Chimeric ILT receptor compositions and methods

Abstract

A chimeric ILT receptor (CIR) is provided, which comprises a targeting region from ILT2 or ILT4, a transmembrane domain, and an intracellular domain (ICD). The ICD includes a signal transduction region (e.g., CD3 zeta (CD3 zeta), DAP10, DAP12), and optionally a co-stimulatory region (e.g., MyD88, TIR domain, etc. Also provided are nucleic acids (e.g., expression vectors) encoding the subject CIR, and genetically modified cells (e.g., immune cells, such as NK cells, NK-T cells, T cells, iNKT cells, macrophages, etc.) expressing the subject CIR.

Description

[0001] Cross-references This application claims the benefit of U.S. Provisional Patent Application No. 63 / 516,288, filed July 28, 2023, and U.S. Provisional Patent Application No. 63 / 607,881, filed December 8, 2023, which are incorporated herein by reference in their entirety.

[0002] Incorporate the sequence list provided in the XML file by reference. This article provides the sequence list as the sequence list XML file "NKLT-002WO_SEQ_LIST.xml", created on July 19, 2024, with a size of 141,995 bytes. The contents of the sequence list XML are incorporated into this article in their entirety by reference.

[0003] I. Introduction Immunotherapy can be used to specifically target diseased cells. This treatment has the potential to cure 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 proliferation of leukemia. Furthermore, genetic modification can direct immune cells (including T cells, natural killer (NK) cells, γd T cells, induced NK-T cells, and macrophages) to target cell populations for therapeutic purposes. For example, chimeric antigen receptor- (CAR-) T cells can be used to specifically redirect T cells to tumor-associated cell surface molecules, independent of human leukocyte antigen (HLA) presentation of peptide antigens to T cell receptors (TCRs). CAR proteins can be engineered to be expressed in other immune cells (including NK cells, γ / d T cells, iNK-T cells, and macrophages). In these cases, the recognition of target proteins by the CAR concentrates the cytotoxic potential of these cells, which typically use so-called innate receptors to recognize foreign or diseased tissues. Numerous preclinical and clinical studies have demonstrated the practicality of CAR technology expressed in T cells, NK cells, iNK-T cells, and macrophages, leading to the approval of six therapies targeting B-cell-derived tumors.

[0004] By altering CAR technology to change the binding mechanism of target recognition, the affinity and specificity of engineered cells for target proteins can be improved. For example, the antibody:antigen binding mechanism present on CAR proteins can be replaced by a receptor:ligand mechanism. An example of this is the engineering of the NKG-2D protein as a modified or native protein for expression on immune cells to recognize multiple ligands of NKG-2D (MICA, MIC-B, ULBP 1-5), which may be expressed in different combinations on diseased tissues. Another example further described in this patent is the expression of a chimeric ILT receptor or CIR, engineered to target HLA-G expressed on tumors. The CIR protein is engineered via binding portions derived from ILT2 or ILT4, the major receptors for immunosuppressive HLA-G. Binding to the native HLA-G receptor domain (ILT2 or ILT4) improves targeting of HLA-G, which is naturally expressed as seven protein isoforms derived from alternative mRNA splicing products. Targeting HLA via antibody / scFv CAR methods is limited by the loss of epitopes in one or more HLA-G isotypes.

[0005] T cells are the most commonly used cellular agents in targeted anticancer CAR therapy. Due to the risk of graft-versus-host disease (GvHD) associated with allogeneic T cell products, they are often used as products derived from autologous or patient cells. Cell types lacking TCR expression reduce the risk of autologous GvHD and can be produced for patients at a lower cost and faster rate. Cell products for anticancer therapy are increasingly being developed from alternative cell types, including NK cells, iNK-T cells, and macrophages. NK and iNK-T CAR-based products often employ CD28.CD3ζ or 4-1BB.CD3ζ signaling strategies to guide target-specific activation of cells and frequently incorporate mechanisms that promote IL-15 signaling. While these constructs do promote target-specific NK and iNK-T cell activation, the CAR mechanism is engineered to mimic the activation of T cells by APCs (especially dendritic cells), whereas NK cells and iNK-T cells are activated through mechanisms distinct from those of T cells. This raises the possibility that NK cell-based chimeric therapies may not be activated to achieve optimal performance when using traditional CAR-T cell constructs. There is a need to improve NK cell activation, particularly to enhance the durability of NK cell products to facilitate the clearance of large tumor burdens in patients and maintain monitoring of tumor escape and recurrence.

[0006] II. Summary of the Invention By altering the binding mechanism of target recognition through CAR technology, the affinity and specificity of engineered cells for target proteins can be improved. For example, the antibody:antigen binding mechanism present on CAR proteins can be replaced by a receptor:ligand mechanism. An example of this is the engineering of the NKG-2D protein as a modified or native protein for expression on immune cells to recognize multiple NKG-2D ligands (MICA, MIC-B, ULBP 1-5), which may be expressed in different combinations on diseased tissues. Another example further described in this article is the expression of a chimeric ILT receptor (CIR), engineered to target HLA-G expressed on tumors. The CIR protein is engineered via binding portions derived from ILT2 or ILT4, the major receptors for immunosuppressive HLA-G. Binding to the native HLA-G receptor domain (ILT2 or ILT4) improves targeting of HLA-G, which is naturally expressed as seven protein isoforms derived from alternative mRNA splicing products. Targeting HLA via antibody / scFv CAR methods is limited by the loss of epitopes in one or more HLA-G isotypes.

[0007] CIR proteins are designed to replace the negative signaling immune receptor tyrosine inhibitory motif (ITIM) of ILT2 or ILT4 with a domain that activates immune cell signaling. Improvements in the performance of non-T-cell therapies can be achieved by altering the signaling domains present in the intracellular domains of chimeric receptors such as CIRs, thereby changing the signal transduction pathways activated upon target binding. In some embodiments, the chimeric protein is a CIR containing a binding element derived from ILT2 or ILT4, each of which is a receptor for HLA-G proteins, which are typically expressed on tumors but not on normal tissues. In other embodiments, the chimeric protein is a CAR, wherein a single-chain variable fragment (scFv) derived from an antibody provides specific targeting of the tumor antigen.

[0008] This disclosure provides genetically modified cells engineered to express chimeric receptor proteins with affinity and specificity, enabling the modified cells to stimulate an immune response in a subject. For example, the chimeric receptor protein can target proteins expressed at high levels in tumor tissue relative to untransformed normal tissue and generate a cytotoxic or inflammatory response against the tumor. For the compositions and methods of this disclosure, affinity and specificity are not maintained by using antibodies or VhH relationships or by binding of randomly generated peptides to the target antigen, but rather by using ligand:receptor interactions, wherein affinity and specificity are maintained through evolution. In some embodiments, the cells expressing the subject chimeric receptor protein are NK cells. In some embodiments, the cells expressing the chimeric protein are iNK-T cells. In some embodiments, the cells expressing the chimeric protein are NK-T cells. In other embodiments, the cells expressing the chimeric protein are macrophages, γ / d T cells, or α / T cells. In each of these embodiments, immune cells can be genetically modified by introducing a vector (e.g., a polycistronic vector), for example, by transduction with gamma retrovirus, lentivirus, adenovirus, adeno-associated virus, or by transposon DNA transfection. For example, in some embodiments, genetically modified cells express a chimeric receptor with high affinity for HLA-G, a target protein present on tumor tissue in one or more of seven known forms, which can be generated through alternative mRNA splicing and post-translational modifications. HLA-G naturally acts as a suppressor of the immune response by binding to immunoglobulin-like transcript 2 (ILT2) and ILT4 receptors, which are negatively signaling on the surface of immune cells. In these embodiments, immune cells, such as T cells, NK cells, NK-T or iNKT cells, or macrophages, are engineered so that the recognition of the active form of HLA-G by ILT2 or ILT4 results in an activation signal. In some embodiments, the intracellular signaling elements of ILT2 or ILT4 are excised and replaced with ITAM-containing signaling domains of the CD3ζ chain (for DAP10 or DAP12), which drive immune cell activation and cytotoxicity. This protein is referred to as a “chimeric ILT receptor” or “CIR”.

[0009] Therefore, chimeric ILT receptors (CIRs) [plus the nucleic acids encoding them and the genetically modified cells expressing them, such as immune cells] are provided, comprising a targeting region, a transmembrane domain, and an intracellular domain (ICD) derived from ILT2 or ILT4. This ICD includes a signaling domain (e.g., CD3 zeta (CD3ζ), DAP10, DAP12) and optional co-stimulatory domains (e.g., a TIR domain, MyD88 protein, a domain stimulating MyD88 signaling, CD28, 4-1BB, OX40, etc.). CD3ζ is naturally expressed in NK cells, γ / δ T cells, and iNK-T cells, but these cell types also express other ITAM-containing signaling adaptors that are specific to innate receptors not naturally expressed by most T cells. In some embodiments, CD3ζ signaling is enhanced by fusion with the signaling domains of DAP10 or DAP12—each of these signaling adaptors containing an ITAM naturally expressed by NK cells. This addition aims to enhance the cytotoxic potency of CIRs or CAR-NK cells. In another embodiment, CD3ζ signaling is replaced by fusion with the signaling domains of DAP10 or DAP12. This replacement aims to optimize the durability of NK cell antitumor efficacy.

[0010] The inventors recognized that this approach has advantages over using antibody-based targeting regions (such as scFv). Antibody-based targeting methods can lead to selection of tumor cells expressing HLA-G isotypes lacking the target epitope—thus allowing cancer to evade treatment. In contrast, chimeric ILT receptors based on the primary ILT2 or ILT4 should target more, and possibly all, HLA-G isotypes, since ILT2 and ILT4 naturally bind to these isotypes. This would significantly reduce, and potentially eliminate, the ability of cancer cells to evade treatment by selecting specific HLA-G isotypes.

[0011] ILT2 and ILT4 have similar extracellular structures and consist of four folded domains (D1, D2, D3, and D4) arranged from distal to proximal relative to the cell membrane. HLA-G interacts with the D1 and D2 domains of ILT2 and ILT4, and these D1 and D2 domains can dissociate from the rest of the ILT protein while maintaining interaction with HLA-G. In some embodiments, in chimeric receptor fusions, ILT2 or ILT4 D3-D4 is replaced by another extracellular domain that serves as a stalk and transmembrane domain to present ILT2 or ILT4 D1-D2 to target cells expressing HLA-G. Examples of stalk proteins used for D1-D2 presentation are derived from CD28, CD8α, the CH2-CH3 region of IgG4, the proximal end of HER2, and mGluR2. In other embodiments, the D3-D4 domains of ILT2 and ILT4 are simply deleted. Therefore, in some embodiments, the subject-chimeric ILT receptor includes ILT2 D1-D2 but lacks ILT2 D3-D4. In some embodiments, the subject-chimeric ILT receptor includes ILT4 D1-D2 but lacks ILT4 D3-D4.

[0012] In CIR-NK constructs, signal transduction domains can be added to the ITAM domain and its cytotoxicity domain to improve cell activation, survival, activation, persistence, cytotoxicity, and the ability to produce pro-inflammatory cytokines. Typically, the signals transduced by these elements promote the PI3 kinase / AKT pro-survival pathway and the NF-κB pathway, the latter often synergistically or collaboratively promoting the expression of activation markers and cytokine release in conjunction with the NF-AT pathway promoted by ITAM domain signaling. In NK cells, these signal transduction elements are referred to as co-activating signals. A typical signal transduction element in many CAR constructs is 4-1BB. While 4-1BB is expressed by activated NK, iNK-T, and T cells, other members of the tumor necrosis factor receptor superfamily are also expressed, and signal transduction derived from alternative signal transduction domains may confer alternative phenotypes of cell products, such as differential cytotoxicity, growth potential, cytokine production, and survival.

[0013] In other embodiments, supporting co-activation signaling from 4-1BB can be replaced or supplemented by signaling from MyD88. This factor naturally serves as a signaling node downstream of innate signals from activated Toll-like receptors (TLRs), which are themselves partially specifically activated by pathogen-derived ligands, including RNA, DNA with altered methylation patterns, and lipid or protein endotoxins. These innate signals are strongly transduced in responding cells via MyD88 activation. Furthermore, MyD88 is a signaling node downstream of the IL-1 receptor superfamily of cytokines, including IL-18R and IL-33R, which are important activators of NK cells, iNK-T cells, and T cells. Incorporation of the death domain of MyD88 alone or in combination with TNF-R-derived signaling domains such as 4-1BB, HVEM, or CD40 increases the target-specific potency of NK cells against AML. Signaling from MyD88 also intrinsically increases the growth potential of NK cells.

[0014] MyD88 signaling is naturally recruited to the plasma membrane via the Toll / interleukin-1 receptor / resistance protein (TIR) ​​domain by IL-1 family cytokine receptors and the TLR. Activation of signaling by both the IL-18 receptor and the TLR is guided by a conformational change in the dimer receptor, leading to MyD88 oligomerization. In some embodiments, the TIR domain may be fused as a co-activating domain with a cytotoxic domain containing the ITAM domain. The purpose of this fusion is to indirectly recruit native MyD88 to the CIR, where it remains inactive until the CIR is activated and dimerized by the HLA-G dimer. When expressed in NK cells, these fusions result in low basal activity of the CIR in the absence of target cells, but produce high cytokine activity in the presence of target cells expressing HLA-G, but not in the presence of target cells lacking HLA-G expression.

[0015] Reagents, compositions, kits / systems, and methods related to chimeric ILT receptors are provided. For example, methods for preparing genetically modified cells and therapeutic methods (e.g., administering immune cells expressing the subject CIR, such as NK cells, T cells, or macrophages, to an individual) are provided.

[0016] III. Description of the Drawings The following detailed description of embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the precise arrangements and means of the embodiments shown in the drawings. The drawings illustrate certain embodiments of the technology and are not restrictive. For clarity and ease of explanation, the drawings are not drawn to scale, and in some cases, various aspects may be exaggerated or enlarged to facilitate understanding of particular embodiments. Figure 1 Interaction of ILT4 D1D2 CIR with HLA-G. (A) Cartoon illustration of the fusion of the D1 and D2 domains of ILT4 as chimeric hybrids with the stalk and transmembrane domains derived from independent proteins, and further with the activation of intracellular signal transduction components. The interaction of D1D2 with HLA-G initiates signal transduction to activate immune cells. Activation domains are contained within intracellular domains (ICDs) derived from signaling proteins that drive immune cell cytotoxicity, proliferation, persistence, and cytokine release. These domains can be a component driving cytotoxicity and activation, or chimeric domains that enhance cytotoxicity and further drive cell proliferation, persistence, and the production of pro-inflammatory cytokines.

[0017] Figure 2 : Retroviral expression constructs in the form of a CIR with alternative intracellular signal transduction domains. The diagram indicates a retroviral construct expressing the ILT2 and ILT4 fusion protein (called a chimeric ILT receptor (CIR)). D1 to D2 indicate the extracellular domains D1 to D2 encoding the native ILT4 domain. STM refers to the stem (S), a connector domain linking the extracellular D domains to the transmembrane domains (TM), which are derived from CD8α (CD8a). Indicates the different intracellular domain components driving activation signals.

[0018] Figure 3 TNF-α production in CIR-NK cells containing alternative ITAM-containing cytotoxic domains. (Top image) TNF-α production in NK cells expressing only CD3ζ, DAP10, or DAP12 as the basis for ITAM-containing signaling domains. Cells were not co-cultured with target cells. (Bottom image) TNF-α production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0019] Figure 4 IFN-γ production in CIR-NK cells containing alternative ITAM-containing cytotoxic domains. (Top image) IFN-γ production is based on the expression of CD3ζ, DAP10, or DAP12 as the primary expression of interferon-γ (IFN-γ) in NK cells containing ITAM signaling domains. Cells were not co-cultured with target cells. (Bottom image) IFN-γ production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0020] Figure 5CIR-NK cells containing alternative ITAM-containing cytotoxic domains were co-cultured with HLA-G-non-expressing KG1 target cells at an effector-target ratio of 1:5. (Top image) KG1 cells were co-cultured with NK cells derived from two donors for seven days. These NK cells either did not express CIR (mimicking) or expressed CIR in their intracellular domains that expressed only CD3ζ, DAP10, or DAP12. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with KG1 cells.

[0021] Figure 6 Co-culturing CIR-NK cells containing alternative ITAM-containing cytotoxic domains with Molm13 target cells expressing HLA-G1 and HLA-G5. (Top image) Molm13 cells were co-cultured for seven days with NK cells derived from two donors at an effector-to-target ratio of 1:10. These NK cells either did not express CIR (mimicking) or expressed CIR in their intracellular domains that expressed only CD3ζ, DAP10, or DAP12. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Molm13 cells.

[0022] Figure 7 TNF-α production in CIR-NK cells containing multiple ITAM-containing cytotoxic domains. (Top image) Basic tumor necrosis factor-α (TNF-α) production in NK cells expressing only CD3ζ, DAP10, and DAP12, or a combination of DAP10 and DAP12 with CD3ζ. Cells were not co-cultured with target cells. (Bottom image) TNF-α production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0023] Figure 8 IFN-γ production in CIR-NK cells containing multiple ITAM-containing cytotoxic domains. (Top image) Basic interferon-γ (IFN-γ) production in NK cells expressing only CD3ζ, DAP10, and DAP12, or a combination of DAP10 and DAP12 with CD3ζ. Cells were not co-cultured with target cells. (Bottom image) IFN-γ production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0024] Figure 9CIR-NK cells containing multiple ITAM-containing cytotoxic domains were co-cultured with HLA-G-non-expressing KG1 target cells at an effector-to-target ratio of 1:5. (Top image) KG1 cells were co-cultured for seven days with NK cells derived from two donors, these NK cells either did not express CIR (mimicking) or expressed CIRs in their intracellular domains that expressed only CD3ζ, DAP10, or DAP12, or a combination of DAP10 and DAP12 with CD3ζ. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with KG1 cells.

[0025] Figure 10 CIR-NK cells containing multiple ITAM-containing cytotoxic domains were co-cultured with Molm13 target cells expressing HLA-G1 and HLA-G5. (Top image) Molm13 cells were cultured for seven days with NK cells derived from two donors at an effector-to-target ratio of 1:10. These NK cells either did not express CIR (mimicking) or expressed CIRs in their intracellular domains that expressed only CD3ζ, DAP10, or DAP12, or a combination of DAP10 and DAP12 with CD3ζ. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Molm13 cells.

[0026] Figure 11 TNF-α production in CIR-NK cells containing co-activation domains and ITAM-containing cytotoxic signaling domains. (Top image) Basal production of tumor necrosis factor-α (TNF-α) in NK cells expressing only CD3ζ or a chimera of CD3ζ with signaling elements from 4-1BB or HVEM. Cells were not co-cultured with target cells. (Bottom image) TNF-α production in CIR-NK cells after 24 hours of co-culture with Kasumi1AML target cells.

[0027] Figure 12 IFN-γ production in CIR-NK cells containing co-activation domains and ITAM-containing cytotoxic signaling domains. (Top image) Basic interferon-γ (IFN-γ) production in NK cells expressing only CD3ζ or a chimera of CD3ζ with signaling elements from 4-1BB or HVEM. Cells were not co-cultured with target cells. (Bottom image) IFN-γ production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0028] Figure 13CIR-NK cells containing co-activating domains and ITAM-containing cytotoxic signaling domains were co-cultured with HLA-G-non-expressing KG1 target cells at an effector-to-target ratio of 1:5. (Top image) KG1 cells were cultured for seven days with NK cells derived from two donors, these NK cells expressing either no CIR (mimicking) or expressing only CD3ζ in their intracellular domains, or a chimera of CD3ζ with signaling elements from 4-1BB or HVEM. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with KG1 cells.

[0029] Figure 14 CIR-NK cells containing co-activation domains and ITAM-containing cytotoxic signaling domains were co-cultured with Molm13 target cells expressing HLA-G1 and HLA-G5. (Top image) Molm13 cells were co-cultured for seven days with NK cells derived from two donors at an effector-to-target ratio of 1:10. These NK cells either did not express CIR (mimicking) or expressed only CD3ζ in their intracellular domains, or a chimeric CIR of CD3ζ and signaling elements from 4-1BB or HVEM. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Molm13 cells.

[0030] Figure 15 TNF-α production in CIR-NK cells containing co-activation domains and multiple ITAM-containing cytotoxic signaling domains. (Top image) Basic tumor necrosis factor-α (TNF-α) production in NK cells expressing only CD3ζ or a chimera of CD3ζ with DAP10 or DAP12 combined with signaling elements from 4-1BB. Cells were not co-cultured with target cells. (Bottom image) TNF-α production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0031] Figure 16 IFN-γ production in CIR-NK cells containing co-activation domains and multiple ITAM-containing cytotoxic signaling domains. (Top image) Basic interferon-γ (IFN-γ) production in NK cells expressing only CD3ζ or a chimera of CD3ζ with DAP10 or DAP12 combined with signaling elements from 4-1BB. Cells were not co-cultured with target cells. (Bottom image) IFN-γ production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0032] Figure 17CIR-NK cells containing co-activating domains and multiple ITAM-containing cytotoxic signaling domains were co-cultured with HLA-G-non-expressing KG1 target cells at an effector-to-target ratio of 1:5. (Top image) KG1 cells were cultured for seven days with NK cells derived from two donors, these NK cells expressing either no CIR (mimicking) or expressing only CD3ζ or a chimera of CD3ζ with DAP10 or DAP12 combined with signaling elements from 4-1BB. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with KG1 cells.

[0033] Figure 18 CIR-NK cells containing co-activating domains and multiple ITAM-containing cytotoxic signaling domains were co-cultured with Molm13 target cells expressing HLA-G1 and HLA-G5. (Top image) Molm13 cells were co-cultured with NK cells derived from two donors at an effector-to-target ratio of 1:10 for seven days. These NK cells either did not express CIR (mimicking) or expressed only CD3ζ or a chimera of CD3ζ with DAP10 or DAP12 combined with signaling elements from 4-1BB in their intracellular domains. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Molm13 cells.

[0034] Figure 19 TNF-α production in CIR-NK cells containing an ITAM-containing cytotoxic domain and a MyD88-derived co-activating signaling element. (Top image) Basal production of tumor necrosis factor-α (TNF-α) in NK cells expressing only CD3ζ or DAP12, or a chimera of CD3ζ and DAP12 (with or without a co-activating domain derived from the MyD88 death domain). Cells were not co-cultured with target cells. (Bottom image) TNF-α production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0035] Figure 20 IFN-γ production in CIR-NK cells containing an ITAM-containing cytotoxic domain and a MyD88-derived co-activating signaling element. (Top image) Basal interferon-γ (IFN-γ) production in NK cells expressing only CD3ζ or DAP12, or a chimera of CD3ζ and DAP12 (with or without a co-activating domain derived from the MyD88 death domain). Cells were not co-cultured with target cells. (Bottom image) IFN-γ production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0036] Figure 21 CIR-NK cells containing MyD88 as a coactivating domain and an ITAM-containing cytotoxic signaling domain were co-cultured with HLA-G-non-expressing KG1 target cells at an effector-to-target ratio of 1:5. (Top image) KG1 cells were co-cultured for seven days with NK cells derived from two donors, which either did not express CIR (mimicking) or expressed CIR with only CD3ζ or DAP12 or a chimera of CD3ζ and DAP12 in their intracellular domains (with or without a coactivating domain derived from the MyD88 death domain). The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with KG1 cells.

[0037] Figure 22 CIR-NK cells containing MyD88 as a coactivating domain and an ITAM-containing cytotoxic signaling domain were co-cultured with Molm13 target cells expressing HLA-G1 and HLA-G5. (Top image) Molm13 cells were co-cultured with NK cells derived from two donors at an effector-to-target ratio of 1:10 for seven days. These NK cells either did not express CIR (mimicking) or expressed CIR whose intracellular domains expressed only CD3ζ or DAP12, or a chimera of CD3ζ and DAP12 (with or without a coactivating domain derived from the MyD88 death domain). The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Molm13 cells.

[0038] Figure 23 TNF-α production in CIR-NK cells containing MyD88 and TNF-R-derived coactivating domains and an ITAM-containing cytotoxic signaling domain. (Top image) Basal production of tumor necrosis factor-α (TNF-α) in NK cells expressing only CD3ζ or DAP12, or a chimera of CD3ζ and DAP12 (with or without a coactivating domain derived from the MyD88 death domain, and with or without additional coactivating domains derived from 4-1BB or HVEM). Cells were not co-cultured with target cells. (Bottom image) TNF-α production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0039] Figure 24TNF-γ production in CIR-NK cells containing MyD88 and IFN-R-derived coactivating domains and an ITAM-containing cytotoxic signaling domain. (Top image) Basal interferon-γ (IFN-γ) production in NK cells expressing only CD3ζ or DAP12, or a chimera of CD3ζ and DAP12 (with or without a coactivating domain derived from the MyD88 death domain, and with or without additional coactivating domains derived from 4-1BB or HVEM). Cells were not co-cultured with target cells. (Bottom image) IFN-γ production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0040] Figure 25 CIR-NK cells containing MyD88 and TNF-R-derived coactivating domains and ITAM-containing cytotoxic signaling domains were co-cultured with HLA-G-non-expressing KG1 target cells. (Top image) KG1 cells were co-cultured for seven days with NK cells derived from two donors at an effector-target ratio of 1:5. These NK cells either did not express CIR (mimicking) or expressed CIR only in their intracellular domains CD3ζ or DAP12, or CD3ζ and DAP12 chimeras (with or without a coactivating domain derived from the MyD88 death domain, and with or without additional coactivating domains derived from 4-1BB or HVEM). The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with KG1 cells.

[0041] Figure 26 CIR-NK cells containing MyD88 and TNF-R-derived coactivation domains and ITAM-containing cytotoxic signaling domains were co-cultured with Molm13 target cells expressing HLA-G1 and HLA-G5. (Top image) Molm13 cells were co-cultured for seven days with NK cells derived from two donors at an effector-to-target ratio of 1:10. These NK cells either did not express CIR (mimicking) or expressed CIR whose intracellular domains expressed only CD3ζ or DAP12, or a chimera of CD3ζ and DAP12 (with or without the coactivation domain derived from the MyD88 death domain). The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Molm13 cells.

[0042] Figure 27TNF-α production in CIR-NK cells containing an ITAM-containing cytotoxic signaling domain and a TIR-containing co-activation domain. (Top image) TNF-α production in NK cells expressing only CD3ζ or DAP12, or a chimera of CD3ζ and DAP12 (with or without a co-activation domain containing a TIR domain derived from MyD88 or the interleukin-18 receptor α chain (IL-18Rα), Toll-like receptor (TLR) 2, or TLR3). Cells were not co-cultured with target cells. (Bottom image) TNF-α production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0043] Figure 28 IFN-γ production in CIR-NK cells containing an ITAM-containing cytotoxic signaling domain and a TIR-containing co-activation domain. (Top image) Basic interferon-γ (IFN-γ) production in NK cells expressing only CD3ζ or DAP12, or a chimera of CD3ζ and DAP12 (with or without a co-activation domain containing a TIR domain derived from MyD88 or the interleukin-18 receptor α chain (IL-18Rα), Toll-like receptor (TLR) 2, or TLR3). Cells were not co-cultured with target cells. (Bottom image) IFN-γ production in CIR-NK cells after 24 hours of co-culture with Kasumi1 AML target cells.

[0044] Figure 29 CIR-NK cells containing an ITAM-containing cytotoxic signaling domain and a TIR-containing co-activation domain were co-cultured with KG1 target cells that did not express HLA-G. (Top image) KG1 cells were co-cultured with NK cells derived from two donors at an effector-target ratio of 1:5 for seven days. These NK cells either did not express CIR (mimicking) or expressed CIRs whose intracellular domains expressed only CD3ζ or DAP12, or a chimera of CD3ζ and DAP12 (with or without a co-activation domain of a TIR domain derived from MyD88 or the interleukin-18 receptor α chain (IL-18Rα), Toll-like receptor (TLR) 2, or TLR3). The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with KG1 cells.

[0045] Figure 30CIR-NK cells containing an ITAM-containing cytotoxic signaling domain and a TIR-containing co-activation domain were co-cultured with Molm13 target cells expressing HLA-G1 and HLA-G5. (Top image) Molm13 cells were cultured with NK cells derived from two donors at an effector-to-target ratio of 1:10 for seven days. These NK cells either did not express CIR (mimicking) or expressed CIRs whose intracellular domains expressed only CD3ζ or DAP12, or a chimera of CD3ζ and DAP12 (with or without a co-activation domain of a TIR domain derived from MyD88 or the interleukin-18 receptor α chain (IL-18Rα), Toll-like receptor (TLR) 2, or TLR3). The growth of tumor cells cultured alone was used as a reference indicator. (Bottom image) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Molm13 cells.

[0046] Figure 31 Retroviral expression constructs in CIR form with alternative intracellular signal transduction domains. The diagram indicates retroviral constructs expressing the extracellular domains of ILT4 (left) or ILT2 (right) D1 and D2, as well as the CD8α stalk and transmembrane domain. The intracellular signal transduction domains are formatted as shown, oriented from the N-terminus (proximal membrane) to the C-terminus (distal membrane).

[0047] Figure 32 Viability of CIR-NK cells transduced with a CIR construct containing alternative signal transduction domains. Cell viability at 8 days post-activation (5 days post-transduction) or 14 days post-activation (11 days post-transduction) was assessed by propidium iodide exclusion assay combined with actinomycin D co-staining. Cell transduction rate was greater than 80%, and viability was high at every time point.

[0048] Figure 33 Expansion of CIR-NK cells in culture after transduction with a CIR construct having alternative signal transduction domains. Transduced CIR-NK cells were counted on day 5 post-activation and again on day 8 (top figure) and day 14 to determine the relative proliferative capacity of NK cells during the expansion period.

[0049] Figure 34 Expression levels of CIR proteins containing alternative signal transduction domains during NK cell expansion. ILT4 CIR protein expression was measured on days 8 and 14 post-activation using flow cytometry with an ILT4 D1 / D2 antibody, and measured by mean fluorescence intensity (MFI) (top panel). Internal normalization of the same cells was performed by quantifying the co-transduced red fluorescent protein (bottom panel).

[0050] Figure 35IFN-γ production in CIR-NK cells containing alternative signal transduction domains. (Top image) Basal production of interferon-γ (IFN-γ) in 200,000 NK cells expressing only CD3ζ or a chimera of CD3ζ with DAP10 or DAP12 combined with signal transduction elements from 4-1BB. Cells were not co-cultured with target cells. (Bottom image) Total IFN production, expressed per cell.

[0051] Figure 36 IFN-γ production when CIR-NK cells containing alternative signal transduction domains are co-cultured with target cells derived from acute myeloid leukemia (AML). (Top image) NK cells transduced solely by RFP (RFP) or CIR-NK cells are co-cultured with Kasumi1 target cells at a 1:10 E:T ratio, and IFN-γ levels are measured by ELISA after 24 hours (top image). (Bottom image) Total IFN production, expressed on a per-cell basis.

[0052] Figure 37 IFN-γ production when CIR-NK cells containing alternative signal transduction domains are co-cultured with target cells derived from solid tumors. (Top image) NK cells transduced solely by RFP (RFP) or CIR-NK cells are co-cultured with HT1376 bladder cancer target cells at a 1:10 E:T ratio, and IFN-γ levels are measured by ELISA after 24 hours (top image). (Bottom image) Total IFN production expressed on a per-cell basis.

[0053] Figure 38 Short-term cytotoxicity of CIR-NK cells containing alternative signaling domains co-cultured with Molm13 target cells expressing HLA-G1 and HLA-G5. (Top panel) Molm13 cells were cultured for two days with NK cells derived from two donors at an effector-to-target ratio of 1:10. These NK cells either did not express CIR (RFP) or expressed CIRs with the indicated signaling domains. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom panel) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Molm13 cells.

[0054] Figure 39Cytotoxicity of CIR-NK cells containing alternative signaling domains co-cultured with Molm13 target cells expressing HLA-G1 and HLA-G5 for 7 days. (Top figure) Molm13 cells were cultured for seven days with NK cells derived from two donors at an effector-to-target ratio of 1:40. These NK cells either did not express CIR (RFP) or expressed CIRs with the indicated signaling domains. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom figure) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Molm13 cells.

[0055] Figure 40 Visual representation of cytotoxicity after 7 days of co-culturing CIR-NK cells containing alternative signaling domains with MOLM13 target cells expressing HLA-G1 and HLA-G5. Molm13-GFP cells were cultured for 7 days with RFP-labeled NK cells that did not express CIR (RFP) or expressed CIRs with the indicated signaling domains. Images were taken with Incucyte. Relative tumor expansion is indicated by green fluorescence, and NK cell expansion is indicated by red fluorescence. Figure 41 Short-term cytotoxicity of CIR-NK cells containing alternative signaling domains co-cultured with Kasumi1 target cells expressing HLA-G1 and HLA-G5. (Top panel) Kasumi1 cells were cultured for two days with NK cells derived from two donors at an effector-to-target ratio of 1:20. These NK cells either did not express CIR (RFP) or expressed the indicated signaling domains. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom panel) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Kasumi1 cells.

[0056] Figure 42 Cytotoxicity of CIR-NK cells containing alternative signaling domains co-cultured with Kasumi1 target cells expressing HLA-G1 and HLA-G5 for 7 days. (Top figure) Kasumi1 cells were cultured for seven days with NK cells derived from two donors at an effector-to-target ratio of 1:40. These NK cells either did not express CIR (RFP) or expressed the indicated signaling domains. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom figure) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with Kasumi1 cells.

[0057] Figure 43HLA-G expression in cell lines derived from solid tumors. HLA-G expression in dissociated HCT-116 colon cancer cells and HT-1376 bladder cancer cells was determined by flow cytometry using the MEMG / 9 antibody. EGFR expression was used as a separate control for population integrity.

[0058] Figure 44 Cytotoxicity of CIR-NK cells containing alternative signaling domains co-cultured with HT-1376-GFPffluc target cells expressing HLA-G1, HLA-G2, and HLA-G5 for 7 days. (Top figure) GFP-labeled cells were cultured for seven days with NK cells derived from two donors at an effector-to-target ratio of 1:10. These NK cells either did not express CIR (RFP) or expressed CIRs with the indicated signaling domains. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom figure) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with HT-1376 cells.

[0059] Figure 45 Cytotoxicity of CIR-NK cells containing alternative signaling domains co-cultured with HCT-116-GFPffluc target cells that do not express HLA-G for 7 days. (Top figure) HCT-116 cells labeled with GFP were cultured with NK cells from two donors at an effector-target ratio of 1:10 for seven days. These NK cells either did not express CIR (RFP) or expressed CIR with the signaling domains shown. The growth of tumor cells cultured alone was used as a reference indicator. (Bottom figure) The levels of RFP-labeled NK cells and CIR-NK cells were measured during co-culture with HCT-116 cells.

[0060] Figure 46 HLA-G expression in SU-8686 cells derived from pancreatic ductal adenocarcinoma. HLA-G expression in dissociated SU8686 cells was determined by flow cytometry using the MEMG / 9 antibody.

[0061] Figure 47Cytotoxicity of CIR-NK cells containing alternative signaling domains co-cultured with SU8686-GFPffluc target cells expressing HLA-G1 and HLA-G2 for 7 days. (Top) SU8686 cells labeled with GFP were cultured for seven days with NK cells derived from two donors at an effector-to-target ratio of 1:10. These NK cells either did not express CIR (RFP) or expressed the indicated signaling domains. 1G indicates a first-generation ILT2 CIR containing only the CD3ζ signaling domain. The growth of tumor cells cultured alone was used as a reference indicator. (Middle) Levels of RFP-labeled NK cells and CIR-NK cells were measured in co-culture with SU8686 cells. (Bottom) The co-culture was harvested, and cell quantification was performed by gating CD56 expression (NK cells) and GFP expression (target cells) using flow cytometry.

[0062] Figure 48 Visual representation of cytotoxicity after 7 days of co-culturing CIR-NK cells containing alternative signaling domains with SU8686 target cells expressing HLA-G1 and HLA-G2. SU8686-GFP cells were cultured for 7 days with RFP-labeled NK cells (E:T 1:5) that did not express CIR (RFP) or expressed the indicated signaling domains. Images representing NK cells from a single donor were captured using Incucyte. Relative tumor expansion is indicated by green fluorescence, and NK cell expansion by red fluorescence.

[0063] Figure 49 Cytotoxicity of CIR-NK cells containing alternative signaling domains co-cultured with Molm13 target cells expressing HLA-G1 and HLA-G5 for 7 days after long-term expansion. (Top image) Molm13 cells were cultured for seven days with NK cells derived from two donors at an effector-to-target ratio of 1:20. These NK cells either did not express CIR (RFP) or expressed the indicated signaling domains. The NK cells in the top image were expanded to standard for 14 days prior to co-culture. The growth of tumor cells cultured alone is used as a reference indicator. The cells in the bottom image were cultured for 26 days to examine the persistence of enhanced cytotoxicity associated with CIR signaling.

[0064] Figure 50The persistence of cytotoxicity of CIR-NK cells containing alternative signal transduction domains in repeated co-cultures with Molm13 target cells over 9 days. Molm13 cells were cultured with NK cells transduced to express only RFP (RFP) or the CIR-NK cells shown at an E:T ratio of 1:10. Co-cultures were expanded for 9 days (1X, top left), or re-seeded with the same number of Molm13 target cells only on day 2 (2X, top right), days 2 and 5 (3X, bottom left), and days 2, 5, and 7 (4X, bottom right).

[0065] Figure 51 The persistence of cytotoxicity of CIR-NK cells containing alternative signal transduction domains co-cultured with Molm13 target cells over 9 days. Molm13 cells were cultured with NK cells transduced to express only RFP (RFP) or the indicated CIR-NK cells (and RFP) at an E:T ratio of 1:10. Cocultures were expanded for 9 days (1X, top left), or re-seeded with the same number of Molm13 target cells only on day 2 (2X, top right), days 2 and 5 (3X, bottom left), and days 2, 5, and 7 (4X, bottom right).

[0066] Figure 52 Expansion of CIR-NK cells containing alternative signal transduction domains and Molm13 target cells by repeated co-culture over 9 days. Molm13 cells were cultured with NK cells transduced to express only RFP (RFP) or the indicated CIR-NK cells (and RFP) at an E:T ratio of 1:10. Co-cultures were expanded for 9 days (1X, top left), or re-seeded with the same number of Molm13 target cells only on day 2 (2X, top right), days 2 and 5 (3X, bottom left), and days 2, 5, and 7 (4X, bottom right).

[0067] Figure 53 Stress test of initial cytotoxicity in co-culture of CIR-NK cells containing alternative signaling domains with Molm13 target cells. Molm13-GFPffluc was co-cultured for two days with NK cells derived from three donors at effector-to-target ratios increasing from 1:40 to 2:1. These NK cells either did not express CIR (RFP) or expressed the indicated signaling domains. On day 2, tumor growth was measured by quantifying GFP fluorescence in Incucyte. The growth of tumor cells cultured alone was used as a reference indicator. CIR-NK cells with ILT4-derived extracellular domains are shown in the left panel, and ILT2-derived CIR-NK cells are shown in the right panel.

[0068] Figure 54Stress assay for cytotoxicity of CIR-NK cells containing alternative signaling domains co-cultured with Molm13 target cells for 7 days. Molm13-GFPffluc was co-cultured for seven days with NK cells derived from three donors at effector-to-target ratios increasing from 1:40 to 2:1. These NK cells either did not express CIR (RFP) or expressed the indicated signaling domains. On day 2, tumor growth was measured by quantifying GFP fluorescence in Incucyte. The growth of tumor cells cultured alone was used as a reference indicator. CIR-NK cells with ILT4-derived extracellular domains are shown in the left panel, and ILT2-derived CIR-NK cells are shown in the right panel.

[0069] Figure 55 Stress assay of initial expansion of CIR-NK cells containing alternative signaling domains co-cultured with Molm13 target cells. Molm13-GFPffluc was co-cultured for two days with NK cells derived from three donors at effector-to-target ratios increasing from 1:40 to 2:1. These NK cells either did not express CIR (RFP) or expressed CIRs with the indicated signaling domains. On day 2, NK cell growth was measured by quantifying RFP fluorescence in Incucyte. CIR-NK cells with ILT4-derived extracellular domains are shown in the left panel, and ILT2-derived CIR-NK cells are shown in the right panel.

[0070] Figure 56 Stress assay of 7-day expansion of CIR-NK cells containing alternative signaling domains co-cultured with Molm13 target cells. Molm13-GFPffluc was co-cultured for seven days with NK cells derived from three donors at effector-to-target ratios increasing from 1:40 to 2:1. These NK cells either did not express CIR (RFP) or expressed CIRs with the indicated signaling domains. On day 2, NK cell growth was measured by quantifying RFP fluorescence in Incucyte. CIR-NK cells with ILT4-derived extracellular domains are shown in the left panel, and ILT2-derived CIR-NK cells are shown in the right panel.

[0071] Figure 57Cytotoxicity of CIR-NK cells containing alternative signaling domains co-cultured with OE19-GFPffluc esophageal cancer target cells expressing low levels of HLA-G1 and HLA-G2 for 7 days. (Left) GFP-labeled OE19 cells were co-cultured with NK cells derived from two donors at an effector-target ratio of 1:5 (left panel) or 1:10 (right panel) for seven days. These NK cells either did not express CIR (simulated) or expressed ILT4 CIRs with the indicated signaling domains. 1G indicates a first-generation ILT4 CIR containing only the CD3ζ signaling domain. The growth of tumor cells cultured alone is used as a reference indicator.

[0072] Figure 58 Timeline of CIR-NK cells containing alternative signal transduction domains co-cultured with target cells after 19 days of continuous exposure to solid tumor target cells. RFP-labeled NK cells or CIR-NK cells derived from two donors were co-cultured with HT-1376 target cells at a 2:1 E:T ratio. After 5 days of co-culture, NK cells were harvested, counted, and reseeded with fresh tumor target cells. This process was repeated twice, with NK cells co-cultured with solid tumor or leukemia target cells at the indicated E:T ratio for 7 days.

[0073] Figure 59 Expansion of CIR-NK cells containing alternative signaling domains during 19 days of continuous exposure to HT-1376 target cells. RFP-labeled NK cells or CIR-NK cells derived from three donors were co-cultured with HT-1376 target cells at a 2:1 E:T ratio. After 5 days of co-culture, NK cells were harvested, counted, and reseeded with fresh tumor target cells. This process was repeated twice. Cell counts relative to previous seeding were plotted to indicate the relative expansion of NK cells with each exposure to the tumor target.

[0074] Figure 60 Cytotoxicity and expansion of Molm13 cells by CIR-NK cells containing alternative signal transduction domains after 19 days of continuous exposure to HT-1376 target cells. RFP-labeled NK cells or CIR-NK cells derived from three donors were co-cultured with HT-1376 target cells at a 2:1 E:T ratio. After 5 days of co-culture, NK cells were harvested, counted, and reseeded with fresh HT-1376 cells. This process was repeated twice. After 19 days and four consecutive exposures to HLA-G-expressing target cells, NK cells were co-cultured with Molm13-GFPffluc target cells at an E:T ratio of 1:10 (left) or 1:20 (right) for 7 days. Tumor cell expansion (top) and NK cell expansion (bottom) were measured using GFP and RFP fluorescence in Incucyte.

[0075] Figure 61 Visual representation of the cytotoxicity of CIR-NK cells containing alternative signaling domains after 7 days of co-culture with Molm13 target cells following 19 days of sustained exposure to HLA-G-expressing target cells. Molm13-GFP cells were cultured for 7 days with RFP-labeled NK cells (E:T = 1:10) that did not express CIR (simulated) or expressed the signaling domains shown. Images representing NK cells from one donor were captured using Incucyte. Relative tumor expansion is indicated by green fluorescence, and NK cell expansion derived from donors 142 and 226 is indicated by red fluorescence.

[0076] Figure 62 Cytotoxicity and expansion of OE19 cells by CIR-NK cells containing alternative signal transduction domains after 19 days of continuous exposure to HT-1376 target cells. RFP-labeled NK cells or CIR-NK cells derived from three donors were co-cultured with HT-1376 target cells at a 2:1 E:T ratio. After 5 days of co-culture, NK cells were harvested, counted, and reseeded with fresh HT-1376 cells. This process was repeated twice. After 19 days and four consecutive exposures to HLA-G-expressing target cells, NK cells were co-cultured with OE19-GFPffluc target cells at an E:T ratio of 1:5 (left) or 1:10 (right) for 7 days. Tumor cell expansion (top) and NK cell expansion (bottom) were measured using GFP and RFP fluorescence in Incucyte.

[0077] Figure 63 Visual representation of the cytotoxicity of CIR-NK cells containing alternative signaling domains after 7 days of co-culture with OE19 target cells following 19 days of sustained exposure to HLA-G-expressing target cells. OE19-GFP cells were cultured for 7 days with RFP-labeled NK cells (E:T 1:5) that did not express CIR (simulated) or expressed the signaling domains shown. Images representing NK cells from one donor were captured using Incucyte. Relative tumor expansion is indicated by green fluorescence, and NK cell expansion derived from donors 142 and 226 is indicated by red fluorescence.

[0078] Figure 64Interferon-γ production in CIR-NK cells containing alternative signal transduction domains during 19 days of continuous exposure to HT-1376 target cells. RFP-labeled NK cells or CIR-NK cells derived from three donors were co-cultured with HT-1376 target cells at a 2:1 E:T ratio. After 5 days of co-culture, NK cells were harvested, counted, and reseeded with fresh tumor target cells. This process was repeated three times. Interferon-γ (IFN-γ) levels were measured 48 hours after each consecutive seeding, and the total level was divided by the number of NK cells at each seeding.

[0079] Figure 65 Control of Molm13-GFPffluc amplification in NSG mice by CIR-NK cells expressing alternative signal transduction domains. Molm13-GFPffluc cells were implanted into immunodeficient NSG mice and amplified for 5 days. NK cells (7.5E6 cells / mouse) were co-transduced with a retrovirus encoding ONLRluc (for bioluminescence (BLI) detection) and a CIR vector (or a mimic transduction) and intravenously injected into NSG mice carrying Molm13 tumors. (Left) BLI of NK cells was measured 14 days after NK cell injection using coelenterate as a substrate. (Right) Systemic BLI of Molm13-GFPffluc was measured 10 days after NK cell injection using fluorescein as a substrate.

[0080] Figure 66 : Control of Molm13-GFPffluc amplification in the bone marrow of NSG mice by CIR-NK cells expressing alternative signal transduction domains. 17 days after NK cell transplantation, from Figure 65 Bone marrow was harvested from selected mice as outlined in the section. Human CD45 was extracted from total bone marrow cells. + Cells were gated and GFP was analyzed by flow cytometry. + Molm13 tumor cells were quantified.

[0081] Figure 67 The migration of CIR-NK cells into the bone marrow of NSG mice and the control of Molm13-GFPffluc amplification by CIR-NK cells expressing alternative signaling domains. Bone marrow was harvested from selected mice 17 days after NK cell transplantation. Human CD45 was analyzed from total bone marrow cells. + Cells were gated and GFP was analyzed by flow cytometry. +Quantification of Molm13 tumor cells and human NK cells (orange nanolanterns (ONL)) is presented. Representative flow cytometry plots of bone marrow derived from group 2 (CIR-less mimicry transduced NK cells), group 3 (first-generation signal transduction domain, ILT4 CIR-CD3ζ), group 7 (ILT4 CIR-BB.DAP10.TLR2), and group 11 (ILT2 CIR-BB.DAP10.TLR2) are shown.

[0082] IV. Detailed Implementation Before further describing the invention, it should be understood that the invention is not limited to the specific embodiments described, as these embodiments can certainly be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention is limited only by the appended claims.

[0083] Where a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (one-tenth of a unit to the lower limit, unless otherwise explicitly stated) and any other stated or intermediate value within the range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in even smaller ranges and are also covered by this invention, subject to any explicitly excluded limits within the stated ranges. Where a stated range contains one or both of the limitations, the range excluding any one or both of the included limitations is also included by this invention.

[0084] The numerical ranges given in this article are preceded by the term "approximately". The term "approximately" is used to provide literal support for the exact number that follows it, as well as numbers that are close to or approximate to the number following the term. In determining whether a number is close to or approximates a specifically listed number, an unlisted number that is close to or approximates can be a number that provides a substantial equivalence to the specifically listed number in its presented context.

[0085] 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 pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, representative illustrative methods and materials are described hereafter.

[0086] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the referenced publications. Reference to any publication is made because it was published prior to the filing date and should not be construed as an admission that the invention is not entitled to precedence over such publications by means of prior art. Furthermore, the publication date provided may differ from the actual publication date, which may require independent verification.

[0087] It should be noted that, unless the context explicitly indicates otherwise, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators. Therefore, the articles “an” and “a” herein refer to one or more (i.e., at least one) grammatical objects of the article. For example, “element” means one or more elements. Thus, for example, a reference to “cell” includes a plurality of such cells, and a reference to “polypeptide” includes a reference to one or more peptides and their equivalents known to those skilled in the art, and so on. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a precondition for the use of exclusive terms such as “solely” or “only” or the use of “negative” restrictions in conjunction with the elements of the claims.

[0088] Those skilled in the art will understand upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any enumerated method may be performed in the order of the enumerated events or in any other logically possible order. For example, it should be understood that certain features of the invention described in separate embodiments for clarity may also be provided in combination form in a single embodiment. Conversely, different features of the invention described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination. All combinations of embodiments belonging to the invention are specifically included in the invention and are disclosed herein as each combination and each combination is individually and clearly disclosed. Furthermore, all sub-combinations of various embodiments and their elements are also specifically included in the invention and disclosed herein as each and each such sub-combination is individually and clearly disclosed herein.

[0089] Although, for the sake of grammatical fluency, the apparatus and method have been or will be described using functional interpretations, it should be clearly understood that, unless explicitly formulated in accordance with 35 USC §112, the claims should not be construed as necessarily being limited in any way to a construction that is limited to “means” or “steps,” but should be given the full meaning and scope of the equivalent as defined by the claims in accordance with the judicial principle of equivalence, and where the claims are explicitly formulated in accordance with 35 USC §112, they should be given full legal equivalence in accordance with 35 USC §112.

[0090] The entire contents of each patent, patent application, publication, and document cited herein are incorporated herein by reference. Such citation of a patent, patent application, publication, or document does not constitute an admission that any of the foregoing content is relevant prior art, nor does it constitute any admission of the content or dates of such publication or document. Citing them does not imply a search of the relevant disclosure. All statements regarding the dates or content of documents are based on available information and do not constitute an admission of their accuracy or correctness.

[0091] Modifications can be made to the above content without departing from the basic aspects of the present technology. Although the present technology has been described in detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes can be made to the embodiments specifically disclosed in this application; however, such modifications and improvements are within the scope and spirit of the present technology.

[0092] The invention described herein can be suitably practiced without any element not specifically disclosed herein. Thus, for example, in each instance herein, any one of the terms “comprising,” “mainly consisting of,” and “consisting of” can be replaced by any of the other two terms. The terms and expressions already used are used as illustrative rather than restrictive terms, and the use of such terms and expressions does not exclude any equivalents to the features or portions thereof shown and described, and various modifications are possible within the scope of the claimed technology. Unless the context clearly indicates that one element or more elements are described, the term “an” or “a” can refer to one or more elements it modifies (e.g., “a reagent” can mean one or more reagents). As used herein, the term “about” refers to a value within 10% of a base parameter (i.e., plus or minus 10%), and the term “about” is used at the beginning of a series of values ​​to modify each value (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 a weight between 90 grams and 110 grams. Furthermore, when this document describes a range of values ​​(e.g., approximately 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate and fractional values ​​(e.g., 54%, 85.4%). Therefore, it should be understood that although the present technology has been specifically disclosed through representative embodiments and optional features, modifications and variations of the concepts disclosed herein can be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the present technology.

[0093] This disclosure provides a chimeric ILT receptor (CIR) comprising a target region derived from ILT2 or ILT4, a transmembrane domain, and an intracellular domain (ICD) having a signal transduction region that, when the target region binds to HLA-G, can transduce a signal into immune effector cells (e.g., NK cells) to induce effector cell function.

[0094] In some cases, the signal transduction region includes a co-stimulatory region containing the MyD88 peptide. In some such cases, the signal transduction region includes a CD3ζ signal transduction domain, a DAP10 signal transduction domain, a DAP12 signal transduction domain, or any combination thereof. For example, in some cases, the signal transduction region includes a CD3ζ signal transduction domain. In some cases, the signal transduction region includes a DAP10 signal transduction domain. In some cases, the signal transduction region includes a DAP12 signal transduction domain. In some cases, the co-stimulatory region also includes a 4-1BB co-stimulatory domain.

[0095] In some cases, the signal transduction region includes the DAP10 or DAP12 signal transduction domain. In some such cases, the signal transduction region also includes the CD3ζ signal transduction domain. In other such cases, the signal transduction region does not include the CD3ζ signal transduction domain. In some cases, the signal transduction region further includes CD40, 4-1BB, or HVEM co-stimulatory domains.

[0096] Intracellular domain (ICD) peptides are also provided, having a signal transduction region capable of transducing signals in immune effector cells to induce effector cell function, wherein the signal transduction region comprises: (i) a CD3ζ signal transduction domain, a DAP10 signal transduction domain, or a DAP12 signal transduction domain, and (ii) a co-stimulatory region comprising a Toll / interleukin-1 receptor / resistance protein (TIR) ​​domain. In some cases, the TIR domain is a TLR2 TIR domain, a TLR3 TIR domain, or an IL18R1 TIR domain. In some cases, the signal transduction region includes a CD3ζ signal transduction domain. CIRs comprising such ICD peptides are also provided.

[0097] 1. HLA-G Non-classical MHC-I protein HLA-G is a major factor in maintaining maternal-fetal developmental immune tolerance [Kovats et al.] Science (1990) 248:220, Ferreira et al., (2017) I 38:272. Its normal expression is highest in the extravillous trophoblast of the fetal placenta, where its function is to block the activation and infiltration of most types of maternal immune cells, especially T cells and NK cells from fetuses with haploidentical MHC haplotypes. It is maintained at much lower levels in other immune-exempt tissues, including the cornea and 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 [LeDiscorede et al. (2003)] Human Immunology 64:1039, Cirulli et al. (2006) Diabetes 55:1214]. HLA-G is expressed in various solid tumor types and leukemia [see review by Lin and Yan (2018)]. Front Imm. 9:Art 2164], including melanoma [Paul et al., (1998)] Proc Natl Acad Sci [95:4510], colorectal cancer, AML, ALL, renal cell carcinoma [Tronik-Le Roux et al., (2017)] Mol.Oncol. [11:1561], breast cancer, and lung cancer. Its function in cancer is to directly evade immune attack, but HLA-G is also expressed in tolerogenic DC-10 dendritic cells, which suppress lymphocyte responses through the secretion of inhibitory cytokines and activate Treg cells and myeloid-derived suppressor cells (MDSCs) to create an immunosuppressive tumor microenvironment [see Carosella et al., review]. Blood (2011)118:6499, Gao et al., (2018) BBA [1869:278]. Therefore, HLA-G can be considered an important checkpoint mediator of tumor promotion.

[0098] The HLA-G gene produces multiple mRNA transcripts that encode at least seven different protein products [Ishitani et al. (1992)]. Proc.Natl.Acad.Sci 89:3947, (HLA-G1 to G5 are SEQ ID NO: 9, 15, 17, 19, 21 (proteins), and encode nucleotides SEQ ID NO: 8, 14, 16, 18, 20, respectively). HLA-G1 contains an α1-α2-α3 domain structure, which has an α-helical peptide-binding groove, a transmembrane domain, and a short intracellular carboxyl-terminal domain (see...). Figure 1 The structure of this domain is typical of MHC-I products. Other expressed splice products delete the entire domain; for example, HLA-G2 encodes α1, α3, and the transmembrane domain, but deletes α2. HLA-G4 deletes the α3 domain, and HLA-G3 encodes only the α1 domain. When expressed as transgenes in M8 cells, each of these forms of HLA-G has been reported to exhibit immunosuppressive activity against NK cell attack [Riteau et al. (2001)]. J. Immunology [166:5018]. Secretory forms derived from splicing include HLA-G5 and HLA-G6, which maintain the domain structures of HLA-G1 and G2, respectively, but instead encode short secretory peptides derived from intron 4, without using the splice donor site of intron 4. Similarly, HLA-G7 uses a three-amino acid peptide derived from intron 2. Other secretory forms of HLA-G1 are produced by cleavage at the transmembrane domain by matrix metalloproteinases, resulting in the detachment of some HLA-G1 from the cell surface [Rizzo et al. (2012)]. Mol Cell Biochem 381:243).

[0099] HLA-G exists in both monomeric and oligomeric forms. The oligomers are primarily dimers guided by disulfide bonds at Cys42 (in α1) or Cys 147 (in α2) [Gonen-Gross et al., (2005)]. J. Imm.175:4866, Boyson et al. Proc.Natl.Acad.Sci [99:16180]. Evidence suggests that the dimeric form of HLA-G is the predominant immunosuppressive form, and that it employs a tortuous quaternary structure relative to the natural 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).

[0100] Different HLA-G forms collectively pose challenges to CAR-based therapies that rely on antibody-derived scFv or VhH domains as targets. Because different splicing forms delete epitopes of a given antibody, CAR therapies can only express epitopes not recognized by the CAR conjugate, while retaining immunosuppressive activity. Furthermore, oligomerization can mask scFv epitopes due to structural changes. Additionally, two commonly used HLA-G antibody reagents, 4H84 and 87G, exhibit cross-reactivity with other HLA species, which could lead to off-target or off-tumor targeting of CAR-T or CAR-NK cells [Attia et al. (2021)]. Int. J. Mol.Sci 21:8678, Polakova et al. (2004) Hum.Imm. 65:157, Swets et al. (2018) Clin.Imm 194:80, Furukawa et al. (2019) Int J. Mol.Sci 20:5947] 2. ILT2 and ILT4 HLA-G acts as a membrane-bound ligand for the inhibitory receptors immunoglobulin-like transcript 2 (ILT2) and ILT4 (also known as LIRB1 and LIRB2 or CD85j and CD85d, respectively) on target immune cells to guide their immunosuppressive activity [Colonna et al. (1998)]. J. Immunology 160:3096 See Gao et al. (2018) for a review. BBA[1869:278]. ILT2 (see Seq ID NO: 29) (encoding nucleotide sequence SEQ ID NO: 28) is expressed in subsets of natural killer cells, iNKT cells, T cells, B cells, and dendritic cells. ILT4 (see Seq ID NO: 53) (encoding nucleotide sequence SEQ ID NO: 52) has a broader expression pattern, primarily expressed in myeloid cells and stem cells, including macrophages, myeloid-derived suppressor cells (a population of less differentiated cells in the monocyte lineage), granulocytes (including neutrophils), monocytes, hematopoietic stem cells, and some neurons.

[0101] ILT2 has an extracellular domain structure consisting of four domains that share sequence and structural homology with the immunoglobulin domain (Ig domain), arranged in a column from the distal membrane D1 to the proximal membrane D4, followed by a transmembrane domain and an intracellular signal transduction domain, which includes four repeating immunoreceptor tyrosine inhibitory motifs (ITIMs). ILT4 has a similar extracellular and transmembrane architecture, but its intracellular domain contains only three ITIMs.

[0102] The D1 and D2 domains of ILT2 (see, for example, Seq ID NO: 37 and 71) (SEQ ID NO: 36 provides the nucleotide sequence encoding SEQ ID NO: 37) and ILT4 (see, for example, SEQ ID NO: 57 and 75) (SEQ ID NO: 56 provides the nucleotide sequence encoding SEQ ID NO: 57) control interactions 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 of the CD3 complex with classical MHC-I and the inhibitory and activating interactions of KIR proteins on NK cells, ILT2 and ILT4 do not bind to the α1-α2 domains containing the peptide-binding groove, but interact with the proximal α3 domain and β2-microglobulin. ILT2 forms extensive contact with β2-M and relatively less contact with the α3 domain of HLA-G, and β2-M association with HLA-G is required to maintain even low-affinity interactions. In contrast, ILT4 forms extensive contact with α3 and can maintain interactions with all known forms of HLA-G with active immunosuppressive activity, possibly excluding HLA-G3 / G7 containing only the α1 domain.

[0103] ILT2 and ILT4 can interact with other MHC-I and MHC-I-like proteins, particularly HLA-A2, HLA-B, HLA-C, HLA-F, CD1d, and UL18. This is in addition to UL18 (a decoy MHC-I from cytomegalovirus) [Wilcox et al. (2002)]. BMC Struct.Biol Beyond 2:6], these are all low-affinity interactions, with a dissociation constant (K). D ) in 2 M and 40 Between M. Such weak affinity has not yet been shown to be associated with immunosuppressive signaling. Similarly, the interactions between ILT2 and ILT4, as well as between monomeric HLA-G, are weak. Within the M range. However, the dimer HLA-G form exhibits high affinity (2–4 nM) for ILT2 and ILT4, which may be due to the presence of more contact sites, or alternatively, due to an affinity effect that reduces the rate of ILT dissociation [Shiroishi et al., (2006)]. J. Biol.Chem 281:10440, Gao et al. (2020) Cell Mol Imm [17:966]. Therefore, the dimer form of HLA-G is most likely to possess biological activity [Gonen-Gross et al., (2005)]. J. Imm. [175:4866], and has immunosuppressive function in the tumor environment, and is also most likely to be a target molecule for chimeric receptor cell-based immunotherapy.

[0104] 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 of soluble ANGPTL2 and ANGPTL5 is thought to provide protective signals from the bone marrow stroma for the self-renewal and survival of ILT4-expressing hematopoietic stem cells. The interaction between ILT4 and ANGLP is synergistically guided by the D1 and D4 domains of ILT4, and specific residues in either D1 or D4 are crucial for maintaining high-affinity interactions. Notably, the tyrosine 96 mutation to alanine reduces ANGPTL2 / 5 binding but does not reduce the interaction between HLA-G1 and full-length ILT4 [Deng et al. (2014)]. Blood 124:924).

[0105] ILT4 interacts with myelin-derived inhibitory Nogo receptor ligands with moderate affinity [Atwal et al. (2008)]. Science 322:967, Matsushita et al., (2014) J. Biol.Chem 286:25739]. The mouse ortholog of the ILT protein, PIRB, is also present in a subset of neurons and can be linked to myelin-based MAG, Nogo, and OMGp [US Patent 20100047232] and Sema4a [Lu et al. (2018)]. Nat. Comm. The interaction [7:742] regulates axon growth. A high-affinity interaction was characterized in the mouse ortholog of ILT4, PIRB, and this interaction did not map to the D1 and D2 domains of HLA-G binding, but rather to the proximal membrane domain of PIRB [Matsushita et al., (2014)]. J. Biol.Chem 286:25739).

[0106] 3. Chimeric receptors targeted by ILT proteins (i.e., "chimeric ILT receptors" or "CIRs") The cytotoxic retargeting of T cells or NK cells can be controlled through antigen-scFv (or TCR) interaction binding, but it can also be controlled through receptor-ligand pairing, allowing the receptor targeting the ligand to form a chimeric protein that maintains a high affinity interaction with the cell while simultaneously maintaining signal transduction to activate immune cells (we will use T cells and NK cells as examples below). The receptor or portion of the receptor used to bind to the target should be target-specific to prevent off-target effects. Similar to scFv and other binding agents, the target protein or ligand should be expressed at high levels on the target tissue (e.g., tumor) relative to normal tissue. Cells expressing CIR on their surface induce cell activation by transducing signals through signal transduction domains (e.g., the CD3 zeta (CD3ζ) chain) upon contact and binding with HLA-G.

[0107] (i) Target region The extracellular domains of ILT2 (D1-D4) (see, for example, SEQ ID NO: 31, which includes D1-D4 plus the transmembrane region of ILT2; encoding the nucleotide sequence SEQ ID NO: 30) can be engineered to target HLA-G-expressing tumor cells and generate activation signal transduction in immune cells expressing a chimeric form of ILT2 that replaces the naturally inhibitory ITIM-containing intracellular domain (ICD) of ILT2 with signal transduction components that drive activation signals.

[0108] Similarly, the extracellular domain (D1-D4) of ILT4 (see, for example, SEQ ID NO: 55, which comprises D1-D4 plus the transmembrane region of ILT4; encoding nucleotides SEQ ID NO: 54) can be engineered to generate an activation signal in immune cells by replacing the ILT4 ICD with the activation signal transduction portion. Using the D1-D4 extracellular domain of ILT2 or ILT4 as the target region will generate an ILT2 D1-D4 chimeric receptor (i.e., ILT2 D1-D4 CIR) or an ILT4 D1-D4 chimeric receptor (i.e., ILT4 D1-D4 CIR) (see... Figure 4 A). Therefore, in some cases, the target region of the subject-chimeric ILT receptor (CIR) includes the ILT2 or ILT4 D1-D4 domains (and thus targets HLA-G).

[0109] ILT4 maintains more contact with α3 on the HLA heavy chain and can interact with the free heavy chain form of HLA-G, while ILT2 D1 / D2 needs to contact with β2-M and α3 to maintain its interaction with HLA-G.

[0110] Because ILT2 and ILT4 maintain a high affinity for the dimer HLA-G (K D It interacts with low-nM proteins and maintains low-affinity interactions with other MHC-I proteins (including monomeric HLA-G and CD1d). D for Therefore, the specificity for tumors carrying high levels of HLA-G increases proportionally to the amount of HLA-G in the dimer form, allowing selection of tumor tissue rather than normal tissue expressing high levels of classical MHC-I but with little or no HLA-G. Mutations in HLA-G1 at the Cys42 to Ser or Cys147 to Ser positions block HLA-G1 dimerization and severely reduce targeting by ILT2 or ILT4 CIR-T cells or CIR-NK cells.

[0111] The inventors recognized that HLA-G exists in several different isotypes. CARs that include antibody-based targeting regions (such as scFv) will only target HLA-G isotypes that include epitopes targeted by antigen-binding regions (e.g., scFv). This could lead to selection for tumor cells expressing HLA-G isotypes lacking target epitopes—allowing cancer to evade treatment. Conversely, chimeric receptor proteins based on ILT2 or ILT4 (which target HLA-G) should target more, and likely all, HLA-G isotypes, as ILT2 and ILT4 naturally bind to these isotypes.

[0112] Construction of ILT2 D1 / D2 CIR and ILT4 D1 / D2 CIR Domains D1 and D2 are sufficient to guide the binding of ILT2 and ILT4 to HLA-G, while domains D3 and D4 may act as scaffolds to expose D1 and D2 to HLA-G [Shiroishi et al., (2006)]. J. Biol.Chem April 14; 281(15):10439-47]. In some embodiments, the D3 and D4 domains can be removed from the ILT2 CIR or ILT4 CIR, and functional interaction with the HLA-G form can be maintained through the D1-D2 domains from ILT2 (see, for example, Seq ID NO: 71) or ILT4 (see, for example, Seq ID NO: 75) (see...). Figure 4 A). Therefore, in some cases, the target region of the subject ILT2 or ILT4 chimeric receptor will include the D1-D2 domain of ILT2 or ILT4 (see, for example, SEQ ID NO: 71 for the D1-D2 of ILT2, SEQ ID NO: 75 for the D1-D2 of ILT4), and in some such cases, the target region will not include (i.e., will lack) the D3-D4 domain.

[0113] In some embodiments, the targeting region (including the region comprising the D1-D2 domain) of the subject ILT2 chimeric receptor comprises an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with respect to the ILT2 sequence shown in any of SEQ ID Nos: 37, 70, 71, and 72, as follows: MHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 37) MHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 70) PKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 71) PKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 72) In some cases, the target region comprises an amino acid sequence having 90% or higher sequence identity (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the sequence shown in any of SEQ ID Nos: 37, 70, 71, and 72. In some cases, the target region comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the sequence shown in any of SEQ ID Nos: 37, 70, 71, and 72. In some cases, the target region comprises an amino acid sequence shown in any of SEQ ID Nos: 37, 70, 71, and 72.

[0114] In some cases, the target region of the ILT2 chimeric receptor (including the region containing the D1-D2 domain) comprises an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the sequence shown in SEQ ID No: 37. In some cases, the target region comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the sequence shown in SEQ ID No: 37. In some cases, the target region comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the sequence shown in SEQ ID No: 37. In some cases, the target region includes the amino acid sequence shown in SEQ ID No: 37.

[0115] In some embodiments, the targeting region (including the region comprising the D1-D2 domain) of the subject ILT4 chimeric receptor comprises an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the ILT4 sequence shown in any of SEQ ID Nos: 57, 74, and 75, as follows: MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDP LVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPG(SEQ ID NO: 57) MTPIVTVLICLGLSLGPRTRVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDP LVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPG(SEQ ID NO: 74) PKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPG (SEQ ID NO: 75) In some cases, the target region comprises an amino acid sequence having 90% or higher sequence identity (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the sequence shown in any of SEQ ID Nos: 57, 74, and 75. In some cases, the target region comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the sequence shown in any of SEQ ID Nos: 57, 74, and 75. In some cases, the target region comprises an amino acid sequence shown in any of SEQ ID Nos: 57, 74, and 75.

[0116] In some cases, the targeting region of the ILT4 chimeric receptor (including the region containing the D1-D2 domain) comprises an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the sequence shown in SEQ ID No: 57. In some cases, the targeting region comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the sequence shown in SEQ ID No: 57. In some cases, the targeting region comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the sequence shown in SEQ ID No: 57. In some cases, the target region includes the amino acid sequence shown in SEQ ID No: 57.

[0117] For any of the embodiments discussed in this section, in some cases, the subject ILT2 or ILT4 chimeric receptor lacks the D3 and D4 domains (i.e., the regions corresponding to the D3-D4 domains of ILT2 (SEQ ID NO: 73) or ILT4 (SEQ ID NO: 76), respectively). For ILT2, the region having the D3-D4 domain is: PLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLT (SEQ ID NO: 73).

[0118] For ILT4, the region with the D3-D4 structural domain is: QPGPVMAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGS 76).

[0119] In some cases, the subject CIR lacks an amino acid sequence that has 85% or higher (e.g., 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100%) sequence identity with the sequence shown in SEQ ID NO: 73. In some cases, the subject CIR lacks an amino acid sequence that has 85% or higher (e.g., 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100%) sequence identity with the sequence shown in SEQ ID NO: 76. In some cases, the subject CIR lacks an amino acid sequence that has 85% or higher (e.g., 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100%) sequence identity with the sequence shown in either SEQ ID NO: 73 or 76.

[0120] In some embodiments, the linker may function as a chimera, fused to the plasma membrane with the D1-D2 domain from ILT2 or ILT4, and as a stalk replacing the D3 and D4 domains. In these embodiments, deletion of D3-D4 prevents the CIR from interacting with proteins other than HLA-G that interact with native ILT2 or ILT4 via the D3 or D4 domains, such as the interaction of ANGPTL2 and ANGPTL5 with ILT4 D4, and the interaction of nogo, Omgp, and MAG with ILT4 D3-D4. Preventing such interactions reduces the potential mistargeting and toxicity of CIR-expressing cells to non-tumor tissues such as bone marrow stroma, myelin, and endothelium.

[0121] In some embodiments, the targeting region of the subject ILT2 chimeric receptor (ILT2 CIR) (including the region comprising the D1-D2 domains) includes domains D1-D4, and thus in some cases includes an amino acid sequence having 80% or higher sequence identity with the ILT2 sequence shown in SEQ ID NO: 94 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%).

[0122] In some embodiments, the targeting region of the subject ILT4 chimeric receptor (ILT4 CIR) (including the region comprising the D1-D2 domains) includes domains D1-D4, and thus in some cases includes an amino acid sequence having 80% or higher sequence identity with the ILT2 sequence shown in SEQ ID NO: 96 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%).

[0123] Handle structural domain In some embodiments, the substitution of D3-D4 can be made with any protein or portion of a protein that appropriately represents an ILT2 or ILT4 D1-D2 conjugate in the context of HLA-G expressed on a single cell. In some embodiments, a short polypeptide linker can form a connection between the transmembrane domain and the intracellular domain of the chimeric ILT receptor. Thus, the chimeric ILT receptor may further include a stalk, which is an extracellular region of amino acids between the extracellular domain and the transmembrane domain. The purpose of the stalk domain is to extend the D1 / D2 domain from the plasma membrane toward the target protein HLA-G. For example, the stalk may be an amino acid sequence naturally associated with a selected transmembrane domain. In some embodiments, the CIR comprises a CD8α transmembrane domain, and in some embodiments, the CIR comprises the CD8α transmembrane domain plus additional amino acids on the extracellular portion of the transmembrane domain. In some embodiments, the CIR comprises a CD8α transmembrane domain and a CD8α stalk (see SEQ ID NO: 43; encoding nucleotide sequence SEQ ID NO: 42). In one specific embodiment, the CD8α transmembrane domain comprises (or is composed of) the sequence disclosed herein (see SEQ ID NO: 100). In another specific embodiment, the CD8α stalk comprises (or is composed of) the sequence disclosed herein (see SEQ ID NO: 43, which includes the stalk and TM). The chimeric ILT receptor may further comprise an amino acid region between the transmembrane domain and the cytoplasmic domain, which is natively associated with a polypeptide derived from the transmembrane domain.

[0124] Examples of such chimeric stalk portions include, but are not limited to, the proximal membrane portion of CD8α (see, for example, SEQ ID NO: 43 and 107), the CH2 / CH3 domain of IgG (e.g., IgG1, IgG4) (see, for example, SEQ ID NO: 51 and 98), the CH3 domain of IgG (e.g., IgG1, IgG4) (see, for example, SEQ ID NO: 102), HER2, mGluR2, CD28 (see, for example, SEQ ID NO: 47 and 106), and CTLA4.

[0125] For example, in some cases, the stalk domain of the subject's CIR is selected from: ILT2, ILT4, CD28, CH2 / CH3, CH3, and CD8α stalk domains. See, for example: VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQ (SEQ ID NO: 39) (SEQ ID NO: 38 is the encoding nucleotide sequence), which includes the ILT2 stalk and TM domain; PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 107), which includes the CD8α handle domain; PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR (SEQ ID NO: 43), which includes a CD8 handle and a TM structural domain; IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 106), which includes a CD28 handle; IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 47), which includes a CD28 handle and a TM structural domain; VDKRVESKYGPPCPSCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL (SEQ ID NO: 98), which includes a CH2CH3 handle; VDKRVESKYGPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL (SEQ ID NO: 102), which includes a CH3 handle; DPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 51), which includes a CH2CH3 handle and a CD28 TM domain; VVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLFLILRHRRQ (SEQ ID NO: 59) includes an ILT4 handle and a TM structural domain.

[0126] In some embodiments, the stalk domain of the subject CIR comprises an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the stalk sequence portion of the amino acid sequence shown in any of SEQ ID NO: 39, 43, 47, 51, and 59. In some embodiments, the stalk domain of the subject CIR comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the stalk sequence portion of the amino acid sequence shown in any of SEQ ID NO: 39, 43, 47, 51, and 59. In some embodiments, the stalk domain of the subject CIR comprises the stalk sequence portion of the amino acid sequence shown in any of SEQ ID NO: 39, 43, 47, 51, and 59.

[0127] In some embodiments, the stem domain of the subject CIR comprises an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the stem sequence shown in any of SEQ ID NO: 98, 102, 106, and 107. In some embodiments, the stem domain of the subject CIR comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the stem sequence shown in any of SEQ ID NO: 98, 102, 106, and 107. In some embodiments, the stem domain of the subject CIR comprises the stem sequence shown in any of SEQ ID NO: 98, 102, 106, and 107.

[0128] The interaction between the CIR containing the D1 / D2 stalk and the dimer HLA-G will have the effect of dimerizing intracellular signal transduction domains, which in some embodiments can stimulate ICD activation and cell signal transduction.

[0129] Mutations of ILT2 D1 / D2 CIR or ILT4 D1 / D2 CIR In another embodiment, mutations can be made within the ILT2 or ILT4 D1 or D2 domains contained within the CIR to increase the specificity of the CIR for HLA-G relative to other potentially interacting proteins. For example, mutations can be made to encode an amino acid other than tyrosine (e.g., Y96A) at the corresponding position of native amino acid 96 (Y96) of ILT4 (SEQ ID NO: 53) or ILT2 (SEQ ID NO: 29). The effect of such mutations is to reduce potential interactions with ANGPTL2 and ANGPTL5 while preserving binding affinity for HLA-G. In yet another embodiment, a similar mutation can be placed in the full-length ILT4 CIR containing the D1-D4 domains, while a mutation is made in the D4 domain (at the position corresponding to tyrosine 394 (Y394) of SEQ ID NO: 53), thereby further disrupting the stability of interactions with ANGPTL2 and ANGPTL5. The corresponding position of ILT2 is tyrosine 395 (Y395) in SEQ ID NO: 29.

[0130] Therefore, in some cases, the subject ILT4 CIR includes a mutation at the amino acid position corresponding to Y96 of SEQ ID NO: 53 (e.g., Y96A). In some cases, the subject ILT2 CIR includes a mutation at the amino acid position corresponding to Y96 of SEQ ID NO: 29 (e.g., Y96A). In some cases, the subject ILT4 CIR includes a mutation at the amino acid position corresponding to Y394 of SEQ ID NO: 53 (e.g., Y394A) (see also SEQ ID NO: 60-61). In some cases, the subject ILT2 CIR includes a mutation at the amino acid position corresponding to Y395 of SEQ ID NO: 29 (e.g., Y395A). In some cases, the subject ILT4 CIR includes a mutation at the amino acid position corresponding to Y96 of SEQ ID NO: 53 (e.g., Y96A) and a mutation at the amino acid position corresponding to Y394 of SEQ ID NO: 53 (e.g., Y394A) (e.g., Y96A / Y394A). In some cases, the subject ILT2 CIR includes mutations at the amino acid position corresponding to Y96 of SEQ ID NO: 29 (e.g., Y96A) and mutations at the amino acid position corresponding to Y395 of SEQ ID NO: 29 (e.g., Y395A) (e.g., Y96A / Y395A).

[0131] Other embodiments following a similar line of thought can restrict interaction with classical HLA proteins or CD1 while preserving binding to HLA-G. These mutations can replace the interaction sites with α3 domains that are specific to the heavy chains of these HLA proteins or on the surface that binds to β2-M.

[0132] (ii) Transmembrane (TM) region CIR can include transmembrane sequences that can be single-pass or multiple-pass (CIR). For example,Single-transmembrane regions (SCRs) are located at the N- or C-terminus of chimeric proteins, or within the protein itself (e.g., linking extracellular targeting regions to intracellular domains). SCRs are present in certain CD molecules, tyrosine kinase receptors, serine / threonine kinase receptors, TGFβ, BMP, activins, and phosphatases. SCRs typically consist of a signal peptide region and a transmembrane region of approximately 20 to 25 amino acids, many of which are hydrophobic and can form α-helices. A short, positively charged amino acid sequence usually follows the transmembrane span to anchor the protein to the membrane. Multi-transmembrane proteins include ion pumps, ion channels, and transporters, and consist of two or more helices that cross the membrane multiple times. Sometimes, all or substantially all of a multi-transmembrane protein is incorporated into a chimeric protein. The sequences of both SCRs and multi-transmembrane regions are known and can be selected for incorporation into chimeric protein molecules.

[0133] In some embodiments, the transmembrane domain is fused with the extracellular domain of the CIR. In some embodiments, the transmembrane domain is fused with both the extracellular and intracellular regions, thereby connecting the extracellular and intracellular regions to each other. In one embodiment, a transmembrane domain natively associated with one of the domains in the CIR is used. In other embodiments, a transmembrane domain not natively associated with one of the domains in the CIR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution. For example (This is usually changed to hydrophobic residues) to avoid such domains binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0134] Transmembrane (TM) domains can, for example, be derived from the α, β, or ζ chain of the T cell receptor, CD3-ε, CD3ζ, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD28, CD33, CD38, CD64, CD80, CD86, CD134, CD137, ILT2, HER2, ILT4, or CD154 — or transmembrane regions containing their functional variants can be used, such as those that retain most of their structural properties (e.g., transmembrane properties). See, for example, 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.

[0135] Alternatively, in some instances, the transmembrane domain can be synthesized de novo, primarily containing hydrophobic residues such as, for example, leucine, isoleucine, phenylalanine, and valine. Examples of suitable CD8 stalk sequences, transmembrane sequences, and CD3ζ sequences are disclosed herein.

[0136] For example, in some cases, the TM domain of the subject CIR is selected from the following TM domains: ILT2 (see, for example, SEQ ID NO: 39), ILT4 (see, for example, SEQ ID NO: 59), CD28 (see, for example, SEQ ID NO: 47 and 104), and CD8 (see, for example, SEQ ID NO: 43 and 100). See, for example: VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQ (SEQ ID NO: 39), which includes an ILT2 handle and a TM structural domain; IYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR (SEQ ID NO: 100), which includes the CD8™ structural domain; PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR (SEQ ID NO: 43), which includes a CD8 handle and a TM structural domain; FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 104), which includes the CD28 TM structural domain; IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 47), which includes a CD28 handle and a TM structural domain; DPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 51), which includes the CH2CH3 handle and the CD28 TM domain; and VVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLFLILRHRRQ (SEQ ID NO: 59) includes an ILT4 handle and a TM structural domain.

[0137] In some embodiments, the TM domain of the subject CIR comprises an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the TM domain sequence portion of the amino acid sequence shown in any of SEQ ID NO: 39, 43, 47, 51, and 59. In some embodiments, the TM domain of the subject CIR comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with the TM domain sequence portion of the amino acid sequence shown in any of SEQ ID NO: 39, 43, 47, 51, and 59. In some embodiments, the TM domain of the subject CIR comprises the TM domain sequence portion of the amino acid sequence shown in any of SEQ ID NO: 39, 43, 47, 51, and 59.

[0138] In some embodiments, the stalk domain plus TM domain (stalk / TM domain) of the subject CIR comprises an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with respect to the amino acid sequence shown in any of SEQ ID NOs: 39, 43, 47, 51, and 59. In some embodiments, the stalk domain plus TM domain (stalk / TM domain) of the subject CIR comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with respect to the amino acid sequence shown in any of SEQ ID NOs: 39, 43, 47, 51, and 59. In some embodiments, the stalk domain plus TM domain (stalk / TM domain) of the subject CIR includes the amino acid sequence shown in any one of SEQ ID NO: 39, 43, 47, 51 and 59.

[0139] In some embodiments, the TM domain of the subject CIR comprises an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with respect to the TM domain sequence shown in either SEQ ID NO: 100 or 104. In some embodiments, the TM domain of the subject CIR comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with respect to the TM domain sequence shown in either SEQ ID NO: 100 or 104. In some embodiments, the TM domain of the subject CIR comprises the TM domain sequence shown in either SEQ ID NO: 100 or 104.

[0140] (iii) Intracellular domains (ICDs) As described above, the subject-specific chimeric ILT receptor (based on ILT2 or ILT4) includes an intracellular region (intracellular domain or ICD) that replaces the native intracellular portion of ILT2 or ILT4 (which is inhibitory) with the CAR's ICD (which is activating). Therefore, the ICD of the subject-specific CIR (ILT2 version or ILT4 version) includes a "signaling region" having at least one signaling domain that leads to cellular activation, and may optionally include a "co-stimulatory region" that may include one or more co-stimulatory domains.

[0141] Signal transmission region T cell activation is guided by the binding of the T cell receptor to the peptide-MHC complex and the activation of the CD3 signaling complex. Cell signaling is triggered by phosphorylation of the ITAM motif present on the CD3ζ, CD3d, CD3γ, and CD3e components of this complex. CD3ζ contains three ITAMs, which recruit the ZAP70 kinase, which initiates a downstream signaling cascade to classically activate T cells by activating NF-AT. NK cells also express CD3ζ, which acts as an intracellular signaling adaptor for the natural cytotoxic receptor (NCR) NKp46. Other NCRs, such as CD94 / NKG2C, bind to the ITAM-containing signaling adaptor DAP12, and NKG2D binds to the DAP10 signaling adaptor. DAP12 contains a single ITAM motif, which can be phosphorylated by Syk or ZAP70 to activate NK cell cytotoxic signaling. Notably, DAP10 does not contain a classical ITAM domain and instead signals via a mechanism more similar to CD28 in T cells.

[0142] A “signal transduction region” (or “intracellular signal transduction domain”), such as the signal transduction region of a CIR, refers to the portion involved in transducing binding signals (e.g., the binding of a CIR to a target molecule (HLA-G in the case of a subject CIR)) into the interior of an immune effector cell to elicit effector cell function, such as activation, cytokine production, proliferation, and / or cytotoxic activity, including the release of cytotoxic factors to target cells bound by the CIR, or other cellular responses elicited by the binding of a target molecule to an extracellular CIR domain. Therefore, the term “signal transduction region” (“intracellular signal transduction domain”) refers to a protein portion that transduces effector function signals and directs the cell to perform a specific function. Regarding the use of a truncated portion of an intracellular signal transduction domain, it can be used in place of the full-length intracellular signal transduction domain as long as that truncated portion transduces effector function signals. The term signal transduction region is intended to include any truncated portion of an intracellular signal transduction domain sufficient to transduce effector function signals. In some cases, the signal transduction region includes a signal transduction motif known as an immune receptor tyrosine activation motif (or “ITAM”). The ITAM motif of CD3ζ is contained on an intracellular domain, such as that of a classic CAR. In some cases, the signal transduction region does not include ITAM. In some cases, the signal transduction region of a subject CIR includes an intracellular domain (e.g., CD3ζ) that includes ITAM. In some cases, the signal transduction region of a subject CIR includes an intracellular domain (e.g., DAP10) that does not include ITAM.

[0143] Examples of intracellular domain sequences that can be used in the signal transduction region of a subject CIR include those derived from the lymphocyte receptor chain, the TCR / CD3 complex protein, the Fc receptor subunit, the IL-2 receptor subunit, CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD278(ICOS), FcsRl, DAP10, and DAP12.

[0144] In some embodiments, the signal transduction region (e.g., the signal transduction region of the subject CIR) includes a CD3 zeta (CD3ζ) signal transduction domain (see, for example, SEQ ID NO: 33). Therefore, in some cases, the ICD (e.g., the ICD of the subject chimeric ILT receptor (ILT2 or ILT4 version)) includes a signal transduction region comprising an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with SEQ ID NO: 33. In some embodiments, the signal transduction region comprises an amino acid sequence having 90% or higher sequence identity (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with SEQ ID NO: 33. In some embodiments, the signal transduction region comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO:33 (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises the amino acid sequence shown in SEQ ID NO:33.

[0145] In some embodiments, the signal transduction region (e.g., the signal transduction region of the subject CIR) includes a DAP10 signal transduction domain (see, for example, SEQ ID NO: 4), which may or may not have a CD3ζ domain. In some embodiments, the signal transduction region (e.g., the signal transduction region of the subject CIR) includes a DAP12 signal transduction domain (see, for example, SEQ ID NO: 5), which may or may not have a CD3ζ domain. In some embodiments, DAP10 of DAP12 (e.g., SEQ ID NO: 4, SEQ ID NO: 5) may be fused to supplement CD3ζ signal transduction. Examples are described herein of DAP10 or DAP12 alone or in combination with other signal transduction motifs that drive NK cell co-activation or T cell co-stimulation replacing or supplementing CD3ζ.

[0146] In some embodiments, the signal transduction region (e.g., the signal transduction region of the subject CIR) includes the DAP10 signal transduction domain (see, for example, SEQ ID NO: 4). Therefore, in some cases, the ICD (e.g., the ICD of the subject chimeric ILT receptor (ILT2 or ILT4 version)) includes a signal transduction region comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 4 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO: 4 (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO: 4 (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises the amino acid sequence shown in SEQ ID NO: 4.

[0147] In some embodiments, the signal transduction region (e.g., the signal transduction region of the subject CIR) includes the DAP12 signal transduction domain (see, for example, SEQ ID NO: 23). Therefore, in some cases, the ICD (e.g., the ICD of the subject chimeric ILT receptor (ILT2 or ILT4 version)) includes a signal transduction region comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 23 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO: 23 (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO: 23 (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises the amino acid sequence shown in SEQ ID NO: 23.

[0148] Co-stimulation zone T cell activation is generated by antigen-presenting cells (APCs), typically dendritic cells that present peptide-MHC to competent T cells. To prevent autoimmunity induced by the low-affinity MHC-peptide-TCR complex, co-stimulatory elements further stimulate T cells that bind to the presenting cells with high affinity. These elements are typically derived from the binding of B7-H1 and B7-H2 to CD28 on T cells, and the interaction of 4-1BB or OX40 with their ligands once the T cell is partially activated. The co-stimulation provided by CD28 or 4-1BB, combined with ITAM activation from the TCR / CD3 complex, drives T cell differentiation into a fully activated state that supports proliferation and persists into a memory cell state. Signaling derived from receptors driving T cell co-stimulation can be incorporated into CIR or CAR products to support differentiation into a fully activated and persistent cellular state. NK cells express 4-1BB but not CD28 and are typically not stimulated by cell-to-cell contact with supporting cells, but rather by direct activation by target cells.

[0149] In an inflammatory environment, co-activation of NK cells is promoted by cytokines such as IL-12, IL-18, IL-21, and IL-1. IL-18 and IL-1 signaling are mediated by the cytoplasmic signaling node MyD88. Activation of Toll-like receptors (TLRs) by pathogenic ligands is also an effective NK cell activation mechanism, which is also guided downstream by MyD88.

[0150] The examples below include the incorporation of signaling elements that drive NK cell activation to a degree exceeding that of co-stimulatory elements currently used in CAR-T cell products and CAR-NK products derived from constructs designed for T cell integration.

[0151] Co-stimulatory peptides may contain one or more co-stimulatory signaling regions, such as truncated MyD88, 4-1BB, or HVEM, or combinations of these or other co-stimulatory motifs. Co-stimulatory peptides may contain one or more suitable co-stimulatory signaling regions that activate signaling pathways activated by MyD88, 4-1BB, or HVEM. Co-stimulatory peptides include any molecule or peptide that activates the NF-κB pathway, MyD88 pathway, STAT5 pathway, STAT1 pathway, Akt pathway, and / or p38 pathway of tumor necrosis factor receptor (TNFR) family members (i.e., CD40, RANK / TRANCE-R, OX40, 4-1BB) and CD28 family members (CD28, ICOS). More than one co-stimulatory peptide or a co-stimulatory peptide cytoplasmic region may be expressed in modified cells. In some embodiments, the ICD (e.g., the ICD of a topic CIR) also includes a co-stimulatory region. The co-stimulatory region includes at least one co-stimulatory domain (e.g., one, two, three, one or more, two or more, or three or more co-stimulatory domains). Examples of co-stimulatory domains include, but are not limited to, the CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, and ICOS co-stimulatory domains. Examples of co-stimulatory domains include, but are not limited to, 4-1BB, OX40, ICOS, CD28, CD27, MyD88, IL-1Rα, HVEM, TRANCE, and IL-1R. , CD70, IL-18Rα, CD40, IL-18R IL-33Rα, CD30 and IL-33R Examples of co-stimulatory domains include, but are not limited to: 4-1BB, OX40, ICOS, RANK, DAP10, DAP12, CD28, CD27, MyD88, IL-1Rα, HVEM, TRANCE, and IL-1R. , CD70, IL-18Rα, CD40, IL-18R IL-33Rα, CD30 and IL-33R In some cases, the costimulatory region includes one or more (e.g., one, two, three, one or more, or two or more) costimulatory domains selected from the group consisting of: CD28 (see, for example, SEQ ID NO: 49), 4-1BB (see, for example, SEQ ID NO: 35), and OX40—or any combination thereof. In some cases, the CD28 costimulatory domain is used. In some cases, the 4-1BB costimulatory domain is used. In some cases, both the CD28 and 4-1BB costimulatory domains are used (i.e., both are used). In some cases, both the CD28 and OX40 costimulatory domains are used.

[0152] In some cases, the co-stimulatory region comprises a truncated MyD88 peptide fused to a signal transduction domain of a co-stimulatory receptor mediator, such as, for example, CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, or ICOS. In other cases, the co-stimulatory region comprises the MyD88 peptide or a truncated MyD88 peptide and a co-stimulatory domain selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40, and DAP10.

[0153] In some embodiments, the ICD (e.g., an ICD of a subject-specific chimeric ILT receptor (ILT2 or ILT4 version)) includes a co-stimulatory region comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 49 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO: 49 (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO: 49 (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region includes the amino acid sequence shown in SEQ ID NO: 49.

[0154] In some embodiments, the ICD (e.g., an ICD of a subject-specific chimeric ILT receptor (ILT2 or ILT4 version)) includes a co-stimulatory region comprising an amino acid sequence having 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with SEQ ID NO: 35. In some embodiments, the signal transduction region comprises an amino acid sequence having 90% or higher sequence identity (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with SEQ ID NO: 35. In some embodiments, the signal transduction region comprises an amino acid sequence having 95% or higher sequence identity (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) with SEQ ID NO: 35. In some embodiments, the signal transduction region includes the amino acid sequence shown in SEQ ID NO: 35.

[0155] In some embodiments, the ICD (e.g., an ICD of a subject-chimeric ILT receptor (ILT2 or ILT4 version)) includes a co-stimulatory region comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 35 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) and an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 49 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO: 35 (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) and an amino acid sequence having 90% or higher sequence identity with SEQ ID NO: 49 (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO: 35 (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) and an amino acid sequence having 95% or higher sequence identity with SEQ ID NO: 49 (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the signal transduction region includes the amino acid sequences shown in SEQ ID NO: 35 and SEQ ID NO: 49.

[0156] In some embodiments, the signal transduction region (e.g., the signal transduction region of the topic CIR) includes a CD3 zeta (CD3ζ) signal transduction domain, and the co-stimulation region includes a CD28 co-stimulation domain. In some embodiments, the signal transduction region includes a CD3 zeta (CD3ζ) signal transduction domain, and the co-stimulation region includes a 4-1BB co-stimulation domain. In some embodiments, the signal transduction region includes a CD3 zeta (CD3ζ) signal transduction domain, and the co-stimulation region includes both a 4-1BB co-stimulation domain and a CD28 co-stimulation domain. In some embodiments, the signal transduction region includes a CD3 zeta (CD3ζ) signal transduction domain, and the co-stimulation region includes both a CD28 co-stimulation domain and an OX40 co-stimulation domain.

[0157] In some embodiments, the signal transduction region (e.g., the signal transduction region of the subject CIR) includes a DAP10 or DAP12 signal transduction domain (without a CD3ζ signal transduction domain), and the co-stimulation region includes a CD28 co-stimulation domain. In some embodiments, the signal transduction region includes a DAP10 or DAP12 signal transduction domain (without a CD3ζ signal transduction domain), and the co-stimulation region includes a 4-1BB co-stimulation domain. In some embodiments, the signal transduction region includes a DAP10 or DAP12 signal transduction domain (without a CD3ζ signal transduction domain), and the co-stimulation region includes a 4-1BB co-stimulation domain and a CD28 co-stimulation domain. In some embodiments, the signal transduction region includes a DAP10 or DAP12 signal transduction domain (without a CD3ζ signal transduction domain), and the co-stimulation region includes a CD28 co-stimulation domain and an OX40 co-stimulation domain. In some embodiments, the signal transduction region includes a DAP12 signal transduction domain (without a CD3ζ signal transduction domain), and the co-stimulation region includes a 4-1BB co-stimulation domain. In some embodiments, the signal transduction region includes a DAP10 signal transduction domain (without a CD3ζ signal transduction domain), and the co-stimulation region includes a 4-1BB co-stimulation domain.

[0158] In some embodiments, the signal transduction region (e.g., the signal transduction region of a topic CIR) includes a DAP10 or DAP12 signal transduction domain (fused with a CD3ζ signal transduction domain), and the co-stimulation region includes a CD28 co-stimulation domain. In some embodiments, the signal transduction region includes a DAP10 or DAP12 signal transduction domain (fused with a CD3ζ signal transduction domain), and the co-stimulation region includes a 4-1BB co-stimulation domain. In some embodiments, the signal transduction region includes a DAP10 or DAP12 signal transduction domain (fused with a CD3ζ signal transduction domain), and the co-stimulation region includes a 4-1BB co-stimulation domain and a CD28 co-stimulation domain. In some embodiments, the signal transduction region includes a DAP10 or DAP12 signal transduction domain (fused with a CD3ζ signal transduction domain), and the co-stimulation region includes a CD28 co-stimulation domain and an OX40 co-stimulation domain. In some embodiments, the signal transduction region includes a DAP12 signal transduction domain (fused with a CD3ζ signal transduction domain), and the co-stimulation region includes a 4-1BB co-stimulation domain. In some embodiments, the signal transduction region includes a DAP10 signal transduction domain (fused with a CD3ζ signal transduction domain), and the co-stimulation region includes a 4-1BB co-stimulation domain.

[0159] Cells may include chimeric signaling peptides, including, for example, chimeric signaling peptides in which a truncated MyD88 peptide is also fused with a signaling domain of a co-stimulatory receptor mediator (such as, for example, 4-1BB or HVEM). The fusion may incorporate other signaling domains, such as those from CD40, CD27, CD28, 4-1BB, OX40, or ICOS. More than one co-stimulatory peptide or a co-stimulatory peptide cytoplasmic region may be expressed in the modified cells.

[0160] Cells may include chimeric signaling peptides, including, for example, chimeric signaling peptides in which a truncated MyD88 peptide has also been fused with the signaling domain of a co-stimulated receptor mediator (such as, for example, CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, or ICOS).

[0161] In some embodiments, the chimeric signaling peptide comprises cytoplasmic signaling regions from two co-stimulatory peptides, such as, for example, 4-1BB and CD28, or cytoplasmic signaling regions from one or more co-stimulatory peptides selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, or OX40. In some embodiments, the chimeric signaling peptide comprises a MyD88 peptide or a truncated MyD88 peptide and a cytoplasmic signaling region from a co-stimulatory peptide selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, and OX40.

[0162] Non-limiting examples of co-stimulatory signaling domains of 4-1BB, CD28, and OX40 can be found in US20130266551, US 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 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); and US Patent Application 2012 / 20148552, all of which are incorporated herein by reference for their teachings relating to co-stimulatory domains.

[0163] Non-limiting examples of chimeric peptides that can be used to induce cell activation, as well as related methods for inducing therapeutic cell activation (including, for example, expression constructs, methods for constructing vectors, and assays for activity or function), can also be found in the following patents and patent applications: US2014-0286987-A1; WO2014 / 151960; US2016 / 0046700; WO2015 / 123527; US2004 / 0209836; US Patent No. 7,404,950; WO2004 / 073641; US2011 / 0033388; US Patent No. 8,691,210; WO2008 / 049113; US2014 / 0087468; US Patent 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 these is incorporated herein by reference in its entirety, including all text, tables and figures, for all purposes, including those relating to the description of cellular activation domains (e.g., cellular signaling and co-stimulatory domains).

[0164] In some embodiments, cells are engineered to deliver constitutively active therapy. In some embodiments, the genetically modified cells comprise nucleic acids containing 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 at the 5' end of the first polynucleotide. In some embodiments, the second polynucleotide is located at the 3' end of the first polynucleotide. In some embodiments, a third polynucleotide encoding a connector polypeptide is located between the first and second polynucleotides. In the case where the third polynucleotide is located at the 3' end of the first polynucleotide and the 5' end of the second polynucleotide, the connector polypeptide may remain intact post-translationally, or may separate the polypeptide encoded by the first and second polynucleotides during or after translation. In some embodiments, the connector polypeptide is a 2A polypeptide (see elsewhere herein) that may separate the polypeptide encoded by the first and second polynucleotides during or after translation. High levels of co-stimulation are constitutively delivered via alternative mechanisms, wherein a leaky 2A co-translational sequence (see elsewhere herein) is used to separate the CAR from the chimeric signaling polypeptide. In cases of incomplete 2A separation, such as due to leaky 2A sequences, most of the expressed chimeric signaling peptide molecules are separated from the chimeric antigen receptor peptide and can remain in the cytoplasm, while some partial or chimeric signaling peptide molecules remain attached to or linked to the CAR.

[0165] The term "constitutive activity" refers to the cellular activating activity of chimeric stimulating peptides, even in the absence of an inducer. One method of generating constitutive active signaling is to anchor activating protein factors to the plasma membrane via transmembrane domains or lipid-targeting moieties. Co-activation via MyD88 signaling MyD88 (encoded by myeloid differentiation primary response gene 88) is a key mediator of downstream signaling for several receptors, particularly Toll-like receptors (TLRs) that guide some innate immune responses. MyD88 is also a major mediator of downstream signaling for the interleukin-1 receptor family (including receptors for IL-1, IL-18, and IL-33). MyD88 contains two domains that direct its activity—an N-terminal death domain that directs oligomerization to form a complex that further directs downstream signaling via the NK-κB pathway to induce cytokine production and co-activation of signals from the ITM-guided pathway, the AKT growth and survival pathway, and the interferon response factor pathway. The C-terminal TIR domain directs recruitment to Toll-like receptors and IL-1 family receptors through interactions with their TIR domains. Similar TIR-TIR interactions can recruit related signaling proteins, such as TRIF, as nodes to TLR3 and TLR4.

[0166] In some embodiments, a TIR domain from an IL1 family receptor or a Toll-like receptor (TLR) can be used to recruit MyD88 or TRIF signaling to an activated chimeric receptor (e.g., a CAR, CIR) by directly fusing the TIR as a co-activating domain to the receptor. For example, a "co-stimulatory region" may include a TIR domain (e.g., from an IL1 family receptor or TLR). Thus, in some cases, the co-stimulatory region (e.g., the co-stimulatory region of a CIR or CAR) includes a TIR domain from an IL1 family receptor or TLR.

[0167] In some cases, the co-stimulatory region comprises a TIR from TLR2. Therefore, in some cases, the co-stimulatory region comprises an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 111 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO: 111 (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO: 111 (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises the amino acid sequence shown in SEQ ID NO: 111.

[0168] In some cases, the co-stimulatory region comprises a TIR from TLR3. Therefore, in some cases, the co-stimulatory region comprises an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 113 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO: 113 (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO: 113 (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises the amino acid sequence shown in SEQ ID NO: 113.

[0169] In some cases, the co-stimulatory region comprises a TIR from IL-18R1 (IL-18 receptor α). Therefore, in some cases, the co-stimulatory region comprises an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 109 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO: 109 (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO: 109 (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region includes the amino acid sequence shown in SEQ ID NO: 109.

[0170] Any of the above-described TIR domains can be used in combination with any desired signal conduction domain (e.g., CD3ζ, DAP10, DAP12). In some cases, the signal conduction region includes the CD3ζ signal conduction domain and a TIR from TLR2. In some cases, the signal conduction region includes the CD3ζ signal conduction domain and a TIR from TLR3. In some cases, the signal conduction region includes the CD3ζ signal conduction domain and a TIR from IL-18R1. In some cases, the signal conduction region includes the DAP10 signal conduction domain and a TIR from TLR2. In some cases, the signal conduction region includes the DAP10 signal conduction domain and a TIR from TLR3. In some cases, the signal conduction region includes the DAP10 signal conduction domain and a TIR from IL-18R1. In some cases, the signal conduction region includes the DAP12 signal conduction domain and a TIR from TLR2. In some cases, the signal conduction region includes the DAP12 signal conduction domain and a TIR from TLR3. In some cases, the signal conduction region includes the DAP12 signal conduction structure domain and the TIR from IL-18R1.

[0171] The truncated MyD88 peptide lacking the TIR domain fuses with the intracellular domain of CD40 to produce a chimeric peptide that amplifies certain MyD88-guided signals. When T cells are transfected or transduced with a combination of MC-encoding nucleic acid and chimeric antigen receptor (CAR), MC delivers potent co-stimulatory signals that enhance T cell and NK cell growth, persistence, and cytotoxic activity against CAR-specifically targeted cells (Foster et al.). Mol.Ther.25:2176 (2017), Duong et al., Mol.Ther.Onc 12:124 (2018), Collinson-Pautz et al., Leukemia 33:2195, Wang et al., Blood Adv. 4:1950).

[0172] MyD88 Fusion In some embodiments, the truncated MyD88 peptide has also been fused with the signaling domain of a co-stimulated receptor mediator, such as, for example, 4-1BB or HVEM. These chimeric signaling peptides produce different phenotypic outcomes in CIR-expressing modified NK cells (CIR-NK cells), particularly in terms of cytotoxicity, growth potential, and the ability to release cytokines upon binding to their targets.

[0173] The chimeric truncated MyD88 peptide described herein, when expressed in, for example, CIR-NK cells, produces significantly fewer certain toxic inflammatory cytokines such as TNF-α compared to CIR-NK cells expressing the MyD88-CD40 chimeric peptide, while retaining potent or even enhanced tumor cell killing ability. Modified chimeric receptors are also provided, wherein the chimeric receptor peptide contains only the truncated MyD88 peptide.

[0174] This article also provides information on immune cells, such as activated NK cells expressing chimeric signaling peptides. Activated cells can be used to enhance the immune response against a disease or to treat cancer by, for example, shrinking tumor size. Treatment regimens using activated NK cells and activated CIR-NK cells can be monitored using various imaging modalities (e.g., CT, bone scan, MRI, PET scan, Trofex scan) to determine tumor size and vascular distribution, using various standard blood biomarkers (e.g., PSA, circulating tumor cells), or by serum levels of various inflammatory, hypoxic cytokines, or other factors in the treated patient. In some embodiments, the co-stimulatory region comprises the MyD88 polypeptide (see, for example, SEQ ID NO: 27). Therefore, in some cases, the co-stimulatory region comprises an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 27 (e.g., 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO: 27 (e.g., 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO: 27 (e.g., 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%). In some embodiments, the co-stimulatory region comprises the amino acid sequence shown in SEQ ID NO: 27. The MyD88 polypeptide in this segment can be combined with other co-stimulatory molecules as part of the co-stimulatory region. For example, in some cases it is combined with CD40. In some cases it is combined with 4-1BB. In some cases it is combined with HVEM (see, for example, SEQ ID NO: 25). Any of these combinations (MyD88, MyD88-CD40, MyD88-4-1BB, MyD88-HVEM) can be used in combination with any desired signal transduction domain, such as CD3ζ, DAP10, DAP12. In some cases, the signal transduction region comprises a CD3ζ signal transduction domain and the MyD88, MyD88-CD40, MyD88-4-1BB, or MyD88-HVEM co-stimulatory region. In some cases, the signal transduction region includes the DAP10 signal transduction domain and the MyD88, MyD88-CD40, MyD88-4-1BB, or MyD88-HVEM co-stimulation regions. In some cases, the signal transduction region includes the DAP12 signal transduction domain and the MyD88, MyD88-CD40, MyD88-4-1BB, or MyD88-HVEM co-stimulation regions. In some cases, the signal transduction region includes the DAP12 signal transduction domain and the MyD88 co-stimulation region. In some cases (e.g., the signal transduction region of a CIR), the signal transduction region includes the CD3ζ signal transduction domain and the MyD88-4-1BB co-stimulation region.

[0175] CIR variants The functional portion of the CIR described herein is provided. When referring to a CIR, the term "functional portion" means any part or fragment of a CIR that retains the biological activity of its parent CIR (parental CIR). The functional portion encompasses, for example, those parts of a CIR that retain the ability to recognize targets (HLA-G) or target cells, or to detect, treat, or prevent disease, to a similar, equal, or greater degree as the parental CIR. In the case of a parental CIR, the functional portion may comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or longer of the parental CIR. ICD peptides are also provided. Such peptides may form part of a CIR as an ICD, but may also form part of another chimeric receptor (such as a CAR) as an ICD.

[0176] The functional portion may contain additional amino acids at the amino-terminus, carboxyl-terminus, or both ends of the portion, which are not found in the amino acid sequence of the parental CIR. Desiredly, the additional amino acids do not interfere with biological functions, such as target cell recognition, cancer detection, cancer treatment, or cancer prevention. More desirously, the additional amino acids enhance biological activity compared to the biological activity of the parental CIR.

[0177] The scope of this disclosure includes functional variants or bioequivalents of the CIRs of the present invention disclosed herein. Functional variants may, for example, comprise the amino acid sequence of a parent polypeptide having at least one conserved amino acid substitution. Alternatively or additionally, functional variants may comprise the amino acid sequence of a parent polypeptide having at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Non-conserved amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is enhanced compared to the parent polypeptide.

[0178] This biological variant (including its functional portion) may contain synthetic amino acids that replace one or more naturally occurring amino acids.

[0179] Such biovariants (including their functional parts) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, for example via disulfide cyclization, or converted to an acid addition salt and / or optionally dimerized or polymerized, or conjugated.

[0180] Such biovariants (including their functional parts) can be obtained by methods known in the art. Peptides can be prepared by any suitable method for preparing peptides or proteins. Suitable methods for de novo synthesis of peptides and proteins are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, edited by Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, edited by Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Furthermore, peptides and proteins can be generated using standard recombinant methods with nucleic acid recombination as described herein. See, for example, 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.

[0181] 4. Safety switch Genetically modified cells expressing the chimeric ILT receptor (CIR) can also express a safety switch (also known as an induced suicide gene or suicide switch) if needed. For example If a GvHD occurs, this safety switch can be used in in vivoEliminating therapeutic cells. In some instances, therapeutic cells may trigger adverse events, such as off-target toxicity due to CIR, or patients may experience negative symptoms during treatment with modified cells, or side effects may occur due to nonspecific attack on healthy tissue; or sometimes, therapeutic cells may no longer be needed, or the therapy is designed to last for a specified amount of time, for example, therapeutic cells may function to reduce tumor cells or tumor size and may no longer be required. Therefore, it may be useful if genetically modified cells can also inducibly express peptides that cause cell death (such as inducible caspase-9 peptide). This switch can be triggered if needed, for example, to reduce the number of therapeutic cells.

[0182] These switches respond to a trigger, such as a pharmacological agent, which is supplied when it is desired to eliminate therapeutic cells, and the trigger causes cell death. For example (By triggering necrosis or apoptosis). These agents can lead to toxic gene products. From the beginning However, if genetically modified cells already express proteins that respond to the reagent by converting them into a toxic form, a faster response can be obtained.

[0183] In some embodiments, the safety switch is based on a pro-apoptotic protein that can be triggered by administration of a trigger molecule (also known as a ligand inducer) to the subject. If the pro-apoptotic protein is fused to a polypeptide sequence that binds to the trigger molecule, delivery of the trigger molecule brings the two pro-apoptotic proteins close together, causing them to trigger 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 reagent vermicomycete (AP1903). Using a safety switch based on a human pro-apoptotic protein, such as, for example, caspase-9, minimizes the risk that cells expressing the switch will be recognized as foreign by the subject's immune system. Thus, delivery of vermicomycete to the subject can trigger apoptosis in cells expressing the caspase-9 switch.

[0184] Other non-limiting examples of chimeric peptides that can be used to induce cell death or apoptosis can be found in the following patents and patent applications, each of which is incorporated herein by reference 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 herein by reference in its entirety for all purposes, including all text, tables, and figures. Further details regarding certain switches and methods are provided below: Inducible caspase 9 (iC9): This pro-apoptotic switch comprises a fusion of caspase-9 with FKBP12 or a derivative thereof. It is latent in the absence of a ligand but drives the dimerization of the initiating caspase (caspase 9) from the intrinsic pathway of apoptosis. Dimerization leads to caspase-9 activation, cleavage and activation of the effector caspase (caspase-3), and rapid cell death via apoptosis. Inducible caspase-9 has specific utility as a safety switch in cell therapy to block toxic responses.

[0185] Caspase-9 switch Examples are described below: Di Stasi et al. (2011) As mentioned above 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 9,434,935; U.S. Patent 9,913,882; U.S. Patent 9,393,292; and Patent Application US2015 / 0328292.

[0186] The safety switch may include a modified Caspase-9 peptide with modifying activity, such as, for example, a reduced basal activity in the absence of a homodimeric ligand. Modified Caspase-9 peptides are discussed, for example, in U.S. Patents 9,913,882 and US2015 / 0328292 (as described above), and may include, for example, an amino acid substitution at position 330 (e.g., D330E or D330A) or, for example, an amino acid substitution at position 450 (e.g., N405Q), or combinations thereof, including, for example, D330E-N405Q and D330A-N405Q. Caspase-9 peptides with lower basal activity have been previously described, for example, in U.S. Patent Nos. 9,434,935, 9,932,572 and 9,913,882, and U.S. Patent Application Nos. 62 / 668,223, 62 / 756,442, 62 / 816,799, 15 / 901,556, and 15 / 888,948.

[0187] In some embodiments, the safety switch may be, for example, as discussed below. iCasp9 Di Stasi et al. (2011) As described above, it consists of the sequence (GenBank AH002818) of human FK506-binding protein (FKBP12) with the F36V mutation, linked via an SGGGS linker to modified human caspase 9 (CASP9) lacking its endogenous caspase activation and recruitment domains. The F36V mutation increases the binding affinity of FKBP12 to the synthetic homodimers AP20187 and lineidoxetine.

[0188] Limiduxe's ​​specific binding to the FKBP12 allele Rimidux binds to the valine 36 allele of FKBP12 with high affinity (~0.1 nM), but to the wild-type phenylalanine 36 FKBP12 allele with low affinity (~500 nM). Rapamycin and rapamycin analogs can bind to either FKBP allele. Rimidux has two identical tail-to-tail protein-binding surfaces, each for the valine 36 form (also known as FKBP12(F36V), FKBP12v36, FKBPV, F...). V36 or simply F v It possesses high affinity and specificity. See Jemal people, 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 to induce higher-order oligomers upon lineidoxetine crosslinking. One or more F...V Linking a domain to one or more cell signaling molecules that typically rely on homodimerization can convert the protein into a vermidoxetine-controlled switch. FKBP12 variants can also be used. These variants can bind to rapamycin or rapamycin analogs, but have a lower affinity for vermidoxetine compared to, for example, FKBP12v36. Examples of FKBP12 variants include variants from many species, including, for example, yeast. In one embodiment, the FKBP12 variant is FKBP12.6 (calstablin).

[0189] The suicide switch can be controlled by a drug composition containing a triggering molecule, such as a dimerized or multimerized ligand. The effective amount of the drug composition containing the triggering molecule is the amount that achieves the desired result of killing genetically modified cells. The degree of killing can be very high ( For example (More than 60%, 70%, 80%, 85%, 90%, 95%, or 97%) or complete kill; conversely, sometimes only partial removal is desired ( For example Less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of genetically modified cells were killed. Therefore, genetically modified cells can exhibit a range of sensitivities to trigger molecules. Thus, trigger molecules can be used to eliminate only a subset of cells. For example At least 10%), while allowing some cells ( For example At least 10% survival. The concentration of the triggering molecule can be selected based on the desired balance between cell death and survival. For example If a higher percentage of cell elimination (or complete elimination) is desired, a higher concentration will be delivered.

[0190] These concentrations can be determined through simple dose-range experiments, thereby monitoring the level of cell death in response to the trigger molecule. Any appropriate assay can be used to determine the percentage of genetically modified cells killed. The assay may include the following steps: obtaining a first sample from the subject before administration of the trigger molecule and a second sample from the subject after administration of the trigger molecule, and comparing the number or concentration of therapeutic cells in the first and second samples to determine the percentage of therapeutic cells killed. Those skilled in the art can determine the effective amount of the particular compositions presented herein empirically without excessive experimentation.

[0191] 5. Chimeric antigen receptor Chimeric antigen receptors (or CARs) are artificial receptors designed to deliver antigen-specific antigens to cells. They typically consist of 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.

[0192] CARs are, for example, chimeric peptides containing a polypeptide sequence (antigen recognition domain) that recognizes a target antigen. This polypeptide sequence is linked to a transmembrane peptide and a peptide selected for activating an intracellular domain, thereby providing specific immunity. The antigen recognition domain can be a single-chain variable fragment (scFv) or can be derived, for example, from other molecules, such as, for example, T-cell receptors or camelid VhH domains. The intracellular domain contains at least one polypeptide that induces cellular activation, such as, for example, but not limited to, CD3 zeta (CD3ζ), and optionally co-stimulatory molecules (e.g., but not limited to, CD28, OX40, and 4-1BB).

[0193] Therefore, in a typical example of CAR use, cells are modified to express a CAR comprising a single-chain antibody variable fragment (scFv) fused to a transmembrane domain containing an adapter region and an intracellular domain derived from CD3 zeta components. In native T cells and NK cells, signals from CD3 zeta drive the initial activation of T cells by signaling NF-ATc transcription factors. These signals drive targeted cell killing in cytotoxic T lymphocytes and synergize with co-stimulatory signaling pathways to drive robust cell proliferation in the T cell immune response. Genetically modified cells can be modified by transduction or transfection with a CAR-expressing nucleic acid and a nucleic acid containing a polynucleotide encoding a chimeric signaling peptide (the same or different) (see below). In other embodiments, the CAR is expressed without simultaneously expressing the chimeric signaling peptide.

[0194] Chimeric antigen receptors can be expressed in NK cells, iNKT cells, or macrophages to produce antigen-specific cytotoxicity.

[0195] CARs include chimeric receptors derived from antibodies, but also chimeric T-cell receptors. These chimeric T-cell receptors may contain polypeptide sequences that recognize target antigens, wherein the recognizing sequence may be, for example, but not limited to, a recognition sequence derived from a T-cell receptor or scFv. Intracellular domain peptides are those that activate T cells. Chimeric T-cell receptors are discussed, for example, in the following: Gross & Eshar FASEB Journal (1992) 6:3370-3378 and Zhang people, (2010) PLOS Pathogens 6:1-13.

[0196] 8. Connector peptides When it is desired to encode two polypeptides in a single gene, such that they encode on a single transcript, the two polypeptides can be linked by a linker polypeptide. For example, these can include between MyD88 and CD40 in a MyD88-CD40 chimeric polypeptide, or between the cytoplasmic signaling region and the CD3ζ portion of a co-stimulatory polypeptide in a CAR or CIR. Where desired, the linker can be located between any of the regions / domains described herein. For example, in some cases, the linker is located between: a TM domain and a signaling or co-stimulatory region, an ILT2 or ILT4 targeting region (e.g., the D1-D2 domain) and a stalk, a signaling region and a co-stimulatory region, two co-stimulatory domains, a co-stimulatory or signaling region and a T2A sequence, or any combination thereof.

[0197] The adapter peptide includes both cleavable and non-cleavable adapter peptides. Examples of adapters include, but are not limited to, SGR, GS, VD, and PRGSG (SEQ ID NO: 67). Anyone skilled in the art will recognize the use of additional adapters and any readily available adapter may be employed.

[0198] Connector peptides include, for example, those composed of about 2 to about 30 amino acids. For example furin cleavage site, (GGGGS) n In some embodiments, the adaptor peptide 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 adaptor peptide consists of about 18 to 22 amino acids. In some embodiments, the adaptor peptide contains 20 amino acids.

[0199] Cleavable adapters include adapters that are cleaved by an enzyme in modified cells. The enzyme may be exogenous to the cell, such as an enzyme encoded by a polynucleotide introduced into the cell simultaneously or at different times via transfection or transduction with the polynucleotide encoding the adapter. In some embodiments, cleavable adapters include adapters cleaved by an endogenous enzyme in the modified cells of a population, including, for example, enzymes naturally expressed in the cell and enzymes encoded by naturally occurring polynucleotides in the cell, such as, for example, lysozyme. The term "cleavable adapter" is also extended to adapters cleaved by any means, including, for example, non-enzymatic methods such as peptide jumping.

[0200] One advantage of cleavable linkers is that they allow two peptides to be expressed in a essentially fixed stoichiometric ratio (1:1 if two mature peptides are linked by a single cleavable linker).

[0201] The linker polypeptide can be a 2A-like sequence, which can be derived from many different viruses, including, for example, those derived from the tea geometrid moth. Thosea asigna Insect viruses. These sequences are sometimes called “peptide skipping sequences.” When this type of sequence is placed in a cistron between two polypeptides intended to be separated, in the case of the tea geometrid moth sequence, the ribosome appears to skip the peptide bond; the bond between the Gly and Pro amino acids at the carboxyl terminus “PGP” is omitted. This may leave two or three polypeptides, such as 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 using this sequence, the polypeptide encoded at the 5' end of the 2A sequence can end with additional amino acids at the carboxyl terminus, including Gly residues and any upstream residues of the 2A sequence. The peptide encoded at the 3' end of the 2A sequence can end with additional amino acids at the N-terminus, including Pro residues and any downstream residues after the 2A sequence.

[0202] In some embodiments, the cleavable linker is a 2A polypeptide derived from porcine cyclovir-1 (P2A). In some embodiments, the 2A co-translated sequence is a 2A-like sequence. In some embodiments, the 2A co-translated sequence is T2A (tea geometrid virus 2A), F2A (foot-and-mouth disease virus 2A), P2A (porcine cyclovir-1 2A), BmCPV 2A (cytoplasmic polyhedrosis virus 2A), BmIFV 2A (bombyx mori softening virus 2A), or E2A (equine rhinitis A virus 2A). In some embodiments, the 2A co-translated sequence is T2A-GSG, F2A-GSG, P2A-GSG, or E2A-GSG. In some embodiments, the 2A co-translated sequence is selected from the group consisting of T2A, P2A, and F2A. In one specific embodiment, 2TA comprises (or consists of) the sequences disclosed herein. It comprises (or consists of) the sequences disclosed herein (e.g., the sequences disclosed in the embodiments below).

[0203] 2A-like sequences are sometimes “leaky” because some polypeptides do not dissociate during translation but remain as a long polypeptide post-translation. One theory for the cause of leaky linkers is that short 2A sequences may sometimes fail to fold into the structure required to facilitate ribosome jumping (“2A fold”). In these cases, the ribosome may not jump the proline peptide bond, which then leads to the formation of a fusion protein. To reduce leakage levels and thus the number of fusion proteins formed, a GSG (or similar) linker can be added to the N-terminal side of the 2A polypeptide; the GSG linker prevents the spontaneous folding of the secondary structure of the newly translated polypeptide and disrupts the “2A fold”. For example, leaky 2A sequences can be used so that the same encoded polypeptide can sometimes be guided to the cell surface but otherwise remain in the cytoplasm.

[0204] In some embodiments, the 2A linker comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the 2A linker further comprises a GSG amino acid sequence at the amino terminus of the peptide, and in other embodiments, the 2A linker comprises a GSGPR (SEQ ID NO: 68) amino acid sequence at the amino terminus of the peptide. Therefore, the term "2A sequence" may refer to the 2A sequence in the embodiments described herein, or it may refer to the 2A sequence listed herein that further comprises a GSG or GSGPR (SEQ ID NO: 68) sequence at the amino terminus of the linker.

[0205] In some embodiments, the adapter (e.g., the 2A adapter) is cleaved in about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the translated polypeptide.

[0206] This article discloses examples of suitable adapter peptides, including the T2A adapter.

[0207] 6. Genetically modified cells Genetically modified cells (such as immune cells expressing, for example, nucleic acids including those encoding a chimeric ILT receptor) can be any cell therapy-compatible cell, such as immune cells. Cells can be, for example, natural killer (NK) cells, iNK-T cells, NKT cells, T cells, B cells, macrophages, peripheral blood cells, hematopoietic progenitor cells, or bone marrow cells. In some embodiments, the modified cells are NK cells, natural killer T cells (NKT cells / NK-T cells), or T cells.

[0208] Genetically modified cells, such as those expressing nucleic acids including, for example, encoding the subject-specific chimeric ILT receptor, as disclosed herein, can be administered to subjects who may benefit from receiving them. For example Subjects who can benefit from the administration of donor lymphocytes. These subjects are typically human, so these procedures are usually performed using human cells.

[0209] cell origin 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 originate from any healthy donor with an appropriate relationship to the intended recipient. Donors are typically adults (at least 18 years old), but children are also suitable as cell donors. For example See Styczynski2018, Transfus Apher Sci 57(3):323-330).

[0210] The term "autologous" refers to a cell derived from the same individual to which it is subsequently administered. The term "allogeneic" refers to an HLA or MHC locus that differs in antigenicity between host and donor cells. Therefore, cells from the same species can differ in antigenicity. The term "homogeneous" refers to cells with the same or sufficiently similar genotypes to allow for tissue transplantation or immunocompatibility. For example, identical twins or close relatives can be homogeneous.

[0211] The cells can be blood cells. For example, the cells could originate from, for instance, cord blood, bone marrow, or peripheral blood, and they could be peripheral blood mononuclear cells (PBMCs). These include lymphocytes (…). For example T cells, B cells, NK cells) or monocytes. As used in this article, the term "peripheral blood" refers to the cellular components of blood (T cells, B cells, NK cells) or monocytes. For example Umbilical cord blood (containing red blood cells, white blood cells, and platelets) is derived from or prepared from the circulating blood pool and is not isolated in the lymphatic system, spleen, liver, or bone marrow. It differs from peripheral blood and blood isolated in the lymphatic system, spleen, liver, or bone marrow, and refers to the blood remaining in the placenta and attached umbilical cord after delivery. Umbilical cord blood typically contains stem cells, including hematopoietic cells.

[0212] Methods for obtaining and expanding NK cells from the human body are described in the following: Cho & Campana (2009). Korean J Lab Med 29:89-96, Somanchi et al. (2011) J Vis Exp 48:2540 and Wang et al. (2020) Blood Adv. 4:1950. A suitable method for obtaining T cells from the human body is described in the following attached published protocol: DiStasi et al. (2011) N Engl J Med 365:1673-83 (“the plan”).

[0213] NK cells NK cells, also known as natural killer cells or large granular lymphocytes (LGLs), are cytotoxic lymphocytes that are crucial to the innate immune system. NK cells function similarly to 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.

[0214] T cells rely on the initiational interaction between the T cell receptor (TCR) and the MHC-peptide complex on target cells as a necessary first step in T cell activation. Therefore, T cells can recognize single antigens, and tumor cells can evade T cell recognition by significantly reducing antigen presentation through mutations. In contrast, NK cells are able to recognize a large number of transformed and infected cells without relying on the presentation of a single antigen. Therefore, therapy using NK cells can bypass some resistance mechanisms to T cell-based therapies.

[0215] As innate cells, NK cells can secrete pro-inflammatory chemokines and cytokines to recruit and activate the body's adaptive immune system, composed of T cells and B cells, thereby generating a second, durable anti-tumor response. Furthermore, NK cells are not associated with certain toxicities of CAR-T cell therapy, such as cytokine release syndrome and central nervous system toxicity.

[0216] Due to their inherent cytotoxic mechanisms, NK cells can serve as a source for antigen- or receptor-guided cell therapies. NK cells comprise approximately 10–15% of lymphocytes in typical donor peripheral blood and can be readily purified, expanded, and virally transduced. In cases of loss of intratumoral cell-guided cell therapy targets (e.g., HLA-G), activated NK cells possess alternative innate mechanisms to direct cytotoxic function, including NKG2D, p46, p44, p30, DNAM, and CD16. CD4+ and CD8+ T cells The subject composition may include CD4+ and CD8+ T cells. While the ratio of CD4+ to CD8+ cells in apheresis products is typically greater than 2, in some embodiments, the ratio of genetically modified CD4+ cells to genetically modified CD8+ cells in the compositions disclosed herein is less than 2. For example Below 1.5. Ideally, the composition should contain more genetically modified CD8+ T cells than genetically modified CD4+ T cells. Right now The ratio is less than 1. For example The concentrations are below 0.9, below 0.8, below 0.7, below 0.6, or preferably even below 0.5. Therefore, the overall procedure starting from donor cells and generating genetically modified T cells ideally enriches CD8+ T cells relative to CD4+ T cells. Preferably, at least 60% of the genetically modified T cells are CD8+ T cells, and more preferably at least 65%. Within the genetically modified CD3+ T cell population, the preferred range for CD8+ T cells is between 55% and 75%. For example 63-73%. The ratio of CD8+ to CD4+ T cells can be easily assessed by flow cytometry, and the methods for sorting and counting CD4+ and CD8+ T cells are routine in the field.

[0217] A subset of memory T cells (see Mahnke et al. (2013) Eur J Immunol 43:2797-809) Genetically modified T cell populations can include terminal effector memory T cells (defined as CD45RA+CD45RO-CCR7- cells; "TEMRA"), T effector memory cells (defined as CD45RA-CD45RO+CCR7- cells; "EM"), T central memory cells (defined as CD45RA-CD45RO+CCR7+ cells; "CM"), and naive T cells (defined as CD45RA+CD45RO-CCR7+ cells). These cells can be assessed by flow cytometry using CD45RA / RO and CCR7 markers. Marker reagents that identify CCR7 and distinguish between CD45RA and CD45RO isoforms are readily available from commercial suppliers.

[0218] Average apheresis products typically contain approximately 20% terminal effector cells and 20% T effector memory cells. The overall process from donor cells to genetically modified T cells can enrich terminal effector memory T cells relative to T effector memory cells.

[0219] In some embodiments, less than 60% of the genetically modified T cells are naive T cells. For example Less than 58%, preferably less than 55%, and more preferably less than 50%. Within the genetically modified CD3+ T cell population, the preferred range for naïve T cells is 30-60%, more preferably 42-49%, and most preferably 43-46%. This proportion of naïve T cells has been observed to correlate with favorable outcomes in T cell recipients. Naïve EM cells can be assessed by flow cytometry using CD45RA / RO and CCR7 markers.

[0220] In genetically modified T cell populations, the proportion of central memory T cells is typically <10%, excluding TEMRA, EM, and naive T cells.

[0221] In some embodiments, the genetically modified T cell population in the composition comprises about 10% to about 40% CD4+ T cells and about 60% to about 90% CD8+ T cells. The genetically modified CD3+ T cell population 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.

[0222] Cellular genetic modification Cellular genetic modification is achieved by transferring expression constructs (e.g., constructs encoding a subject-specific chimeric ILT receptor) into cells. This transfer can be performed using viral or non-viral gene transfer methods. This section provides a discussion of gene transfer methods and compositions.

[0223] Expression vectors can be introduced into cells using various methods. The terms "transfection" and "transduction" are used interchangeably and refer to the process of introducing a foreign nucleic acid sequence into a eukaryotic host cell. Transfection (or transduction) can be achieved through a variety of methods, including electroporation, microinjection, gene gun delivery, retroviral infection, lipid transfection, and supertransfection.

[0224] Any appropriate method can be used to transfect or transform cells. For example (T cells, NKT cells, or NK cells). This document presents certain non-limiting examples. In some embodiments, the viral vector is an SFG-based viral vector, as discussed below: Tey et al. (2007) Biol Blood Marrow Transpl 13:913-24 and Di Stasi et al. (2011) N Engl J Med 365:1673-83.

[0225] Cells can be transduced using viral vectors encoding the polypeptides described herein. Suitable transduction techniques may involve the fibronectin fragment CH296. As an alternative to transduction using viral vectors, any suitable method known in the art can be used, such as transfecting cells with DNA encoding the relevant polypeptide, for example using calcium phosphate, cationic polymers (such as PEI), magnetic beads, electroporation, and commercial lipid-based reagents such as Lipofectamine™ and Fugene™. One result of the transduction / transfection steps is that various donor cells will now become genetically modified cells capable of expressing CIR and any other desired polypeptides.

[0226] In some embodiments, the viral vector used for transduction is a retroviral vector disclosed below: Tey et al. (2007) Biol Blood Marrow Transpl 13:913-24 and Di Stasi et al. (2011) As mentioned above This vector is based on a gibberish leukemia virus (GalV) pseudotyped retrovirus encoding the iCasp9 suicide switch and the ΔCD19 cell surface transgenic marker (see further below—and SEQ ID NO: 12-13). It can be generated in PG13 packaging cell lines, as discussed below: Tey et al. (2007) As mentioned aboveOther viral vectors encoding the desired protein may also be used. In some embodiments, transduction is performed using a retroviral vector that can provide a high copy number of proviral integrons per cell.

[0227] Following transduction / transfection, cells can be separated from the transduction / transfection material and cultured again to allow for the expansion of genetically modified cells. Cell expansion can be achieved to reach the desired minimum number of genetically modified cells.

[0228] Genetically modified cells can then be selected from the obtained cell population. CIRs may not be suitable for positive selection of desired cells; therefore, in some embodiments, the genetically modified cells should express a target cell surface transgenic marker (see below). Cells expressing this surface marker can... For example Selection can be made using immunomagnetic techniques. For example, paramagnetic beads conjugated to monoclonal antibodies that recognize transgenic markers on the surface of target cells can be used, for instance, using the CliniMACS system (available from Miltenyi Biotec).

[0229] In an alternative procedure, genetically modified cells are selected, cultured, and then fed after the transduction step. Therefore, the order of transduction, feeding, and selection can be varied.

[0230] The result of these procedures is a composition containing genetically modified cells, and therefore capable of expressing chimeric ILT receptors (as well as any other desired peptides). For example Co-stimulatory peptides, suicide switches, cell surface transgenic markers wait These genetically modified cells can be administered to recipients, but may optionally be preserved first after further expansion before administration. For example (Freeze-freezing).

[0231] Selectable markers Cells can be modified to express their expression. in vitro or in vivo The identified peptides allow for the selection of genetically modified cells. For example This allows them to be separated from unmodified cells. Such markers impart recognizable changes to cells, thus allowing for easy identification of cells containing the desired expression construct.

[0232] Including drug selection markers aids in the selection of clones and transformants. For example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, bleomycin, and histamine are useful selection markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (TK) are employed.

[0233] Immunosurface markers containing extracellular non-signal transduction domains or various proteins (e.g., CD34, CD19, LNGFR) can also be used, allowing for simple methods of magnetic or fluorescent antibody-mediated sorting. These markers can be detected. For example Use labeled antibodies that bind to proteins.

[0234] The optional markers used are considered unimportant, as long as they can be associated with the gene product encoding the desired gene product. For example Simultaneous expression of the nucleic acids of the subject CIR is sufficient. Furthermore, ideally, the biomarker should be a polypeptide not expressed by the primordial (donor) cells, although differences in expression levels can be utilized even if the biomarker is indeed endogenous in the primordial cells.

[0235] Ideally, the biomarker is based on a human protein, as this minimizes the risk that cells expressing the biomarker will be recognized as foreign by the subject's immune system. For example (After they are administered therapeutically). For example, when T cells are the desired cell type, human CD proteins, which are not naturally expressed by T cells, can be used for this purpose.

[0236] The genetically modified cells described herein can express cell surface transgenic markers that are present on expression vectors expressing the subject CIR, and / or in some embodiments, they are present on expression vectors encoding proteins other than CIRs, such as, for example, CARs, pro-apoptotic peptide safety switches, or co-stimulatory peptides.

[0237] In one embodiment, the cell surface transgenic marker is a truncated CD19 (ΔCD19) polypeptide (DiStatis). et al. (2011, as described above), which contains human CD19 truncated at amino acid 333 to remove most of the intracytoplasmic domain (see, for example, SEQ ID NO: 12 (nucleotide) and SEQ ID NO: 13 (protein)). The extracellular CD19 domain is still recognizable ( For example In flow cytometry, FACS, or MACS, the likelihood of triggering intracellular signaling is minimized. CD19 is typically expressed by B cells, rather than by T or NK cells, so selecting CD19+ cells allows for the separation of genetically modified cells (e.g., T cells, NK cells, or NKT cells) from unmodified cells.

[0238] Another useful marker is CD34, which has a minimal epitope of 16 amino acids that can be used as a marker.

[0239] By encoding the desired protein at the 5' end of the coding gene and a marker at the 3' end, the selection of peptides that do not have the desired protein can be minimized. For example The risk to cells (due to premature termination of translation). In this way, the expression of biomarkers and desired peptides proceeds in parallel.

[0240] 7. Engineering-based representation constructs Provided are chimeric ILT receptors (CIRs) encoding the subject (and optionally other desired peptides, such as chimeric antigen receptors, signal transduction peptides, safety switches, IL-15). wait Nucleic acid containing the nucleotide sequence of [a specific protein]. In some cases, this nucleic acid is an expression construct. This article provides methods for expressing chimeric ILT receptors (and optionally other desired peptides, such as chimeric antigen receptors, signal transduction peptides, safety switches, IL-15, etc.). wait The expression construct of ). In some embodiments, one or more polypeptides are referred to as being "operably linked" to a promoter, which means that the promoter sequence is functionally linked to a second sequence, wherein the promoter sequence is in the correct position and orientation relative to the second sequence to control the initiation of RNA polymerase and the transcription of DNA corresponding to the second sequence, thereby resulting in a transcript that encodes the target polypeptide.

[0241] A promoter is a DNA sequence recognized by the cell's synthetic mechanisms or introduced synthetic mechanisms; it is the DNA sequence required to initiate the specific transcription of a gene. In some embodiments, the promoter is a developmental regulatory promoter. Right now Promoters act as the initial binding site for RNA polymerases to transcribe genes expressed under certain conditions controlled, initiated, or influenced by developmental programs or pathways.

[0242] The term "expression construct" refers to any type of genetic construct containing nucleic acids encoding a gene product, wherein some or all of the nucleic acid coding sequence can be transcribed. The transcript may be translated into a protein, but is not required to. In some embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression includes only transcription of the nucleic acid encoding the target gene. Expression vectors may contain a variety of control sequences, which are nucleic acid sequences operatively linked in a particular host organism that are necessary for transcription and, possibly, translation of the coding sequence.

[0243] A "vector" is a device that can transfer nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate vectors, and liposomes). Generally, "vector construct," "expression vector," "expression construct," and "gene transfer vector" refer to any nucleic acid construct that can direct the expression of a target nucleic acid and transfer the nucleic acid sequence to target cells. Therefore, the term includes cloning and expression agents, as well as viral vectors.

[0244] In some instances, the polynucleotide encoding a CIR and the polynucleotide encoding a second polypeptide are included in the same vector, such as, for example, a viral vector or a plasmid vector. This second polypeptide can be, for example (and as described elsewhere herein), a downregulator of an endogenous protein, a blocking antibody or scFv of an inhibitory receptor, a signaling polypeptide, an inducible suicide switch, or a biomarker polypeptide. In other instances, the added expressed transcript may not encode a protein, but instead produce a short hairpin RNA product designed to eliminate the expression of certain endogenous RNAs encoding unwanted proteins in the cellular product.

[0245] The construct can be designed to have a promoter operatively linked to a nucleic acid containing a polynucleotide encoding a fusion protein via a linker polypeptide. For example Cleavable linker polypeptides, such as polypeptide 2A. In this example, the first and second polypeptides are generated during a single translation event but can subsequently be separated. In other examples, the two polypeptides can be expressed separately from the same vector, in which the individual nucleic acids encoding the polynucleotide of one polypeptide are operatively linked to separate promoters. In other examples, a promoter can be operatively linked to two polynucleotides, thereby directing the production of two separate RNA transcripts and thus two polypeptides; in one example, the promoter can be bidirectional, and the coding regions can be in opposite 5'-3' orientations. Therefore, the expression constructs discussed herein can contain at least one or at least two promoters.

[0246] In other instances, two peptides (such as, for example, CIR and marker proteins) can be expressed in cells using two separate vectors. Cells can be co-transfected or co-transformed with the vectors, or the vectors can be introduced into cells at different times.

[0247] Any combination of these methods can be used to achieve the desired expression of the polypeptide in genetically modified cells.

[0248] In some embodiments, the nucleic acid construct is contained within a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is an adenovirus vector or a lentiviral vector. It should be understood that in some embodiments, in... In vitro Under certain conditions, cells are contacted with a viral vector, and in some embodiments, under in vivo conditions, cells are contacted with a viral vector. Thus, expression constructs can be inserted into vectors, such as viral vectors or plasmids. The steps of the provided methods can be performed using any suitable method; these methods include, but are not limited to, the methods described herein for transducing, transforming, or otherwise delivering nucleic acids to cells.

[0249] The specific promoter used to control the expression of the target polynucleotide sequence is generally not particularly important, as long as it can guide the expression of the polynucleotide in the desired cells. Therefore, when targeting human cells, the coding region of the polynucleotide sequence can, for example, be located near and controlled by a promoter capable of expression in human cells. Generally, such a promoter can include human promoters or viral promoters. Promoters suitable for the vectors used to express the CIR and other peptides presented herein can be selected.

[0250] In various embodiments, such as when the expression vector is a retrovirus, an example of a suitable promoter is the murine Moronnievus leukemia virus promoter. In other embodiments, the promoter may be, for example, the CMV early gene promoter, the SV40 early promoter, or a Rous sarcoma virus long terminal repeat sequence. 2-Microglobulin, ribosomal protein 31, phosphoglycerate kinase, EF1α, β-actin, rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the target coding sequence. Other viral or mammalian cell promoters well-known in the art are also considered for achieving expression of the target coding sequence, provided the expression level is sufficient for a given purpose. By employing promoters with known properties, the expression level and pattern of the target peptide after transfection or transformation can be optimized.

[0251] In other embodiments, the expression vector is a transposon, such that genetic elements encoding CIR and related marker proteins, coactivators, or endogenous factor inhibitors or the tumor microenvironment are carried on a plasmid vector carrying elements recognized by a transiently co-expressed transposase. The transposase catalyzes the fusion of the transgene with the cellular genome between the repetitive elements recognized by the transposase. Examples of transposon systems that can be used in these embodiments are... sleeping Beauty System and Piggyback The system. The promoter elements carried within the transposon direct transgene expression. Promoters can be, for example, the CMV early gene promoter, the SV40 early promoter, or long terminal repeat sequences of Rous sarcoma virus. 2-Microglobulin, ribosomal protein 31, phosphoglycerate kinase, EF1α, β-actin, rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase. The method of introducing plasmids containing transposons and transposases into cells is transfection rather than viral transduction.

[0252] Promoters and other regulatory elements are selected to enable them to function in the desired cells or tissues. Furthermore, this list of promoters should not be interpreted as exhaustive or restrictive; other promoters may be used in conjunction with the promoters and methods disclosed herein.

[0253] It should be understood that the sequence of polynucleotides can vary and can be tested to determine the suitability of the construct for any particular method. Therefore, nucleic acids can include polynucleotides with different sequences, which also takes into account variations in the sequence of components.

[0254] In some embodiments, cells are transfected or transduced with nucleic acids encoding two polynucleotides, and the cells further contain nucleic acids containing polynucleotides encoding a third polypeptide, and / or the cells further contain nucleic acids containing polynucleotides encoding a fourth polypeptide. In some embodiments, cells are transfected or transduced with nucleic acids encoding three polynucleotides, and the cells further contain nucleic acids containing polynucleotides encoding a fourth polypeptide. For example, cells may contain nucleic acids containing first, second, and third polynucleotides, and cells may also contain nucleic acids containing polynucleotides encoding a chimeric Caspase-9 polypeptide. Furthermore, cells may contain nucleic acids containing first, second, and fourth polynucleotides, and cells may also contain nucleic acids containing polynucleotides encoding a chimeric ILT receptor, a scFv regulator of native ILT2 function, or interleukin-15.

[0255] 8. Methods used to treat diseases Methods for treating or preventing diseases are also provided, in which, for example, the infusion of cells (e.g., cells expressing a subject CIR) may be beneficial. Cells can be used, for example, for regeneration, such as to replace the function of diseased cells. The genetically modified cells described herein can be used for cell therapy.

[0256] As used herein, the terms “treatment” and “treating” refer to achieving the desired pharmacological and / or physiological effect. For a disease and / or adverse effects attributable to said disease, said effect may be preventative in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of partially or completely curing it. As used herein, “treatment” covers any treatment of a disease in mammals (particularly humans) and includes: (a) preventing the occurrence of the disease in subjects who are susceptible to the disease or at risk of developing it but have not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e. causing the disease to regress.

[0257] The terms “individual,” “subject,” and “patient” are used interchangeably in this document and refer to mammals, including but not limited to human and non-human primates, including apes and humans; mammalian competitive animals (e.g., horses); mammalian agricultural animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0258] "Effective amount" or "sufficient amount" means, alone or in combination with one or more other compositions (therapeutic agents such as drugs), treatments, protocols, or medical protocols, an amount that provides a detectable response for any duration (long or short), any measurable or detectable degree, or any duration (e.g., minutes, hours, days, months, years, or cure) to a subject (e.g., an effective amount of cells).

[0259] While alleviating, reducing, suppressing, curbing, limiting, or controlling the progression or worsening of a disease is also a satisfactory outcome, the “effective amount” or “sufficient amount” of dosage used for treatment (e.g., to improve or provide therapeutic benefit or improvement) generally provides a response to one, many, or all adverse symptoms, consequences, or complications of the disease to a measurable extent, such as one or more adverse symptoms, symptoms, diseases, pathologies, or complications caused by or associated with the disease.

[0260] The genetically modified cells (i.e., cells expressing the subject CIR) provided herein can be used in methods for treating human subjects in need of this treatment, and can also be used to prepare medicaments for treating such subjects. The cells will typically be delivered to the recipient subject via infusion.

[0261] Genetically modified cells can be T cells, iNKT cells, macrophages, or NK cells. Typical doses of T cells or NK cells used in subject therapy are in the range of 10... 5 10 7 Between [number] cells / kg.

[0262] In general, the genetically modified T cells and NK cells of this disclosure can be used in the same manner as known donor leukocyte infusion (DLI), but they have the additional benefits of CIR.

[0263] Recipients can undergo lymphocyte depletion pretreatment before receiving genetically modified lymphocytes (and before receiving allogeneic grafts). Therefore, the recipient's own α / β T cells (and B cells) can be depleted before receiving genetically modified T cells or NK cells.

[0264] The recipient may have a blood cancer (such as a refractory blood cancer) or a hereditary blood disorder. For example, the recipient may have acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), severe combined immunodeficiency (SCID), Wiscot-Aldrich syndrome (WA), Fanconi anemia, chronic myeloid leukemia (CML), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), or multiple myeloma.

[0265] Receptors of T cells or NK cells that express CIR may have non-hematologic cancers that express HLA-G. For example, receptors may have renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer, neuroblastoma, hepatocellular carcinoma (HCC), ovarian cancer, endometrial cancer, or prostate cancer.

[0266] Several cell types can also be used in therapy, including any cells administered to the patient to achieve the desired therapeutic outcome. Therapeutic cells can be, for example, immune cells such as T cells, natural killer cells (NK cells), NK-T cells, B cells, tumor-infiltrating lymphocytes or macrophages, or combinations thereof; therapeutic cells can also be, for example, peripheral blood cells, hematopoietic progenitor cells, bone marrow cells, or tumor cells. To further improve the tumor microenvironment and make it more immunogenic, treatment can be combined with one or more adjuvants (…). For example Combinations of IL-12, checkpoint inhibitors, IDO inhibitors, etc. In some embodiments, cells can be delivered to treat solid tumors, such as by delivering cells directly to a tumor bed. In some embodiments, cells can be delivered to treat liquid tumors, such as by delivering cells to treat leukemias, such as AML.

[0267] In some embodiments, nucleic acids that can be applied to the subject are also provided, thereby enabling... in vivo Transformation or transduction of target cells to In situ Genetically modified cells are formed.

[0268] An effective amount of genetically modified cells is administered. To determine whether an effective amount of ligand or modified cells has been administered, any means of determining or measuring the number of target cells, the amount of target antigen, or the tumor size can be used to determine whether the number of target cells, the amount of target antigen, or the tumor size has increased, decreased, or remained unchanged. Samples, images, or other measurements obtained before the administration of the modified cells or ligand can be compared with samples, images, or other measurements obtained after the administration of the modified cells or ligand. Thus, for example, to determine whether the amount or concentration of cells expressing the target antigen has increased, decreased, or remained unchanged, a first sample can be obtained from the subject before the administration of the ligand or modified cells, and a second sample can be obtained from the subject after the administration of the ligand or modified cells. The amount or concentration of cells expressing the target antigen in the first sample can be compared with 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 has increased, decreased, or remained unchanged after the administration of the ligand or modified cells.

[0269] The effective amount for any particular application can vary depending on factors such as the disease or condition being treated, the specific compound being administered, the size of the subject, and / or the severity of the disease or condition. Those skilled in the art can determine the effective amount of the particular compositions presented herein through experience.

[0270] To increase the effectiveness of the modified cells presented herein, it may be desirable to combine these compositions and methods with pharmaceutical agents effective in treating diseases.

[0271] The administration of the pharmaceutical composition can be done before, simultaneously with, and / or after other agents, with intervals ranging from minutes to weeks. In embodiments where the pharmaceutical composition and other agents are administered separately to cells, tissues, or organisms, it is generally ensured that the time interval between each delivery does not exceed a significant period, allowing the pharmaceutical composition and agents to still exert a beneficial combined effect on the cells, tissues, or organisms. For example, in such cases, it is considered that the pharmaceutical composition may be administered substantially simultaneously with the other agents in two, three, four, or more ways (i.e., at the same time). Right now Contact with cells, tissues, or organisms (within approximately one minute). In other respects, one or more agents may be administered before and / or after the application of the expression vector, ranging from substantially simultaneously, approximately one minute, approximately 24 hours, approximately 7 days, approximately 1 to approximately 8 weeks, or any range derived therefrom. Furthermore, various combinations of the pharmaceutical compositions and one or more agents presented herein may be employed.

[0272] Diseases that can be treated or prevented include those caused by viruses, bacteria, yeast, parasites, protozoa, cancer cells, etc. Examples of treatable and / or preventable diseases include, but are not limited to, infections caused by viral pathogens such as HIV, influenza, herpes, viral hepatitis, EB virus, poliomyelitis, viral encephalitis, measles, chickenpox, and human papillomavirus; or infections caused by bacterial pathogens such as pneumonia, tuberculosis, and syphilis; or infections caused by parasites such as malaria, trypanosomiasis, leishmaniasis, trichomoniasis, and amebiasis. Pretumoral or proliferative conditions that can be treated or prevented using pharmaceutical compositions (transduced cells, expression vectors, expression constructs, etc.) include, but are not limited to, pretumoral or proliferative conditions such as colonic polyps, Crohn's disease, ulcerative colitis, and breast lesions.

[0273] Cancers for which cell therapy can be used (including solid tumors and / or liquid tumors) 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 lymphoma, Hodgkin lymphoma, leukemia (e.g., chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia), uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, NPC, bladder cancer, cervical cancer, etc.

[0274] Other hyperproliferative diseases that can be treated with the therapeutic cells and other therapeutic cell activation systems presented in this article include, but are not limited to, rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, precancerous lesions (such as adenomatous hyperplasia and prostatic intraepithelial neoplasia), carcinoma in situ, oral hairy leukoplakia, or psoriasis.

[0275] This method can be used to treat solid tumors originating from any tissue or organ, including, for example, any tumor that expresses a target antigen (e.g., HLA-G) in the vascular system, such as solid tumors present in the lungs, bones, liver, prostate, or brain, as well as solid tumors also present in the breast, ovaries, intestines, testes, colon, pancreas, kidneys, bladder, neuroendocrine system, soft tissues, bone masses, and lymphatic system. Other solid tumors that can be treated include, for example, glioblastoma and malignant multiple myeloma.

[0276] This method can be used to treat liquid tumors (leukemias and lymphomas, such as chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and acute lymphoblastic leukemia), including any cancer (such as any leukemia or lymphoma) in which cancer cells express target antigens (such as HLA-G).

[0277] Zinc supplements may be given to subjects to ensure that any zinc-dependent factors contained in the CIR or cofactors expressed in cell therapy products that include the CIR have an adequate source of the ion to allow for their full activity.

[0278] Methods for preparing the cells of this disclosure are also provided. In some embodiments, such methods include transfecting or transducing cells with nucleic acids or expression vectors of this disclosure (e.g., nucleic acids or expression vectors encoding the subject CIR). The term “transfection” is used to refer to the uptake of exogenous DNA by cells. Cells are “transfected” when exogenous DNA has been introduced into the cell membrane. Various transfection techniques are generally well 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 motifs into suitable host cells. The term refers to both stable and transient uptake of genetic material.

[0279] In some embodiments, the cells of this disclosure are generated by transducing cells with a viral vector encoding a CIR. In some aspects, the polypeptide comprises a CIR and the cells are T cells, making a method for generating CIR T cells provided. In some embodiments, such a method includes activating a T cell population (e.g., T cells obtained from an individual to be administered CIR T cell therapy), stimulating the proliferation of the T cell population, and transducing the T cells with a viral vector encoding a polypeptide comprising a 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), a lentiviral vector, or an AAV encoding a CIR. In some aspects, immune cells T cells are transduced with a lentiviral vector encoding a polypeptide. In some aspects, the polypeptide comprises a CIR and the cells are NK cells, making a method for generating CIR NK cells provided (e.g., by using a viral vector such as an AAV, lentivirus, or retroviral vector).

[0280] 9. Overview The term "comprising" encompasses both "including" and "consisting of". For example A composition that "contains" X may consist of X alone or may include additional content. For example X+Y.

[0281] With numerical values x The related term "about" is optional and means, for example... x + 10%.

[0282] The word "basically" does not exclude "completely". For example Compositions that are "substantially free" of Y can be completely free of Y. The word "substantially" can be omitted from the definition if necessary.

[0283] The term "between" includes both values ​​when it refers to two values. For example The range “between” 10 mg and 20 mg covers Especially 10, 15 and 20 mg.

[0284] Unless otherwise specified, methods that include the step of mixing two or more components do not require any specific mixing order. Therefore, the components can be mixed in any order. When three components are present, two components can be combined with each other, and then that combination can be combined with a third component, and so on.

[0285] The steps of a method may be performed at the same or different times, in the same or different geographical locations (e.g., countries), and by the same or different people or entities.

[0286] The degree of similarity between two sequences can be based on a percentage of sequence identity. In this paper, "sequence identity" refers to the degree to which two nucleotide or amino acid sequences remain unchanged. "Sequence alignment" refers to the process of aligning two or more sequences to achieve the maximum level of identity in order to assess similarity. Many methods for aligning sequences and assessing similarity / identity are known in the art, such as, for example, clustering methods (where similarity is based on the MEGALIGN algorithm) and BLASTN, BLASTP, and FASTA. When using any of these procedures, settings that produce the highest sequence similarity can be selected.

[0287] 10. Examples of specific nucleic acid and amino acid sequences The following sections and tables include examples of polypeptide and nucleotide sequences encoding chimeric signal transduction peptides. It should be understood that the sequences of individual polypeptides provided in these examples, such as, for example, truncated ILT4 D1-D2 polypeptides, cytoplasmic signal transduction regions of co-stimulatory polypeptides, ITAM-containing cytotoxic regions, IL-15, or safety switches, can be used to construct other expression vectors encoding the chimeric signal transduction peptides of this embodiment. The sequences used in the following experimental examples include: ILT4.CD3ζ ILT4.DAP10 ILT4.DAP12 ILT4.DAP10.CD3ζ ILT4.DAP12.CD3ζ ILT4.4-1BB.CD3ζ ILT4.HVEM.CD3ζ ILT4.4-1BB.DAP10 ILT4.4-1BB.DAP10.CD3ζ ILT4.4-1BB.DAP12 ILT4.4-1BB.DAP12.CD3ζ ILT4.MyD88.CD3ζ ILT4.MyD88.4-1BB.CD3ζ ILT4.MyD88.DAP12 ILT4.MyD88.DAP12.CD3ζ ILT4.MyD88.4-1BB.CD3ζ ILT4.MyD88.HVEM.CD3ζ ILT4.MyD88.CD40.CD3ζ ILT4.IL18R1TIR.CD3ζ ILT4.TLR2TIR.CD3ζ ILT4.TLR3TIR.CD3ζ For the following constructs, see, for example, Examples 6-7 below. 241 ILT4,CD3ζ.2A'.IL-15 242 ILT4.4-1BB.DAP10.CD3ζ.2A'.IL-15 243 ILT4.4-1BB.DAP10.2A'.IL-15 244 ILT4.DAP10.CD3ζ.2A'.IL-15 245 ILT4.DAP10.2A'.IL-15 246 ILT4.MyD88.DAP10.CD3ζ.2A'.IL-15 247 ILT4.MyD88.DAP10.2A'.IL-15 248 ILT4.4-1BB.DAP12.CD3ζ.2A'.IL-15 249 ILT4.4-1BB.DAP12.2A'.IL-15 250 ILT4.4-1BB.CD3ζ(1XX).2A'.IL-15 251 ILT4.CD3ζ.2A'.IL-15 252 ILT4.CD3ζ.P2A.MyD88.CD40.T2A'.IL-15 253 ILT4.DAP10.TLR2.2A'.IL-15 254 ILT4.CD3ζ.P2A.MyD88.4-1BB.2A'.IL-15 255 ILT4.CD3ζ.P2A.MyD88.HVEM.2A'.IL-15 256 ILT4.CD3ζ.P2A.IL18R1.T2A'.IL-15 257 ILT4.CD3ζ.P2A.TLR2.T2A'.IL-15 258 ILT4.CD3ζ.P2A.TLR3.T2A'.IL-15 259 ILT4.CD3ζ.P2A.TLR5.T2A'.IL-15 260 ILT4.CD3ζ.P2A.TLR8.T2A'.IL-15 261 ILT4.CD3ζ.MyD88.CD40.T2A'.IL-15 262 ILT4.CD3ζ.MyD88.T2A'.IL-15 263 ILT4.CD3ζ.MyD88.4-1BB.T2A'.IL-15 264 ILT4.CD3ζ.MyD88.HVEM.T2A'.IL-15 265 ILT4.CD3ζ.IL18R1.T2A'.IL-15 266 ILT4.CD3ζ.TLR2.T2A'.IL-15 267 ILT4.4-1BB.DAP10.TLR2.T2A'.IL-15 268 ILT4.CD3ζ.TLR5.T2A'.IL-15 269 ILT4.CD3ζ.TLR8.T2A'.IL-15 270 ILT4.DAP12.TLR2.T2A'.IL-15 271 ILT4.4-1BB.DAP12.TLR2.T2A'.IL-15 272 ILT4.TLR2.DAP12.CD3ζ.T2A'.IL-15 273 ILT4.TLR2.DAP12.T2A'.IL-15 274 ILT4.DAP10.CD3ζ.P2A.TLR2.T2A'.IL-15 275 ILT4.4-1BB.DAP10.CD3ζ.P2A.TLR2.T2A'.IL-15 276 ILT4.DAP12.CD3ζ.P2A.TLR2.T2A'.IL-15 277 ILT4.4-1BB.DAP12.CD3ζ.P2A.TLR2.T2A'.IL-15 278 ILT4.DAP10.P2A.TLR2.T2A'.IL-15 279 ILT4.4-1BB.DAP10.P2A.TLR2.T2A'.IL-15 280 ILT4.DAP12.P2A.TLR2.T2A'.IL-15 281 ILT4.4-1BB.DAP12.P2A.TLR2.T2A'.IL-15 282 ILT2.CD3ζ.2A'.IL-15 283 ILT2.4-1BB.CD3ζ.2A'.IL-15 284 ILT2.4-1BB.DAP10.CD3ζ.2A'.IL-15 285 ILT2.4-1BB.DAP10.2A'.IL-15 286 ILT2.4-1BB.DAP12.2A'.IL-15 287 ILT2.MyD88.DAP10.CD3ζ.2A'.IL-15 288 ILT4.MyD88.DAP10.2A'.IL-15 289 ILT2.MyD88.DAP12.CD3ζ.2A'.IL-15 290 ILT2.MyD88.DAP12.2A'.IL-15 291 ILT2.4-1BB.DAP10.P2A.TLR2.T2A'.IL-15 Examples of non-limiting aspects of this disclosure The various aspects of the subject matter described above (including embodiments) may be beneficial, alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of this disclosure are provided below. As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects can be used or combined with any of the preceding or following individually numbered aspects. This is intended to support all such combinations of the aspects and is not limited to the combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

[0289] 1. A chimeric receptor protein comprising: (a) Targeting region, which targets HLA-G and includes the D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) A transmembrane (TM) region comprising a transmembrane amino acid sequence; and (c) Intracellular domain (ICD), wherein the ICD contains a signal transduction region capable of transducing signals into the interior of immune effector cells after binding to HLA-G in the target region to induce effector cell function. The signal transduction region includes a co-stimulation region containing the MyD88 polypeptide.

[0290] 2. The chimeric receptor protein according to claim 1, wherein the MyD88 polypeptide comprises an amino acid sequence having 85% or more sequence identity with the sequence of SEQ ID NO: 27.

[0291] 3. The chimeric receptor protein according to 1 or 2, wherein the MyD88 polypeptide is fused with a CD40, 4-1BB or HVEM co-stimulatory domain.

[0292] 4. The chimeric receptor protein according to 3, wherein the MyD88 polypeptide is fused with a 4-1BB co-stimulatory domain.

[0293] 5. The chimeric receptor protein according to any one of 1 to 4, wherein the signal transduction region comprises a CD3ζ signal transduction domain, a DAP10 signal transduction domain, a DAP12 signal transduction domain, or any combination thereof.

[0294] 6. The chimeric receptor protein according to claim 4, wherein the signal transduction region comprises a CD3ζ signal transduction domain.

[0295] 7. The chimeric receptor protein according to 1 or 2, wherein the signal transduction region comprises the DAP12 signal transduction domain.

[0296] 8. The chimeric receptor protein according to 7, wherein the signal transduction region does not contain a CD3ζ signal transduction domain.

[0297] 9. The chimeric receptor protein according to 1 or 2, wherein the signal transduction region comprises a DAP12 signal transduction domain and a CD3ζ signal transduction domain.

[0298] 10. An intracellular domain (ICD) polypeptide comprising a signal transduction region capable of transducing signals in immune effector cells to induce effector cell function, wherein the signal transduction region comprises: (i) a CD3ζ signal transduction domain, a DAP10 signal transduction domain, or a DAP12 signal transduction domain, and (ii) a co-stimulatory region comprising a Toll / interleukin-1 receptor / resistance protein (TIR) ​​domain.

[0299] 11. The ICD polypeptide according to 10, wherein the TIR domain is a TLR2 TIR domain, a TLR3 TIR domain, or an IL18R1 TIR domain.

[0300] 12. The ICD polypeptide according to claim 10, wherein the TIR domain comprises an amino acid sequence having 85% or higher sequence identity with the TLR2TIR domain of SEQ ID NO: 111.

[0301] 13. The ICD polypeptide of claim 10, wherein the TIR domain comprises an amino acid sequence having 85% or higher sequence identity with the TLR3TIR domain of SEQ ID NO: 113.

[0302] 14. The ICD polypeptide of claim 10, wherein the TIR domain comprises an amino acid sequence having 85% or higher sequence identity with the IL18R1TIR domain of SEQ ID NO: 109.

[0303] 15. The ICD polypeptide according to any one of 10 to 15, wherein the signal transduction region comprises the CD3ζ signal transduction domain.

[0304] 16. The ICD polypeptide according to any one of 10 to 15, wherein the co-stimulatory polypeptide is contained in a chimeric receptor protein, the chimeric receptor protein comprising: (a) Targeting region, which targets HLA-G and includes the D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) A transmembrane (TM) region comprising a transmembrane amino acid sequence; and (c) The ICD polypeptide.

[0305] 17. A chimeric receptor protein comprising: (a) Targeting region, which targets HLA-G and includes the D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) A transmembrane (TM) region comprising a transmembrane amino acid sequence; and (c) Intracellular domain (ICD), wherein the ICD contains a signal transduction region capable of transducing signals into the interior of immune effector cells after binding to HLA-G in the target region to induce effector cell function. The signal conduction region includes either the DAP10 signal conduction structure domain or the DAP12 signal conduction structure domain.

[0306] 18. The chimeric receptor according to 17, wherein the DAP10 signaling domain comprises an amino acid sequence having 85% or higher sequence identity with SEQ ID NO: 4.

[0307] 19. The chimeric receptor according to 17, wherein the DAP12 signaling domain comprises an amino acid sequence having 85% or higher sequence identity with SEQ ID NO: 4.

[0308] 20. The chimeric receptor according to any one of 17 to 19, wherein the signal transduction region further comprises a CD3ζ signal transduction domain.

[0309] 21. The chimeric receptor according to any one of 17 to 19, wherein the signal transduction region does not include the CD3ζ signal transduction domain.

[0310] 22. The chimeric receptor according to any one of 17 to 21, wherein the signal transduction region further comprises a CD40, 4-1BB, or HVEM co-stimulatory domain.

[0311] 23. The chimeric receptor according to any one of 17 to 21, wherein the signal transduction region further comprises a 4-1BB co-stimulatory domain.

[0312] 24. The chimeric receptor of claim 17, comprising a DAP12 signal transduction domain, wherein the signal transduction domain does not include a CD3ζ signal transduction domain, and wherein the signal transduction domain further includes a 4-1BB co-stimulatory domain.

[0313] 25. The chimeric receptor according to 24, wherein the DAP12 signaling domain comprises an amino acid sequence having 85% or higher sequence identity with SEQ ID NO: 4.

[0314] 26. The chimeric receptor protein according to any one of 1 to 25, wherein the D1-D2 extracellular domain is the ILT4 D1-D2 extracellular domain.

[0315] 27. The chimeric receptor protein according to 26, wherein the targeting region comprises the D3-D4 extracellular domain of ILT4.

[0316] 28. The chimeric receptor protein according to 26, wherein the targeting region does not contain the D3-D4 extracellular domain of ILT4 and contains the stalk domain.

[0317] 29. The chimeric receptor protein according to any one of 1 to 25, wherein the D1-D2 extracellular domain is the ILT2 D1-D2 extracellular domain.

[0318] 30. The chimeric receptor protein according to 29, wherein the targeting region comprises the D3-D4 extracellular domain of ILT2.

[0319] 31. The chimeric receptor protein according to 29, wherein the targeting region does not contain the D3-D4 extracellular domain of ILT4 and contains the stalk domain.

[0320] 32. The chimeric receptor protein according to 28 or 31, wherein the stalk domain comprises an ILT2, ILT4, CD28, CH2 / CH3, CH3, or CD8α stalk domain.

[0321] 33. The chimeric receptor protein according to any one of 1 to 33, wherein the TM domain is an ILT2, ILT4, CD28, or CD8α TM domain.

[0322] 34. The chimeric receptor protein according to any one of 1 to 25, wherein: The D1-D2 extracellular domain is the ILT4 D1-D2 extracellular domain. The extracellular domain lacks the ILT4 D3-D4 extracellular domain. The chimeric receptor protein includes a CD8α stalk domain, and The TM region is CD8α TM.

[0323] 35. The chimeric receptor protein according to any one of 1 to 25, wherein: The D1-D2 extracellular domain is the ILT2 D1-D2 extracellular domain. The extracellular domain lacks the ILT2 D3-D4 extracellular domain. The chimeric receptor protein includes a CD8α stalk domain, and The TM region is CD8α TM.

[0324] 36. A nucleic acid comprising a nucleotide sequence encoding a chimeric receptor protein according to any one of 1 to 35.

[0325] 37. The nucleic acid according to 36, wherein the nucleotide sequence is operatively linked to a constitutive promoter.

[0326] 38. The nucleic acid according to 36, wherein the nucleotide sequence is operatively linked to an inducible promoter.

[0327] 39. The nucleic acid according to any one of 36 to 38, wherein the nucleic acid is an expression vector.

[0328] 40. The nucleic acid according to 39, wherein the expression vector is a retroviral vector, a lentiviral vector, or a plasmid vector.

[0329] 41. A genetically modified cell that expresses the chimeric receptor protein according to any one of 1 to 35.

[0330] 42. The genetically modified cell according to 41, wherein the genetically modified cell is an immune cell.

[0331] 43. The genetically modified cell according to 42, wherein the immune cell is a natural killer (NK) cell, NK-T cell, T cell, iNKT cell or macrophage.

[0332] 44. The genetically modified cell according to 42, wherein the immune cell is a natural killer (NK) cell.

[0333] 45. A treatment method comprising administering to an individual in need the genetically modified cells according to any one of 41 to 44.

[0334] 46. ​​The method according to 45, wherein the genetically modified cell is autologous to the individual.

[0335] 47. The method according to 45, wherein the genetically modified cell is an allogeneic species to the individual.

[0336] 48. The method according to any one of 45 to 47, wherein the individual suffers from cancer.

[0337] 49. The method according to 48, wherein the individual has a solid tumor.

[0338] 50. The method according to 48, wherein the individual suffers from a liquid tumor.

[0339] 51. A method for producing genetically modified cells, the method comprising: Nucleic acids according to any one of 36 to 40 are introduced into cells to produce genetically modified cells.

[0340] 52. The method according to 51, wherein the genetically modified cell is an immune cell.

[0341] 53. The method according to 52, wherein the immune cell is a natural killer (NK) cell, NK-T cell, T cell, iNKT cell or macrophage.

[0342] 54. The method according to 52, wherein the immune cell is a natural killer (NK) cell.

[0343] 55. The method according to 52, wherein the immune cell is a T cell.

[0344] Experimental Examples The following examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the invention should in no way be construed as limited to the following examples, but rather as including any and all variations that become apparent as a result of the teachings provided herein.

[0345] Without further description, it is believed that those skilled in the art can use the foregoing description and the following illustrative examples to make and utilize the invention and practice the claimed methods. Therefore, the following working examples should not be construed as limiting the remainder of this disclosure in any way.

[0346] General methods of molecular and cellular biochemistry can be found in standard textbooks such as *Molecular Cloning: A Laboratory Manual*, 3rd edition (Sambrook et al., HaRBor Laboratory Press 2001); *Short Protocols in Molecular Biology*, 4th edition (edited by Ausubel et al., John Wiley & Sons 1999); *Protein Methods* (Bollag et al., John Wiley & Sons 1996); *Nonviral Vectors for Gene Therapy* (edited by Wagner et al., Academic Press 1999); *Viral Vectors* (edited by Kaplift & Loewy, Academic Press 1995); *Immunology Methods Manual* (edited by I. Lefkovits, Academic Press 1997); and *Cell and Tissue Culture: Laboratory Procedures in Biotechnology* (Doyle & Griffiths, John Wiley & Sons 1998), the contents of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits used in the methods mentioned in or related to this disclosure are available from commercial suppliers such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and depository institutions such as Addgene, Inc., and the American Type Culture Collection (ATCC).

[0347] Example 1. Expression of engineered CIR constructs in primary human NK cells To demonstrate the practicality of chimeric receptor expression that enhances NK cell signaling in a target-specific manner, a gamma-retrovirus was created that targets HLA-G by binding to its native ILT receptor. A cartoon illustration of the resulting CIR construct is depicted in... Figure 1In the universally depicted CIR construct, the D1 and D2 domains of ILT4 are fused with the CD8α stalk and transmembrane domain for stable presentation of the conjugate to the HLA-G target. All other constructs described in these examples contain the same extracellular components to allow for unbiased cross-referencing of altered intracellular signaling domains between constructs. The recombinant DNA constructs were engineered in an SFGγ retroviral vector (Rivere, Brose, and Mulligan, Proc. Natl. Acad. Sci. USA 15:6733-6737 (1995)), where transgene expression was driven by a Moloney mouse sarcoma virus long terminal repeat (LTR). Schematic diagrams of the constructs evaluated in this example and the following examples are depicted in [reference to a specific image / image / image]. Figure 2 In each embodiment of these constructs, DNA encoding CIR is cloned at the 5' end of the gene encoding interleukin-15 (IL-15), a growth factor essential for sustained NK cell growth and survival, which is provided autocrinely to support CIR-NK cells in culture. Cistrons encoding ILT4, CIR, IL-15, and CD19 are separated by a T2A co-translational cleavage site derived from Thoseaasigna virus or a P2A sequence derived from porcine Cheddar virus to allow for separate expression of the proteins from a single mRNA molecule.

[0348] Gamma retroviruses were generated by transfecting these DNA constructs along with helper plasmids encoding reverse transcriptase, viral capsid, and envelope proteins into HEK293 cells. These retroviral vectors were used to transduce CD56-expressing primary human NK cells selected from peripheral blood mononuclear cells (PBMCs) from two healthy donors. All NK cells used in the comparative experiments were derived from the same donor. Transduction efficiency was characterized by the presence of ILT4 expression on the CIR as detected by flow cytometry, although ILT4 is not typically expressed by NK cells. The stability of transgene expression was monitored weekly.

[0349] Methods: DNA constructs were designed using SnapGene software, and DNA sequences were synthesized from GBlock fragments provided by IDT Laboratories. The synthesized DNA fragments were cloned using standard cloning techniques and used to assemble the recombinant DNA into SFG DNA vectors.

[0350] Example 2. Antitumor efficacy of CIR-NK cells with altered ITAM-containing cytotoxic domains To generate tumor cell lines suitable for assessing CIR target-specific cytotoxicity, three derivatives of acute myeloid leukemia (AML) cell lines were created to express a fusion of the biomarker protein green fluorescent protein (GFP) and the individual biomarker firefly luciferase (GFP-ffluc). KG1-GFPffluc cells do not express HLA-G protein detectable by flow cytometry, nor do they express HLA-G1 and HLA-G2 mRNAs encoding the most common membrane-bound HLA-G isotypes, as detected by quantitative PCR. This cell line was used as a negative control for CIR-specific targeting and as a reference for hypothetically overactive innate NK cell cytotoxicity, possibly resulting from basal CIR activity. Molm13-GFPffluc and Kasumi1-GFPffluc cells are AML cell lines expressing low but measurable levels of HLA-G1 protein and HLA-G1 and HLA-G5 mRNAs. These cells were used for conjugation and activation of CIR-NK cells.

[0351] To evaluate the relative signaling efficacy of CIR constructs containing alternative ITAM-containing signaling motifs, primary human NK cells from two healthy donors were transduced with γ-retroviruses encoding the extracellular and stalk / transmembrane domains described in Example 1, as well as the intracellular domains of CD3ζ, DAP10, or DAP12. Notably, DAP10 does not contain the classic ITAM domain but instead signals via a mechanism similar to CD28 in T cells. The expressed CIRs lacked co-activation domains. Separate γ-retroviruses encoding red fluorescent protein (RFP) were co-transduced to assess NK cell growth during co-culture experiments. Simulated transduction (cells manipulated identically to transduced cells but without the virus) and cells transduced with RFP using retroviruses without CIRs served as negative controls.

[0352] Two × 10⁵ CIR-NK cells were cultured without a target to assess the potential basal NK cell activity induced by signal transduction domains present on the CIR construct. Additionally, two × 10³ identical NK cells were co-cultured with HLA-G+ Kasumi1 cells at an effector-to-target ratio of 1:5 (E:T). Production of the cytokines tumor necrosis factor-α (TNF-α) and interferon-γ was measured to assess basal and post-stimulation NK cell activity (see [link to relevant documentation]). Figure 3 and Figure 4 The results were normalized to a cell count of 2 × 10⁵ for comparison and evaluation against NK cell stimulation.

[0353] Compared to NK cells lacking CIR expression, CIR-NK cells exhibited increased basal TNF-α and IFN-γ secretion. Stimulation with Kasumi1 target cells increased total cytokine secretion in mimicked or RFP-transduced NK cells, indicating an innate targeting effect on Kasumi1 cells. TNF-α production in CIR-NK cells was not further stimulated, but after Kasumi1 stimulation, IFN-γ production in ILT4.DAP12 CIR-NK cells was significantly increased compared to CIR-NK cells expressing DAP10 or CD3ζ, or control NK cells. IFN-γ production is a commonly used surrogate marker for NK cell activation. These results suggest that DAP12 may be a superior signaling modality promoting inflammation at tumor sites.

[0354] To assess the innate and target-specific cytotoxic potential of CIR-NK cells, co-culture with HLA-G-KG1-GFPffluc cells was established at an E:T ratio of 1:5. Figure 5 Co-culture with HLA-G+ Molm13- GFPffluc cells was established at an E:T ratio of 1:10. Figure 6 The cytotoxic effect of NK cells in co-cultures was assessed over seven days by imaging GFP fluorescence light in an Incucyte microscope / incubator as an indicator of target cell overgrowth. In the NK cell control culture expressing RFP but not CIR, KG1 cell growth was not controlled compared to the NK cell-deficient culture (tumor only), and the innate activity of CIR-NK cells was lower. CIR-NK cells containing DAP12 enhanced control over Molm13 cell growth compared to CIR-NK cells expressing DAP10 or CD3ζ. NK cell proliferation over seven days was measured by imaging RFP markers expressed in NK cells using Incucyte. Each CIR-NK cell type with a CIR activation domain exhibited enhanced proliferation compared to NK cells expressing only RFP. These results further suggest that DAP12 provides superior NK cell performance compared to CD3ζ or DAP10 when not expressed in conjunction with co-activating signaling modalities.

[0355] To evaluate whether incorporating multiple ITAM-containing elements could further enhance CIR-NK cell potency, constructs simultaneously including DAP10 and CD3ζ or DAP12 and CD3ζ in intracellular domains were prepared, and CIR-NK cell performance was compared with CIR-NK cells expressing each signaling element alone. Basal and Kasumi1 target-stimulated cytokine production were compared, and binding CD3ζ signaling to DAP10 or DAP12 did not enhance TNF-α (…). Figure 7 ) or IFN-γ ( Figure 8 The production of IFN-γ was observed. Compared to the ILT4.DAP12 CIR-NK cell co-culture, the IFN-γ level in the IFN.DAP12.CD3ζ CIR-NK cell co-culture with Kasumi1 was significantly lower. No innate targeting of KG1 cells was found in any CIR-NK cell co-culture. Figure 7 However, binding CD3ζ to DAP10 or DAP12 enhanced HLA-G-specific cytotoxicity against Molm13-GFPffluc cells. These results indicate that adding an additional ITAM domain to the intracellular signaling domains of CIR can enhance the killing performance of CIR-NK cells, but attenuates the signaling pathway that stimulates the production of pro-inflammatory cytokines.

[0356] Example 3. Effects of co-activation signals on CIR-NK cell performance 4-1BB (TNFRS9) and HVEM (TNFRS14) are members of the TNF receptor superfamily, and each can signal through NF-κB and other pathways to promote cytokine gene transcription and cytokine release. These pathways synergize with ITAM-directed signaling pathways to promote the growth and survival of immune cells. These properties are referred to as co-stimulation in T cells and co-activation in NK cells. Retroviral vectors containing 4-1BB or HVEM and a CD3ζ cytotoxic domain were prepared, and CIR-NK cells transduced with these constructs were evaluated relative to CIR-NK cells expressing only CD3ζ signaling elements. The inclusion of 4-1BB or HVEM did not affect basal NK cell signaling that produces TNF-α, but the signaling that drives IFN-γ production was enhanced when 4-1BB was included, but not when HVEM was included. Figure 12 and Figure 13 The presence of 4-1BB or HVEM did not affect the production of TNF-α and IFN-γ in CIR-NK cells co-cultured with Kasumi1 targets. The enhancement of CD3ζ signaling in CIR-NK cells by 4-1BB or HVEM did not affect cytotoxicity against control KG1 cells that do not express HLA-G. Figure 13 However, compared to CD3ζ with or without HVEM enhancement, 4-1BB / CD3ζ signaling enhanced target-specific cytotoxicity against Molm13 cells. Figure 14 These results indicate that 4-1BB signaling enhances CIR-NK cell performance.

[0357] These findings were extended by constructing retroviruses comprising 4-1BB elements with or without DAP10 or DAP12 (with or without CD3ζ elements). Co-activation of 4-1BB reduced basal production of TNF-α and IFN-γ upon binding to DAP12 signaling (with or without further CD3ζ signaling), while DAP10 signaling for cytokine production was relatively unaffected upon binding to 4-1BB. Figure 15 , Figure 16 In co-cultures with Kasumi1 cells, the presence of 4-1BB (with or without further CD3ζ content) significantly enhanced target-specific cytokine production in DAP12-containing CIR-NK cells. These results indicate that 4-1BB does not lead to overall overactivation of CIR-NK cells, but increases their responsiveness to HLA-G-activated CIR when bound to DAP12 signaling. The addition of 4-1BB, alone or in combination with DAP10, DAP12, or CD3ζ, did not affect cytotoxicity against HLA-G-KG1 cells. Figure 17 Adding 4-1BB with DAP10 and CD3ζ (ILT4.4-1BB.DAP10.CD3ζ) improved target-specific cytotoxicity against Molm13 cells, but other combinations of DAP10 and DAP12 with 4-1BB did not improve cytotoxicity against Molm13 cells. Figure 18 ).

[0358] Example 4. Effects of MyD88 co-activation on CIR-NK cell performance MyD88 is a cytoplasmic protein recruited to receptors including IL-1 receptors and Toll-like receptors. Activation of these receptors via ligand-guided dimerization leads to MyD88 activation and downstream signaling via multiple pathways, including the interferon response factor, MAPK, NF-κB, and AKT growth and survival pathways. These signaling pathways partially overlap with the signaling pathways of TNFR family proteins.

[0359] A retroviral construct encoding a CIR was created, which added only the death domain signaling element of MyD88, rather than the TIR domain of the binding receptor, as a replacement for the 4-1BB co-activated element along with CD3ζ, DAP12, or DAP12 / CD3ζ. Basal TNF-α production was only affected in the combination of MyD88 and DAP12, and MyD88 signaling had almost no effect on target-specific TNF-α production. Figure 19 When MyD88 is included together with CD3ζ, DAP12, or DAP12 / CD3ζ, basal IFN-γ production in CIR-NK cells is higher ( Figure 20In contrast, when CIR-NK cells were co-cultured with Kasumi1 targets, MyD88 co-activation did not lead to enhanced IFN-γ or TNF-α production. No cytotoxicity against HLA-G-negative KG1 cells was observed with or without MyD88 signaling, but enhanced NK cell proliferation was observed when MyD88 bound to each of the ITM-containing cytotoxic domains (ILT4.MyD88.CD3ζ, ILT4.MyD88.DAP12, ILT4.MyD88.DAP12.CD3ζ). Figure 21 This finding indicates that MyD88 signaling stimulates CIR-NK cell growth in the absence of CIR activation, and that MyD88 signaling also enhances NK cell proliferation in co-cultures with the HLA-G-expressing Molm13 target. Figure 22 When bound to CD3ζ, DAP12, or DAP12 / CD3ζ, CIR-NK cells containing the MyD88 signaling element showed significantly improved cytotoxicity against Molm13. Figure 22 These results indicate that MyD88 co-activation enhances the cytotoxicity and growth performance of CIR-NK cells, both of which are key elements for effective cell therapy.

[0360] The effects of combining different co-activating signaling elements were evaluated in CIR-NK cells. Retroviral constructs were created that combined MyD88 with 4-1BB, HVEM, or CD40 along with the CD3ζ cytotoxic element into the intracellular signaling domain of the ILT4 CIR. CIR-NK cells expressing these so-called third-generation CIR constructs (ILT4.MyD88.4-1BB.CD3ζ, ILT4.MyD88.HVEM.CD3ζ, ILT4.MyD88.CD40.CD3ζ) were compared with CIR-NK cells containing only the intracellular domains of CD3ζ, MyD88.CD3ζ, 4-1BB.CD3ζ, and HVEM.CD3ζ. In all CIR-NK cells containing the MyD88 co-activating element, basal cytokine production was enhanced. However, TNF-α production was enhanced in Kasumi1 cell co-cultures only when the intracellular domain of the CIR contained the MyD88.CD40.CD3ζ combination. Figure 23 Compared to controls expressing only CD3ζ, 4-1BB.CD3ζ, or HVEM.CD3ζ, all CIR-NK cells containing MyD88 showed enhanced IFN-γ production. Figure 24 These findings further support the hypothesis that MyD88 signaling on the chimeric receptor drives enhanced cytokine production.

[0361] No cytotoxicity was observed against HLA-G-negative KG1 cells, but co-cultures of these cells with all CIR-NK cells containing the MyD88 signaling domain showed enhanced proliferation compared to controls without the MyD88 element. Figure 25 When co-cultured with HLA-G-expressing Molm13 cells at an E:T ratio of 1:10, each CIR-NK cell containing MyD88 showed significantly enhanced potency compared to controls expressing only CD3ζ, 4-1BB.CD3ζ, or HVEM.CD3ζ. These results further suggest that the combination of MyD88 signaling with ITAM domain cytotoxic signaling provides enhanced CIR-NK cell potency.

[0362] Example 5. Recruiting MyD88 signal transduction to a CIR expressing a TIR domain Natural MyD88 is recruited to IL-1 family receptors and TLRs via interactions between the TIR domains on receptors and the TIR domains on MyD88 itself. Downstream signaling is directed by a separate MyD88 death domain, which is incorporated into the CIR described in Example 4. To recruit MyD88 signaling to the CIR domain, a construct (ILT4.IL18R1.CD3ζ) was created in which the CD3ζ cytotoxic domain is fused with the TIR domain of the interleukin-18 receptor 1 (or α) chain. Further constructs for indirect recruitment of MyD88 were created. The intracellular TIR domain of TLR2 interacts with the TIR domain of MAL, which further interacts in a ternary complex containing MyD88. A CIR construct fusing the TLR2 CIR with CD3ζ was created. Another CIR construct fusing the TIR domain of TLR3 with CD3ζ was created. The TIR domain of TLR3 does not interact with MyD88, but instead recruits TRIF through its TIR domain. TRIF signaling has downstream signaling pathways that overlap with and differ from MyD88. These constructs were expressed in CIR-NK cells and compared with CIR-NK cells containing only CD3ζ and those with direct fusion of MyD88 and CD3ζ.

[0363] In CIR-NK cells expressing the TIR domain, basal cytokine production was lower, significantly lower than that of the direct ILT4.MyD88.CD3ζ CIR fusion, and TNF-α production in activated TIR-containing CIR-NK cells co-cultured with Kasumi1 cells was not enhanced relative to the direct ILT4.MyD88.CD3ζ co-culture. Figure 27 In contrast, co-culturing CIR-NK cells containing each TIR domain with Kasumi1 cells significantly stimulated CIR conjugation and IFN-γ (…). Figure 28CIR-NK cells containing the TIR domain did not enhance their innate killing activity against Molm13 cells. Figure 29 ) and target-specific killing ( Figure 30 This may be due to insufficient expression to support cytotoxic signaling. MyD88 signaling is activated by oligomerization of the signaling death domain. The low basal signaling produced by IFN-γ and the high inducibility of CIR-target binding support the hypothesis that the TIR domain receptor is bound to the dimer HLA-G and self-dimerizes. This creates CIR-NK cells with low basal activity and high inducibility potential.

[0364] Example 6: Comparison of ILT4 and ILT2 conjugates with alternative signal transduction domains.

[0365] The CIR construct described in the previous embodiment, which exhibited the highest level of enhancing potency when expressed in NK cells, was selected, and the ILT4 conjugate was replaced with ILT2. The signal transduction domains described in this example are listed below. Figure 31 Each construct contained CIR and IL-15 separated by a 2A sequence, as well as RFP encoding a cistron to label cells and assess transduction efficiency. These constructs were transduced into NK cells to generate CIR-NK cells, and CIR expression levels, viability of NK cells with different signal transduction capabilities, ability to produce cytokines in the presence and absence of targets, and cytotoxicity against a range of HLA-G-expressing targets were measured.

[0366] NK cell viability was measured on days 8 and 14 by staining with actinomycin D and propidium iodide, ensuring the integrity of the cell membrane to exclude the dye, thus measuring single-cell viability. Figure 32 CIR-NK cells containing all activation domains showed >80% viability on day 14, comparable to control NK cells transduced to express only RFP. The growth potential of the transduced cells was examined by counting populations at days 5, 8, and 14 and comparing cell expansion levels at day 8 or 14 relative to day 5. Figure 33All CIR-NK cells expressing ILT4 conjugates expanded well to day 14, but CIR-NK cells with ILT2 conjugates and only first-generation (1G) CARs (ILT2.CD3ζ), 4-1BB domains linked to DAP10 and CD3ζ, or MyD88 domains failed to expand significantly between day 8 and day 14. Other ILT2 CIR-NK cells expanded readily. At day 8 and day 14, the average level of ILT4 CIR expression in CIR-NK cells with each alternative signaling domain was determined by flow cytometry using a specific antibody targeting the ILT4 D1 / D2 domain. CIR expression was higher at day 8 (five days post-transduction) and generally stabilized to a considerable mean fluorescence intensity (MFI) by day 14. Figure 34 Overall transduction efficiency and stability were assessed using co-transduced RFP proteins. The CIR construct containing MyD88 showed the lowest overall expression.

[0367] Despite low CIR expression levels, MyD88-linked CIR-NK cells readily produce basal IFN-γ after expansion to day 14, a good marker of signal transduction via the NF-κB pathway. Figure 35 Compared to the first-generation CAR (1G) lacking co-activation, the level is elevated and comparable to constructs that express 4-1BB, DAP12, DAP10 in various combinations and recruit endogenous MyD88 through fusion with the TIR domain of TLR2.

[0368] When interacting with target cells expressing HLA-G (Kasumi1 AML cells) Figure 36 ) and HT-1376 bladder cancer cells ( Figure 37 When co-cultured, CIR-NK cells expressing MyD88 exhibited stronger IFN-γ production than CIR-NK cells without MyD88, although all CIR-NK cells with co-activation domains showed enhanced cytokine production relative to 1G CIR-NK cells. On a single-cell basis, cytokine production levels following CIR stimulation were significantly increased relative to baseline levels. Figure 35 ).

[0369] CIR-NK cells containing ILT2 and ILT4 enhancement activation domains exhibit potent cytotoxicity against a range of HLA-G-expressing target cells, but not against HLA-G-target cells. In a short-term (2-day) assay targeting Molm13 AML cells, approximately one-third of the target cells were killed, with the strongest short-term cytotoxicity observed in the MyD88.DAP10 activation domain linked to either ILT2 or ILT4. Figure 38Short-term NK cell expansion was variable in the ILT2 CIR construct, but the enhancement was most significant in the ILT4-linked MyD88.DAP12 fusion compared to other ILT4-containing constructs. Figure 38 During a 7-day co-culture, CIR-NK cells demonstrated significant efficacy against AML target cells. At a NK cell (effective cell) to target cell ratio of 1:40, CIR-NK cells exhibited continuous killing activity, controlling the expansion of Molm13 cells compared to the case with only tumor cells. Figure 39 and Figure 40 Similar results were observed when the same CIR-NK cell group was co-cultured with Kasumi1 cells, which also expressed HLA-G but were more difficult to kill. Figure 41 and Figure 42 ).

[0370] CIR-NK cells also exhibited potent cytotoxicity and selectivity against target cell lines derived from solid tumors. HLA-G RNA levels were measured in HCT-116 cells derived from human colon cancer and HT-1376 cells derived from human bladder cancer. HCT-116 cells expressed low to no HLA-G RNA, while HT-1376 cells expressed HLA-G1, HLA-G2, and HLA-G5 isotypes. Cell surface expression of HLA-G was measured by flow cytometry using the MEMG / 9 antibody, and HLA-G was readily detectable in HT-1376 cells but not in HCT-116 cells. Figure 43 CIR-NK cell cytotoxicity was readily observed in HT-1376 cells. Figure 44 ), but not observed in HCT-116 cells ( Figure 45 ).

[0371] SU8686 cells are derived from pancreatic ductal adenocarcinoma (PDAC), and it has been previously noted that despite robust expression of this target, CAR-T cells targeting PDAC remain particularly difficult to kill. SU8686 cells strongly express HLA-G1 on their cell surface (… Figure 46 Furthermore, isotype-specific qPCR detected abundant HLA-G1 (9359 copies / cell) and HLA-G2 (77,668 copies / cell) expression in SU8686 cells. Although speculative, the cell line's resistance to targeted cell therapy may be due to HLA-G2-induced immunosuppression via ILT2. In co-culture of mimic transduced or CIR-NK cells with SU8686 cells, mimic transduced cells lacking CIR expression showed almost no innate cytotoxicity, while ILT2 and ILT4-expressing CIR-NK cells effectively recognized and targeted SU8686 cells. Figure 47 and Figure 48).

[0372] Enhanced signaling provided effective cytotoxicity of CIR-NK against SU8686. While first-generation (ILT2-CD3ζ) CIR-NK cells recognized HLA-G targets and expanded NK cells, they failed to control the expansion of SU8686 in co-cultures. However, enhanced co-activation via a combination of 4-1BB, DAP10, and TLR2 (BB.DAP10.TLR2) was particularly effective for both tumor killing and cell expansion. Figure 47 and Figure 48 ).

[0373] These findings demonstrate that CIR-NK cells with enhanced cytotoxic domains are functional when linked to ILT2 or ILT4 binding domains to target HLA-G. They also show that CIR-NK cells have potent efficacy at effector-to-target ratios up to 1:40, demonstrating that many or most CIR-NK cells possess so-called “serial killing” capabilities. CIR-NK cells are also effective in controlling the growth of target cells derived from solid tumors, suggesting the potential of this technology for therapies targeting a broad range of leukemia and solid tumor indications.

[0374] Example 7. Extending the persistence of CIR-NK cell potency using enhanced activation domains. NK cells have a limited functional lifespan in the natural environment, typically around 2 weeks, and are continuously replenished through hematopoiesis. As cell therapy products, maximizing the durability of NK cell potency to treat high tumor burden is advantageous. To examine the durability of CIR-NK cell function and evaluate the ability of different activating domains to prolong this potency, ILT4 CIR-NK cells grown to a standard 14 days or extended to 26 days were co-cultured with Molm13 target cells at an E:T ratio of 1:20. Different combinations of cytotoxic domains, including CD3ζ, DAP10, and DAP12, were included as activating domains and compared to the same combination with 4-1BB co-activation or TIR domains of TLR2 (with different amino-to-carboxyl orientations of TLR2 relative to the cytotoxic domains). Figure 49 At 14 days of expansion, each CIR-NK cell line exhibited high cytotoxicity against the Molm13 target. This potency was reduced in co-cultures containing "older" first-generation CIR-NK cells grown for 26 days, and in several CIR-NK cell lines with alternative activation domains. CIR-NK cells containing a combination of 4-1BB with DAP10 or DAP12 maintained potency at 26 days, as did the combination of 4-1BB with DAP10 and TLR2 (BB.DAP10.TLR2).

[0375] To further demonstrate the efficacy of enhanced signal transduction in CIR-NK cells in resisting NK cell exhaustion, simulated transduced or CIR-NK cells were repeatedly stimulated with Molm13 tumor target cells up to four times during a 9-day culture period. Figure 50 The transduced NK cells exhibited relatively strong innate cytotoxicity to the target cell line, as demonstrated when Molm13 was cultured with NK cells only once, and were rapidly depleted if more Molm13 targets were added on day 2. First-generation CIR-NK cells showed decreased potency after three or more consecutive additions of the tumor target. After three or four additions of the Molm13 target, enhanced signaling elements showed differentiation, and it was evident that the combination of BB.DAP10, and particularly BB.DAP10.TLR2 signaling elements, bound to ILT2 or ILT4 “bindings,” maintained NK cell potency even under repeated tumor challenges. This finding was confirmed and extended in similar experiments using a select candidate CIR-NK cell cohort. Similarly, the combination of BB.DAP10.TLR2 on ILT2 and ILT4 CIRs showed cytotoxicity against NK cells compared to other CIRs and NK cells transduced only with RFP markers. Figure 51 ) and proliferation ( Figure 52 The most effective maintenance of ).

[0376] The differential expression of CIR signaling domains was also examined in a potency stress test. Molm13 cells were co-cultured with NK cells at increasing effector-to-target ratios (E:T) from 1:40 to 2:1. Short-term (2-day) determination was performed using quantitative imaging in an Incucyte culture microscope. Figure 53 ) and longer (7 days, Figure 54 Cytotoxicity. Similarly, the combination of BB.DAP10 and BB.DAP10.TLR2 was superior to other combinations in terms of cytotoxicity and NK cell proliferation. Figure 54 and Figure 55 Interestingly and surprisingly, this combination of signal transduction nodes so effectively enhances cytotoxicity in NK cells because the TIR domains of 4-1BB, DAP10, or TLR2 do not contain the classic sequence elements of the ITAM domain that drive CD3ζ cytotoxicity in T cells.

[0377] These findings enhance the potential of CIR-NK cells as a highly durable cell therapy in the patient setting. More broadly, fusing these activation domains with scFv to generate non-classical CARs could increase the therapeutic potential of CAR-NK cells against targets beyond HLA-G.

[0378] Example 8. Long-term resistance to CIR-NK cell exhaustion using enhanced activation domains.

[0379] Effective cell therapies for cancer patients should maintain their potency over extended periods to overcome potentially large tumor burdens and minimize the chance of tumor recurrence. In order to achieve this... in vitro An attack model was designed to mimic the long-term efficacy of CIR-NK cells. OE19 cells were derived from esophageal tumors and expressed relatively low levels of HLA-G1 (2080 RNA copies / cell) and HLA-G2 (899 copies / cell). Despite the relatively low HLA-G expression levels, ILT4 CIR-NK cells specifically targeted OE19 cells, with CIR-NK cells carrying the BB.DAP10.TLR2 activation domain being particularly effective at an E:T ratio of 1:10. Figure 57 ).

[0380] To promote NK cell exhaustion within three weeks, simulated transduced NK cells and CIR-NK cells were continuously exposed to HT1376 cells (adherent and expressing high levels of HLA-G) through co-culture on day 0. Semi-adherent NK cells were carefully harvested and counted on day 5, and the NK cell population was subsequently co-cultured with fresh HT1376 targets. This process was repeated on days 9 and 14 of the initial co-culture. Figure 58 NK cell proliferation was monitored in each round of continuous co-culture, and it was found that NK cell proliferation was most robust between the second and third rounds of HT1376 exposure, and CIR-NK cells with the BB.DAP10 activation domain generally exhibited the strongest proliferation. Figure 59 There is significant variation among NK cell donors.

[0381] For the fifth round of co-culture, NK cells were co-cultured with the same or different tumor targets expressing different levels of HLA-G. In co-culture with Molm13 target cells, CIR-NK cells containing BB.DAP10 or BB.DAP10.TLR2 were most effective in tumor control. Figure 60 and Figure 61 In this model system, donor variability was significant, and this was somewhat expected. NK cell expansion was most pronounced in the BB.DAP10 combination during co-culture, while expansion was relatively poor in BB.DAP10.TLR2 after 26 days of tumor exposure. Similar findings were observed when CIR-NK cells continuously exposed to tumor were cultured with OE19 solid tumor target cells. Figure 62 and Figure 63 Cytokine production in first-generation CIR-NK cells is lost after the first round of continuous HT1376 exposure, but is maintained in the presence of enhanced activation domains. Figure 64 ).

[0382] Surprisingly, CIR-NK cells with the BB.DAP10.TLR2 combination maintained a strong ability to control Molm13 in the absence of NK cell growth, supporting the hypothesis that this combination of signaling elements promotes particularly effective cytotoxicity and maintains this cytotoxicity despite continuous encounter with tumor targets.

[0383] Example 9. CIR-NK cells with enhanced signal transduction in vivo Anti-tumor effects.

[0384] To determine the activity of CIR-NK cells in in vivo The efficacy and dynamics of this study were investigated using a xenograft model. NSG mice are immunodeficient due to mutations in the non-obese diabetes mellitus (NOD) MHC haplotype, the severe combined immunodeficiency (Scid) locus, and homozygous deletion of the IL-2 receptor γ-chain. This mouse model exhibited severely limited numbers of T, B, and NK cells, and supported the implantation of human cells. 2 × 10⁻⁶ cells were used. 5 Molm13-GFPffluc cells were transplanted into NSG mice via tail vein and allowed to implant and establish for 5 days. Then, 7.5 × 10⁶ cells were used. 6 Mice were challenged with NK cells transduced to express an orange lantern-infused fusion of orange nanolantern and Renilla luciferase (ONL-Rluc), which used a substrate orthogonal to firefly luciferase (ffluc). Tumor and NK cell graft expansion was monitored over 17 days using bioluminescence imaging (BLI) with the orthogonal substrate. Figure 65 NK cells expressing only ONL-Rluc (simulated) failed to [achieve certain results]. in vivo Expansion, and first-generation ILT4 CIR-NK cells also showed poor expansion. CIR-NK cells in in vivo Strong initial expansion was observed, except for ILT2 CIR-NK cells with the BB.DAP10.TLR2 combination. ILT4-BB.DAP10.TLR2 cells expanded well. Both ILT2 and ILT4 CIR-NK cells with the BB.DAP10.TLR2 activation domain effectively controlled Molm13 expansion compared to the control group.

[0385] To examine the dynamics of NK cell migration to the tumor implantation site, mice were euthanized on day 17, and blood, spleen, and bone marrow were collected from selected groups, including two control populations (simulated and first-generation CIR-NK cells) and ILT2 and ILT4 CIR-NK cell groups with BB.DAP10.TLR2. Molm13-GFPffluc cells were widely present in the femoral bone marrow of mice with simulated transduction or first-generation CIR-NK cells, but not in CIR-NK cells with enhanced BB.DAP10.TLR2 signaling. Figure 66 Further examination by flow cytometry showed that the transduced NK cells failed to effectively engraft and migrate to the bone marrow. Figure 67 First-generation (CD3ζ) CIR-NK cells were abundant in the bone marrow but failed to control Molm13. In contrast, ILT2 and ILT4 migrated efficiently to the bone marrow and had sufficient cytotoxicity to control the expansion of Molm13 in the bone marrow.

[0386] Molm13 cells are derived from acute myeloid leukemia (AML), a type of tumor known to proliferate in the bone marrow and disrupt hematopoietic function. The efficacy of enhanced activated CIR-NK cells migrating to the bone marrow and targeting HLA-G-expressing AML suggests that the combination of CIR technology with these activating domains could provide an effective NK cell therapy option for AML patients.

[0387] Although the foregoing invention has been described in detail by way of illustration and examples for the purpose of clarity, it will be apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0388] Therefore, the foregoing has merely illustrated the principles of the invention. It will be understood that those skilled in the art will be able to design various arrangements that, while not expressly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all instances and conditional language described herein are primarily intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to the field, and should be construed as not being limited to these specifically described instances and conditions. Moreover, all statements herein describing the principles, aspects, and embodiments of the invention and their specific examples are intended to cover both structural and functional equivalents. Furthermore, it is intended that such equivalents include both currently known equivalents and future-developed equivalents, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be offered to the public, whether or not such disclosure is expressly stated in the claims.

[0389] Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, 35 USC §112(f) or 35 USC §112(6) is explicitly defined as being invoked only when such limitation in the claim begins with the exact phrase “means for…” or the exact phrase “steps for…”; if such an exact phrase is not used in the limitation of the claim, then 35 USC §112(f) or 35 USC §112(6) is not invoked.

Claims

1. A chimeric receptor protein comprising: (a) Targeting region, which targets HLA-G and includes the D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) A transmembrane (TM) region comprising a transmembrane amino acid sequence; and (c) Intracellular domain (ICD), wherein the ICD contains a signal transduction region capable of transducing signals into the interior of immune effector cells after binding to HLA-G in the target region to induce effector cell function. The signal transduction region includes a co-stimulation region containing the MyD88 polypeptide.

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

27.

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

4. The chimeric receptor protein according to claim 3, wherein the MyD88 polypeptide is fused with a 4-1BB co-stimulatory domain.

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

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

7. The chimeric receptor protein according to claim 1 or claim 2, wherein the signal transduction region comprises the DAP12 signal transduction domain.

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

9. The chimeric receptor protein according to claim 1 or claim 2, wherein the signal transduction region comprises a DAP12 signal transduction domain and a CD3ζ signal transduction domain.

10. An intracellular domain (ICD) polypeptide comprising a signal transduction region capable of transducing signals in immune effector cells to induce effector cell function, wherein the signal transduction region comprises: (i) a CD3ζ signal transduction domain, a DAP10 signal transduction domain, or a DAP12 signal transduction domain, and (ii) a co-stimulatory region comprising a Toll / interleukin-1 receptor / resistance protein (TIR) ​​domain.

11. The ICD peptide according to claim 10, wherein the TIR domain is a TLR2 TIR domain, a TLR3 TIR domain, or an IL18R1 TIR domain.

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

111.

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

113.

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

109.

15. The ICD polypeptide according to any one of claims 10 to 15, wherein the signal transduction region comprises the CD3ζ signal transduction domain.

16. The ICD polypeptide according to any one of claims 10 to 15, wherein the co-stimulatory polypeptide comprises a chimeric receptor protein, the chimeric receptor protein comprising: (a) Targeting region, which targets HLA-G and includes the D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) A transmembrane (TM) region comprising a transmembrane amino acid sequence; and (c) The ICD polypeptide.

17. A chimeric receptor protein comprising: (a) Targeting region, which targets HLA-G and includes the D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) A transmembrane (TM) region comprising a transmembrane amino acid sequence; and (c) Intracellular domain (ICD), wherein the ICD contains a signal transduction region capable of transducing signals into the interior of immune effector cells after binding to HLA-G in the target region to induce effector cell function. The signal conduction region includes either the DAP10 signal conduction structure domain or the DAP12 signal conduction structure domain.

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

4.

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

4.

20. The chimeric receptor according to any one of claims 17 to 19, wherein the signal transduction region further comprises a CD3ζ signal transduction domain.

21. The chimeric receptor according to any one of claims 17 to 19, wherein the signal transduction region does not include the CD3ζ signal transduction domain.

22. The chimeric receptor according to any one of claims 17 to 21, wherein the signal transduction region further comprises a CD40, 4-1BB, or HVEM co-stimulatory domain.

23. The chimeric receptor according to any one of claims 17 to 21, wherein the signal transduction region further comprises a 4-1BB co-stimulatory domain.

24. The chimeric receptor of claim 17, comprising a DAP12 signal transduction domain, wherein the signal transduction domain does not include a CD3ζ signal transduction domain, and wherein the signal transduction domain further includes a 4-1BB co-stimulatory domain.

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

4.

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

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

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

29. The chimeric receptor protein according to any one of claims 1 to 25, wherein the D1-D2 extracellular domain is the ILT2 D1-D2 extracellular domain.

30. The chimeric receptor protein of claim 29, wherein the targeting region comprises the D3-D4 extracellular domain of ILT2.

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

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

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

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

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

36. A nucleic acid comprising a nucleotide sequence encoding a chimeric receptor protein according to any one of claims 1 to 35.

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

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

39. The nucleic acid according to any one of claims 36 to 38, wherein the nucleic acid is an expression vector.

40. The nucleic acid according to claim 39, wherein the expression vector is a retroviral vector, a lentiviral vector, or a plasmid vector.

41. A genetically modified cell that expresses the chimeric receptor protein according to any one of claims 1 to 35.

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

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

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

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

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

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

48. The method according to any one of claims 45 to 47, wherein the individual suffers from cancer.

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

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

51. A method for producing genetically modified cells, the method comprising: The nucleic acid according to any one of claims 36 to 40 is introduced into the cell to produce genetically modified cells.

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

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

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

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

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