human AML cell lines. Molm13-GFP and Molm14-GFP cells were stained with a fluorescently labeled antibody specific for HLA-G and subjected to flow cytometry. Gating was set by staining with a labeled isotype control antibody. [Figure 13] Figures 13A-13B: Antitumor efficacy of CIR constructs against Molm-14 tumor targets. (Figure 13A) HLA-G positive Molm14 cells stably transduced with GFPffluc were co-cultured with non-transduced primary human T cells, CIR-T cells, or HLA-G directed CAR-T cells at an effector to target ratio of 5:1 (N=2 donors). Molm14 proliferation at 48 hours was measured by GFP fluorescence in an Incucyte microscope incubator. (Figure 13B) Short-term cytotoxicity of CIR-T cells against Molm14 tumor targets. Primary human T cells transduced with ILT4 CIR constructs or HLA-G CAR (N=2 donors) were co-cultured with Molm14-GFPffluc cells for 24 hours. Molm14 killing was measured by loss of luciferase activity from tumor targets. [Figure 14]Figures 14A-14B: Co-culture of ILT4 CIR with THP1 cells expressing HLA-G isoform. (Figure 14A) Primary human T cells (NT), ILT4 CIR-T cells, or HLA-G CAR-T cells were cultured with THP1 cells stably transduced with HLA-G1 isoform. Activation of T cells upon CIR or CAR engagement was monitored by secretion of interferon-gamma (IFN-γ) into the culture medium. (Figure 14B) Secretion of interferon-γ produced in co-culture of CIR-T cells and CAR-T cells with THP1 cells stably transduced to express HLA-G2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] IV. Detailed Description Chimeric ILT receptors (CIRs) are provided that include a targeting region, a transmembrane domain, and an intracellular domain (ICD) from ILT2 or ILT4. The ICD includes a signaling region (e.g., CD3 zeta (CD3ζ)) and optionally a costimulatory region (e.g., CD28, 4-1BB, OX40, etc.). Nucleic acids (e.g., expression vectors) encoding the subject CIRs are also provided, as well as genetically modified cells (e.g., immune cells such as NK cells, T cells, iNKT cells, macrophages, etc.) that express the subject CIRs. Genetically modified immune cells, such as NK cells, that include nucleic acids encoding ILT2 or ILT4 CIRs, are also provided. Methods of making genetically modified cells and methods of treatment (e.g., administering to an individual immune cells such as NK cells, T cells, or macrophages that express the subject CIRs) are also provided.

[0017] 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.

[0018] Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intervening value is included, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value in that stated range. 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 specific 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.

[0019] Certain ranges are presented herein with the term "about" preceding the numerical values. In this specification, the term "about" is used to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately a specifically recited number, the near or approximate unrecited number may be a number that provides the substantial equivalent of the specifically recited number in the context in which it is presented.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are now described.

[0021] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein 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 date of the present application 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.

[0022] It should be 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. Thus, the articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more elements. Thus, for example, a reference to "a cell" includes a plurality of such cells, and a reference to "the polypeptide" includes one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely" and "only" in connection with the recitation of claim elements, or for the use of a "negative" limitation.

[0023] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that can 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 may be carried out in the order of events recited or in any other order that is logically possible. It is understood that certain features of the invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination. All combinations of the embodiments according to the present invention are also specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed herein. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0024] Although the apparatus and methods have been or will be described in functional descriptions for grammatical fluidity, it is expressly understood that the claims should not be construed as necessarily limited in any way by the syntax of "means" or "step" limitations unless expressly recited under 35 U.S.C. 112, but should be accorded the full scope of meaning and equivalents of the definitions provided by the claims under the doctrine of judicial equivalents, and that the claims, when expressly recited under 35 U.S.C. 112, should be accorded the full statutory equivalents under 35 U.S.C. 112.

[0025] The entirety of each patent, patent application, publication, and document referenced herein is incorporated herein by reference. Citation of patents, patent applications, publications, and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or dates of such publications or documents. Such citations do not indicate a search for the relevant disclosures. All statements as to the dates or contents of documents are based on the information available and do not constitute an admission as to their accuracy or correctness.

[0026] Modifications can be made to the foregoing without departing from the fundamental aspects of the technology. Although the technology has been described in substantial detail with reference to one or more specific embodiments, those skilled in the art will recognize that modifications can be made to the embodiments specifically disclosed in this application. However, these modifications and improvements are within the scope and spirit of the technology.

[0027] The technology illustratively described herein may be suitably practiced in the absence of any element 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 used are used as terms of description, not of limitation, and the use of such terms and expressions does not exclude any equivalent of the features shown and described or portions thereof, and various modifications are possible within the scope of the claimed technology. The term "a" or "an" may refer to one or more of the elements it modifies, unless one or more of the elements described is clear from the context (e.g., "a reagent" may mean one or more reagents). The term "about" as used herein refers to a value within 10% (i.e., plus or minus 10%) of the underlying parameter, and the use of the term "about" at the beginning of a series 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. Furthermore, when a list of values ​​is set forth herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate and fractional values ​​thereof (e.g., 54%, 85.4%). Thus, while the present technology has been specifically disclosed by exemplary embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the present technology.

[0028] 1. MHC class I ("MHC-I") molecules MHC-I molecules in humans include classical HLA-A, HLA-B, or HLA-C α chains, and non-classical HLA-E, HLA-F, HLA-G, and HLA-H α chains. Classical MHC-I molecules present peptide fragments to cytotoxic T cells from within cells. When non-self antigens are presented on the MHC-I complex, cytotoxic T cells specific for the MHC:peptide complex can recognize and kill the MHC-I presenting cell. These cytotoxic signals also activate the proliferative capacity of T cells in combination with costimulatory signaling.

[0029] MHC class I molecules are heterodimers consisting of two polypeptide chains (α and β2-microglobulin β2-M). The two chains are non-covalently linked through the interaction of the β2-M and α3 domains. The α chain (encoded by the HLA genes) is highly polymorphic, whereas the β2-M subunit (encoded by the β2-microglobulin gene) is not. The α3 domain is plasma membrane transmembrane and interacts with the CD8+ coreceptor of T cells. The α3-CD8 interaction holds the MHC-I molecule in place, and the T cell receptor (TCR) on the surface of cytotoxic T cells binds to its α1-α2 heterodimeric ligand and checks the antigenicity of the coupled peptide. The α1 and α2 domains fold to form an α-helical groove for peptide binding [Bjorkman et al, (1987) Nature 329:506].

[0030] Although it is possible to match donors and recipients based on their HLA subtypes (e.g., A, B, or C), MHC-I:peptide complexes presented on the cell surface may still be recognized as non-self. For allogeneic T cell therapy, it is beneficial to match classical HLA types to prevent allorecognition of normal host tissues leading to graft-versus-host disease (GvHD) and improve the persistence of transplanted cells by mitigating host-versus-graft (HvG) responses. However, allogeneic natural killer cell therapy for cancer benefits from classical HLA subtype mismatches, especially on HLA-C1, HLA-C2, and HLA-Bw4, which contain alleles that can promote mismatches in KIR recognition by NK cells and the enhancement of their innate cytotoxic responses.

[0031] Non-classical MHC-I proteins (also called MHC class Ib proteins) are composed of similar domain structures and generally bind to β2-M [Clements et al., (2005) Proc Natl Acad Sci 102:3360]. They also bind peptide fragments from inside cells, but the diversity of bound peptides is limited and they do not present these peptides to the CD3-TCR complex to direct cytotoxicity. Instead, these proteins engage inhibitory receptors expressed on a subset of immune cells to inhibit or attenuate immune cell function. HLA-E engages with the CD94 / NKG2A complex on cytotoxic T cells and NK cells, and HLA-G interacts with ILT proteins on a diverse group of immune effectors.

[0032] 2. HLA-G The nonclassical MHC-I protein HLA-G is a major factor in maintaining immune tolerance during maternal-fetal development [Kovats et al. Science (1990) 248:220, Ferreira et al, (2017) I 38:272]. Its normal expression is highest in the extravillous trophoblast cells of the fetal placenta, where it functions to block the activation and infiltration of most types of maternal immune cells, particularly T and NK cells from the fetus with a haploidentical MHC haplotype. It is maintained at much lower levels in other immune-privileged 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 the 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 on tolerogenic DC-10 dendritic cells, which suppress lymphocytic responses by secreting inhibitory cytokines and activate Treg cells and myeloid-derived suppressor cells (MDSCs) to generate an immunosuppressive tumor microenvironment [reviewed in Carosella et al, Blood (2011) 118:6499; Gao et al, (2018) BBA 1869:278]. HLA-G can therefore be considered as a key checkpoint mediator of tumor promotion.

[0033] The HLA-G gene produces multiple mRNA transcripts that code for at least seven different protein products [Ishitani et al (1992) Proc. Natl. Acad. Sci 89:3947] (SEQ ID NOs: 9, 15, 17, 19, 21). HLA-G1 contains an α1-α2-α3 domain structure with an α-helical peptide-binding groove, a transmembrane domain, and a short intracellular carboxy-terminal domain (see FIG. 1). This domain structure is standard for MHC-I products. Other expressed splice products lack entire domains, e.g., HLA-G2 codes for α1, α3, and transmembrane domains, and lacks α2. HLA-G4 lacks the α3 domain, and HLA-G3 codes for only the α1 domain. When expressed in M8 cells as a transgene, 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 maintain the domain structure of HLA-G1 and G2, respectively, but do not use the splice donor site in intron 4, instead encoding a short secreted peptide derived from intron 4. Similarly, HLA-G7 uses a three amino acid peptide derived from intron 2. Additional secreted forms of HLA-G1 are generated by cleavage at the transmembrane domain by matrix metalloproteinases to remove some of the HLA-G1 from the cell surface [Rizzo et al (2012) Mol Cell Biochem 381:243].

[0034] HLA-G exists in monomeric and oligomeric forms. The oligomers are primarily dimers oriented by disulfide bonds at Cys42 (in α1) or Cys147 (in α2) [Gonen-Gross et al, (2005) J.Imm.175:4866, Boyson et al, Proc.Natl.Acad.Sci 99:16180]. There is evidence that the dimeric form of HLA-G is the predominant immunosuppressive form and adopts a distorted quaternary structure compared to the dimeric form of the native monomer [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].

[0035] Together, the different HLA-G forms create challenges for CAR-based therapies that rely on the binding of antibody-derived scFv or VhH domains as targeting agents. Different splice forms are selected for by CAR therapies to eliminate epitopes of a given antibody and therefore express only epitopes that are not recognized by the CAR binder while retaining immunosuppressive activity. Furthermore, oligomerization can mask the epitopes of scFvs due to conformational changes. Similarly, two of the commonly used antibody reagents for HLA-G 4H84 and 87G show cross-reactivity to other HLA types that may lead to off-target, extratumor 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].

[0036] 3. ILT2 and ILT4 HLA-G directs its immunosuppressive activity as a membrane-bound ligand of the inhibitory receptors immunoglobulin-like transcript 2 (ILT2) and ILT4 (also called LIRB1 and LIRB2, or CD85j and CD85d, respectively) on target immune cells [reviewed in Colonna et al (1998) J. Immunology 160:3096, Gao et al (2018) BBA 1869:278]. ILT2 (SEQ ID NO:29) is expressed in a subset of natural killer cells, iNKT cells, T cells, B cells, and dendritic cells. ILT4 (SEQ ID NO:53) has a broader expression pattern, mainly in myeloid cells and stem cells (including macrophages, myeloid-derived suppressor cells) (a population of less differentiated cells on the monocytic lineage), granulocytes (including neutrophils, monocytes, hematopoietic stem cells), and some neurons.

[0037] ILT2 has an extracellular domain structure consisting of four domains with sequence and structural homology to immunoglobulin domains (Ig domains) arranged in a row from the membrane distal D1 to the most membrane proximal D4, followed by a transmembrane domain and an intracellular signaling domain containing four repeated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). ILT4 has a similar extracellular and transmembrane architecture, but has only three ITIMs in its intracellular domain.

[0038] The D1 and D2 domains of ILT2 (see, e.g., SEQ ID NOs: 37 and 71) and ILT4 (see, e.g., SEQ ID NOs: 57 and 75) govern the 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 the activating interactions formed by the CD3 complex with classical MHC-I, the inhibitory and activating interactions formed by the KIR proteins of ILT2 and ILT4 of NK cells do not bind the α1-α2 domains that contain the peptide-binding groove, but instead interact with the membrane-proximal α3 domain and β2-microglobulin (see Figure 1B). ILT2 makes extensive contacts with β2-M and relatively few contacts with α3 of HLA-G, and requires the association of β2-M with HLA-G even to maintain a low affinity interaction. Conversely, ILT4 makes extensive contacts with α3 and is probably able to maintain interactions with all known active immunosuppressive forms of HLA-G, except for HLA-G3 / G7, which contains only the α1 domain.

[0039] ILT2 and ILT4 can interact with other MHC-I and MHCI-like proteins, in particular 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 have dissociation constants (K) ranging from 2 μM to 40 μM. D) is a low affinity interaction. The relevance of immunosuppressive signaling has not been demonstrated with such weak affinities. Similarly, the interaction of ILT2 and ILT4 with the monomeric form of HLA-G is weak, in the μM range. However, the dimeric HLA-G form shows high affinity (2-4 nM) interactions with ILT2 and ILT4, possibly due to the presentation of additional contact sites or, alternatively, an avidity effect that reduces the dissociation rate of ILT dissociation [Shiroishi et al, (2006) J. Biol. Chem 281:10440, Gao et al (2020) Cell Mol Imm 17:966]. Therefore, the dimeric form of HLA-G is most likely to be biologically active [Gonen-Gross et al, (2005) J. Imm. 175:4866] and function for immunosuppression in the tumor environment, and furthermore is most likely relevant as a targeted molecule for cell-based immunotherapy via chimeric receptors.

[0040] 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 the self-renewal and survival of ILT4-expressing hematopoietic stem cells. The interaction between ILT4 and ANGLP is directed by the D1 domain in concert with the D4 domain of ILT4, and specific residues in either 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].

[0041] ILT4 interacts with moderate affinity to myelin-derived inhibitory Nogo receptor ligands [Atwal et al (2008) Science 322:967, Matsushita et al, (2014) J. Biol. Chem 286:25739]. PIRB, the mouse orthologue of ILT proteins, is also found in a subset of neurons and may regulate axonal outgrowth through interactions with myelin-based MAG, Nogo, and OMgp [US 2010 / 0047232], as well as Sema4a [Lu et al. (2018) Nat. Comm. 7:742]. A high affinity interaction was characterized in ILT4, the mouse orthologue for PIRB, which did not map to the HLA-G binding D1 and D2 domains but rather to the membrane proximal domain of PIRB [Matsushita et al, (2014) J. Biol. Chem 286:25739].

[0042] 4. Chimeric Antigen Receptor Chimeric antigen receptors (or CARs) are artificial receptors designed to confer antigen specificity to cells. They generally contain an antigen-specific component, a transmembrane component, and an intracellular component selected to activate the cell. Cells expressing CARs can be used in a variety of therapies, including cancer therapies.

[0043] CARs are chimeric polypeptides that include a polypeptide sequence that recognizes a target antigen (antigen recognition domain) linked to, for example, a transmembrane polypeptide and an intracellular domain polypeptide that is selected to activate the cell, thereby providing specific immunity. The antigen recognition domain may be a single chain variable fragment (scFv) or may be derived from other molecules, such as, for example, T cell receptors or camelid VhH domains. The intracellular domain includes at least one polypeptide that causes activation of the cell ("signaling domain"), such as, for example, but not limited to, CD3 zeta (CD3ζ) (see, for example, SEQ ID NO: 33), and optionally, a costimulatory molecule ("costimulatory domain") (such as, but not limited to, CD28 (see, for example, SEQ ID NO: 49), OX40, and 4-1BB (see, for example, SEQ ID NO: 35)).

[0044] Thus, in a typical example of the use of CARs, cells are engineered to express a CAR comprising a single chain antibody variable fragment (scFv) fused to a transmembrane domain including a linker region and an intracellular domain derived from a CD3 zeta component (see, for example, SEQ ID NO: 33). In natural T cells and NK cells, signals from CD3 zeta drive the initial activation of T cells 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 robust cell proliferation in T cell immune responses. Genetically engineered cells can be engineered by transduction or transfection with a nucleic acid (same or different) that expresses the CAR and a polynucleotide that encodes a chimeric signaling polypeptide (see below). In other embodiments, the CAR is expressed without also expressing a chimeric signaling polypeptide.

[0045] Chimeric antigen receptors can be expressed in NK cells, iNKT cells, or macrophages to generate antigen-specific cytotoxicity.

[0046] CARs include chimeric receptors derived from antibodies, but also chimeric T cell receptors. These chimeric T cell receptors may include a polypeptide sequence that recognizes a target antigen, and the recognition sequence may be, for example, but not limited to, a recognition sequence derived from a T cell receptor or scFv. The intracellular domain polypeptide acts to activate T cells. Chimeric T cell receptors are discussed, for example, in Gross & Eshar FASEB Journal (1992) 6:3370-3378 and Zhang et al., (2010) PLOS Pathogens 6:1-13.

[0047] 5. Chimeric Receptors Targeted by ILT Proteins (i.e., "Chimeric ILT Receptors" or "CIRs") The redirection of the cytotoxic specificity of T cells or NK cells can be controlled by the engagement of antigen-scFv (or TCR) interactions, but also by receptor-ligand pairing, such that the receptor of the target ligand can be formed into a chimeric protein that can maintain high affinity interaction with the cell while still allowing the transmission of signals to activate the immune cell (T cells and NK cells are used as examples below). Essential for the use of such receptors as binders of CAR-like proteins is a high affinity interaction with the target protein (less than 50 nM), allowing stable adhesion of the immune cell to the target and stable signaling to activate the T cells or NK cells. Moreover, in a highly preferred embodiment, the receptor or part of the receptor used to engage the target is specific only to the target to prevent extratumoral targeting. Third, as with scFv and similar binders, it is essential that a high level of expression of the target protein or ligand is maintained on the target tissue (e.g., tumor) compared to normal tissue.

[0048] (i) Targeting area The extracellular domains of ILT2 (D1-D4) (e.g., including D1-D4 plus the transmembrane region of ILT2, see SEQ ID NO: 31) can be engineered to target HLA-G expressing tumor cells and generate transduction of activation signals 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 activation signals (e.g., the ICDs of CD3ζ and 4-1BB).

[0049] Similarly, the extracellular domains (D1-D4) of ILT4 (e.g., including the transmembrane region of ILT4 in addition to D1-D4, see SEQ ID NO:55) can be engineered to generate activation signals in immune cells by replacing the ILT4 ICD with an activation signaling moiety (e.g., the ICDs of CD3ζ and 4-1BB). Using the D1-D4 extracellular domains of ILT2 or ILT4 in the targeting region generates 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 FIG. 4A). Thus, in some cases, the targeting region of the subject chimeric ILT receptor (CIR) includes an ILT2 or ILT4 D1-D4 domain (thus targeting HLA-G).

[0050] ILT4 maintains more contacts with α3 on the HLA heavy chain and can interact with the free heavy chain form of HLA-G, whereas ILT2 D1 / D2 requires contacts with β2-M and α3 to maintain interaction with HLA-G (Figure 4B).

[0051] ILT2 and ILT4 bind dimeric HLA-G and low affinity (K D has a high affinity (K of 1 μM) for interactions with monomeric HLA-G and other MHC-I proteins, including CD1d. DSpecificity for tumors carrying high levels of HLA-G is achieved by proportionally increasing the amount of HLA-G in dimeric form to maintain the interaction at low nM, allowing selection of tumor tissue over normal tissue that expresses 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 dimerization of HLA-G1 and significantly reduces targeting by ILT2 or ILT4 CIR-T or CIR-NK cells.

[0052] The inventors recognize that HLA-G exists in several different isoforms. A CAR containing an antibody-based targeting region (such as scFv) would be able to target only the HLA-G isoform that contains the epitope targeted by the antigen-binding region (e.g., scFv). This may result in the selection of tumor cells expressing HLA-G isoforms that lack the targeted epitope, thus allowing the cancer to evade treatment. In contrast, the subject ILT2 or ILT4-based chimeric receptor protein (targeting HLA-G) should target many more, and possibly all, HLA-G isoforms, since ILT2 and ILT4 naturally bind to those isoforms.

[0053] Construction of ILT2 D1 / D2 CIR and ILT4 D1 / D2 CIR The D1 and D2 domains are sufficient for direct binding of ILT2 and ILT4 to HLA-G, while the D3 and D4 domains likely function as a scaffold to present 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 may be deleted from the ILT2 CIR or ILT4 CIR and maintain functional interaction via 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 morphology (see FIG. 4A). Thus, in some cases, the targeting region of a subject ILT2 or ILT4 chimeric receptor will comprise 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 cases, the targeting region will not comprise (i.e., lack) the D3-D4 domains.

[0054] In some embodiments, the targeting region of a subject ILT2 chimeric receptor (the region including the D1-D2 domains) comprises an amino acid sequence having 80% or more (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%) sequence identity to an ILT2 sequence set forth in any one of SEQ ID NOs: 37, 70, 71, and 72 (the sequences of which are as follows): MHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 37) MHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 70) PKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 71) PKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 72)

[0055] In some cases, the targeting region comprises an amino acid sequence having 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to a sequence set forth in any one of SEQ ID NOs: 37, 70, 71, and 72. In some cases, the targeting region comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to a sequence set forth in any one of SEQ ID NOs: 37, 70, 71, and 72. In some cases, the targeting region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 37, 70, 71, and 72.

[0056] In some cases, the targeting region of the ILT2 chimeric receptor (the region including the D1-D2 domain) comprises an amino acid sequence having 80% or more (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%) sequence identity to the sequence set forth as SEQ ID NO: 37. In some cases, the targeting region comprises an amino acid sequence having 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the sequence set forth as SEQ ID NO: 37. In some cases, the targeting region comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the sequence set forth as SEQ ID NO: 37. In some cases, the targeting region comprises an amino acid sequence set forth as SEQ ID NO: 37.

[0057] In some embodiments, the targeting region of a subject ILT4 chimeric receptor (the region including the D1-D2 domains) comprises an amino acid sequence having 80% or more (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%) sequence identity to an ILT4 sequence set forth in any one of SEQ ID NOs: 57, 74, and 75 (the sequences of which are as follows): MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPG (SEQ ID NO: 57) MTPIVTVLICLGLSLGPRTRVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPG (SEQ ID NO: 74) PKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPG (SEQ ID NO: 75)

[0058] In some cases, the targeting region comprises an amino acid sequence having 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to a sequence set forth in any one of SEQ ID NOs: 57, 74, and 75. In some cases, the targeting region comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to a sequence set forth in any one of SEQ ID NOs: 57, 74, and 75. In some cases, the targeting region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 57, 74, and 75.

[0059] In some cases, the targeting region of the ILT4 chimeric receptor (the region including the D1-D2 domain) comprises an amino acid sequence having 80% or more (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%) sequence identity to the sequence set forth as SEQ ID NO: 57. In some cases, the targeting region comprises an amino acid sequence having 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the sequence set forth as SEQ ID NO: 57. In some cases, the targeting region comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the sequence set forth as SEQ ID NO: 57. In some cases, the targeting region comprises an amino acid sequence set forth as SEQ ID NO: 57.

[0060] For any of the above embodiments discussed in this section, in some cases, the subject ILT2 or ILT4 chimeric receptors lack the D3 and D4 domains (i.e., lack the regions corresponding to the D3-D4 domains of ILT2 (SEQ ID NO: 73) or ILT4 (SEQ ID NO: 76), respectively). In the case of ILT2, the regions having the D3-D4 domains are as follows: PLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLT (sequence number 73). In the case of ILT4, the region containing the D3 to D4 domains is as follows: QPGPVMAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLEL (sequence number 76).

[0061] 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 to 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 to 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 to the sequence set forth in any one of SEQ ID NOs: 73 and 76.

[0062] In some embodiments, the linker may be fused as a chimera with the D1-D2 domains from ILT2 or ILT4 and the plasma membrane, functioning as a stalk replacing the D3 and D4 domains. In these embodiments, deletion of D3-D4 may prevent interaction of CIR with proteins other than HLA-G that interact with native ILT2 or ILT4 through the D3 or D4 domains, such as, for example, the interaction 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 cells expressing CIR to non-tumor tissues such as bone marrow stroma, myelin, and endothelium.

[0063] Stork Domain In the above embodiments, the replacement of D3-D4 can be with any protein or part of a protein that adequately presents ILT2 or ILT4 D1-D2 binders in the context of HLA-G expressed on a separate cell. In certain embodiments, a short polypeptide linker can form the bond between the transmembrane domain and the intracellular domain of the chimeric ILT receptor. Thus, the chimeric ILT receptor can further comprise a stalk (i.e., 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 domain away from the plasma membrane and towards the target protein HLA-G. For example, the stalk can be a sequence of amino acids that naturally associate with the selected transmembrane domain. In some embodiments, the chimeric ILT receptor comprises a CD8 transmembrane domain, and in certain embodiments, the chimeric ILT receptor comprises a CD8 transmembrane domain with additional amino acids in the extracellular portion of the transmembrane domain. In certain embodiments, the CAR comprises a CD8 transmembrane domain and a CD8 stalk. In a particular embodiment, the CD8 transmembrane domain comprises (or consists of) a sequence disclosed herein (see Tables 4-42). In another particular embodiment, the CD8 stalk comprises (or consists of) a sequence disclosed herein (see Tables 4-42). The chimeric ILT receptor may further comprise a region of amino acids between the transmembrane domain and the cytoplasmic domain that is naturally associated with the polypeptide from which the transmembrane domain is derived.

[0064] After transduction, the cells now express the CIR on their surface and, upon contact and ligation with HLA-G, signal via the CD3 zeta chain that induces cytotoxicity and cell activation. It is important that such domains have no affinity for other proteins, thereby causing potential mistargeting of immune cells expressing the CIR. Examples of such chimeric stalk moieties include, but are not limited to, the membrane proximal portion 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 domains 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 (see FIG. 5A).

[0065] For example, in some cases, the stalk domain of a subject CIR is selected from an ILT2, ILT4, CD28, CH2 / CH3, CH3, and CD8 stalk domain. VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQ (SEQ ID NO: 39), which includes the ILT2 stalk and TM domains; PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 107), which includes the CD8 stalk domain; PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR (SEQ ID NO: 43), which includes the CD8 stalk and TM; IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 106), which includes the CD28 stalk; IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 47), which includes the CD28 stalk and TM domains; VDKRVESKYGPPCPSCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL (SEQ ID NO: 98), which includes the CH2CH3 stalk; VDKRVESKYGPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL (SEQ ID NO: 102), which includes the CH3 stalk; DPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:51), which includes the CH2CH3 stalk and CD28 TM domain; VVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLFLILRHRRQ (SEQ ID NO:59), which includes the ILT4 stalk and TM domains.

[0066] In some embodiments, the stalk of a subject CIR domain comprises an amino acid sequence having 80% or more (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%) sequence identity to a 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 a subject CIR domain comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to a 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 a subject CIR domain comprises a stalk sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.

[0067] In some embodiments, the stalk of a subject CIR domain comprises an amino acid sequence having 80% or more (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%) sequence identity to the stalk sequence set forth in any one of SEQ ID NOs: 98, 102, 106, and 107. In some embodiments, the stalk of a subject CIR domain comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the stalk sequence set forth in any one of SEQ ID NOs: 98, 102, 106, and 107. In some embodiments, the stalk of a subject CIR domain comprises a stalk sequence set forth in any one of SEQ ID NOs: 98, 102, 106, and 107.

[0068] Interaction of D1 / D2 stalk-containing CIR with dimeric HLA-G will have the effect of dimerizing intracellular signaling domains, which in certain embodiments can stimulate activation of the ICD and cell signaling (Figure 5B).

[0069] Mutant forms of ILT2 D1 / D2 CIR or ILT4 D1 / D2 CIR In further embodiments, mutations may be made in the ILT2 or ILT4 D1 or D2 domains contained within the CIR to increase the specificity of the CIR for HLA-G over other potentially interacting proteins. For example, mutations may be made to encode an amino acid other than tyrosine at the naturally occurring amino acid 96 (Y96) of ILT4 (SEQ ID NO: 57) or the corresponding position of ILT2 (SEQ ID NO: 31) (e.g., Y96A). The effect of such mutant forms is to reduce potential interactions with ANGPTL2 and ANGPTL5 while retaining binding affinity for HLA-G. In yet another embodiment, similar mutations may be placed in the full length ILT4 CIR, including the D1-D4 domains, along with a mutation in domain D4 (position corresponding to tyrosine 394 (Y394) of SEQ ID NO: 55) to further destabilize interactions with ANGPTL2 and ANGPTL5 (e.g., Y394A mutation, see SEQ ID NO: 61). The corresponding position in ILT2 is tyrosine 395 (Y395) of SEQ ID NO: 31.

[0070] Thus, in some cases, a subject ILT4 CIR comprises a mutation at an amino acid position corresponding to Y96 in SEQ ID NO: 57 (e.g., Y96A). In some cases, a subject ILT2 CIR comprises a mutation at an amino acid position corresponding to Y96 in SEQ ID NO: 31 (e.g., Y96A). In some cases, a subject ILT4 CIR comprises a mutation at an amino acid position corresponding to Y394 in SEQ ID NO: 55 (e.g., Y394A). In some cases, a subject ILT2 CIR comprises a mutation at an amino acid position corresponding to Y395 in SEQ ID NO: 31 (e.g., Y395A). In some cases, a subject ILT4 CIR comprises a mutation at an amino acid position corresponding to Y96 in SEQ ID NO: 55 (e.g., Y96A) and a mutation at an amino acid position corresponding to Y394 in SEQ ID NO: 55 (e.g., Y394A) (e.g., Y96A / Y394A). In some cases, a subject ILT2 CIR includes a mutation at an amino acid position corresponding to Y96 in SEQ ID NO: 31 (e.g., Y96A) and a mutation at an amino acid position corresponding to Y395 in SEQ ID NO: 31 (e.g., Y395A) (e.g., Y96A / Y395A).

[0071] Other embodiments may similarly limit interactions with classical HLA proteins or CD1 while retaining binding to HLA-G. These mutations may be specific to the heavy chains of these HLAs or may replace interaction sites with the α3 domain bound to β2-M on the surface.

[0072] (ii) Transmembrane (TM) region The CIR may contain 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 an extracellular targeting region to an intracellular domain). Single-pass transmembrane regions are found in certain CD molecules, tyrosine kinase receptors, serine / threonine kinase receptors, TGFβ, BMP, activins, and phosphatases. Single-pass transmembrane regions often contain 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 is often followed by a transmembrane span that anchors the protein to the membrane. Multiple-pass transmembrane proteins include ion pumps, ion channels, and transporters, and contain two or more helices that span the membrane multiple times. All or substantially all of the multiple-pass transmembrane proteins are incorporated into the chimeric protein. The sequences of single-pass and multiple-pass transmembrane regions are known and can be selected for incorporation into the chimeric protein molecule.

[0073] 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 each other. In one embodiment, a transmembrane domain that is naturally associated with one of the domains of the CIR is used. In other embodiments, a transmembrane domain that is not naturally associated with one of the domains of the CIR is used. In some cases, the transmembrane domain may be selected or modified by amino acid substitution (e.g., typically changed to hydrophobic residues) to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins and minimize interactions with other members of the receptor complex.

[0074] A transmembrane (TM) domain can be, and can be used, a transmembrane region comprising, for example, the alpha, beta, or zeta chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD28, CD33, CD38, CD64, CD80, CD86, CD134, CD137, ILT2, HER2, ILT4, or CD154, or a functional variant thereof, such as one that retains a substantial portion of its structural (e.g., transmembrane) properties. 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.

[0075] Alternatively, in some instances, the transmembrane domain may be synthesized de novo, containing a majority of hydrophobic residues, such as, for example, leucine, isoleucine, phenylalanine, and valine. Suitable CD8 stalk, transmembrane, and CD3 zeta sequences for use in the present invention are disclosed in Tables 4-42.

[0076] For example, in some cases, the TM domain of a subject CIR is selected from the TM domains of 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). VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQ (SEQ ID NO: 39), which includes the ILT2 stalk and TM domains; IYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR (SEQ ID NO: 100), which contains the CD8 TM domain; PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR (SEQ ID NO: 43), which includes the CD8 stalk and TM domains; FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 104), which includes the CD28TM domain; IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 47), which includes the CD28 stalk and TM domains; DPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:51), which includes the CH2CH3 stalk and the CD28 TM domain; and VVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLFLILRHRRQ (SEQ ID NO:59), which includes the ILT4 stalk and TM domains.

[0077] In some embodiments, the TM domain of a subject CIR comprises an amino acid sequence having 80% or more (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%) sequence identity to 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 comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to 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 comprises 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.

[0078] In some embodiments, the stalk domain plus the TM domain (stalk / TM domain) of a subject CIR comprises an amino acid sequence having 80% or more (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%) sequence identity to 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 comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to 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 comprises an amino acid sequence having an amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.

[0079] In some embodiments, the TM domain of a subject CIR comprises an amino acid sequence having 80% or more (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%) sequence identity to 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 comprises 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% sequence identity) to 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 comprises a TM domain sequence set forth in any one of SEQ ID NOs: 100 and 104.

[0080] (iii) Intracellular domain (ICD) As noted above, a subject chimeric ILT receptor (based on ILT2 or ILT4) comprises an intracellular region (intracellular domain or ICD) that replaces the natural intracellular portion of ILT2 or ILT4, which is inhibitory, with the ICD of an activating CAR. Thus, the ICD of a subject CIR (ILT2 version or ILT4 version) comprises a "signaling region" having at least one signaling domain that causes activation of the cell, and may optionally comprise a "co-stimulatory region" that may include one or more costimulatory domains.

[0081] Signal Transduction Region The "signaling region" (or "intracellular signaling domain") of a CIR refers to the portion of the CIR that is involved in transmitting a signal from CIR binding to a target molecule (HLA-G in the case of the subject CIR) to the interior of an immune effector cell to induce effector cell function (e.g., activation, cytokine production, proliferation, and / or cytotoxic activity, including release of cytotoxic factors to the CIR-bound target cell or other cellular response elicited by the target molecule binding to the extracellular CIR domain). Thus, the term "signaling region" ("intracellular signaling domain") refers to the portion of a protein that transmits an effector function signal and instructs a cell to perform a particular function. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the full-length intracellular signaling domain, so long as such a truncated portion transmits an effector function signal. The term signaling region is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal. In some cases, the signaling region includes a signaling motif known as an immunoreceptor tyrosine-based activation motif (or "ITAM").

[0082] Examples of intracellular domain sequences that may be used in the signaling region of a subject CIR include those derived from the intracellular signaling domains of lymphocyte receptor chains, TCR / CD3 complex proteins, Fc receptor subunits, IL-2 receptor subunits, CD3zeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, CD66d, CD278 (ICOS), FcsRl, DAP10, and DAP12.

[0083] In some embodiments, a signaling region of a subject CIR comprises a CD3 zeta (CD3ζ) signaling domain (see, e.g., SEQ ID NO: 33). Thus, in some cases, an ICD of a subject chimeric ILT receptor (ILT2 version or ILT4 version) comprises a signaling region that comprises an amino acid sequence having 80% or more (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%) sequence identity to SEQ ID NO: 33. In some embodiments, a signaling region comprises an amino acid sequence that has 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to SEQ ID NO: 33. In some embodiments, a signaling region comprises an amino acid sequence that has 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to SEQ ID NO: 33. In some embodiments, the signaling region comprises the amino acid sequence set forth as SEQ ID NO:33.

[0084] In some embodiments, the signaling region of a subject CIR comprises a DAP10 signaling domain. In some embodiments, the signaling region of a subject CIR comprises a DAP12 signaling domain.

[0085] costimulation area In some embodiments, the ICD of a subject CIR also comprises a costimulatory region. The costimulatory region comprises at least one costimulatory domain (e.g., 1, 2, 3, more than 1, more than 2, or more than 3 costimulatory domains). Examples of costimulatory domains include, but are not limited to, CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, DAP10, 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 comprises one or more (e.g., 1, 2, 3, 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 are used (i.e., they are both used). In some cases, a CD28 costimulatory domain and an OX40 costimulatory domain are used.

[0086] In some cases, the costimulatory region comprises a truncated MyD88 polypeptide fused to a signaling domain of a receptor mediator of costimulation, such as, for example, CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, or ICOS. In some cases, the costimulatory region comprises 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.

[0087] In some embodiments, the ICD of a subject chimeric ILT receptor (ILT2 version or ILT4 version) comprises a costimulatory region comprising an amino acid sequence having 80% or more (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%) sequence identity to SEQ ID NO:49. In some embodiments, the signaling region comprises an amino acid sequence having 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to SEQ ID NO:49. In some embodiments, the signaling region comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to SEQ ID NO:49. In some embodiments, the signaling region comprises the amino acid sequence set forth as SEQ ID NO:49.

[0088] In some embodiments, the ICD of a subject chimeric ILT receptor (ILT2 version or ILT4 version) comprises a costimulatory region comprising an amino acid sequence having 80% or more (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%) sequence identity to SEQ ID NO:35. In some embodiments, the signaling region comprises an amino acid sequence having 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to SEQ ID NO:35. In some embodiments, the signaling region comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to SEQ ID NO:35. In some embodiments, the signaling region comprises an amino acid sequence set forth as SEQ ID NO:35.

[0089] In some embodiments, the ICD of a subject chimeric ILT receptor (ILT2 version or ILT4 version) comprises a costimulatory region comprising an amino acid sequence having 80% or more (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%) sequence identity to number 35 and an amino acid sequence having 80% or more (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%) sequence identity to number 49. In some embodiments, the signaling region comprises an amino acid sequence having 90% or more sequence identity to number 35 (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) and an amino acid sequence having 90% or more sequence identity to number 49 (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the signaling region comprises an amino acid sequence having 95% or more sequence identity to number 35 (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) and an amino acid sequence having 95% or more sequence identity to number 49 (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the signaling region comprises an amino acid sequence set forth in SEQ ID NO:35 and an amino acid sequence set forth in SEQ ID NO:49.

[0090] In some embodiments, the signaling region of a subject CIR comprises a CD3 zeta (CD3ζ) signaling domain and the costimulatory region comprises a CD28 costimulatory domain. In some embodiments, the signaling region of a subject CIR comprises a CD3 zeta (CD3ζ) signaling domain and the costimulatory region comprises a 4-1BB costimulatory domain. In some embodiments, the signaling region of a subject CIR comprises a CD3 zeta (CD3ζ) signaling domain and the costimulatory region comprises a 4-1BB costimulatory domain and a CD28 costimulatory domain. In some embodiments, the signaling region of a subject CIR comprises a CD3 zeta (CD3ζ) signaling domain and the costimulatory region comprises a CD28 costimulatory domain and an OX40 costimulatory domain.

[0091] Non-limiting examples of 4-1BB, CD28, and OX40 costimulatory signaling domains are described in US2013 / 0266551, U.S. Patent No. 5,686,281, Geiger, TL 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 NM et al., J Immunol 169:5780-5786(2002), Haynes NM et al., Blood 100:3155-3163 (2002), US2012 / 20148552, all of which are incorporated by reference herein for their teachings related to costimulatory domains.

[0092] 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 activity or functional assays, 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, U.S. Patent No. 6,223,236, US2007 / 0209836 ... No. 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 in its entirety, including all text, tables, and figures, for all purposes, including those related to the description of cell activation domains (e.g., cell signaling domains and costimulatory domains).

[0093] Example CIR The different regions / domains of the subject CIRs are modular and can be conveniently combined. Figures 6A and 10 provide examples of the various forms that the subject CIRs can take, and those shown in the figures have been generated and tested in various assays throughout the Examples section. For example, in Figure 6A, formats CIR1, CIR2, CIR3, and CIR4 are introduced, and in Figure 10, formats CIR6, CIR7, CIR8, CIR9, and CIR10 are introduced. Figure 10 shows only these formats for ILT4, but one skilled in the art will understand that ILT2 CIRs can be generated using the same formats, if desired.

[0094] In some cases, a subject CIR comprises an ILT2 CIR4 or an ILT4 CIR4 (i.e., an ILT2 or ILT4 D1-D2 targeting domain, a CD8 stalk and TM, a CD3 zeta (CD3ζ) signaling domain, and a 4-1BB costimulatory domain (the T2A-delta-CD19 region is optional, i.e., absent in some cases). See, e.g., FIG. 6A.

[0095] In some cases, a subject CIR comprises an ILT2 CIR3 or an ILT4 CIR3 (i.e., an ILT2 or ILT4 D1-D4 targeting domain, a CD8 stalk and TM, a CD3 zeta (CD3ζ) signaling domain, and a 4-1BB costimulatory domain (the T2A-delta-CD19 region is optional, i.e., absent in some cases). See FIG. 6A.

[0096] In some cases, a subject CIR comprises an ILT2 CIR2 or an ILT4 CIR2 (i.e., an ILT2 or ILT4 D1-D2 targeting domain, an ILT2 or ILT4 stalk and TM, a CD3 zeta (CD3ζ) signaling domain, and a 4-1BB costimulatory domain (the T2A-delta-CD19 region is optional, i.e., absent in some cases). See FIG. 6A.

[0097] In some cases, a subject CIR comprises an ILT2 CIR1 or an ILT4 CIR1 (i.e., an ILT2 or ILT4 D1-D4 targeting domain, an ILT2 or ILT4 stalk and TM, a CD3 zeta (CD3ζ) signaling domain, and a 4-1BB costimulatory domain (the T2A-delta-CD19 region is optional, i.e., absent in some cases). See FIG. 6A.

[0098] CIR Variations The scope of the present invention includes functional portions of the CIRs of the present invention described herein. The term "functional portion", when used in reference to a CIR, refers to any portion or fragment of a CIR of the present invention, which portion or fragment retains the biological activity of the CIR of which it is a part (the parent CIR). A functional portion encompasses, for example, a portion of a CIR that retains the ability to recognize a target (HLA-G) or target cell, or to detect, treat, or prevent a disease, to a similar extent, to the same extent, or to a greater extent than the parent CIR. With respect to the parent CIR, a functional portion can include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CIR.

[0099] A functional portion can include additional amino acids at the amino or carboxy terminus, or both, of the portion, that are not found in the amino acid sequence of the parent CIR. Desirably, the additional amino acids do not interfere with a biological function, such as target cell recognition, cancer detection, cancer treatment, or prevention, etc. More desirably, the additional amino acids enhance a biological activity compared to the biological activity of the parent CIR.

[0100] The scope of the present invention includes functional variants or biological equivalents of the CIRs of the present invention disclosed herein. A functional variant may, for example, comprise the amino acid sequence of a parent polypeptide with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant may comprise the amino acid sequence of a parent polypeptide with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does 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 compared to the parent polypeptide.

[0101] Such biological variants (including functional portions thereof) may contain synthetic amino acids in place of one or more naturally occurring amino acids.

[0102] Such biological variants (including functional portions thereof) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0103] Such biological variants (including functional parts thereof) can be obtained by methods known in the art. Polypeptides can be produced by any suitable method of producing polypeptides or proteins. Suitable methods for synthesizing polypeptides and proteins de novo are described, for example, in Chan et al., Fmoc 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. Patent No. 5,449,752. Polypeptides and proteins can also be produced recombinantly using the nucleic acids described herein using standard recombinant methods. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

[0104] 6. Costimulatory Polypeptides A genetically modified cell expressing a subject chimeric ILT receptor (CIR) may further express a costimulatory polypeptide (e.g., in addition to expressing the CIR). Expression of the costimulatory polypeptide may be inducible or constitutive.

[0105] A costimulatory polypeptide can include one or more costimulatory signaling regions, such as CD27, ICOS, RANK, IL-18R, DAP12, HVEM, TRANCE, CD28, 4-1BB, IL-1R, OX40, DAP10, IL-33R, MyD88, or CD40, or, for example, a cytoplasmic region thereof. A costimulatory polypeptide can include one or more suitable costimulatory signaling regions that activate a signaling pathway activated by CD27, ICOS, RANK, IL-18R, DAP-12, HVEM, TRANCE, CD28, 4-1BB, IL-1R, OX40, DAP10, IL-33R, MyD88, or CD40. Costimulatory polypeptides include any molecule or polypeptide that activates the NF-κB pathway, the MyD88 pathway, the STAT5 pathway, the STAT1 pathway, the Akt pathway, and / or the p38 pathway of the tumor necrosis factor receptor (TNFR) family (i.e., CD40, RANK / TRANCE-R, OX40, 4-1BB), and CD28 family members (CD28, ICOS). Multiple costimulatory polypeptides or cytoplasmic domains of costimulatory polypeptides can be expressed in the engineered cells.

[0106] Suitable CD28, OX40, 4-1BB, and ICOS sequences for use with the present invention are disclosed in Tables 4d-36.

[0107] The cells can contain chimeric signaling polypeptides, including, for example, chimeric signaling polypeptides in which a truncated MyD88 polypeptide is fused to a signaling domain of a costimulatory receptor mediator, such as, for example, CD40, CD27, CD28, 4-1BB, OX40, or ICOS.

[0108] 7. Safety switch The genetically modified cells expressing the subject chimeric ILT receptors (CIRs) can also express a safety switch, also known as an inducible suicide gene or suicide switch. This can be used to eradicate the therapeutic cells in vivo, if necessary, for example, when GvHD develops. In some instances, the therapeutic cells may induce adverse events, such as off-target toxicity due to the CIR, or the patient may experience negative symptoms during treatment using the modified cells, or there may be side effects due to non-specific attack on healthy tissue, or sometimes the therapeutic cells may no longer be needed, or the therapy is intended for a certain period of time, for example, the therapeutic cells may function to reduce tumor cells or tumor size, and may no longer be needed. Thus, it may be useful if the genetically modified cells can also inducibly express a polypeptide that kills cells, such as an inducible caspase 9 polypeptide. For example, the switch can be induced when the number of therapeutic cells needs to be reduced.

[0109] These switches are delivered when it is desired to eradicate therapeutic cells and respond to inducers, such as pharmacological agents, that cause cell death (e.g., cause necrosis or apoptosis). These agents may result in de novo expression of toxic gene products, but a more rapid response can be obtained if the genetically modified cells already express proteins that are switched to a toxic form in response to the agent.

[0110] In some embodiments, the safety switch is based on a pro-apoptotic protein that can be triggered by administering a triggering molecule (also referred to as a ligand inducer) to a subject. If the pro-apoptotic protein is fused to a polypeptide sequence that binds to the triggering molecule, delivery of the triggering molecule can bring the two pro-apoptotic proteins into close proximity, thereby inducing apoptosis. For example, caspase 9 can be fused to a modified human FK binding protein that can be induced to dimerize in response to the pharmacological agent rimiduside (AP1903). For example, the use of a safety switch based on a human pro-apoptotic protein, such as caspase 9, minimizes the risk that cells expressing the switch will be recognized as foreign by the immune system of a human subject. Thus, delivery of rimiduside to a subject can induce apoptosis of cells expressing the caspase 9 switch.

[0111] Further non-limiting examples of chimeric polypeptides useful for inducing cell death or apoptosis can be found in the following patents and patent applications (each of which is incorporated by reference herein in its entirety for all purposes): US Patent Application US2011 / 0286980, US Patent 9,089,520, US Patent Application US2014 / 0255360, US 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 in its entirety, including all text, tables, and figures, for all purposes). Details of some specific switches and approaches are also provided below.

[0112] Inducible caspase 9 (iC9)This pro-apoptotic switch comprises a fusion of caspase 9 with FKBP12 or a derivative. It drives dimerization of the initiator caspase, caspase 9, from the intrinsic pathway of apoptosis of cells, which is latent in the absence of ligand. Dimerization leads to activation of caspase 9, cleavage and activation of the effector caspase, caspase-3, and rapid cell death by apoptosis. Inducible caspase 9 is particularly useful as a safety switch in cell therapy to block toxic responses.

[0113] Caspase-9 switch 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, U.S. Patent No. 9,434,935, U.S. Patent No. 9,913,882, U.S. Patent No. 9,393,292, and U.S. Patent Application 2015 / 0328292.

[0114] The safety switch can include a modified caspase 9 polypeptide having altered activity, e.g., reduced basal activity in the absence of a homodimeric ligand. Modified caspase 9 polypeptides are described, e.g., in U.S. Pat. No. 9,913,882 and U.S. Pat. No. 2015 / 0328292, supra, and can include, e.g., an amino acid substitution at position 330 (e.g., D330E or D330A) or, e.g., an amino acid substitution at position 450 (e.g., N405Q), or combinations thereof (e.g., including D330E-N405Q and D330A-N405Q). Caspase 9 polypeptides with low basal activity have been previously described, for example, in U.S. Pat. Nos. 9,434,935, 9,932,572, and 9,913,882, and U.S. patent application Ser. Nos. 62 / 668,223, 62 / 756,442, 62 / 816,799, 15 / 901,556, and 15 / 888,948.

[0115] In some embodiments, the safety switch can be, for example, iCasp9, discussed in Di Stasi et al. (2011) supra, which consists of the sequence of human FK506 binding protein (FKBP12) (GenBank AH002 818) with an F36V mutation connected via a SGGGS linker to a modified human caspase 9 (CASP9) that lacks its endogenous caspase activation and recruitment domains. The F36V mutation increases the binding affinity of FKBP12 to the synthetic homodimers AP20187 and rimducaside.

[0116] FKBP12 allele-specific binding by rimiduside Rimiducide binds with high affinity (approximately 0.1 nM) to the valine 36 allele of FKBP12, but with low affinity (approximately 500 nM) to the wild-type phenylalanine 36 FKBP12 allele. Rapamycin and rapalogs can bind to either FKBP allele. Rimiducide binds to the valine 36 form (FKBP12(F36V), FKBP12v36, FKBPV, F V36 , or simply F vTwo identical protein-binding surfaces are arranged in opposite orientation, each with high affinity and specificity for the rimiducide (variously known as rimiducide). 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 can also be used in the construct, such that higher order oligomers are induced upon cross-linking with rimiduside. One or more Fs can be linked to one or more cell signaling molecules that normally rely on homodimerization. V Binding the domains can convert the protein into a rimiduside-controlled switch. FKBP12 variants can also be used. The variants can bind to rapamycin or rapalogs, but have a lower affinity for rimiduside 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 (calstabulin).

[0117] The suicide switch may be controlled by a pharmaceutical composition containing a triggering molecule (such as a dimerization or multimerization ligand). An effective amount of a pharmaceutical composition containing a triggering molecule is an amount that achieves the desired result of killing the genetically modified cells. The degree of killing may be high (e.g., more than 60%, 70%, 80%, 85%, 90%, 95%, or 97%) or complete; conversely, sometimes only partial removal (e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the genetically modified cells are killed) may be desired. Thus, genetic modifications may exhibit different sensitivities to the triggering molecule. Thus, triggering molecules can be used to eradicate only some cells (e.g., at least 10%) while allowing some cells (e.g., at least 10%) to survive. The concentration of the inducer molecule can be selected according to the desired balance of cell life and death, eg, a higher concentration is delivered if eradication (or complete eradication) of a higher percentage of cells is desired.

[0118] These concentrations can be determined by simple dose range experiments that monitor the level of cell death in response to the triggering molecule. Any suitable assay can be used to determine the percentage of genetically modified cells that are killed. The assay can include obtaining a first sample from the subject before administration of the triggering molecule, obtaining a second sample from the subject after administration of the triggering molecule, and comparing the number or concentration of therapeutic cells in the first sample and the second sample to determine the percentage of therapeutic cells that are killed. The effective amount of a particular composition presented herein can be empirically determined without undue experimentation.

[0119] The cells can contain chimeric signaling polypeptides, including, for example, chimeric signaling polypeptides in which a truncated MyD88 polypeptide is fused to a signaling domain of a costimulatory receptor mediator, such as, for example, CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, or ICOS.

[0120] In some embodiments, a chimeric signaling polypeptide comprises a cytoplasmic signaling region from two costimulatory polypeptides, e.g., 4-1BB and CD28, or one or more costimulatory polypeptide cytoplasmic signaling regions selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40, DAP10. In some embodiments, a 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, and DAP10.

[0121] As noted above, 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 activity or functional assays, may also be found in the following patents and patent applications (each of which is incorporated by reference herein in its entirety for all purposes): US2014-0286987-A1, WO2014 / 151960, US2016 / 0046700, WO2015 / 123527, US2004 / 0209836, U.S. Pat. No. 7,404,950, WO2004 / 073363, U.S. Pat. No. 6,223,950, and WO2004 / 073363. No. 641, US2011 / 0033388, U.S. Pat. No. 8,691,210, WO2008 / 049113, US2014 / 0087468, U.S. Pat. No. 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 in its entirety, including all text, tables, and figures, for all purposes.

[0122] In some embodiments, the cells are engineered to provide a constitutively active therapy. In some embodiments, the 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 located 5' of the first polynucleotide. In some embodiments, the second polynucleotide is located 3' of the first polynucleotide. In some embodiments, a third polynucleotide encoding a linker polypeptide is located between the first and second polynucleotides. When the third polynucleotide is located 3' of the first polynucleotide and 5' of the second polynucleotide, the linker polypeptide may remain intact after 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) that can separate the polypeptides encoded by the first and second polynucleotides during or after translation. High levels of costimulation are constitutively provided through an alternative mechanism that segregates the CIR from the chimeric signaling polypeptide using a leaky 2A cotranslational sequence (see elsewhere herein). If segregation of 2A (e.g., from the leaky 2A sequence) is incomplete, the majority of the expressed chimeric signaling polypeptide molecule may be separated from the chimeric antigen receptor polypeptide and remain in the cytoplasm, with some portions or chimeric signaling polypeptide molecules remaining bound or linked to the CIR.

[0123] "Constitutively active" means that the cell activation activity of the chimeric stimulatory polypeptide is active even in the absence of an inducer. One way to generate constitutively active signaling is to tether an activator protein factor to the plasma membrane via a transmembrane domain or lipid targeting moiety.

[0124] Immune cell therapies can also be designed to provide a constitutively active therapy, such as constitutively active T cells or NK cells, but provide an inducible safety switch to stop therapy or reduce levels if needed (see above). In some embodiments, immune cells, such as CIR-T cells or CIR-NK cells, express a chimeric antigen receptor and a chimeric signaling polypeptide.

[0125] 8. Linker Polypeptides If it is desired that the two polypeptides are encoded in a single gene such that they are encoded in a single transcript, the two polypeptides may be linked by a linker polypeptide. For example, these may be included between MyD88 and CD40 in a MyD88-CD40 chimeric polypeptide, or between a costimulatory polypeptide cytoplasmic signaling region and the CD3ζ portion of a CAR or CIR. Optionally, the linker may be located between any of the regions / domains described herein. For example, in some cases, the linker is placed between the TM domain and the signaling or costimulatory region, between an ILT2 or ILT4 targeting region (e.g., D1-D2 domains) and the stalk, between a signaling region and a costimulatory region, between two costimulatory domains, between a costimulatory or signaling region and a T2A sequence, or any combination thereof.

[0126] 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 are known to those skilled in the art, and any convenient linker may be used.

[0127] The linker polypeptide may be, for example, a polypeptide having about 2 to about 30 amino acids (e.g., a furin cleavage site (GGGGS) nIn 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 18-22 amino acids. In some embodiments, the linker polypeptide consists of 20 amino acids.

[0128] Cleavable linkers include linkers that are cleaved by an enzyme in the engineered cell. The enzyme may be exogenous to the cell. For example, the enzyme is encoded by a polynucleotide that is introduced into the cell by transfection or transduction, either at the same time or at a different time as the polynucleotide encoding the linker. In some embodiments, cleavable linkers include linkers that are cleaved by an enzyme endogenous to the engineered cells in the population, including, for example, an enzyme naturally expressed in the cell and an enzyme encoded by a polynucleotide native to the cell (e.g., lysozyme, etc.). The term "cleavable linker" also extends to linkers that are cleaved by any means, including, for example, non-enzymatic means such as peptide skipping.

[0129] One advantage of a cleavable linker is that it allows for an essentially fixed stoichiometry of expression of the two polypeptides (a 1:1 ratio when the two mature polypeptides are linked by a single cleavable linker).

[0130] The linker polypeptide may be a 2A-like sequence, which may be derived from many different viruses, including, for example, Thosea asigna insect virus. These sequences are sometimes 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 the peptide bond, and in the case of the Thosea asigna sequence, the bond between the Gly and Pro amino acids at the carboxy-terminal "PGP" 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 encoded 5' of the 2A sequence may end with additional amino acids at the carboxy-terminus, including a Gly residue and any upstream residues in the 2A sequence. The peptide encoded 3' of the 2A sequence may end with additional amino acids at the amino-terminus, including a Pro residue and any downstream residues following the 2A sequence.

[0131] 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 (Tosea asignavirus 2A), F2A (Foot and Mouth Disease virus 2A), P2A (porcine teschovirus-1 2A), BmCPV 2A (cytoplasmic polyhedrosis virus 2A), BmIFV 2A (B. mori flacherie virus 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 certain embodiments, the 2TA comprises (or consists of) a sequence disclosed herein. It comprises (or consists of) a sequence disclosed herein (eg, a sequence disclosed in the Examples below).

[0132] 2A-like sequences are sometimes "leaky" in that some of the polypeptides are not separated during translation, but instead remain as one long polypeptide after translation. One theory on the cause of leaky linkers is that short 2A sequences may not fold into the necessary structure that promotes ribosome skipping ("2A fold"). In these cases, the ribosome does not miss the proline peptide bond, and then a fusion protein results. To reduce the leaky level and therefore the number of fusion proteins formed, a GSG (or similar) linker may be added to the amino-terminal side of the 2A polypeptide. This GSG linker blocks the secondary structure of the newly translated polypeptide from spontaneously folding and disrupting the "2A fold". For example, a leaky 2A sequence can be used so that the same encoded polypeptide can be directed to the cell surface but otherwise remains in the cytoplasm.

[0133] In certain embodiments, the 2A linker comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the 2A linker further comprises a GSG amino acid sequence at the amino terminus of the polypeptide, and in other embodiments, the 2A linker comprises a GSGPR (SEQ ID NO: 68) amino acid sequence at the amino terminus of the polypeptide. Thus, by "2A" sequence, the term may refer to the 2A sequences in the examples described herein, or may refer to the 2A sequences listed herein that further comprise a GSG or GSGPR (SEQ ID NO: 68) sequence at the amino terminus of the linker.

[0134] In some embodiments, the linker, e.g., a 2A linker, is cleaved at about 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% of the translated polypeptide.

[0135] Suitable linker polypeptides (including T2A linkers) for use in the present invention are disclosed in Tables 4-42 (including the "flex" linker polypeptide sequences).

[0136] 9. Membrane targeting sequences The membrane targeting sequence provides for transport of the chimeric protein to the cell surface membrane. Molecules that associate with the cell membrane contain certain regions that promote membrane association, and such regions can be incorporated into the chimeric protein molecule to generate membrane-targeted molecules. In some embodiments, the CIR can be transported to the cell surface through signal-dependent translation into the lumen of the endoplasmic reticulum via the native signal peptide of ILT2 or ILT4. In other embodiments, this native signal peptide can be replaced by that of other secreted proteins. In these embodiments, the signal peptide can be derived from GM-CSF or immunoglobulin light chain, but is not limited to being derived from these proteins.

[0137] For example, some proteins contain sequences at the acylated N- or C-terminus, and these acyl moieties promote membrane association. Such sequences are recognized by acyltransferases and often fit specific sequence motifs. Certain acylation motifs can be modified with a single acyl moiety (often followed by several positively charged residues to improve association with anionic lipid head groups (e.g., human c-Src: MGSNKSKPKDASQRRR, SEQ ID NO: 69)), while other acylation motifs can be modified with multiple acyl moieties. For example, the N-terminal sequence of protein tyrosine kinase Src can contain a single myristoyl moiety. Dual acylation regions are located within the N-terminal regions of certain protein kinases (e.g., a subset of Src family members (e.g., Yes, Fyn, Lck)) and G protein alpha subunits. Such dual acylation regions are often located within the first 18 amino acids of such proteins and conform to the sequence motif Met-Gly-Cys-Xaa-Cys, where Met is truncated, Gly is N-acylated, and one of the Cys residues is S-acylated. Often, Gly is myristoylated and Cys can be palmitoylated. Acylation regions conforming to the sequence motif Cys-Ala-Ala-Xaa (the so-called "CAAX box") can be modified with C15 or C10 isoprenyl moieties from the C-terminus of G protein gamma subunits and other proteins (e.g., https: / / www.ebi.ac.uk / interpro / entry / InterPro / IPR031771 / or https: / / prosite.expasy.org / PS00294) and can also be used. These and other acylation motifs include those discussed in, for example, Gauthier-Campbell et al., Molecular Biology of the Cell 15:2205-2217 (2004), Glabati et al., Biochem. J. 303:697-700 (1994), and Zlakine et al., J. Cell Science 110:673-679 (1997), and can be incorporated into chimeric molecules to direct membrane localization.

[0138] In some embodiments, the membrane targeting region comprises a myristoylation region. In some embodiments, the membrane targeting region is selected from the group consisting of a myristoylation targeting sequence from a receptor, a palmitoylation targeting sequence, a prenylation sequence (i.e., farnesylation, geranyl-geranylation, CAAX box), a protein-protein interaction motif, or a transmembrane sequence (using a signal peptide). Examples include those discussed in, for example, ten Klooster et al, Biology of the Cell (2007) 99, 1-12, or Vincent et al., Nature Biotechnology 21:936-40, 1098 (2003).

[0139] When a polypeptide does not include a membrane targeting region or lacks a membrane targeting region, such as certain chimeric polypeptides provided herein, the polypeptide does not include a region that provides transport of the chimeric protein to the cell membrane.The polypeptide may, for example, not include a sequence that transports the polypeptide to the cell surface membrane, or the polypeptide may, for example, include a dysfunctional membrane targeting region that does not transport the polypeptide to the cell surface membrane (e.g., a myristoylation region that includes a proline that destroys the function of the myristoylation targeting region).(See, for example, Resh, MD, Biochim. Biophys. Acta. 1451:1-16 (1999)).Polypeptides that are not transported to the membrane are considered to be cytoplasmic polypeptides.

[0140] Myristoylation sequences suitable for use in the present invention are disclosed in Tables 4-42.

[0141] 10. Genetically modified cells The genetically modified cell (e.g., a cell such as an immune cell that expresses a subject chimeric ILT receptor (including an encoding nucleic acid)) can be any cell useful for cell therapy, e.g., an immune cell. The cell can be, for example, a T cell, a natural killer (NK) cell, a B cell, a macrophage, a peripheral blood cell, a hematopoietic progenitor cell, or a bone marrow cell. In a preferred embodiment, the modified cell is a T cell, a natural killer cell, or a natural killer T cell.

[0142] Cells that have been genetically modified as disclosed herein (e.g., cells such as immune cells that express a subject chimeric ILT receptor (e.g., comprising an encoding nucleic acid)) are useful for administration to subjects who may benefit from their administration (e.g., who may benefit from the administration of donor lymphocytes). These subjects are typically humans, and as such, the invention will typically be carried out using human cells.

[0143] Cell Source The cells to be genetically modified can be autologous, syngeneic, or allogeneic. Allogeneic cells can be derived from any healthy donor, and syngeneic cells can be derived from any healthy donor that is appropriately related to the intended recipient. Donors are generally adults (at least 18 years old), although children are also suitable as cell donors (see, for example, Styczynski 2018, Transfus Apher Sci 57(3):323-330).

[0144] The term "autologous" refers to cells derived from the same individual to whom they are subsequently administered. The term "allogeneic" refers to HLA or MHC loci that are antigenically different between host and donor cells. Thus, cells from the same species can be antigenically different. The term "syngeneic" refers to cells that have sufficiently identical or closely related genotypes or are immunologically compatible to allow tissue transplantation. For example, identical twins or close relatives can be syngeneic.

[0145] 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. As used herein, the term "peripheral blood" refers to the cellular components of blood (e.g., red blood cells, white blood cells, and platelets) that are obtained or prepared from the circulating pool of blood and are not sequestered in the lymphatic system, spleen, liver, or bone marrow. Umbilical cord blood refers to the blood that remains in the placenta and attached umbilical cord after birth, unlike peripheral blood and blood that is sequestered in the lymphatic system, spleen, liver, or bone marrow. Umbilical cord blood often contains stem cells, including hematopoietic cells.

[0146] A suitable process for obtaining T cells from humans is described in the published protocol accompanying Di Stasi et al. (2011) N Engl J Med 365:1673-83 ("Protocol"). Generally speaking, T cells can be obtained, subjected to genetic modification and selection, and then administered to a recipient subject. A useful source of T cells is human peripheral blood. A peripheral blood sample will generally be subjected to leukopheresis to provide a sample enriched for white blood cells. This enriched sample (also known as a leukopack) can be composed of various blood cells including monocytes, lymphocytes, platelets, plasma, and red blood cells. Leukopacks typically contain a higher concentration of cells compared to venipuncture or buffy coat products.

[0147] Although the sample may be subjected to allodepletion (as discussed in the protocol), it is preferred that the sample is not subjected to allodepletion. Thus, the preferred sample is alloreplete, as discussed in Zhou et al. (2015) Blood 125:4103-13. These populations can provide a more robust T cell repertoire for therapeutic purposes. Thus, the preferred composition of the present invention is not T cell allodepleted and has not been subjected to a step of allodepletion.

[0148] T cells are generally cultured (usually under activating conditions, e.g., using anti-CD3 and / or anti-CD28 antibodies, optionally with IL-2) before being genetically modified. This step results in a higher yield of T cells at the end of the modification process.

[0149] Processes for obtaining and expanding NK cells from humans are 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.

[0150] CD4+ and CD8+ T cells The subject compositions may comprise CD4+ T cells and CD8+ T cells. The ratio of CD4+ cells to CD8+ cells in a leukopack is typically greater than 2, but in some embodiments, the ratio of genetically modified CD4+ cells to genetically modified CD8+ cells in the compositions of the invention is less than 2, e.g., less than 1.5. Ideally, there are more genetically modified CD8+ T cells in the composition than genetically modified CD4+ T cells, 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 less than 0.5. Thus, starting from donor cells, the overall procedure for producing genetically modified T cells ideally enriches for CD8+ T cells compared to CD4+ T cells. Preferably, at least 60% of the genetically modified T cells are CD8+ T cells, more preferably at least 65%. Within a population of genetically modified CD3+ T cells, a preferred range for CD8+ T cells is 55-75% (e.g., 63-73%). The percentage of CD8+ and CD4+ T cells can be readily assessed by flow cytometry, and methods for sorting and enumerating CD4+ and CD8+ T cells are conventional in the art.

[0151] Memory T cell subsets (see Mahnke et al. (2013) Eur J Immunol 43:2797-809) The population of genetically modified T cells may 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 naive T cells (defined as CD45RA+CD45RO-CCR7+ cells). These cells can be assessed by flow cytometry using CD45RA / RO and CCR7 markers. Labeling reagents that recognize CCR7 and can distinguish between CD45RA and CD45RO isoforms are readily available from commercial suppliers.

[0152] An average leukopack typically contains about 20% each of terminal effector cells and T effector memory cells. The entire process from donor cells to genetically modified T cells may enrich for terminal effector memory T cells compared to T effector memory cells.

[0153] In some embodiments, less than 60% of the genetically modified T cells are naive T cells, e.g., less than 58%, preferably less than 55%, more preferably less than 50%. Within a population of genetically modified CD3+ T cells, the preferred range of naive T cells is 30-60%, more preferably 42-49%, most preferably 43-46%. This percentage of naive T cells has been found to correlate with a good outcome in the T cell recipient. Naive EM cells can be assessed by flow cytometry using CD45RA / RO and CCR7 markers.

[0154] Within a population of genetically modified T cells, the proportion of T central memory cells, in addition to TEMRA, EM, and naive T cells, is generally less than 10%.

[0155] In some embodiments, the population of genetically modified T cells in the 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 may 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.

[0156] NK cells NK cells, also known as natural killer cells or large granular lymphocytes (LGLs), are cytotoxic lymphocytes important to the innate immune system. The role of NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virus-infected cells and respond to tumor formation.

[0157] T cells rely on a priming interaction between the T cell receptor (TCR) on a target cell and the MHC-peptide complex as the first step required for T cell activation. As a result, T cells can recognize a single antigen, and tumor cells can evade T cell recognition by mutations that greatly reduce antigen presentation. In contrast, NK cells can recognize a large number of transformed and infected cells without relying on the presentation of a single antigen. Thus, treatment with NK cells can bypass some of the resistance mechanisms to T cell-based therapy.

[0158] 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, to generate a second wave of sustained anti-tumor responses. 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.

[0159] NK cells may be useful as a source of antigen or receptor-based directed cell therapy due to their innate cytotoxicity mechanisms. NK cells comprise approximately 10-15% of lymphocytes in the peripheral blood of a typical donor and can be easily purified, expanded, and virally transduced. When targets for directed cell therapy to cells within a tumor (e.g., HLA-G) are lost, activated NK cells have alternative innate mechanisms for directing cytotoxic function, including NKG2D, p46, p44, p30, DNAM, and CD16.

[0160] Genetic modification of cells Cells are genetically modified by introducing into them an expression construct (encoding a subject chimeric ILT receptor). Such introduction can employ viral or non-viral methods of gene transfer. This section describes gene transfer methods and compositions.

[0161] Expression vectors can be introduced into cells by a variety of means. The terms "transfection" and "transduction" are interchangeable and refer to the process by which exogenous nucleic acid sequences are introduced into eukaryotic host cells. Transfection (or transduction) can be accomplished by any one of several means, including electroporation, microinjection, gene gun delivery, retroviral infection, lipofection, superfection, and the like.

[0162] Any suitable method can be used to transfect or transform cells (e.g., T cells or NK cells). Certain non-limiting examples are provided herein. In some embodiments, the viral vector is a SFG-based viral vector, as discussed in Tey et al. (2007) Biol Blood Marrow Transpl 13:913-24 and Di Stasi et al., (2011) N Engl J Med 365:1673-83.

[0163] The cells can be transduced using a viral vector encoding the polypeptides described herein. A suitable transduction technique may include the fibronectin fragment CH-296. As an alternative to transduction using a viral vector, the cells can be transfected by any suitable method known in the art, such as with DNA encoding the relevant polypeptide, using, for example, calcium phosphate, cationic polymers (e.g., PEI), magnetic beads, electroporation, and commercially available lipid-based reagents (e.g., Lipofectamine™ and Fugene™). One result of the transduction / transfection step is that the various donor cells become genetically modified cells capable of expressing CIR and any other desired polypeptides.

[0164] In some embodiments, the viral vector used for transduction is a retroviral vector as disclosed by Tey et al. (2007) Biol Blood Marrow Transpl 13:913-24 and Di Stasi et al. (2011) supra. This vector is based on a Gibbon Ape Leukemia Virus (Gal-V) pseudotyped retrovirus that encodes the iCasp9 suicide switch and the ΔCD19 cell surface transgene marker (see further below). It can be produced in the PG13 packaging cell line as discussed by Tey et al. (2007) supra. Other viral vectors encoding desired proteins can also be used. In some embodiments, a retroviral vector capable of providing a high copy number of proviral integrants per cell is used for transduction.

[0165] After transduction / transfection, the cells can be separated from the transduction / transfection material and cultured again to grow the genetically modified cells. The cells can be grown to achieve the desired minimum number of genetically modified cells.

[0166] Genetically modified cells can then be selected from the resulting cell population. Because CIR may not be suitable for positive selection of desired cells, in some embodiments, genetically modified cells must express the cell surface transgene marker of interest (see below). Cells expressing this surface marker can be selected, for example, using immunomagnetic techniques. For example, paramagnetic beads conjugated to monoclonal antibodies that recognize the cell surface transgene marker of interest can be used (for example, using the CliniMACS system (available from Miltenyi Biotec)).

[0167] In an alternative procedure, the genetically modified cells are selected, cultured and then fed after the transduction step, thus the order of transduction, feeding and selection can be altered.

[0168] The result of these procedures is a composition that contains genetically modified cells and is therefore capable of expressing the chimeric ILT receptor (and any other desired polypeptides (e.g., costimulatory polypeptides), suicide switches, cell surface transgene markers, etc.). These genetically modified cells may be administered to a recipient, although they may first be stored (e.g., cryopreserved) before being administered, optionally after further expansion.

[0169] Selection marker Cells can be engineered to express a polypeptide whose expression can be identified in vitro or in vivo, thereby allowing for the selection of genetically engineered cells, e.g., to separate genetically engineered cells from unmodified cells. Such markers confer an identifiable change to the cells, allowing easy identification of cells containing the desired expression construct.

[0170] The inclusion of a drug selection marker aids in cloning and selection of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) are used.

[0171] Immunological surface markers can also be used, including extracellular non-signaling domains, or various proteins (e.g., CD34, CD19, LNGFR), allowing for straightforward methods for magnetic or fluorescent antibody-mediated sorting. These markers can be detected, for example, using labeled antibodies that bind to the proteins.

[0172] 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 the desired gene product (e.g., a subject CIR). Further, the marker should ideally be a polypeptide that is not expressed by the initial (donor) cells, although in situations where the marker is, in fact, endogenous to the initial cells, differences in expression levels can be exploited.

[0173] Ideally, the markers are based on human proteins to minimize the risk that cells expressing the markers will be recognized as foreign by the immune system of a human subject (e.g., after they are administered therapeutically). For example, if 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.

[0174] The genetically modified cells provided herein can express a cell surface transgene marker present on an expression vector expressing a subject CIR, and / or in some embodiments may be present on an expression vector encoding a protein other than a CIR, such as, for example, a CAR, a pro-apoptotic polypeptide safety switch, or a costimulatory polypeptide.

[0175] In one embodiment, the cell surface transgene marker is a truncated CD19 (ΔCD19) polypeptide (Di Stasi et al. (2011) supra) that comprises human CD19 truncated at amino acid 333 to remove most of the cytoplasmic domain (see, e.g., SEQ ID NOs: 12 (nucleotide) and 13 (protein)). The extracellular CD19 domain can still be recognized (e.g., in flow cytometry, FACS, or MACS), but the potential to induce intracellular signaling is minimized. Because CD19 is typically expressed by B cells, but not T cells or NK cells, selection of CD19+ cells allows for the separation of genetically modified cells (e.g., T cells, NK cells, or NKT cells) from unmodified cells.

[0176] Another useful marker is CD34, which has a minimal epitope of 16 amino acids (SEQ ID NO:41) that is useful as a marker.

[0177] By encoding the desired protein at the 5' end of the encoding gene and the marker at the 3' end, the risk of selecting cells that do not have the desired polypeptide (e.g., due to premature termination of translation) is minimized. In this way, expression of the marker and the desired polypeptide is carried out in parallel.

[0178] Suitable CD34 marker sequences for use in the present invention are disclosed in Tables 4-42.

[0179] 11. Engineered Expression Constructs Nucleic acids are provided that include a nucleotide sequence encoding a subject chimeric ILT receptor (CIR). In some cases, such nucleic acids are expression constructs. Expression constructs for expressing chimeric ILT receptors (and optionally other desired polypeptides, such as chimeric antigen receptors, signaling polypeptides, safety switches, etc.) are provided herein. In some examples, one or more polypeptides are said to be "operably linked" to a promoter. This indicates that the promoter sequence is operably linked to a second sequence and is in the correct position and orientation with respect to the second sequence such that the promoter sequence controls RNA polymerase initiation and transcription of DNA in response to the second sequence, whereby the resulting transcript encodes a polypeptide of interest.

[0180] A "promoter" is a DNA sequence recognized by or introduced into the synthetic machinery of a cell required to initiate specific transcription of a gene. In some embodiments, the promoter is a developmentally regulated promoter, i.e., a promoter that serves as an initial binding site for RNA polymerase to transcribe a gene that is expressed under certain conditions that are controlled, initiated, or influenced by a developmental program or pathway.

[0181] The term "expression construct" refers to any type of genetic construct that contains a nucleic acid encoding a gene product where some or all of the nucleic acid coding sequence can be transcribed. The transcript can be translated into a protein, but need not be. In certain embodiments, expression includes both transcription of the gene and translation of the mRNA into a gene product. In other embodiments, expression includes only transcription of the nucleic acid encoding the gene of interest. Expression vectors can contain various control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism.

[0182] A "vector" is capable of introducing a nucleic acid sequence into a target cell (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct", "expression vector", "expression construct", and "gene transfer vector" refer to any nucleic acid construct capable of inducing expression of a nucleic acid of interest and introducing a nucleic acid sequence into a target cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0183] In certain instances, the polynucleotide encoding the CIR is included in the same vector (e.g., a viral or plasmid vector) as a polynucleotide encoding a second polypeptide. This second polypeptide can be, for example (and as described elsewhere herein), a downregulator of an endogenous protein, a blocking antibody or scFv against an inhibitory receptor, a signaling polypeptide, an inducible suicide switch, or a marker polypeptide. In other instances, the additional expressed transcript does not code for a protein, but instead may generate a short hairpin RNA product designed to eliminate expression of certain endogenous RNAs that code for undesirable proteins in the cellular product.

[0184] The construct may be designed with one promoter operably linked to a nucleic acid comprising a polynucleotide encoding a fusion protein of 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, with each nucleic acid comprising a polynucleotide encoding one of the polypeptides being operably linked to a separate promoter. In yet other examples, one promoter may be operably linked to two polynucleotides that direct the production of two separate RNA transcripts (and thus two polypeptides), in one example, the promoter may be bidirectional, with the coding regions in opposite orientations 5'-3'. Thus, the expression constructs discussed herein may comprise at least one, or at least two promoters.

[0185] In yet other examples, two polypeptides (such as a CIR and a marker protein) can be expressed in a cell using two separate vectors. The cell may be co-transfected or co-transformed with the vectors, or the vectors may be introduced into the cell at different times.

[0186] Any combination of these approaches can be used to achieve expression of the desired polypeptide in the genetically modified cells.

[0187] In some embodiments, the 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, the cell is contacted with the viral vector ex vivo, and in some embodiments, the cell is contacted with the viral vector in vivo. Thus, the expression construct may be inserted into a vector (e.g., a viral vector or a plasmid). The steps of the provided methods may be performed using any suitable method, including, but not limited to, the methods of transducing, transforming, or otherwise providing a cell with a nucleic acid described herein.

[0188] The specific promoter used to control the expression of the polynucleotide sequence of interest is generally not particularly important, so long as it can direct the expression of the polynucleotide in the desired cell.Thus, when a human cell is targeted, the coding region of the polynucleotide sequence can be adjacent to and under the control of, for example, a promoter that can be expressed in a human cell.Generally speaking, such promoters can include either human promoters or viral promoters.A suitable promoter can be selected for the vector used to express the CIR and other polypeptides provided herein.

[0189] For example, in various embodiments where the expression vector is a retrovirus, an example of a suitable promoter is the mouse Moloney leukemia virus promoter. In other embodiments, the promoter can 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, the rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase, to obtain high levels of expression of the coding sequence of interest. The use of other viral or mammalian cell promoters that are well known in the art to achieve expression of the coding sequence of interest is also contemplated, provided that the expression level is sufficient for a given purpose. By using a promoter with well-known properties, the expression level and pattern of the polypeptide of interest after transfection or transformation can be optimized.

[0190] In other embodiments, the expression vector is a transposon in which genetic elements encoding CIR and associated marker proteins, coactivator proteins or inhibitors of endogenous factors, or tumor microenvironment are carried on a plasmid vector carrying an element recognized by a transiently coexpressed transposase. The action of the transposase is to catalyze the fusion of the carried transgene between the repeat element recognized by the transposase and the cell genome. Examples of transposon systems that can be used in these embodiments are the Sleeping Beauty system and the Piggyback system. A promoter element carried within the transposon directs the expression of the transgene. The promoter can 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, β-actin, rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase. The method for introducing the transposon and transposase plasmids into cells is transfection rather than viral transduction.

[0191] Promoters and other regulatory elements are selected such that they are functional in the desired cells or tissues. Furthermore, this list of promoters should not be construed as exhaustive or limiting. Other promoters for use in conjunction with the promoters and methods disclosed herein.

[0192] It is understood that the order of the polynucleotides may be varied and tested to determine the suitability of the construct for any particular method, and thus the nucleic acid may comprise various orders of polynucleotides, where variations in the order of components (a)-(d) are also considered. And the nucleic acid may comprise first through fourth polynucleotides in any of the following orders, where 1, 2, 3, and 4 (representing the arrangement of components in the intracellular region, e.g., (1) signaling region, (2) first costimulatory domain, (3) second costimulatory domain, and (4) surface marker) indicate the first, second, third, and fourth order of polynucleotides in the nucleic acid in the 5' to 3' direction. It is understood that other polynucleotides, such as those encoding 2A polypeptides, may be present among the listed polynucleotides, if applicable.

[0193] (Table 1) TIFF2025505546000002.tif44143

[0194] Similarly, the nucleic acid may include only three polynucleotides encoding the three polypeptides provided in the table above. In some examples, the cell is transfected or transduced with a nucleic acid that includes four polynucleotides included in Table 1 above. In other examples, the cell is transfected or transduced with a nucleic acid that encodes three polynucleotides encoding, for example, the three polypeptides provided in Table 2.

[0195] (Table 2) TIFF2025505546000003.tif47128

[0196] Likewise, the nucleic acid may include only two polynucleotides that encode the two polypeptides provided in the table above.

[0197] (Table 3) TIFF2025505546000004.tif25128

[0198] Permutations of the various polypeptide elements can be seen, for example, in Tables 4-42.

[0199] In some embodiments, the cell is transfected or transduced with a nucleic acid encoding two polynucleotides, the cell also comprises a nucleic acid comprising a polynucleotide encoding a third polypeptide, and / or the cell also comprises a nucleic acid comprising a polynucleotide encoding a fourth polypeptide. In some embodiments, the cell is transfected or transduced with a nucleic acid encoding three polynucleotides, the cell also comprises a nucleic acid comprising a polynucleotide encoding a fourth polypeptide. For example, the cell may comprise a nucleic acid comprising a first, second, and third polynucleotide, the cell may also comprise a nucleic acid comprising a polynucleotide encoding a chimeric caspase 9 polypeptide. The cell may also comprise a nucleic acid comprising a first, second, and fourth polynucleotide, the cell may also comprise a nucleic acid comprising a polynucleotide encoding a chimeric ILT receptor, an scFv modulator of native ILT2 function, or interleukin 15.

[0200] 12. Methods for Treating Disease Also provided are methods of treating or preventing diseases in which administration of cells (e.g., cells expressing a subject CIR), e.g., by injection, may be beneficial. The cells may be used, for example, for regeneration, e.g., to replace the function of diseased cells. The genetically modified cells described herein may be used for cell therapy.

[0201] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic in that it partially or completely cures the disease and / or the deleterious effects caused by the disease. As used herein, "treatment" encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the onset of disease in a subject predisposed to or at risk of acquiring the disease, but not yet 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.

[0202] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates (including monkeys and humans), mammalian sports animals (e.g., horses), mammalian farm animals (e.g., sheep, goats, etc.), mammalian pets (dogs, cats, etc.), and rodents (e.g., mice, rats, etc.).

[0203] An "effective amount" or a "sufficient amount" refers to an amount (e.g., an effective amount of cells) that, in single or multiple doses, alone or in combination with one or more other compositions (therapeutic agents, e.g., drugs), treatments, protocols, or therapeutic regimens, provides an expected or desired outcome in a subject, or benefit to a subject, of any duration (long-term or short-term), of any measurable or detectable degree, or of any duration (e.g., minutes, hours, days, months, years, or cure).

[0204] An "effective amount" or "sufficient amount" of a dose for treatment (e.g., to ameliorate or provide a therapeutic benefit or improvement) is typically effective to provide a measurable degree of response to one, more than one or all adverse symptoms, consequences or complications of a disease, e.g., one or more adverse symptoms, disorders, diseases, conditions, or complications caused by or associated with a disease, although a decrease, reduction, inhibition, suppression, limiting, or control of the progression or worsening of the disease is also a satisfactory outcome.

[0205] The 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 injection.

[0206] The genetically modified cells can be T cells, iNKT cells, macrophages, or NK cells. A typical dose of T cells or NK cells for therapy in a subject is 10 5 ~10 7 Pediatric patients generally receive approximately 10 6 Although adult patients receive a dose of 3×10 cells / kg, 6 A higher dose of cells / kg will be administered.

[0207] Generally speaking, the genetically modified T cells and NK cells of the present invention can be used in the same manner as known donor leukocyte infusions (DLI), but they have the added advantage of CIR.

[0208] A subject receiving genetically modified T cells or NK cells will typically also receive other tissues from an allogeneic donor, for example, hematopoietic cells and / or hematopoietic stem cells (e.g., CD34+ cells). The allogeneic tissue and genetically modified T cells are ideally derived from the same donor, such that they are genetically matched. Furthermore, the donor and recipient are preferably, for example, matched unrelated donors, or suitable family members. For example, the donor can be a parent or child of the recipient. Thus, when a subject is identified as needing genetically modified T cells, a suitable donor can be identified as the T cell donor.

[0209] The recipient may undergo lymphodepletion conditioning prior to receiving the genetically modified T cells (and prior to receiving the allograft), thus depleting the recipient's own α / β T cells (and B cells) prior to receiving the genetically modified T cells or NK cells.

[0210] The recipient may have a blood cancer (such as a treatment-resistant blood cancer) or a genetic blood disorder. For example, the recipient may have acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WA), Fanconi anemia, chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma (HL), or multiple myeloma.

[0211] A recipient of T or NK cells expressing CIR may have a non-hematological cancer expressing HLA-G, for example, the recipient may have renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer, neuroblastoma, or hepatocellular carcinoma (HCC).

[0212] Other cell types can also be used in therapy, including any cell administered to a patient for a desired therapeutic outcome. The therapeutic cell can be, for example, an immune cell, such as a T cell, a natural killer cell, a B cell, a tumor-infiltrating lymphocyte, or a macrophage, or a combination thereof, and the therapeutic cell can be, for example, a peripheral blood cell, a hematopoietic progenitor cell, a bone marrow cell, or a tumor cell. To further improve the tumor microenvironment to be more immunogenic, the therapy can be combined with one or more adjuvants (e.g., IL-12, TLR, IDO inhibitors, etc.). In some embodiments, the cells can be delivered to treat a solid tumor, such as, for example, delivery of the cells to the tumor bed.

[0213] In some embodiments, a nucleic acid is also provided that can be administered to a subject, thereby transforming or transducing target cells in vivo to form genetically modified cells in situ.

[0214] An effective amount of genetically modified cells is administered. To determine whether an effective amount of ligand or modified cells is administered, any means of assaying or measuring the number of target cells, or the amount of target antigen, or the size of the tumor can be used to determine whether the number of target cells, the amount of target antigen, or the size of the tumor is increased, decreased, or remains the same. A sample, image, or other measurement taken before administration of the modified cells or ligand may be used to compare with a sample, image, or other measurement taken after administration of the modified cells or ligand. Thus, for example, to determine whether the amount or concentration of cells expressing the target antigen is increased, decreased, or remains the same, a first sample may be obtained from the subject before administration of the ligand or modified cells, and a second sample may be obtained from the 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 to the amount or concentration of cells expressing the target antigen in the second sample to determine whether the amount or concentration of cells expressing the target antigen is increased, decreased, or remains the same after administration of the ligand or modified cells.

[0215] The effective amount for any particular application may vary depending on factors such 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. Effective amounts of particular compositions provided herein can be determined empirically.

[0216] To enhance the effectiveness of the modified cells presented herein, it may be desirable to combine these compositions and methods with agents effective in the treatment of disease.

[0217] Administration of the pharmaceutical composition may precede, be concurrent with, and / or follow the other agent by intervals ranging from minutes to weeks. In embodiments in which the pharmaceutical composition and the other agent are applied separately to the cell, tissue, or organism, it is generally ensured that no significant period of time expires between the time of each delivery, so that the pharmaceutical composition and the agent can still exert their advantageously combined effect on the cell, tissue, or organism. For example, in such cases, it is contemplated that the cell, tissue, or organism can be contacted with the pharmaceutical composition in two, three, four, or more modalities substantially simultaneously (i.e., within about one minute). In other aspects, the one or more agents can be administered substantially simultaneously, before and / or after administration of the expression vector, for about one minute to about 24 hours to about seven days to about one week to about eight weeks or more, and any range derivable therein. Additionally, various combination regimens of pharmaceutical compositions and one or more agents presented herein may be used.

[0218] Diseases that may be treated or prevented include diseases caused by viruses, bacteria, yeasts, parasites, protozoa, cancer cells, and the like. Exemplary diseases that may 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, chickenpox, papillomavirus, and the like; or infections of bacterial etiology, such as pneumonia, tuberculosis, syphilis, and the like; or infections of parasitic etiology, such as malaria, trypanosomiasis, leishmaniasis, trichomoniasis, amebiasis, and the like. Preneoplastic or hyperplastic conditions that may be treated or prevented using pharmaceutical compositions (transduced cells, expression vectors, expression constructs, and the like) include, but are not limited to, preneoplastic or hyperplastic conditions, such as colonic polyps, Crohn's disease, ulcerative colitis, breast lesions, and the like.

[0219] Cancers, including solid tumors, that may be treated using the cells include, but are not limited to, primary or metastatic melanoma, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, NPC, bladder cancer, cervical cancer, and the like.

[0220] Other hyperproliferative diseases, including solid tumors, that may be treated using the therapeutic cells and other therapeutic cell activation systems presented herein include, but are not limited to, rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, preneoplastic lesions (e.g., adenomatous hyperplasia and prostatic intraepithelial neoplasia), carcinoma in situ, oral hairy leukoplakia, or psoriasis.

[0221] Any tumor expressing a target antigen, e.g., HLA-G, in the vasculature, e.g., lung, bone, liver, prostate, or brain, as well as solid tumors of any tissue or organ origin, including solid tumors in the breast, ovary, intestine, testis, colon, pancreas, kidney, bladder, neuroendocrine system, soft tissue, bone mass, and lymphatic system, can be treated using this method. Other solid tumors that can be treated include, e.g., glioblastoma, and malignant multiple myeloma.

[0222] The subject may be administered zinc supplements to ensure that any zinc-dependent factors contained in the CIR or cofactors expressed in a cell therapy product containing the CIR have an adequate source of this ion to allow for full activity.

[0223] 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., encoding a subject CIR). The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell is "transfected" when exogenous DNA is introduced into the cell membrane. Several transfection techniques are generally known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell. The term refers to both stable and transient uptake of genetic material.

[0224] In some embodiments, the cells of the disclosure are produced by transducing the cells with a viral vector encoding the CIR. In certain aspects, the polypeptide comprises the CIR and the cells are T cells, thus providing methods of producing CIR T cells. In some embodiments, such methods include activating a population of T cells (e.g., T cells obtained from an individual to whom CIR T cell therapy will be administered), stimulating the population of T cells to proliferate, and transducing the T cells with a viral vector encoding a polypeptide comprising the CIR. In some embodiments, immune cells (e.g., T cells, NK cells, macrophages) are transduced with a retroviral vector (e.g., a gamma retroviral vector or a lentiviral vector) or an AAV encoding the CIR. In certain aspects, immune cells, T cells, are transduced with a lentiviral vector encoding the polypeptide. In certain aspects, the polypeptide comprises the CIR and the cells are CIR NK cells, thus providing methods of producing CIR NK cells (e.g., by using a viral vector such as an AAV, lentivirus, or retroviral vector).

[0225] 13.General The term "comprising" encompasses "including" as well as "consisting." For example, a composition "comprising" X may consist exclusively of X or may include additional items (e.g., X+Y).

[0226] The term "about" in reference to a numerical value x is optional and means, for example, x±10%.

[0227] The word "substantially" does not exclude "completely". For example, a composition that is "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the invention.

[0228] The term "between" in reference to two values ​​means that a range "between" those two values, for example 10 mg to 20 mg, includes 10, 15, and 20 mg, among others.

[0229] Unless otherwise specified, a method that includes mixing two or more components does not require any particular order of mixing.Thus, the components can be mixed in any order.When there are three components, two components can be combined with each other, and then the combination can be combined with the third component, etc.

[0230] The various steps of the method may be performed at the same or different times, in the same or different geographic locations (eg, countries), and by the same or different people or entities.

[0231] The term "pharmacologically acceptable" refers to molecular entities and compositions that do not produce unwanted, allergic, or other adverse reactions when administered to an animal or human.

[0232] The degree of similarity between two sequences can be based on the percent sequence identity. "Sequence identity" in this specification refers to the degree to which two nucleotide or amino acid sequences are invariant. "Sequence alignment" refers to the process of aligning two or more sequences to achieve the highest level 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, similarity based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA. When using any of these programs, the settings that result in the highest sequence similarity can be selected.

[0233] 14. Examples of specific nucleic acid and amino acid sequences The following sections and tables contain examples of polypeptide and nucleotide sequences that encode chimeric signaling polypeptides. It is understood that the sequences of the individual polypeptides provided in these examples, such as the truncated ILT2 and ILT4 polypeptides, the costimulatory polypeptide cytoplasmic signaling domain, the safety switch, etc., can be used to construct other expression vectors that encode the chimeric signaling polypeptides of the present embodiments. Table 4 contains an example of a complete vector sequence for delivery of a gamma-retrovirus containing an HLA-G1 sequence along with the marker protein ΔCD19. Tables 5-20 contain only the transgenes expressed by the gamma-retroviral vectors of the same example.

[0234] (Table 4) Plasmid A: pNT101-SFG-HLAG1-T2A-ΔCD19 TIFF2025505546000005.tif110134

[0235] (Table 5) Plasmid B: pNT102-SFG-HLAG2-T2A-ΔCD19 TIFF2025505546000006.tif41134

[0236] (Table 6) Plasmid C: pNT103-SFG-HLAG3-T2A-ΔCD19 TIFF2025505546000007.tif42132

[0237] (Table 7) Plasmid D: pNT104-SFG-HLAG4-T2A-ΔCD19 TIFF2025505546000008.tif40128

[0238] (Table 8) Plasmid E: pNT105-SFG-HLAG5-T2A-ΔCD19 TIFF2025505546000009.tif39128

[0239] (Table 9) Plasmid F: pNT106-SFG-HLAG5C42S-T2A-ΔCD19 TIFF2025505546000010.tif46128

[0240] (Table 10) Plasmid G: pNT107-SFG-HLAG1C42S-T2A-ΔCD19 TIFF2025505546000011.tif44128

[0241] (Table 11) Plasmid H: pNT108-SFG-HLAG1C42SC147S-T2A-ΔCD19 TIFF2025505546000012.tif43128

[0242] (Table 12) Plasmid I: pNT109-SFG-HLAG1C147S-T2A-ΔCD19 TIFF2025505546000013.tif45128

[0243] (Table 13A) Plasmid K: pNT-110 SFG-ILT2-T2A-ΔCD19 TIFF2025505546000014.tif40134

[0244] (Table 13B) Plasmid K: pNT-110 SFG-ILT2-T2A-ΔCD19 TIFF2025505546000015.tif68150

[0245] (Table 14) Plasmid L: pNT-111 SFG-ILT2.ζ-T2A-ΔCD19 TIFF2025505546000016.tif65131

[0246] (Table 15A) Plasmid M: pNT-112 SFG-ILT2.BB.ζ-T2A-ΔCD19 TIFF2025505546000017.tif70133

[0247] (Table 15B) Plasmid M: pNT-112 SFG-ILT2.BB.ζ-T2A-ΔCD19 TIFF2025505546000018.tif103148

[0248] (Table 16A) Plasmid N: pNT-113 SFG-ILT2D1D2STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000019.tif81128

[0249] (Table 16B) Plasmid N: pNT-113 SFG-ILT2D1D2STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000020.tif95129

[0250] (Table 17) Plasmid 0: pNT-114 SFG-ILT2D1D2.Q.8STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000021.tif76128

[0251] (Table 18) Plasmid P: pNT-115 SFG-ILT2D1D2.Q.28STM.CD28.ζ-T2A-ΔCD19 TIFF2025505546000022.tif82128

[0252] (Table 19) Plasmid R: pNT-116 SFG-ILT2D1D2.Q.CH2CH3.8TM.BB.ζ-T2A-ΔCD19 TIFF2025505546000023.tif69133

[0253] (Table 20A) Plasmid S: pNT-117 SFG-ILT2D1D2.CD8STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000024.tif78132

[0254] (Table 20B) Plasmid S: pNT-117 SFG-ILT2D1D2.CD8STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000025.tif94135

[0255] (Table 21A) Plasmid T: pNT-118 SFG-ILT4-T2A-ΔCD19 TIFF2025505546000026.tif36128

[0256] (Table 21B) Plasmid T: pNT-118 SFG-ILT4-T2A-ΔCD19 TIFF2025505546000027.tif67149

[0257] (Table 22) Plasmid U: pNT-119 SFG-ILT4.ζ-T2A-ΔCD19 TIFF2025505546000028.tif58135

[0258] (Table 23A) Plasmid V: pNT-120 SFG-ILT4.BB.ζ-T2A-ΔCD19 TIFF2025505546000029.tif69130

[0259] (Table 23B) Plasmid V: pNT-120 SFG-ILT4.BB.ζ-T2A-ΔCD19 TIFF2025505546000030.tif98149

[0260] (Table 24A) Plasmid W: pNT-121 SFG-ILT4D1D2STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000031.tif83128

[0261] (Table 24B) Plasmid W: pNT-121 SFG-ILT4D1D2STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000032.tif94128

[0262] (Table 25) Plasmid X: pNT-122 SFG-ILT4D1D2.Q.8STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000033.tif88128

[0263] (Table 26) Plasmid Y: pNT-123 SFG-ILT4D1D2.Q.28STM.CD28.ζ-T2A-ΔCD19 TIFF2025505546000034.tif94128

[0264] (Table 27) Plasmid Z: pNT-124 SFG-ILT4D1D2.Q.CH2CH3.8TM.BB.ζ-T2A-ΔCD19 TIFF2025505546000035.tif75134

[0265] (Table 28A) Plasmid AA: pNT-124 SFG-ILT4D1D2.CD8STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000036.tif79131

[0266] (Table 28B) Plasmid AA: pNT-124 SFG-ILT4D1D2.CD8STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000037.tif93133

[0267] (Table 29) Plasmid U: pNT-119 SFG-ILT4Y394A.ζ-T2A-ΔCD19 TIFF2025505546000038.tif57137

[0268] (Table 30) Plasmid AC:pNT-126 SFG-ILT4Y394A.BB.ζ-T2A-ΔCD19 TIFF2025505546000039.tif69137

[0269] (Table 31) Plasmid A-D: pNT-128 SFG-ILT4D1D2Y96A.STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000040.tif86134

[0270] (Table 32) Plasmid AE: pNT-129 SFG-ILT4D1D2Y96A.Q.8STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000041.tif94137

[0271] (Table 33) Plasmid AF: pNT-130 SFG-ILT4D1D2Y96A.Q.28STM.CD28.ζ-T2A-ΔCD19 TIFF2025505546000042.tif84130

[0272] (Table 34) Plasmid AG: pNT-131 SFG-ILT4D1D2Y96A.Q.CH2CH3.8TM.BB.ζ-T2A-ΔCD19 TIFF2025505546000043.tif72131

[0273] (Table 35) Plasmid AH: pNT-132 SFG-ILT4D1D2Y96A.CD8STM.BB.ζ-T2A-ΔCD19 TIFF2025505546000044.tif89136

[0274] (Table 36) pNT133-pSFG-ILT2(D1-D4).CD8stalkTM.BB.z.T2A-dCD19 TIFF2025505546000045.tif83128

[0275] (Table 37) pNT134-pSFG-ILT4(D1-D4).CD8 stalk TM.BB.z.T2A-dCD19 TIFF2025505546000046.tif87131

[0276] (Table 38) pNT138 SFG-ILT4(D1-D2).CH2CH3(4-2NQ).CD8TM.BB.z.T2A-dCD19 TIFF2025505546000047.tif84128

[0277] (Table 39) pNT139 SFG- ILT4(D1-D2).CH3.CD8tm.BB.z.T2A-dCD19 TIFF2025505546000048.tif87128

[0278] (Table 40) pNT-159 SFG-ILT4(D1-D2).CH2CH3(4-2NQ)s.CD28tm.BB.z.T2A-dCD19 TIFF2025505546000049.tif91128

[0279] (Table 41) pNT-160 SFG-ILT4(D1-D2).CH3s.CD28tm.BB.z.T2A-dCD19 TIFF2025505546000050.tif84128

[0280] (Table 42) pNT-158 SFG- ILT4(D1-D2).CD28s.CD28tm.BB.z.T2A-dCD19 TIFF2025505546000051.tif86128

[0281] Exemplary Non-Limiting Aspects of the Disclosure The above aspects, including the embodiments of the subject matter, may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting aspects of the present disclosure are provided below (see Set A and Set B). Each individually numbered aspect may be used or combined with any of the individually numbered aspects that precede or follow it, as will be apparent to a person skilled in the art upon reading this disclosure. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below. It will be apparent to a person skilled in the art that various changes and modifications may be made without departing from the spirit or scope of the present invention. Set A 1. A composition in which genetically engineered cells express a chimeric receptor protein that specifically targets a protein or carbohydrate having a binding domain that is not derived from an antibody, a VhH domain, or a synthetically derived polypeptide. 2. The composition of 1, wherein the chimeric receptor protein employs a recognition or binding domain that targets HLA-G that is not derived from an antibody, a VhH domain, or a synthetically derived polypeptide. 3. The composition of 1 or 2, wherein the chimeric receptor proteins employ human receptor proteins that naturally recognize target proteins, including HLA-G, as part of their biological mechanism of action. 4. The composition according to 3, wherein the chimeric receptor protein uses ILT2 derived from human or monkey sequences. 5. The composition according to 3, wherein the chimeric receptor protein uses ILT4 derived from human or monkey sequences. 6. The composition according to 4, wherein the chimeric receptor protein uses naturally occurring polymorphic forms of ILT2 derived from human or monkey sequences. 7. The composition according to 5, wherein the chimeric receptor protein uses naturally occurring polymorphic forms of ILT4 derived from human or monkey sequences. 8. The composition according to 4 and 6, wherein the chimeric receptor protein uses the D1 and D2 domains derived from ILT2. 9. The composition of 5 and 7, wherein the chimeric receptor protein uses the D1 and D2 domains derived from ILT4. 10. A composition according to any one of the preceding paragraphs, wherein the D1 or D2 domain of ILT2 or ILT4 is mutated to encode alternative amino acids in order to retain sufficient affinity for HLA-G to activate the chimeric receptor, whilst reducing affinity for proteins other than HLA-G. 11. The composition according to 10, wherein the mutation generates an amino acid substitution at a position corresponding to amino acid 96 or 394 in ILT4. 12. A composition according to any one of the preceding claims, wherein the genetically modified cell further expresses a safety switch. 13. The composition according to claim 12, wherein the safety switch is inductive. 14. The composition according to 12, wherein the safety switch is triggered by rimiduside or by rapamycin or an analogue of rapamycin. 15. The composition of any one of the preceding claims, wherein the genetically modified cell further expresses a second chimeric antigen receptor. 16. The composition according to any one of 1 to 11, wherein the genetically modified cell further expresses, as a fusion element with the composition according to any one of 1 to 11, a cytoplasmic signaling domain that drives activation of cytotoxicity against NK cells or T cells. 17. The composition of claim 16, wherein the fused cytotoxic signaling element comprises an ITAM sequence. 18. The composition of 16 or 17, wherein the fused signaling element is derived from CD3zeta, DAP10, or DAP12. 19. A composition according to any one of the preceding claims, wherein the genetically modified cell further expresses a costimulatory polypeptide as part of the chimeric receptor. 20. A composition according to any one of the preceding claims, wherein the genetically modified cell further expresses a costimulatory polypeptide as a separate entity from the chimeric receptor protein. 21. The composition of 19 and 20, wherein the costimulatory polypeptide comprises a signaling element derived from 4-1BB, OX40, ICOS, CD28, CD27, MyD88, IL-1Rα, HVEM, TRANCE, IL-1Rβ, CD70, IL-18Rα, CD40, IL-18Rβ, IL-33Rα, CD30, or IL-33Rβ. 22. The composition of any one of the preceding claims, wherein the genetically modified cell is (i) a T cell, (ii) a NK cell, (iii) an iNKT cell, or (iv) a macrophage. 23. A method of treating a human subject, comprising administering to the patient a composition according to any one of the preceding claims. 24. A method of treating a human subject, wherein the subject is a cancer patient, comprising administering to the patient a composition described in any preceding paragraph. 25. An expression construct comprising a polynucleotide encoding a chimeric receptor protein according to any one of 1 to 21. 26. A vector comprising the expression construct according to 25, which is a retroviral vector, a lentiviral vector, or a plasmid vector. Set B 1. (a) a targeting region that targets HLA-G and includes the D1-D2 extracellular domains of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region comprising a transmembrane amino acid sequence; (c) an intracellular domain (ICD) that includes a signaling region that can transmit a signal to the inside of an immune effector cell upon binding of the targeting region to HLA-G to induce a function of the effector cell; A chimeric receptor protein comprising: 2. The chimeric receptor protein according to 1, wherein the targeting region comprises an amino acid mutation at a position corresponding to Y96 in the ILT4 amino acid sequence shown in SEQ ID NO:57 or Y96 in the ILT2 amino acid sequence shown in SEQ ID NO:31. 3. The chimeric receptor protein according to 1 or 2, wherein the D1 to D2 extracellular domains are ILT2 D1 to D2 extracellular domains. 4. The chimeric receptor protein according to 1 or 2, wherein the D1 to D2 extracellular domains are ILT4 D1 to D2 extracellular domains. 5. A chimeric receptor protein according to any one of 1 to 4, wherein the targeting region comprises the D3 to D4 extracellular domains of ILT2 or ILT4. 6. The chimeric receptor protein according to 5, wherein the targeting region comprises an amino acid mutation at a position corresponding to Y394 in the ILT4 amino acid sequence shown in SEQ ID NO: 55 or Y395 in the ILT2 amino acid sequence shown in SEQ ID NO: 31. 7. A chimeric receptor protein according to any one of 1 to 4, wherein the targeting region lacks the ILT2 or ILT4 D3-D4 extracellular domains. 8. The chimeric receptor protein according to 5, comprising a stalk domain. 9. The chimeric receptor protein of 8, wherein the stalk domain comprises an ILT2, ILT4, CD28, CH2 / CH3, CH3, or CD8 stalk domain. 10. The chimeric receptor protein according to any one of 1 to 9, wherein the TM domain is an ILT2, ILT4, CD28, or CD8 TM domain. 11. A chimeric receptor protein according to any one of 1 to 10, wherein the signalling region comprises an immunoreceptor tyrosine-based activation motif (ITAM). 12. The chimeric receptor protein according to any one of 1 to 10, wherein the signaling region comprises a CD3ζ signaling domain, a DAP10 signaling domain, a DAP12 signaling domain, or any combination thereof. 13. A chimeric receptor protein according to any one of 1 to 10, wherein the signal transduction region comprises a CD3ζ signal transduction domain. 14. The chimeric receptor protein of any one of 1 to 13, wherein the ICD further comprises a costimulatory region comprising at least one costimulatory domain. 15. The chimeric receptor protein of 14, wherein the at least one costimulatory domain comprises a CD28 costimulatory domain. 16. The chimeric receptor protein of 14 or 15, wherein the at least one costimulatory domain comprises a 4-1BB costimulatory domain. 17. The chimeric receptor protein of 14, wherein the at least one costimulatory domain comprises a 4-1BB, OX40, CD28, ICOS, RANK, DAP10, DAP12, CD27, MyD88, IL-1Rα, HVEM, TRANCE, IL-1Rβ, CD70, IL-18Rα, CD40, IL-18Rβ, IL-33Rα, CD30, or IL-33Rβ costimulatory domain, or any combination thereof. 18. the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain; The extracellular domain lacks the ILT2 D3-D4 extracellular domain, the chimeric receptor protein comprises a CD8 stalk domain; the TM region is CD TM; the signaling region comprises a CD3ζ signaling domain; The chimeric receptor protein comprises a 4-1BB costimulatory domain. 2. A chimeric receptor protein according to claim 1. 19. the D1-D2 extracellular domain is an ILT4 D1-D2 extracellular domain; The extracellular domain lacks the ILT4 D3 to D4 extracellular domains; the chimeric receptor protein comprises a CD8 stalk domain; the TM region is CD TM; the signaling region comprises a CD3ζ signaling domain; The chimeric receptor protein comprises a 4-1BB costimulatory domain. 2. A chimeric receptor protein according to claim 1. 20. the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain; the extracellular domain comprises the ILT2 D3-D4 extracellular domain; the chimeric receptor protein comprises a CD8 stalk domain; the TM region is CD TM; the signaling region comprises a CD3ζ signaling domain; The chimeric receptor protein comprises a 4-1BB costimulatory domain. 2. A chimeric receptor protein according to claim 1. twenty one. the D1-D2 extracellular domain is an ILT4 D1-D2 extracellular domain; the extracellular domain comprises the ILT4 D3-D4 extracellular domain; the chimeric receptor protein comprises a CD8 stalk domain; the TM region is CD TM; the signaling region comprises a CD3ζ signaling domain; The chimeric receptor protein comprises a 4-1BB costimulatory domain. 2. A chimeric receptor protein according to claim 1. 22. A nucleic acid comprising a nucleotide sequence encoding a chimeric receptor protein according to any one of 1 to 21. 23. The nucleic acid of 22, wherein the nucleotide sequence is operably linked to a constitutive promoter. 24. The nucleic acid of 22, wherein the nucleotide sequence is operably linked to an inducible promoter. 25. The nucleic acid according to any one of 22 to 24, which is an expression vector. 26. The nucleic acid according to 25, wherein the expression vector is a retroviral vector, a lentiviral vector, or a plasmid vector. 27. A genetically modified cell expressing a chimeric receptor protein according to any one of 1 to 22. 28. The genetically modified cell according to 27, which is an immune cell. 29. The genetically modified cell according to 28, wherein the immune cell is a natural killer (NK) cell, a T cell, an iNKT cell, or a macrophage. 30. The genetically modified cell according to 28, wherein the immune cell is a natural killer (NK) cell. 31. The system according to 28, wherein the immune cell is a T cell. 32. A genetically modified cell according to any one of 27 to 31, which expresses a safety switch. 33. The genetically modified cell according to 32, wherein the safety switch is inducible. 34. The genetically modified cell according to 33, wherein the safety switch is triggered by rimiduside or by rapamycin or an analogue of rapamycin. 35. A genetically modified cell according to any one of 27 to 34, which further expresses a chimeric antigen receptor. 36. A genetically modified cell according to any one of 27 to 35, which further expresses a costimulatory polypeptide that is not fused to the chimeric receptor protein. 37. The genetically modified cell of 36, wherein the costimulatory polypeptide not fused to the chimeric receptor protein comprises a 4-1BB, OX40, CD28, ICOS, RANK, DAP10, DAP12, CD27, MyD88, IL-1Rα, HVEM, TRANCE, IL-1Rβ, CD70, IL-18Rα, CD40, IL-18Rβ, IL-33Rα, CD30, or IL-33Rβ costimulatory domain, or any combination thereof. 38. A method of treatment comprising administering to an individual in need thereof a genetically modified cell according to any one of 27 to 37. 39. The method of claim 38, wherein the genetically modified cells are autologous to the individual. 40. The method of claim 38, wherein the genetically modified cells are allogeneic to the individual. 41. The method according to any one of 38 to 40, wherein the individual has cancer. 42. The method of claim 41, wherein the individual has a solid tumor. 43. A method for producing a genetically modified cell, comprising: 27. A method comprising introducing the nucleic acid according to any one of claims 22 to 26 into a cell, thereby producing a genetically modified cell. 44. The method according to claim 43, wherein the genetically modified cell is an immune cell. 45. The method according to 44, wherein the immune cell is a natural killer (NK) cell, a T cell, an iNKT cell, or a macrophage. 46. ​​The method according to 44, wherein the immune cells are natural killer (NK) cells. 47. The method according to claim 44, wherein the immune cell is a T cell. EXAMPLES

[0282] Experimental Example The following examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Thus, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0283] 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 use the present invention and practice the claimed methods, and therefore the following examples are not to be construed as limiting in any way the remainder of the disclosure.

[0284] General methods in molecular and cellular biochemistry are described in 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 Reagents, cloning vectors, cells, and kits for the methods mentioned 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 repositories such as, for example, Addgene, Inc., the American Type Culture Collection (ATCC), and the like.

[0285] Example 1: Expression of engineered CIR constructs in primary human T cells To demonstrate the utility of expressing a chimeric receptor targeting HLA-G, via binding to its native receptor altered to generate a signal that activates immune function versus inhibitory signaling, recombinant DNA constructs were engineered in the SFG γ-retroviral vector (see schematic in Figure 6A). In one example, a full-length gene encoding ILT4 was cloned 5' to a gene encoding the extracellular domain of CD19 (ΔCD19) (see Table 21B). Full-length ΔCD19 was useful for marking the expression of transgenes introduced with this vector. The cistrons encoding ILT4 and CD19 were separated by a T2A cotranslational cleavage site derived from the Tosea asigna virus to allow expression of distinct proteins from individual mRNA molecules. All other examples were similarly marked ΔCD19, separated by a T2A sequence.

[0286] Another example of an expression construct encoded a chimeric ILT receptor (CIR) encoding the D1-D4 and transmembrane domains of ILT4, but replacing the native intracellular domain of ILT4 with a costimulatory domain from human 4-1BB and a signaling domain from human CD3ζ (BB.ζ). This construct encoded a CIR protein, designated CIR1 in this example (see Table 23B). A further example encoded CIR2, which contains sequences encoding only the D1 and D2 domains that directly interact with HLA-G, but not the D3 and D4 domains. These sequences were fused to sequences encoding the ILT4 transmembrane domain and the costimulatory domain of 4-1BB and the CD3ζ signaling domain (see Table 24B). A further example replaced sequences encoding the ILT4 stalk and transmembrane domains with sequences encoding the stalk and transmembrane domains of CD8α. These constructs encoded ILT4 CIR3 (see Table 37) and CIR4 (see Table 28B).

[0287] In yet further examples, sequences encoding the D1-D4 domains of ILT4 were replaced with sequences encoding ILT2 or only the D1 and D2 domains of ILT2. These constructs encoded ILT2 CIR1, CIR2, CIR3, and CIR4 (see Tables 15B, 16B, 36, and 20B, respectively). For ILT2 CIR1 and CIR2, sequences encoding the stalk and transmembrane domains of ILT2 were used, while for CIR3 and CIR4, these sequences were replaced with sequences encoding the CD8α stalk and transmembrane domains. In further experiments, two additional constructs were generated as positive controls. They encoded the binding elements of the HLA-G specific antibody 15E7 fused to the CD8α stalk and transmembrane domains of the CD8α and BB.ζ intracellular domains (see US Pat. No. 1,312,774 B2). This together encodes an HLA-G chimeric antigen receptor (HLA-G CAR). The second positive control construct encoded a CAR directed against CD33 (Duong et al, Mol. Ther. Onc. 12:124), a target protein commonly expressed by acute myeloid leukemia cells (AML).

[0288] Gamma-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 T cells derived from two healthy donors. Transduction efficiency was characterized by the expression of the ΔCD19 marker protein detected by flow cytometry (see FIG. 6B). Transduction efficiency exceeded 80% for each recombinant retrovirus (FIG. 6C). Expression of CIR constructs containing the extracellular domains of ILT4 and ILT2 was determined by flow cytometry using antibodies specific for ILT4 (FIG. 6D) and ILT2 (FIG. 6E), quantified by the mean fluorescence intensity of the population of stained transduced cells. Although the expression level of ILT2 CIR4 was significantly reduced compared to the other CIR proteins, stable expression of CIR proteins was readily detectable.

[0289] The experiments described in this Example demonstrated that recombinant retroviruses encoding CIR proteins derived from ILT2 and ILT4 can be generated, and that primary human immune cells can be transduced with these retroviruses to generate immune cells that stably express the CIR proteins.

[0290] Methods: DNA constructs were designed with SnapGene software and DNA sequences were synthesized from GBlock fragments by IDT Laboratories. The synthetic DNA fragments were cloned by standard cloning techniques for the assembly of recombinant DNA into SFG DNA vectors for the production of Moloney Murine Leukemia Virus-based retroviruses.

[0291] Example 2: Antitumor efficacy of CIR-T cells To generate tumor cell lines stably expressing different isoforms of HLA-G, recombinant retroviruses were generated encoding the HLA-G isoforms HLA-G1, HLA-G2, and HLA-G5. Each of these retroviruses also encodes a marker protein, ΔEGFR, which is separated from HLA-G using a T2A cotranslational cleavage element to detect the efficiency of viral transduction. These γ-retroviruses were used to transduce THP1 AML cells for stable high-level expression of HLA-G isoforms. Transduction efficiency, characterized by staining with an antibody against EGFR, was detected by flow cytometry and was high for each recombinant retrovirus. Expression of HLA-G in transduced THP1 cells was also determined by flow cytometry using the MEM-G / 9 antibody, which is specific for HLA-G1 but not HLA-G2, and expression was at a high level in the transduced cells.

[0292] The expression of GFP-ffluc marker protein in THP1-GFP cells, which also express HLA-G1, was at a high level (Figure 7B). The expression of GFPff luciferase was useful to indirectly measure the loss of viability of THP1 cells in co-culture experiments with CIR-T cells by the decrease in green fluorescence over time or the loss of luciferase enzyme activity. Thus, THP1 cells expressing HLA-G were suitable target cells for the determination of the cytotoxicity of CIR-expressing T cells.

[0293] Primary human T cells from two healthy donors were transduced with the same cohort of retroviruses encoding CIR and CAR as described in Example 1. CIR-T, CAR-T, or mock-transduced (NT) T cells were co-cultured with HLA-G1 expressing THP1-GFP cells at an effector-to-target ratio of 5:1 and placed in an Incucyte incubator equipped with a microscope capable of periodic imaging over a 2-day period. Green fluorescence from THP1-HLAG1 GFP cells was quantified every 12 hours during the co-culture period as an indirect measure of tumor growth or tumor cell killing (Figure 8). Co-culture with mock-transduced T cells or with T cells transduced to express full-length ILT2 or ILT4 containing the native inhibitory signaling domain (negative control, Neg ctl) showed stable expression of GFP in tumor targets. Co-culture with HLA-G CAR-T cells or CD33 CAR-T cells showed a decrease in GFP fluorescence starting from the first 12 hours time point indicating tumor control. These CAR-T cells served as positive controls in this experiment. ILT2 CIR-T cells derived from each donor also showed reduced GFP fluorescence and effective tumor control. Note that the CIR4 construct had reduced antitumor efficacy, likely due to reduced expression levels of this construct (see FIG. 6E). ILT4 CIR-T cells also showed effective control of THP-1 HLA-G1 proliferation.

[0294] Antitumor efficacy over the same short 24-hour time course against THP-1 HLA-G1 was determined by measuring luciferase activity associated with GFP-luciferase expression in tumor targets in co-culture with transduced human T cells (Figure 9). Co-culture with T cells expressing full-length ILT2 (Neg Ctl) (E:T - 5:1 and 1:1) had measurable luciferase activity of over 100,000 light units and served as a reference for the ability of other ILT2 CIR constructs to kill THP1-HLA-G1-GFPffluc targets. ILT2 CIR-T cell co-culture significantly reduced luciferase activity, demonstrating the killing ability of ILT2 CIR-T cells. Note again that ILT2 CIR4 cells showed reduced antitumor cytotoxicity compared to other CIR-T cells and CAR-T cell positive controls, likely due to reduced CIR expression. Co-culture with ILT4 CIR-T cells also showed enhanced anti-THP1-HLAG1 cytotoxicity compared to full-length ILT4 (Neg Ctl). Note that in this case, CIR4-T cells have superior anti-tumor cytotoxicity compared to other CIR-T cells and CD33 CAR-T cells.

[0295] The experiments described in this example demonstrate that expression of CIR proteins in human immune cells (T cells in this particular exemplary embodiment) can redirect the specificity of immune cells (e.g., T cells) to HLA-G expressing tumor targets. Furthermore, these experiments demonstrate that binding interactions between HLA-G and ILT2 or ILT4 sequences are not sufficient for anti-tumor efficacy, but rather replacement of native ILT2 or ILT4 intracellular signaling domains with domains that activate T cell function is necessary for enhanced cytotoxicity. Furthermore, it has been demonstrated that the D1 and D2 domains of ILT2 or ILT4 are sufficient to target HLA-G in chimeric ILT receptor (CIR) constructs.

[0296] Example 3: Modification of stalk and transmembrane domains in CIR constructs In engineered chimeric proteins designed to facilitate both binding interactions with target cells and transduction of intracellular signals upon engagement, the selection of an optimal linker domain can be important for obtaining improved efficiency. The stalk domain extends the target-specific binding element from the cell membrane and presents the binding element in a context that does not inhibit engagement with the target protein. The transmembrane domain facilitates transduction of signals to the intracellular domain upon engagement and maintains stable expression in the plasma membrane.

[0297] Examples 1 and 2 show that ILT4 CIR4 showed adequate expression levels upon transduction in human T cells and enhanced anti-tumor cytotoxicity compared to other CIR constructs. This construct design, containing the D1 and D2 domains of ILT4 together with the BB.ζ signaling element, was used in a "combination" experiment in which the CD8α stalk and transmembrane domain were replaced with the stalk and transmembrane domain of other signaling receptors. The constructs created are shown in FIG. 10. As an example, CIR6 replaced the CD8α stalk with the CH2 and CH3 domains derived from human immunoglobulin protein IgG4. A second example reduced the length of the stalk by encoding only the CH3 domain of human IgG4 fused to the CD8α transmembrane domain. Another example fused the CH3CH2 stalk with the transmembrane domain from human CD28, and a fourth example also reduced the length of the stalk to only the CH3 domain, also fused to CD28 TM. Yet further examples encoded short CD28-derived stalks fused to the CD28 transmembrane domain. These CIR constructs were designated CIR6, CIR7, CIR8, CIR9, and CIR10 (see Tables 38-42, respectively). Retroviral constructs encoding these alternative ILT4 CIR derivatives also encoded a ΔCD19 marker protein separated by a T2A cotranslational cleavage site.

[0298] Gamma-retroviruses encoding ILT4 CIR constructs were generated and primary human T cells were transduced. For low and high multiplicity of infection, transduction was performed with 2 or 8 milliliters (mL) of retroviral supernatant. Overall transduction efficiency was monitored by flow cytometry to detect expression of the ΔCD19 marker 7 and 14 days after transduction (Figure 11A). Transduction efficiency was greater than 60% for ILT2 CIR4, ILT4 CIR4 (parental construct), and a control construct encoding an HLA-G CAR. Transduction efficiency was comparable to ILT4 CIR derivatives containing a CD28 transmembrane domain. However, transduction efficiency was relatively low for CIR constructs encoding a CD8α transmembrane domain matching the CH2CH3 or CH3 stalks. Expression levels of ILT4 CIR derivatives were examined by flow cytometry using an ILT4-specific antibody (Figure 11B, Figure 11C). Expression of CIR4 containing the CD8α stalk and transmembrane domain was robust when examined as % of gated cells above background (FIG. 11B) or as mean fluorescence intensity (MFI) of the signal. Conversely, the MFI of ILT4 CIR derivatives with alternative stalk or transmembrane domains was not elevated above background signal observed in T cells transduced with control constructs such as HLA-G CAR or ILT2 CIR4 that do not express ILT4 CIR.

[0299] The experiments described in this Example indicate that the context of presentation of the D1 and D2 domains of ILT4 may be important for stable expression of CIR and thus optimal functionality of CIR-T cells.

[0300] Example 4: Broad specificity of HLA-G isoforms for CIR-T cell targeting Chimeric antigen receptors (CARs) typically engage their macromolecular targets through a binding domain derived from an antibody, a VhH domain similar to an antibody, or, less commonly, a synthetic peptide selected for target affinity and screened for target specificity. In each of these cases, the binding domain achieves specificity and affinity randomly, and binders are selected from a wide diversity of a pool of potential binders. Importantly, the epitopes on the target engaged by the binders are most frequently linear groups of amino acids that can be removed or reconstructed by changes in sequence identity of different functional isoforms of the target protein produced by alternative mRNA splicing or post-translational modifications. In the case of HLA-G, the HLA-G2 isoform removes the entire α2 domain, and any antibody using an epitope that requires exposure of α2 will be masked from interacting with HLA-G2. The inventors have noticed that, conversely, receptor-ligand interactions between proteins tend to have broad regions of binding interactions that would be naturally selected. In the example of HLA-G interaction with ILT2 and ILT4, we realized that the functional performance of HLA-G as an agent of immune evasion by fetal placenta or tumor cells likely requires each functional isoform to interact with ILT2 and ILT4. Therefore, experiments were designed to test whether this natural selection for broad specificity means that chimeric ILT receptors (CIRs) have broader specificity for HLA-G isoforms than typical HLA-G-targeted chimeric antigen receptors (CARs).

[0301] Molm13-GFPffluc and Molm14-GFPffluc cells were derived from AML tumors and each was stably transduced to express GFP as a fusion with firefly luciferase. Each was assessed for HLA-G expression by flow cytometry, and Molm14 cells were found to be HLA-G positive (Figure 12). Primary human T cells from two donors were transduced with ILT4 CIR4, CIR6, and CIR7 constructs encoding ILT4 D1 and D2 domains as HLA-G binders with CD8α transmembrane domain and CD8α stalk (CIR4), CH2CH3 stalk (CIR6), or CH3 stalk (CIR7). In these experiments, HLA-G CAR-T cells were also generated.

[0302] Co-cultures of transduced T cells with Molm14 targets expressing HLAG were performed at an E:T ratio of 5:1. Co-cultures were evaluated for the ability of CIR-T or CAR-T cells to control tumor growth by monitoring GFP fluorescence in an Incucyte incubator / microscope (Figure 13A). Mock-transduced T cells not expressing CIR or CAR did not control the growth of Molm14 over 48 hours, whereas ILT4 CIR4-T co-cultures showed much reduced GFP fluorescence compared to mock-transduced cells. Given the poor expression of these constructs shown in Example 3, CIR6 and CIR7-T cells showed poor control of Molm14 cells, as expected. Surprisingly, HLA-G CAR-T cells also showed poor ability to control the growth of Molm14-GFPffluc cells.

[0303] Similar co-cultures were performed to determine the cytotoxicity of CIR-T and HLA-G CAR-T cells against Molm14-GFPffluc cells over a 24-hour period by the reduction in luciferase activity (Figure 13B). The luciferase activity produced by Molm14-GFPffluc cells co-cultured with mock-transduced T cells was identical to that of tumor cells cultured alone. Co-cultures with produced HLA-G CAR-T cells had very similar luciferase activity to co-cultures with mock-transduced T cells. This indicates that in an independent assay for cytotoxicity, HLA-G CAR-T cells were unable to target Molm14 AML cells. Conversely, ILT4 CIR4-T cells had substantial cytotoxicity against Molm14-GFPffluc targets. These results led to the hypothesis that HLA-G CAR-T cells may not recognize the HLA-G isoforms present on Molm14 cells that are instead specified and targeted by ILT4 D1D2 CIR.

[0304] To determine whether ILT4 CAR-T cells show broad specificity for targeting HLA-G isoforms, ILT4 CIR-T cells expressing optimal stalk and transmembrane domains (CIR4) or suboptimal domains with reduced expression (CIR6, CIR7), or HLA-G CAR-T cells were co-cultured with THP1-GFPluc cells transduced to express HLA-G1 only (Figure 14A) or HLA-G2 only (Figure 14B). Upon engagement of the binder with the target protein and activation of signal transduction, production of proinflammatory cytokines is induced. Secretion of interferon gamma (IFN-γ) into the medium was used as a surrogate for the activation state of T cells in the co-culture. As expected from the results shown in Example 2, co-culture of HLA-G1-expressing target cells with both ILT4 CIR4-T cells and HLA-G CAR-T cells showed enhanced IFN-γ production (Figure 14A). Interestingly, IFN-γ production was stimulated only in ILT4 CIR4-T cells cocultured with THP1-HLA-G2-GFPffluc target cells, and IFN-γ secretion was not supported by coculture of HLA-G CAR-T cells with target cells expressing only HLA-G2 (Figure 14B).

[0305] The results presented in this example showed that the specificity of HLA-G isoform targeting can be increased (e.g., the range of isoforms that can be targeted can be expanded) by using ILT D1 and D2 containing binders (ILT4 D1-D2 were used in this particular exemplary embodiment) compared to the specificity of antibody-derived binders in classical chimeric antigen receptors (CARs).

[0306] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of this invention, certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0307] Thus, the above merely illustrates the principles of the invention. It will be appreciated that those skilled in the art can devise various arrangements that embody the principles of the invention and are within the spirit and scope of the invention, although not expressly described or shown herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to further the art, and should not be construed as limitations to such specifically recited examples and conditions. Furthermore, all statements herein that describe the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be made public, regardless of whether such disclosure is expressly recited in the claims.

[0308] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the 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 a claim only when the precise phrase "means for" or the precise phrase "step for" is recited at the beginning of such limitation in the claim, and 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked if such precise phrase is not used in the limitation in the claim.

Claims

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

2. 2. The chimeric receptor protein of claim 1, wherein the targeting region comprises an amino acid mutation at a position corresponding to Y96 of the ILT4 amino acid sequence set forth in SEQ ID NO: 57 or Y96 of the ILT2 amino acid sequence set forth in SEQ ID NO:

31.

3. 2. The chimeric receptor protein of claim 1, wherein the D1-D2 extracellular domain is an ILT4 D1-D2 extracellular domain.

4. 2. The chimeric receptor protein of claim 1, wherein the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain.

5. 2. The chimeric receptor protein of claim 1, wherein the targeting region lacks the ILT4 or ILT2 D3-D4 extracellular domains.

6. The chimeric receptor protein of claim 5 , comprising a stalk domain.

7. 7. The chimeric receptor protein of claim 6, wherein the stalk domain comprises an ILT4, ILT2, CD28, CH2 / CH3, CH3, or CD8 stalk domain.

8. The chimeric receptor protein of claim 1, wherein the targeting region comprises the D3-D4 extracellular domain of ILT4 or ILT2.

9. 9. The chimeric receptor protein of claim 8, wherein the targeting region comprises an amino acid mutation at a position corresponding to Y394 of the ILT4 amino acid sequence set forth in SEQ ID NO: 55 or Y395 of the ILT2 amino acid sequence set forth in SEQ ID NO:

31.

10. The chimeric receptor protein of claim 1 , wherein the TM domain is an ILT4, ILT2, CD28, or CD8 TM domain.

11. The chimeric receptor protein of claim 1 , wherein the signaling region comprises an immunoreceptor tyrosine-based activation motif (ITAM).

12. The chimeric receptor protein of claim 1 , wherein the signaling region comprises a CD3ζ signaling domain, a DAP10 signaling domain, a DAP12 signaling domain, or any combination thereof.

13. The chimeric receptor protein of claim 1 , wherein the signaling region comprises a CD3ζ signaling domain.

14. The chimeric receptor protein of claim 1 , wherein the ICD further comprises a costimulatory region comprising at least one costimulatory domain.

15. The chimeric receptor protein of claim 14, wherein the at least one costimulatory domain comprises a CD28 costimulatory domain.

16. The chimeric receptor protein of claim 14, wherein the at least one costimulatory domain comprises a 4-1BB costimulatory domain.

17. 15. The chimeric receptor protein of claim 14, wherein the at least one costimulatory domain comprises a 4-1BB, OX40, CD28, ICOS, RANK, DAP10, DAP12, CD27, MyD88, IL-1Rα, HVEM, TRANCE, IL-1Rβ, CD70, IL-18Rα, CD40, IL-18Rβ, IL-33Rα, CD30, or IL-33Rβ costimulatory domain, or any combination thereof.

18. the D1-D2 extracellular domain is an ILT4 D1-D2 extracellular domain; the extracellular domain lacks the ILT4 D3-D4 extracellular domain; the chimeric receptor protein comprises a CD8 stalk domain; the TM region is a CD TM; the signaling region comprises a CD3ζ signaling domain; the chimeric receptor protein comprises a 4-1BB costimulatory domain; The chimeric receptor protein of claim 1.

19. the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain; the extracellular domain lacks the ILT2 D3-D4 extracellular domain; the chimeric receptor protein comprises a CD8 stalk domain; the TM region is a CD TM; the signaling region comprises a CD3ζ signaling domain; the chimeric receptor protein comprises a 4-1BB costimulatory domain; The chimeric receptor protein of claim 1.

20. the D1-D2 extracellular domain is an ILT4 D1-D2 extracellular domain; the extracellular domain comprises an ILT4 D3-D4 extracellular domain; the chimeric receptor protein comprises a CD8 stalk domain; the TM region is a CD TM; the signaling region comprises a CD3ζ signaling domain; the chimeric receptor protein comprises a 4-1BB costimulatory domain; The chimeric receptor protein of claim 1.

21. the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain; the extracellular domain comprises an ILT2 D3-D4 extracellular domain; the chimeric receptor protein comprises a CD8 stalk domain; the TM region is a CD TM; the signaling region comprises a CD3ζ signaling domain; the chimeric receptor protein comprises a 4-1BB costimulatory domain; The chimeric receptor protein of claim 1.

22. A nucleic acid comprising a nucleotide sequence encoding the chimeric receptor protein of claim 1.

23. 23. The nucleic acid of claim 22, wherein the nucleotide sequence is operably linked to a constitutive promoter.

24. 23. The nucleic acid of claim 22, wherein the nucleotide sequence is operably linked to an inducible promoter.

25. 23. The nucleic acid of claim 22, which is an expression vector.

26. 26. The nucleic acid of claim 25, wherein the expression vector is a retroviral vector, a lentiviral vector, or a plasmid vector.

27. A genetically modified cell that expresses the chimeric receptor protein of claim 1.

28. 28. The genetically modified cell of claim 27, which is an immune cell.

29. 29. The genetically modified cell of claim 28, wherein the immune cell is a natural killer (NK) cell, a T cell, an iNKT cell, or a macrophage.

30. 29. The genetically modified cell of claim 28, wherein the immune cell is a natural killer (NK) cell.

31. 29. The genetically modified cell of claim 28, wherein the immune cell is a T cell.

32. 28. The genetically modified cell of claim 27, which expresses a safety switch.

33. 33. The genetically modified cell of claim 32, wherein the safety switch is inducible.

34. 34. The genetically modified cell of claim 33, wherein the safety switch is triggered by rimiducid or by rapamycin or an analog of rapamycin.

35. 28. The genetically modified cell of claim 27, further expressing a chimeric antigen receptor.

36. 28. The genetically modified cell of claim 27, further expressing a costimulatory polypeptide that is not fused to the chimeric receptor protein.

37. 37. The genetically modified cell of claim 36, wherein the costimulatory polypeptide that is not fused to the chimeric receptor protein comprises a 4-1BB, OX40, CD28, ICOS, RANK, DAP10, DAP12, CD27, MyD88, IL-1Rα, HVEM, TRANCE, IL-1Rβ, CD70, IL-18Rα, CD40, IL-18Rβ, IL-33Rα, CD30, or IL-33Rβ costimulatory domain, or any combination thereof.

38. 28. A composition for use in a method of treatment, the method comprising administering the composition to an individual in need thereof, the composition comprising the genetically modified cell of claim 27.

39. 39. The composition of claim 38, wherein the genetically modified cells are autologous to the individual.

40. 39. The composition of claim 38, wherein the genetically modified cells are allogeneic to the individual.

41. 39. The composition of claim 38, wherein the individual has cancer.

42. 42. The composition of claim 41, wherein the individual has a solid tumor.

43. A method for producing genetically modified cells in vitro, comprising:

23. The method comprising introducing the nucleic acid of claim 22 into a cell, thereby producing a genetically modified cell.

44. 44. The method of claim 43, wherein the genetically modified cell is an immune cell.

45. 45. The method of claim 44, wherein the immune cell is a natural killer (NK) cell, a T cell, an iNKT cell, or a macrophage.

46. 45. The method of claim 44, wherein the immune cells are natural killer (NK) cells.

47. 45. The method of claim 44, wherein the immune cell is a T cell.

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