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WO2025029597A1 - Compositions et méthodes de récepteur ilt chimérique - Google Patents

Compositions et méthodes de récepteur ilt chimérique Download PDF

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Publication number
WO2025029597A1
WO2025029597A1 PCT/US2024/039649 US2024039649W WO2025029597A1 WO 2025029597 A1 WO2025029597 A1 WO 2025029597A1 US 2024039649 W US2024039649 W US 2024039649W WO 2025029597 A1 WO2025029597 A1 WO 2025029597A1
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cells
domain
signaling
cell
cir
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Inventor
Joseph Henri Bayle
MyLinh Thi DUONG
Jihyun Park
Raphaël OGNAR
Simon Wain-Hobson
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Nkilt Therapeutics Inc
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Nkilt Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/15—Non-antibody based
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • donor lymphocyte infusions, allogeneic T cells and allogeneic Natural Killer (NK) cells can be used to control the outgrowth of leukemias.
  • gene modification can direct the specificity of immune cells including T cells, Natural Killer (NK) cells, ⁇ d T cells, inducible NK-T cells and macrophages toward a target cell population for therapeutic purposes.
  • Chimeric antigen receptor- (CAR-) T cells can be used to redirect T cell specificity to tumor-associated cell surface molecules independent of Human Leukocyte Antigen (HLA) presentation of peptide antigens to a T cell receptor (TCR).
  • HLA Human Leukocyte Antigen
  • CAR proteins can be engineered for expression other immune cells including NK cells, ⁇ /d T cells, iNK-T cells and macrophages.
  • recognition of the target protein by a CAR focuses the cytotoxic potential of these cells that normally use so-called innate receptors to recognize foreign or diseased tissue.
  • Numerous pre-clinical and clinical studies have demonstrated the utility of CAR technology expressed in T cells, NK cells, iNK-T cells and macrophages such that six therapies against B cell-derived tumors have received marketing approval.
  • Variation of CAR technology to alter the binding mechanism for target recognition can improve the affinity and specificity of an engineered cell for target proteins.
  • the antibody:antigen binding mechanism present on a CAR protein can be replaced with a receptor:ligand mechanism.
  • a receptor:ligand mechanism is the engineering of the NKG-2D protein a modified or native protein for expression on immune cells with the purpose of recognizing the multiple ligands (MICA, MIC-B, ULBPs 1-5) for NKG- Atty Docket No.: NKLT-002WO 2D that may be expressed on diseased tissues in varying combinations.
  • MICA multiple ligands
  • NKLT-002WO 2D NKG- Atty Docket No.: NKLT-002WO 2D that may be expressed on diseased tissues in varying combinations.
  • NKLT-002WO 2D NKG- Atty Docket No.: NKLT-002WO 2D that may be expressed on diseased tissues in varying combinations.
  • NKLT-002WO 2D a Chimeric ILT Receptor or CIR that are engineered to target HLA-G expressed on tumors
  • T cells are the most commonly employed cell vehicle for targeted anti-cancer CAR therapy. They are generally employed as autologous, or patient-cell derived, products due to the risk of graft versus host disease with allogeneic T cell products.
  • NK and iNK-T CAR-based products frequently employ a CD28.CD3 ⁇ or 4-1BB.CD3 ⁇ signaling strategy to direct target- specific activation of the the cells frequently with the addition of mechanisms to promote IL-15 signaling. While these constructs do promote target-specific NK and iNK-T cell activation, the CAR mechanisms are designed to mimic T cell activation by APCs, particularly dendritic cells while NK cells and iNK-T cells are activated by mechanisms distinct from T cells.
  • NK cell-based therapies with chimeric proteins may not be activated for optimal performance with traditional CAR-T cell construct designs.
  • Variation of CAR technology to alter the binding mechanism for target recognition can improve the affinity and specificity of an engineered cell for target proteins.
  • the antibody:antigen binding mechanism present on a CAR protein can be replaced with a receptor:ligand mechanism.
  • NKG-2D protein a modified or native protein for expression on immune cells with the purpose of recognizing the multiple ligands (MICA, MIC-B, ULBPs 1-5) for NKG- 2D that may be expressed on diseased tissues in varying combinations.
  • MICA multiple ligands
  • MIC-B multiple ligands
  • ULBPs 1-5 multiple ligands
  • a further example described further herein is the expression of a Chimeric ILT Receptor (CIR) engineered to target HLA-G expressed on tumors.
  • CIR proteins are engineered with a Atty Docket No.: NKLT-002WO binding moiety derived from ILT2 or ILT4 which are the principal receptors for the immunosuppressive HLA-G.
  • HLA-G The binding of the natural receptor domains for HLA-G (ILT2 or ILT4) improves the targeting for HLA-G which is expressed naturally as 7 protein isoforms derived from alternative mRNA splice products.
  • An antibody/scFv CAR approach to targeting HLA would be limited by the loss of epitopes in one or more HLA-G isoforms.
  • CIR proteins are designed to replace the negatively signaling Immunoreceptor Tyrosine-based Inhibitory Motives (ITIM) of ILT2 or ILT4 with domains that activate immune cell signaling.
  • the chimeric protein is a CIR containing binding elements derived from ILT2 or ILT4, each receptors for the HLA-G protein that is commonly expressed on tumors but not on normal tissues.
  • the chimeric protein is a CAR in which a single chain variable fragment (scFv) derived from an antibody gives specific targting for a tumor antigen.
  • the chimeric receptor protein may target a protein expressed at high level in tumor tissue relative to untransformed, normal tissue and generate a cytotoxic or inflammatory response against the tumor.
  • affinity and specificity are not maintained by use of antibody- or VhH-relationship or by binding of randomly generated peptides to a target antigen, but rather by use of ligand:receptor interactions where affinity and specificity are maintained by evolution.
  • the cell expressing a subject chimeric receptor protein is a NK cell.
  • the cell expressing the chimeric protein is an iNK-T cell. In some embodiments, the cell expressing the chimeric protein is an NK-T cell. In further embodiments, the cell expressing the chimeic protein are macrophages, ⁇ /d T cells or ⁇ / ⁇ T cells.
  • the immune cell can be genetically-modified by introduction of a vector (e.g., multi-cistronic vector) by, e.g., transduction with a ⁇ -retrovirus, lentivirus, adenovirus, adeno-associated virus, or DNA transfection of a transposon [0009]
  • a vector e.g., multi-cistronic vector
  • the genetically modified cells express a chimeric receptor that has high affinity for HLA-G, a target protein that can exist on tumor tissues in one or more of seven known forms generated by alternative mRNA splicing and post-translational modifications.
  • HLA-G naturally acts as an agent to suppress Atty Docket No.: NKLT-002WO immune responses through engagement with the negatively signaling Immunoglulin- like Transcript 2 (ILT2) and ILT4 receptors on the surface of immune cells.
  • immune cells such as T cells, NK cells, NK-T, or iNKT cells or macrophages are engineered such that recognition of active forms HLA-G by ILT2 or ILT4 instead generate activating signals.
  • the intracellular signalling elements of ILT2 or ILT4 are excised and replaced with ITAM-containing signalling domains of the CD3 ⁇ chain (for DAP10 or DAP12) that drive immune cell activation and cytotoxicity.
  • CIR Chimeric ILT Receptor
  • nucleic acids encoding them and genetically modified cells, such as immune cells, expressing them
  • ICD intracellular domain
  • a signaling region e.g., CD3 zeta (CD3 ⁇ )
  • DAP10 DAP12
  • costimulatory region e.g., a TIR domain, a MyD88 protein, a domain that stimulates MyD88 signaling, CD28, 4-1BB, OX40, and the like.
  • CD3 ⁇ is naturally expressed in NK cells, ⁇ / ⁇ T cells and in iNK-T cells, but these cell types also express other ITAM-containing signaling adaptors specific for innate receptors that are not naturally expressed by most T cells.
  • the CD3 ⁇ signaling is augmented by fusion with the signaling domain of DAP10 or DAP12 – each signaling adaptor proteins containing one ITAM that are naturally expressed by NK cells. The purpose of this addition is to enhance the cytotoxic potency of CIR or CAR-NK cells.
  • CD3 ⁇ signaling is replaced by fusion with the signaling domain of DAP10 or DAP12. The purpose of this replacement is to optimize the persistence of NK cell anti-tumor potency.
  • an antibody-based targeting region such as an scFv.
  • An antibody-based targeting approach could cause a selection for tumor cells that express HLA-G isoforms that lack the targeted epitope – thus allowing a cancer to evade treatment.
  • a subject ILT2 or ILT4 based chimeric ILT receptor should target many more, and perhaps all, HLA-G isoforms because ILT2 and ILT4 naturally bind those isoforms. This will greatly reduce, and perhaps eliminate, the ability of cancer cells to evade treatment by selection for a particular HLA-G isoform.
  • ILT2 and ILT4 are structurally similar in the extracellular region and are composed of four folded domains (D1, D2, D3 and D4) arranged in a distal-to-proximal fashion relative to the plasma membrane of the cell.
  • HLA-G interacts with the D1 and D2 domains of ILT2 and ILT4 and these D1 and D2 domains can be separated from the rest of the ILT proteins while maintaining interaction with HLA-G.
  • ILT2 or ILT4 D3-D4 is replaced in the chimeric receptor fusion with Atty Docket No.: NKLT-002WO another extracellular domain that serves as a stalk and transmembrane domain to present ILT2 or ILT4 D1-D2 to HLA-G expressing target cells.
  • stalk proteins to present D1-D2 are derived from CD28, CD8 ⁇ , the CH2-CH3 region of IgG4, HER2 membrane proximal, and mGluR2.
  • the D3-D4 domains of ILT2 and ILT4 are simply deleted.
  • a subject chimeric ILT receptor includes D1-D2 of ILT2, but lacks D3-D4 of ILT2.
  • a subject chimeric ILT receptor includes D1-D2 of ILT4, lacks D3- D4 of ILT4.
  • signaling domains can be added to ITAM domain cytotoxicity domains to improve the survival, activation, persistence of cell activation, cytotoxicity and capacity to produce pro-inflammatory cytokines.
  • these elements transduce signals that promote the PI3Kinase/AKT prosurvival pathway and the NF- ⁇ B pathway that promotes activation marker expression and cytokine release frequently in cooperation or synergy with the NF-AT pathway promoted by ITAM- domain signaling.
  • these signaling elements are termed coactivation signals.
  • a canonical signaling element for many CAR constructs is 4-1BB. While 4- 1BB is expressed by activated NK, iNK-T and T cells, other members of the Tumor Necrosis Factor Receptor superfamily are also expressed and the signaling derived from alternate signaling domains are likely to give alternate phenotypes to cell products such as differential cytotoxicity, growth potential, cytokine production and survival.
  • the signaling from 4-1BB to support coactivation can be replaced or supplemented by signaling from MyD88.
  • This factor naturally acts as a signaling node downstream of innate signals from activated Toll-like Receptors (TLR) that are themselves activated with partial specificity by ligands derived from pathogens including RNA, DNA with altered methylation pattern and lipid or protein endotoxins. These innate signals are transduced strongly in responding cells by MyD88 activation.
  • MyD88 is a signaling node downstream of the IL-1 receptor superfamily of cytokines (including IL-18R and IL-33R) that are important activators of NK cells, iNK- T cells and T cells.
  • Incorporation of the signaling Death Domain of MyD88 alone or in combination with TNF-R-derived signaling domains such as 4-1BB, HVEM or CD40 increased the target-specific potency of NK cells against AML. Signaling from MyD88 also increased the growth potential of NK cells intrinsically.
  • MyD88 signaling is naturally recruited to the plasma membrane by IL-1 family cytokine receptors and TLRs through Toll/Interleukin-1 Receptor/Resistance Protein (TIR) domains. Activation of signaling by both IL-18 receptor and TLRs is directed by conformational change in dimeric receptors leading to MyD88 oligomerization.
  • TIR Toll/Interleukin-1 Receptor/Resistance Protein
  • TIR domains can be fused as a coactivation domain with an Atty Docket No.: NKLT-002WO ITAM domain-containing cytotoxicity domain.
  • the purpose of this fusion is to indirectly recruit native MyD88 to a CIR where it is inactive until activation and dimerization of the CIR by dimeric HLA-G.
  • NKLT-002WO ITAM domain-containing cytotoxicity domain When expressed in NK cells, these fusions led to low tonic activity of the CIR in the absence of target cells, but high cytokine production in the presence HLA-G expressing target cells but not target cells lacking HLA-G expression.
  • kits for making genetically modified cells and methods of treatment e.g., administering an immune cell, such as an NK cell, a T cell, or a macrophage, that expresses a subject CIR to an individual.
  • FIG.1 Interaction of an ILT4 D1D2 CIR with HLA-G.
  • A Cartoon depiction of the D1 and D2 domains of ILT4 fused as a chimeric hybrid with a stalk and transmembrane domain derived from a separate protein and further fused with an activating intracellular signaling moiety.
  • Interaction of D1D2 with HLA-G initiates signaling to activate immune cells.
  • Activation domains are contained in the intracellular domain (ICD) derived from signaling proteins that drive immune cell cytotoxicity, proliferation, persistence and release of cytokines.
  • ICD intracellular domain
  • FIG.2 Retroviral expression constructs of CIR forms with alternate intracellular signaling domains. Chart indicated retroviral constructs expressing ILT2 and ILT4 fusion proteins referred to as Chimeric ILT Receptors (CIRs). D1 through D2 indicate the encoding of extracellular domains D1 through D2 derived from native ILT4. STM refers to the stem (S), a linker domain linking extracellular D domains with the transmembrane spanning domain (TM) these are derived from CD8 ⁇ (CD8a).
  • FIG.3 TNF- ⁇ production of CIR-NK cells containing alternative ITAM-containing cytotoxicity domains.
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • FIG.4 IFN- ⁇ production of CIR-NK cells containing alternative ITAM-containing cytotoxicity domains.
  • FIG.5 Coculture of CIR-NK cells containing alternative ITAM-containing cytotoxity domains with KG1 target cells that do not express HLA-G.
  • FIG.6 Coculture of CIR-NK cells containing alternative ITAM-containing cytotoxity domains with Molm13 target cells that express HLA-G1 and HLA-G5.
  • Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3 ⁇ , DAP10, or DAP12 in their intracellular domains at an effector-to-target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells. [0024] FIG.7: TNF- ⁇ production of CIR-NK cells containing multiple ITAM-containing cytotoxicity domains.
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • NK cells expressing only CD3 ⁇ , DAP10 and DAP12 or DAP10 and DAP12 in combination with CD3 ⁇ . Cells were not cultured with target cells.
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • FIG.8 IFN- ⁇ production of CIR-NK cells containing multiple ITAM-containing cytotoxicity domains.
  • IFN- ⁇ Interferon- ⁇
  • FIG.9 Coculture of CIR-NK cells containing multiple ITAM-containing cytotoxicity domains with KG1 target cells that do not express HLA-G.
  • KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR Atty Docket No.: NKLT-002WO (Mock) or CIRs expressing only CD3 ⁇ , DAP10, or DAP12 or as a combination of DAP10 or DAP12 with CD3 ⁇ in their intracellular domains at an effector-to-target ratio of 1:5.
  • Outgrowth of the tumor cells cultured alone is indicated for reference.
  • FIG.10 Coculture of CIR-NK cells containing multiple ITAM-containing cytotoxicity domains with Molm13 target cells that express HLA-G1 and HLA-G5.
  • Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3 ⁇ , DAP10, or DAP12 or as a combination of DAP10 or DAP12 with CD3 ⁇ in their intracellular domains at an effector-to-target ratio of 1:10.
  • Outgrowth of the tumor cells cultured alone is indicated for reference.
  • FIG.11 TNF- ⁇ production of CIR-NK cells containing a co-activation domain and an ITAM-containing cytotoxicity signaling domain.
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • FIG.12 IFN- ⁇ production of CIR-NK cells containing a co-activation domain and an ITAM-containing cytotoxicity signaling domain.
  • Upper Tonic production of Interferon- ⁇ (IFN- ⁇ ) by NK cells expressing only CD3 ⁇ or a chimera of CD3 ⁇ with signaling elements from 4-1BB or HVEM. Cells were not cultured with target cells.
  • Lower IFN- ⁇ production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.
  • FIG.13 Coculture of CIR-NK cells containing a co-activation domain and an ITAM- containing cytotoxicity signaling domain with KG1 target cells that do not express HLA-G.
  • FIG.14 Coculture of CIR-NK cells containing a co-activation domain and an ITAM- containing cytotoxicity signaling domain with Molm13 target cells that express HLA-G1 and HLA-G5.
  • FIG.15 TNF- ⁇ production of CIR-NK cells containing a co-activation domain and multiple ITAM-containing cytotoxicity signaling domains.
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • NK cells expressing only CD3 ⁇ or a chimera of CD3 ⁇ with DAP10 or DAP12 in combination with signaling elements from 4-1BB. Cells were not cultured with target cells.
  • TNF- ⁇ production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.
  • FIG.16 IFN- ⁇ production of CIR-NK cells containing a co-activation domain and multiple ITAM-containing cytotoxicity signaling domains.
  • FIG.17 Coculture of CIR-NK cells containing a co-activation domain and multiple ITAM-containing cytotoxicity signaling domains with KG1 target cells that do not express HLA-G.
  • KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3 ⁇ or a chimera of CD3 ⁇ with DAP10 or DAP12 in combination with signaling elements from 4-1BB in their intracellular domains at an effector-to-target ratio of 1:5. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with KG1 cells.
  • FIG.18 Coculture of CIR-NK cells containing a co-activation domain and multiple ITAM-containing cytotoxicity signaling domains with Molm13 target cells that express HLA-G1 and HLA-G5.
  • Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3 ⁇ or a chimera of CD3 ⁇ with DAP10 or DAP12 in combination with signaling elements from 4-1BB in their intracellular domains at an effector-to-target ratio of 1:10.
  • Outgrowth of the tumor cells cultured alone is indicated for reference.
  • FIG.19 TNF- ⁇ production of CIR-NK cells containing an ITAM-containing cytotoxicity domain and co-activation signaling elements derived from MyD88.
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • FIG.20 IFN- ⁇ production of CIR-NK cells containing an ITAM-containing cytotoxicity domain and co-activation signaling elements derived from MyD88.
  • IFN- ⁇ Interferon- ⁇
  • NK cells expressing only CD3 ⁇ or DAP12 or a CD3 ⁇ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88. Cells were not cultured with target cells.
  • FIG.21 Coculture of CIR-NK cells containing MyD88 as a co-activation domain and ITAM-containing cytotoxicity signaling domains with KG1 target cells that do not express HLA-G.
  • KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3 ⁇ or DAP12 or a CD3 ⁇ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88 in their intracellular domains at an effector-to-target ratio of 1:5.
  • Outgrowth of the tumor cells cultured alone is indicated for reference.
  • FIG.22 Coculture of CIR-NK cells containing MyD88 as a co-activation domain and ITAM-containing cytotoxicity signaling domains with Molm13 target cells that express HLA-G1 and HLA-G5.
  • Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3 ⁇ or DAP12 or a CD3 ⁇ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88 in their intracellular domains at an effector- to-target ratio of 1:10.
  • FIG.23 TNF- ⁇ production of CIR-NK cells containing MyD88 and TNF-R derived co- activation domains and an ITAM-containing cytotoxicity signaling domain.
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • FIG.24 IFN- ⁇ production of CIR-NK cells containing MyD88 and TNF-R derived co- activation domains and an ITAM-containing cytotoxicity signaling domain.
  • NKLT-002WO Tonic production of Interferon- ⁇ (IFN- ⁇ ) by NK cells expressing only CD3 ⁇ or DAP12 or Atty Docket No.: NKLT-002WO a CD3 ⁇ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88 with or without a further coactivation domain derived from 4- 1BB or HVEM. Cells were not cultured with target cells. (Lower) IFN- ⁇ production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.
  • FIG.25 Coculture of CIR-NK cells containing MyD88 and TNF-R derived co-activation domains and an ITAM-containing cytotoxicity signaling domain with KG1 target cells that do not express HLA-G.
  • KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3 ⁇ or DAP12 or a CD3 ⁇ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88 with or without a further coactivation domain derived from 4-1BB or HVEM in their intracellular domains at an effector-to-target ratio of 1:5.
  • FIG.26 Coculture of CIR-NK cells containing MyD88 and TNF-R derived co-activation domains and an ITAM-containing cytotoxicity signaling domain with Molm13 target cells that express HLA-G1 and HLA-G5.
  • Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3 ⁇ or DAP12 or a CD3 ⁇ chimera with DAP12 with or without a co- activation domain derived from the death domain of MyD88 in their intracellular domains at an effector-to-target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells. [0044] FIG.27: TNF- ⁇ production of CIR-NK cells containing an ITAM-containing cytotoxicity signaling domain and a TIR containing co-activation domain.
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • NK cells expressing only CD3 ⁇ or DAP12 or a CD3 ⁇ chimera with DAP12 with or without a co-activation domain derived from MyD88 or the TIR domain of the Interleukin 18 Receptor ⁇ chain (IL-18R ⁇ ), Toll-like Receptor (TLR) 2 or TLR3.
  • IL-18R ⁇ Interleukin 18 Receptor ⁇ chain
  • TLR Toll-like Receptor
  • FIG.28 IFN- ⁇ production of CIR-NK cells containing an ITAM-containing cytotoxicity signaling domain and a TIR containing co-activation domain.
  • IFN- ⁇ Interferon- ⁇
  • NK cells expressing only CD3 ⁇ or DAP12 or a CD3 ⁇ chimera with DAP12 with or without a co-activation domain derived from MyD88 or the TIR domain of the Interleukin 18 Receptor ⁇ chain (IL-18R ⁇ ), Toll-like Receptor (TLR) 2 or TLR3.
  • IL-18R ⁇ Interleukin 18 Receptor ⁇ chain
  • TLR Toll-like Receptor
  • FIG.29 Coculture of CIR-NK cells containing an ITAM-containing cytotoxicity signaling domain and a TIR containing co-activation domain with KG1 target cells that do not express HLA-G.
  • KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3 ⁇ or DAP12 or a CD3 ⁇ chimera with DAP12 with or without a co-activation domain derived from MyD88 or the TIR domain of the Interleukin 18 Receptor ⁇ chain (IL-18R ⁇ ), Toll- like Receptor (TLR) 2 or TLR3 in their intracellular domains at an effector-to-target ratio of 1:5. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with KG1 cells.
  • FIG.30 Coculture of CIR-NK cells containing an ITAM-containing cytotoxicity signaling domain and a TIR containing co-activation domain with Molm13 target cells that express HLA-G1 and HLA-G5.
  • Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3 ⁇ or DAP12 or a CD3 ⁇ chimera with DAP12 with or without a co- activation domain derived from MyD88 or the TIR domain of the Interleukin 18 Receptor ⁇ chain (IL-18R ⁇ ), Toll-like Receptor (TLR) 2 or TLR3 in their intracellular domains at an effector-to-target ratio of 1:10.
  • IL-18R ⁇ Interleukin 18 Receptor ⁇ chain
  • TLR Toll-like Receptor
  • FIG.31 Retroviral expression constructs of CIR forms with alternate intracellular signaling domains. Chart indicated retroviral constructs expressing ILT4 (left) or ILT2 (right) D1 and D2 extracellular domains together with a CD8 ⁇ stalk and transmembrane domain. Formats for the intracellular signaling domains are as indicated oriented from N-terminal (membrane proximal) to C-terminal (distal). [0049] FIG.32: Viability of CIR-NK cells following transduction with CIR constructs with alternate signaling domains.
  • FIG.33 Expansion of CIR-NK cells in culture following transduction with CIR constructs with alternate signaling domains. Transduced CIR-NK cells were counted at day 5 post-activation and again at day 8 (upper) and day 14 to determine the relative proliferative ability of NK cells during the expansion period.
  • FIG.34 Expression level of transduced CIR proteins containing alternate signaling domains during expansion of NK cells.
  • FIG.35 IFN- ⁇ production of CIR-NK cells containing alternate signaling domains. (Upper) Tonic production of Interferon- ⁇ (IFN- ⁇ ) by 200,000 NK cells expressing only CD3 ⁇ or a chimera of CD3 ⁇ with DAP10 or DAP12 in combination with signaling elements from 4-1BB. Cells were not cultured with target cells.
  • IFN- ⁇ Interferon- ⁇
  • FIG.36 IFN- ⁇ production of CIR-NK cells containing alternate signaling domains when cocultured with target cells derived from an Acute Myeloid Leukemia (AML).
  • AML Acute Myeloid Leukemia
  • Upper NK cells transduced with RFP alone (RFP) or CIR-NK cells were co-cultured with Kasumi1 target cells at an E:T ratio of 1:10 and IFN- ⁇ levels were measured 24 hours later by ELISA (upper).
  • FIG.37 IFN- ⁇ production of CIR-NK cells containing alternate signaling domains when cocultured with target cells derived from a solid tumor.
  • NK cells transduced with RFP alone (RFP) or CIR-NK cells were co-cultured with HT1376 bladder carcinoma target cells at an E:T ratio of 1:10 and IFN- ⁇ levels were measured 24 hours later by ELISA (upper).
  • ELISA ELISA
  • FIG.38 Short-term cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells that express HLA-G1 and HLA-G5.
  • Molm13 cells were cultured for two days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference.
  • Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells.
  • FIG.39 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells that express HLA-G1 and HLA-G5.
  • FIG.40 Visual representation of 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with MOLM13 target cells that express HLA-G1 and HLA-G5.
  • FIG.41 Short-term cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Kasumi1 target cells that express HLA-G1 and HLA-G5.
  • FIG.42 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Kasumi1 target cells that express HLA-G1 and HLA-G5.
  • FIG.43 Expression of HLA-G in cell lines derived from solid tumors. HLA-G expression was determined in dissociated HCT-116 colon carcinoma cells and in HT- 1376 bladder carcinoma cells by flow cytometry with the MEMG/9 antibody. EGFR expression was determined as a separate control for integrity of the population.
  • FIG.44 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with HT-1376-GFPffluc target cells that express HLA-G1, HLA-G2 and HLA-G5.
  • (Upper) cells labelled with GFP were cultured for seven days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference.
  • RFP CIR-activated protein
  • Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with HT-1376 cells.
  • FIG.45 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with HCT-116-GFPffluc target cells that do not express HLA-G.
  • HCT-116 cells labelled with GFP were cultured for seven days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference.
  • RFP CIR-activated protein
  • Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with HCT-116 cells.
  • FIG.46 Expression of HLA-G in cell SU-8686 cells derived from a pancreatic ductal adenocarcinoma. HLA-G expression in was determined in dissociated SU8686 cells by flow cytometry with the MEMG/9 antibody. Atty Docket No.: NKLT-002WO [0064] FIG.47: 7-day cytotoxicity of CIR-NK cells containing alternate signalling domains cocultured with SU8686-GFPffluc target cells that express HLA-G1 and HLA-G2.
  • NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signalling domains at an effector to target ratio of 1:10.1G indicates a first-generation ILT2 CIR containing only a CD3 ⁇ signalling domain. Outgrowth of the tumor cells cultured alone is indicated for reference.
  • RFP CIR
  • Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with SU8686 cells.
  • Cocultures were harvested, and cells were quantitated by flow cytometry gating for CD56 expression (NK cells) and GFP expression (target cells).
  • FIG.48 Visual representation of 7-day cytotoxicity of CIR-NK cells containing alternate signalling domains cocultured with SU8686 target cells that express HLA-G1 and HLA-G2.
  • SU8686-GFP cells were cultured for 7 days with RFP-labelled NK cells (E:T of 1:5) expressing no CIR (RFP), or CIRs expressing the indicated signalling domains.
  • Images representative of one donor’s NK cells were taken in the Incucyte. Relative expansion of tumor is indicated by green fluorescence and NK cell expansion indicated by red fluorescence.
  • FIG.49 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells that express HLA-G1 and HLA-G5 following long term expansion.
  • (Upper) Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:20.
  • NK cells in the upper panel were expanded for the standard 14 days prior to coculture. Outgrowth of the tumor cells cultured alone is indicated for reference.
  • Cells in the lower panel were cultured for 26 days to examine the persistence of enhanced cytotoxicity associated with CIR signaling.
  • FIG.50 Persistence of cytotoxicity of CIR-NK cells containing alternate signaling domains repeatedly cocultured with Molm13 target cells over 9 days.
  • Molm13 cells were cultured with NK cells transduced to express RFP alone (RFP) or the indicated CIR-NK cells at an E:T ratio of 1:10.
  • Co-cultures were allowed to expand for 9 days (1X, top left) or were reseeded with an identical amount of Molm13 target cells at day 2 only (2X, Top right), at day 2 and day 5 (3X, bottom left), and days 2, 5 and 7 (4X, bottom right).
  • FIG.51 Persistence of cytotoxicity of CIR-NK cells containing alternate signaling domains repeatedly cocultured with Molm13 target cells over 9 days.
  • Molm13 cells were cultured with NK cells transduced to express RFP alone (RFP) or the indicated CIR-NK cells (and RFP) at an E:T ratio of 1:10. Co-cultures were allowed to expand for 9 days (1X, top left) or were reseeded with an identical amount of Molm13 target Atty Docket No.: NKLT-002WO cells at day 2 only (2X, Top right), at day 2 and day 5 (3X, bottom left), and days 2, 5 and 7 (4X, bottom right). [0069] FIG.52: Expansion of CIR-NK cells containing alternate signaling domains repeatedly cocultured with Molm13 target cells over 9 days.
  • Molm13 cells were cultured with NK cells transduced to express RFP alone (RFP) or the indicated CIR-NK cells (and RFP) at an E:T ratio of 1:10. Co-cultures were allowed to expand for 9 days (1X, top left) or were reseeded with an identical amount of Molm13 target cells at day 2 only (2X, Top right), at day 2 and day 5 (3X, bottom left), and days 2, 5 and 7 (4X, bottom right). [0070] FIG.53: Stress test of initial cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells.
  • Molm13-GFPffluc were cultured for two days with NK cells derived from three donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at increasing effector to target ratio from 1:40 to 2:1. Outgrowth of tumor was measured at 2 days by quantitation of GFP fluorescence in the Incucyte. Outgrowth of the tumor cells cultured alone is indicated for reference. CIR-NK cells with an ILT4-derived extracellular domain are indicated on the left and ILT2-derived CIR-NK cells are represented on the right. [0071] FIG.54: Stress test of 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells.
  • Molm13-GFPffluc were cultured for seven days with NK cells derived from three donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at increasing effector to target ratio from 1:40 to 2:1. Outgrowth of tumor was measured at 2 days by quantitation of GFP fluorescence in the Incucyte. Outgrowth of the tumor cells cultured alone is indicated for reference. CIR-NK cells with an ILT4-derived extracellular domain are indicated on the left and ILT2-derived CIR-NK cells are represented on the right. [0072] FIG.55: Stress test of initial expansion of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells.
  • Molm13-GFPffluc were cultured for two days with NK cells derived from three donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at increasing effector to target ratio from 1:40 to 2:1. Outgrowth of NK cells was measured at 2 days by quantitation of RFP fluorescence in the Incucyte. CIR-NK cells with an ILT4-derived extracellular domain are indicated on the left and ILT2-derived CIR-NK cells are represented on the right. [0073] FIG.56: Stress test of 7-day expansion of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells.
  • Molm13-GFPffluc were cultured for seven days with NK cells derived from three donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at increasing effector to target ratio from 1:40 to 2:1. Outgrowth of NK cells was measured at 2 days by quantitation of RFP fluorescence in the Incucyte. CIR-NK cells with an ILT4-derived extracellular domain are indicated on the left and ILT2-derived CIR-NK cells are represented on the right.
  • FIG.57 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with OE19-GFPffluc esophageal carcinoma target cells that express low levels of HLA-G1 and HLA-G2.
  • OE19 cells labelled with GFP were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or ILT4 CIRs expressing the indicated signaling domains at an effector to target ratio of 1:5 (left graph) or 1:10 (right graph).1G indicates a first-generation ILT4 CIR containing only a CD3 ⁇ signaling domain.
  • FIG.58 Timeline of co-culture of CIR-NK cells containing alternate signaling domains with target cell cells following constant exposure to solid tumor target cells for 19 days.
  • NK cells or CIR-NK cells labelled with RFP and derived from 2 donors were cocultured with HT-1376 target cells at an E:T ratio of 2:1.
  • E:T ratio of 2:1.
  • NK cells were harvested, counted and reseeded with fresh tumor target cells. This process was repeated two times and NK cells were cocultured with solid or leukemic target cells at the indicated E:T ratios for 7 days.
  • FIG.59 Expansion of CIR-NK cells containing alternate signaling domains during constant exposure to HT-1376 target cells for 19 days.
  • NK cells or CIR-NK cells labelled with RFP and derived from 3 donors were cocultured with HT-1376 target cells at an E:T ratio of 2:1. After 5 days of coculture, NK cells were harvested, counted and reseeded with fresh tumor target cells. This process was repeated two times. Cell counts relative to the previous seeding are plotted to indicate the relative expansion of NK cells with each exposure to tumor target.
  • FIG.60 Cytotoxicity and expansion of CIR-NK cells containing alternate signaling domains against Molm13 cells following constant exposure to HT-1376 target cells for 19 days.
  • NK cells or CIR-NK cells labelled with RFP and derived from 3 donors were cocultured with HT-1376 target cells at an E:T ratio of 2:1. After 5 days of coculture, NK cells were harvested, counted and reseeded with fresh HT-1376 cells. This process was repeated two times. After 19 days and four successive exposures to HLA-G expressing target cells, NK cells were cocultured with Molm13-GFPffluc target cells for 7 days at an E:T ratio of 1:10 (left) or 1:20 (right). Tumor cell expansion (upper graphs) and NK cell expansion (lower graphs) was measured by GFP and RFP fluorescence in the Incucyte.
  • FIG.61 Visual representation of 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells following 19 days of constant exposure to HLA-G expressing target cells.
  • Molm13-GFP cells were cultured for 7 days with RFP-labelled NK cells (E:T of 1:10) expressing no CIR (Mock), or CIRs expressing the indicated signaling domains. Images representative of one donor’s NK cells were taken in the Incucyte. Relative expansion of tumor is indicated by green Atty Docket No.: NKLT-002WO fluorescence and NK cell expansion of cells derived from donors 142 and 226 indicated by red fluorescence.
  • FIG.62 Cytotoxicity and expansion of CIR-NK cells containing alternate signaling domains against OE19 cells following constant exposure to HT-1376 target cells for 19 days.
  • NK cells or CIR-NK cells labelled with RFP and derived from 3 donors were cocultured with HT-1376 target cells at an E:T ratio of 2:1.
  • NK cells were harvested, counted and reseeded with fresh HT-1376 cells. This process was repeated two times.
  • NK cells were cocultured with OE19-GFPffluc target cells for 7 days at an E:T ratio of 1:5 (left) or 1:10 (right).
  • FIG.63 Visual representation of 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with OE19 target cells following 19 days of constant exposure to HLA-G expressing target cells.
  • OE19-GFP cells were cultured for 7 days with RFP-labelled NK cells (E:T of 1:5) expressing no CIR (Mock), or CIRs expressing the indicated signaling domains. Images representative of one donor’s NK cells were taken in the Incucyte.
  • FIG.64 Interferon- ⁇ production from CIR-NK cells containing alternate signaling domains during constant exposure to HT-1376 target cells for 19 days. NK cells or CIR-NK cells labelled with RFP and derived from 3 donors were cocultured with HT- 1376 target cells at an E:T ratio of 2:1. After 5 days of coculture, NK cells were harvested, counted and reseeded with fresh tumor target cells. This process was repeated three times. Interferon- ⁇ (IFN- ⁇ ) levels were determined at 48 hours following each successive seeding with the total levels divided by the number of NK cells at each seeding.
  • IFN- ⁇ Interferon- ⁇
  • FIG.65 Control of Molm13-GFPffluc expansion in NSG mice with CIR-NK cells expressing alternative signaling domains.
  • Molm13-GFPffluc cells were implanted into immunodeficient NSG mice and expanded for 5 days.
  • NK cells 7.5E6 cells/mouse
  • ONLRluc for bioluminescence (BLI) detection
  • CIR vectors or Mock transduced
  • Left BLI for NK cells was measured 14 days post NK cell injection with coelenterazine as substrate.
  • FIG.66 Control of Molm13-GFPffluc expansion in the bone marrow of NSG mice with CIR-NK cells expressing alternative signaling domains. Bone marrow was harvested Atty Docket No.: NKLT-002WO from select groups of mice outlined in FIG.65 at 17 days post NK cell transplantation. Human CD45 + cells were gated from total bone marrow cells and GFP + Molm13 tumor cells quantitated by flow cytometry.
  • FIG.67 Trafficking of CIR-NK cells to bone marrow of NSG mice and control of Molm13-GFPffluc expansion with CIR-NK cells expressing alternative signaling domains. Bone marrow was harvested from select mice at 17 days post NK cell transplantation. Human CD45 + cells were gated from total bone marrow cells and GFP + Molm13 tumor cells and human NK cells (orangenanolantern (ONL)) quantitated by flow cytometry.
  • ONL rangenanolantern
  • a weight of “about 100 grams” can include weights between 90 grams and 110 grams.
  • a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) Atty Docket No.: NKLT-002WO the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%).
  • CIRs chimeric ILT receptors
  • ICD intracellular domain
  • the signaling region includes a costimulatory region with a MyD88 polypeptide.
  • the signaling region includes a CD3 ⁇ signaling domain, a DAP10 signaling domain, a DAP12 signaling domain, or any combination thereof.
  • the signaling region includes a CD3 ⁇ signaling domain.
  • the signaling region includes a DAP10 signaling domain.
  • the signaling region includes a DAP12 signaling domain.
  • the costimulatory region also includes a 4-1BB costimulatory domain.
  • the signaling region includes a DAP10 signaling domain or a DAP12 signaling domain.
  • the signaling region also includes a CD3 ⁇ signaling domain. In other such cases, the signaling region does not include a CD3 ⁇ signaling domain. In some cases, the signaling region further includes a CD40, 4-1BB, or HVEM costimulatory domain.
  • ICD intracellular domain
  • the signaling region comprises (i) a CD3 ⁇ signaling domain, a DAP10 signaling domain, or a DAP12 signaling domain, and (ii) a costimulatory region that comprises a Toll/Interleukin-1 Receptor/Resistance Protein (TIR) domain.
  • TIR Toll/Interleukin-1 Receptor/Resistance Protein
  • the TIR domain is a TLR2 TIR domain, TLR3 TIR domain, or a IL18R1 TIR domain.
  • the signaling region includes the CD3 ⁇ signaling domain.
  • HLA-G The non-classical MHC-I protein HLA-G is a major factor in the maintenance of immunological tolerance to maternal-fetal development [Kovats et al. Science (1990) 248:220, Ferreira et al, (2017) I 38:272]. Its normal expression is highest in the Atty Docket No.: NKLT-002WO extravillous trophoblasts of the fetal placenta where it functions to block activation and infiltration of maternal immune cells of most types, but particularly T cells and NK cells from the fetus which has a haploidentical MHC haplotype.
  • HLA-G is expressed in a diverse set of solid tumor types and leukemias [Reviewed in Lin and Yan (2016) 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.
  • HLA-G Its function in cancer is to directly evade immune attack, but HLA-G is also expressed in tolerogenic DC-10 dendritic cells that inhibit lymphocytic responses by suppressive cytokine secretion and activate Treg cells and myeloid derived suppressor cells (MDSC) to create an immunosuppressive tumor microenvironment [Reviewed in Carosella et al, Blood (2011) 118:6499, Gao et al, (2016) BBA 1869:278]. HLA-G can thereby be considered an important checkpoint mediator of tumor promotion. [00101] The HLA-G gene produces multiple mRNA transcripts that encode at least seven different protein products [Ishitani et al (1992) Proc. Natl. Acad.
  • HLA-G1 through G5 are SEQ ID NOs: 9, 15, 17, 19, 21 (protein) and encoding nucleotides are SEQ ID NOs: 8, 14, 16, 18, 20, respectively).
  • HLA-G1 contains the ⁇ 1- ⁇ 2- ⁇ 3 domain structure with an alpha helical peptide binding cleft and a transmembrane domain and short intracellular carboxy terminal domain (see Figure 1). This domain structure is canconical to MHC-I products. Other expressed splice products delete entire domains, for example HLA-G2 encodes ⁇ 1, ⁇ 3 and the transmembrane domain, deleting ⁇ 2.
  • HLA-G4 deletes the ⁇ 3 domain and HLA-G3 encodes only the ⁇ 1 domain.
  • HLA-G When expressed in M8 cells as transgenes, each of these forms of HLA-G was reported to exhibit immunosuppressive activity toward NK cell attack [Riteau et al (2001) J. Immunology 166:5018].
  • Secreted forms generated by alternative splicing include HLA-G5 and HLA-G6 which maintains the domain structure of HLA-G1 and G2 respectively but do not use the splice donor site for intron 4 and instead encode a short secreted peptide derived from intron 4.
  • HLA-G7 uses a three amino acid peptide derived from intron 2.
  • HLA-G exists in monomeric and oligomeric forms. Oligomers are chiefly dimers directed by disulphide linkages at Cys42 (in ⁇ 1) or Cys 147 (in ⁇ 2) [Gonen-Gross et al, (2005) J. Imm.175:4866, Boyson et al, Proc. Natl. Acad. Sci 99:16180].
  • NKLT-002WO exists that the dimeric form of HLA-G is the principal immunosuppressive form and that it adopts a kinked quaternary structure relative to that of native monomers [Shiroishi et al (2006) Proc Natl Acad Sci 103:10095, Clements et al (2005) Proc Natl Acad Sci 102:3360, Wang et al (2020) Cel and Mol. Imm.17:966]. [00103] The different HLA-G forms together create a challenge for CAR-based therapy that relies on binding of an antibody-derived scFv or VhH domain as the targeting agent.
  • ILT2 and ILT4 HLA-G directs its immunosuppressive activity as a membrane bound ligand for inhibitory receptors Immunoglobulin-like transcript 2 (ILT2) and ILT4 (also called LIRB1 and LIRB2 or CD85j and CD85d respectively) on target immune cells [Colonna et al (1998) J. Immunology 160:3096 reviewed in Gao et al (2016) BBA 1869:278].
  • ILT2 and ILT4 also called LIRB1 and LIRB2 or CD85j and CD85d respectively
  • ILT2 (see Seq ID NO: 29)(an encoding nucleotide sequence is SEQ ID NO: 28) is expressed in a subset of Natural Killer cells, iNKT cells, T cells, B cells and dendritic cells.
  • ILT4 (see Seq ID NO: 53) (an encoding nucleotide sequence is SEQ ID NO: 52) has a more broad expression pattern primarily in myeloid and stem cells including macrophage, myeloid derived suppressor cells (a population of less differentiated cells on the monocytic lineage), granulocytes including neutrophils, monocytes, hematopoietic stem cells and some neurons.
  • ILT2 has an extracellular domain structure consisting of four domains that have sequence and structural homology to Immunoglobulin domains (Ig domains) arranged in a column from membrane-distal D1 through to most membrane-proximal D4 followed by a transmembrane domain and an intracellular signaling domain that includes four iterated Immunoreceptor Tyrosine-based Inhibitory Motives (ITIMs).
  • ITIMs Immunoglobulin domains
  • ILT4 has a similar extracellular and transmembrane architecture but only three ITIMs in its intracellular domain.
  • the D1 and D2 domains of ILT2 (see, e.g., Seq ID NO: 37 and 71) (SEQ ID NO: 36 provides a nucleotide sequence encoding SEQ ID NO: 37) and ILT4 (see, e.g., SEQ ID NO: 57 and 75)(SEQ ID NO: 56 provides a nucleotide sequence encoding SEQ ID Atty Docket No.: NKLT-002WO NO: 57) govern interaction with HLA-G and can be separated from the D3 and D4 domains [Donadi et al (2011) Cell. Mol. Life Sci.68:369, Morales (2007) 122:179, HoWanYin et al (2012) Cell.
  • ILT2 and ILT4 do not bind with the ⁇ 1- ⁇ 2 domains that contain the peptide binding cleft, but instead interact with the membrane proximal ⁇ 3 domain and with ⁇ 2-microglobulin. ILT2 makes extensive contact with ⁇ 2-M and relatively few contacts with ⁇ 3 of HLA-G and requires ⁇ 2-M association with HLA-G to maintain even a low affinity interaction.
  • ILT4 makes extensive contact with ⁇ 3 and can maintain interaction with all known actively immunosuppressive forms of HLA-G, possibly excluding HLA-G3/G7 that contains only the ⁇ 1 domain.
  • ILT2 and ILT4 can interact with other MHC-I and MHCI-like proteins, notably HLA-A2, HLA-B, HLA-C, and HLA-F, CD1d and UL18.
  • MHC-I and MHCI-like proteins notably HLA-A2, HLA-B, HLA-C, and HLA-F, CD1d and UL18.
  • UL18 a decoy MHC-I from cytomegalovirus [Wilcox et al (2002) BMC Struct. Biol 2:6], these are low affinity interactions with dissociation constants (K D ) between 2 ⁇ M and 40 ⁇ M.
  • HLA-G Relevance for immunosuppressive signalling has not been demonstrated with affinities this weak.
  • interaction between ILT2 and ILT4 with monomeric forms of HLA-G are weak, in the ⁇ M range.
  • dimeric HLA-G forms display high affinity (2-4 nM) interaction with ILT2 and ILT4 possibly due to display of further contact sites or, alternatively, due to an avidity effect reducing the off rate for ILT dissociation [Shiroishi et al, (2006) J. Biol. Chem 281: 10440, Gao et al (2020) Cell Mol Imm 17:966].
  • the dimeric forms of HLA-G are therefore most likely to be bioactive [Gonen-Gross et al, (2005) J.
  • ILT4 is a receptor for non-MHC ligands including Angiopoietin-like proteins 2 and 5 [Zheng et al (2012) Nature 485:656, Deng et al (2014) Blood 124:924] Regulation by soluble ANGPTL2 and ANGPTL5 is thought to provide a protective signal from bone marrow stroma for self-renewal and survival of ILT4-expressing hematopoietic stem cells.
  • Interaction between ILT4 and ANGLPs is directed by the D1 domain in concert with the D4 domain of ILT4 and specific residues in either the D1 or D4 are essential to maintain high affinity interaction.
  • mutation of tyrosine 96 to alanine reduced ANGPTL2/5 binding but did not reduce HLA-G1 interaction with full-length ILT4 [Deng et al (2014) Blood 124:924].
  • ILT4 interacts with moderate affinity to inhibitory Nogo receptor ligands derived from myelin [Atwal et al (2008) Science 322:967, Matsushita et al, (2014) J. Biol. Chem Atty Docket No.: NKLT-002WO 286:25739].
  • the mouse ortholog of ILT proteins, PIRB is also found in subsets of neurons and may regulate axonal outgrowth by interaction with myelin-based MAG, Nogo and OMgp [US patent 20100047232] and Sema4a [Lu et al. (2016) Nat. Comm. 7:742]. High affinity interactions were characterized in the mouse ortholog for ILT4, PIRB and did not map to the HLA-G binding D1 and D2 domains, but rather to the membrane proximal domains of PIRB [Matsushita et al, (2014) J. Biol. Chem 286:25739]. 3.
  • Chimeric receptors targeted by ILT proteins i.e., a “Chimeirc ILT Receptor” or “CIR”
  • Redirection of the cytotoxic specificity of T or NK cells can be controlled by engagement of an antigen-scFv (or TCR) interaction, but can also be controlled by a receptor-ligand pairing such that the receptor for the targeted ligand can be formed into a chimeric protein that can maintain a high affinity interaction with the cell while capably maintaining signal transduction to activate the immune cell (we use T cells and NK cells as an example hereafter).
  • the receptor or portions of the receptor used to engage the target should be specific for the target, thereby preventing off-tumor targeting.
  • target protein or ligand should be present on target tissue (for example, a tumor) relative to normal tissues.
  • target tissue for example, a tumor
  • cells that express the CIR on their surface upon contact and ligation with HLA-G, signal through the signaling domain (e.g., CD3 zeta (CD3 ⁇ ) chain), inducing cellular activation.
  • CD3 zeta (CD3 ⁇ ) chain e.g., CD3 zeta (CD3 ⁇ ) chain
  • the extracellular domains (D1-D4) of ILT2 can be engineered to target HLA-G expressing tumor cells and generate activating signal transduction in immune cells expressing a chimeric version of ILT2 that replaces the naturally inhibitory ITIM-containing ILT2 intracellular domain (ICD) with signaling components that drive activating signals.
  • the extracellular domains (D1-D4) of ILT4 can be engineered to generate activating signals in immune cells by replacement of the ILT4 ICD with activation signaling moieties.
  • the targeting region of a Atty Docket No.: NKLT-002WO subject chimeric ILT receptor (CIR) includes an ILT2 or ILT4 D1-D4 domain (which therefore targets HLA-G).
  • ILT4 maintains more contacts with ⁇ 3 on the HLA heavy chain and can interact with free heavy chain forms of HLA-G while ILT2 D1/D2 requires contact with ⁇ 2-M and ⁇ 3 to maintain interaction with HLA-G.
  • ILT2 and ILT4 maintain high affinity (KD low nM) interactions with dimeric HLA-G and low affinity (K D is ⁇ M for interaction with other MHC-I proteins including monomeric HLA-G and CD1d)
  • K D is ⁇ M for interaction with other MHC-I proteins including monomeric HLA-G and CD1d
  • HLA-G exists in several different isoforms.
  • CARs which include an antibody-based targeting region (such as an scFv), would only be able to target HLA-G isoforms that include the epitope targeted by the antigen binding region (e.g., scFv). This could place a selection for tumor cells expressing HLA-G isoforms that lack the targeted epitope – thus allowing the cancer to evade treatment.
  • a subject ILT2 or ILT4 based chimeric receptor protein (which targets HLA-G) should target many more, and perhaps all, HLA-G isoforms because ILT2 and ILT4 naturally bind those isoforms.
  • Construction of an ILT2 D1/D2 CIR and an ILT4 D1/D2 CIR [00115] The D1 and D2 domains are sufficient to direct binding of ILT2 and ILT4 to HLA-G while the D3 and D4 domains are likely to serve as a scaffold to display D1 and D2 to HLA-G [Shiroishi et al, (2006) J. Biol. Chem Apr 14; 281(15):10439-47].
  • the D3 and D4 domains can be deleted from an ILT2 CIR or and ILT4 CIR and maintain functional interactions through the D1-D2 domains from ILT2 (see, e.g., Seq ID NO: 71) or ILT4 (see, e.g., Seq ID NO: 75) with HLA-G forms (see Figure 4A).
  • the targeting region of a subject ILT2 or ILT4 chimeric receptor will include the D1-D2 domains of ILT2 or ILT4 (see, e.g., SEQ ID NO: 71 for D1-D2 of ILT2 and SEQ ID NO: 75 for D1-D2 of ILT4), and in some such cases the targeting region will not include (i.e., will lack) the D3-D4 domains.
  • the targeting region (the region including the D1-D2 domains) of a subject ILT2 chimeric receptor includes an amino acid sequence that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the ILT2 sequence set forth in any one of SEQ ID Nos: 37, 70, 71, and 72, which sequences are as follows: Atty Docket No.: NKLT-002WO MHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKG QFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGN VTLQCDSQVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRC YAYDSNSPYE
  • the targeting region includes an amino acid sequence that has 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth in any one of SEQ ID Nos: 37, 70, 71, and 72. In some cases, the targeting region includes the amino acid sequence set forth in any one of SEQ ID Nos: 37, 70, 71, and 72.
  • the targeting region (the region including the D1-D2 domains) of an ILT2 chimeric receptor includes an amino acid sequence that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 37.
  • the targeting region includes an amino acid sequence that has 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 37.
  • the targeting region includes an amino acid sequence that has 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 37. In some cases, the targeting region includes the amino acid sequence set forth as SEQ ID No: 37.
  • the targeting region (the region including the D1-D2 domains) of a subject ILT4 chimeric receptor includes an amino acid sequence that has 80% or Atty Docket No.: NKLT-002WO more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the ILT4 sequence set forth in any one of SEQ ID Nos: 57, 74, and 75, which sequences are as follows: MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRL YREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTG AYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSS RAIFSVGPVSPNRR
  • the targeting region includes an amino acid sequence that has 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth in any one of SEQ ID Nos: 57, 74, and 75. In some cases, the targeting region includes the amino acid sequence set forth in any one of SEQ ID Nos: 57, 74, and 75.
  • the targeting region (the region including the D1-D2 domains) of an ILT4 chimeric receptor includes an amino acid sequence that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 57.
  • the targeting region includes an amino acid sequence that has 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 57.
  • the targeting region includes an amino acid sequence that has 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 57. In some cases, the targeting region includes the amino acid sequence set forth as SEQ ID No: 57.
  • the subject ILT2 or ILT4 chimeric receptor lacks a D3 and D4 domain (i.e., lacks a region Atty Docket No.: NKLT-002WO corresponding to the D3-D4 domains of ILT2 (SEQ ID NO: 73) or ILT4 (SEQ ID NO: 76), respectively).
  • the region with the D3-D4 domains is: PLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLR STYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLT (SEQ ID NO: 73).
  • the region with the D3-D4 domains is: QPGPVMAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPV SRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLC QSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSD PYLLSHPSEPLEL (SEQ ID NO: 76).
  • a subject CIR lacks an amino acid sequence having 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) sequence identity with the sequence set forth as SEQ ID NO: 73. In some cases, a subject CIR lacks an amino acid sequence having 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) sequence identity with the sequence set forth as SEQ ID NO: 76.
  • a subject CIR lacks an amino acid sequence having 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) sequence identity with the sequence set forth in any one of SEQ ID NOs: 73 and 76.
  • a linker may be fused as a chimera with D1-D2 domains from ILT2 or ILT4 to the plasma membrane and serves as a stalk that replaces D3 and D4 domains.
  • deletion of D3-D4 may prevent interaction of a CIR with proteins other than HLA-G that interact with native ILT2 or ILT4 through the D3 or D4 domains, for examples the interactions of ANGPTL2 and ANGPTL5 with ILT4 D4 and the interaction of nogo, Omgp and MAG with ILT4 D3-D4. Prevention of such interactions may reduce potentially toxic mistargeting of a CIR-expressing cell with non-tumor tissue such as bone marrow stroma, myelin and endothelium.
  • the targeting region (the region including the D1-D2 domains) of a subject ILT2 chimeric receptor (an ILT2 CIR) includes domains D1-D4, and as such in some cases includes an amino acid sequence that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the ILT2 sequence set forth in SEQ ID NO: 94).
  • the targeting region (the region including the D1-D2 domains) of a subject ILT4 chimeric receptor (an ILT4 CIR) includes domains D1-D4, and as such in some cases includes an amino acid sequence that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the ILT2 sequence set forth in SEQ ID NO: 96).
  • NKLT-002WO Stalk domains replacement of D3-D4 can be made with any protein or portion of a protein that properly displays the ILT2 or ILT4 D1-D2 binder in a context for HLA- G expressed on a separate cell.
  • a short polypeptide linker may form the linkage between the transmembrane domain and the intracellular domain of the chimeric ILT receptor.
  • the chimeric ILT receptors may further comprise a stalk, that is, an extracellular region of amino acids between the extracellular domain and the transmembrane domain.
  • the stalk domain is to extend the D1/D2 domains away from the plasma membrane and toward the target protein HLA-G.
  • the stalk may be a sequence of amino acids naturally associated with a selected transmembrane domain.
  • the CIR comprises a CD8 ⁇ transmembrane domain
  • the CIR comprises a CD8 ⁇ transmembrane domain together with additional amino acids on the extracellular portion of the transmembrane domain.
  • a CIR comprises a CD8 ⁇ transmembrane domain and a CD8 ⁇ stalk (see SEQ ID NO: 43; an encoding nucleotide sequence is SEQ ID NO: 42).
  • a CD8 ⁇ transmembrane domain comprises (or consists of) a sequence disclosed herein (see SEQ ID NO: 100).
  • a CD8 ⁇ stalk comprises (or consists of) a sequence disclosed herein (see SEQ ID NO: 43, which includes a stalk and TM).
  • the chimeric ILT receptor may further comprise a region of amino acids between the transmembrane domain and the cytoplasmic domain, which are naturally associated with the polypeptide from which the transmembrane domain is derived.
  • Examples of such chimeric stalk moieties include, but are not limited to membrane proximal portions of CD8 ⁇ (see, e.g., SEQ ID NOs: 43 and 107), the CH2/CH3 domains of IgG (e.g., IgG1, IgG4) (see, e.g., SEQ ID NOs: 51 and 98), the CH3 domain of IgG (e.g., IgG1, IgG4)(see, e.g., SEQ ID NO: 102), HER2, mGluR2, CD28 (see, e.g., SEQ ID NOs: 47 and 106) and CTLA4.
  • CD8 ⁇ see, e.g., SEQ ID NOs: 43 and 107
  • the CH2/CH3 domains of IgG e.g., IgG1, IgG4
  • the CH3 domain of IgG e.g., IgG1, IgG4
  • HER2, mGluR2, CD28 see, e
  • the stalk domain of a subject CIR is selected from: an ILT2, ILT4, CD28, CH2/CH3, CH3, and CD8 ⁇ stalk domain.
  • VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLL LLFLILRHRRQ (SEQ ID NO: 39) (SEQ ID NO: 38 is an encoding nucleotide sequence), which includes an ILT2 stalk and TM domain; PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 107), which includes a CD8 ⁇ stalk domain; Atty Docket No.: NKLT-002WO PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VITLYCNHRNRRRVCKCPR (SEQ ID NO: 43), which includes a CD8 stalk and TM domain; IEVMYP
  • the stalk of domain of a subject CIR includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the stalk sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.
  • the stalk of domain of a subject CIR includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the stalk sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59. In some embodiments, the stalk of domain of a subject CIR includes the stalk sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.
  • the stalk of domain of a subject CIR includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the stalk sequence set forth in any one of SEQ ID NOs: 98, 102, 106, and 107.
  • the stalk of domain of a subject CIR includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the stalk sequence set forth in any one of SEQ ID NOs: 98, 102, 106, and 107. In some embodiments, the stalk of domain of a subject CIR includes the stalk sequence set forth in any one of SEQ ID NOs: 98, 102, 106, and 107. [00132] Interaction of the D1/D2 stalk-containing CIR with dimeric HLA-G will have the effect of dimerizing the intracellular signaling domains that in certain embodiments can stimulated ICD activation and cell signaling.
  • mutations may be made within the ILT2 or ILT 4 D1 or D2 domains contained within a CIR to increase the specificity of the CIR toward HLA-G over other potentially interacting proteins.
  • a mutation may be made to encode an amino acid other than tyrosine at the corresponding position of native amino acid 96 (Y96) of ILT4 SEQ ID NO: 53) or ILT2 (SEQ ID NO: 29) (e.g., Y96A). The effect of such a mutant form is to reduce potential interaction with ANGPTL2 and ANGPTL5 while retaining binding affinity for HLA-G.
  • a similar mutation may be placed in a full-length ILT4 CIR containing D1- D4 domains together with a mutation in domain D4 (at a position corresponding to tyrosine394 (Y394) of SEQ ID NO: 53) that further destabilizes interaction with ANGPTL2 and ANGPTL5.
  • the corresponding position of ILT2 is tyrosine395 (Y395) of SEQ ID NO: 29.
  • a subject ILT4 CIR includes a mutation at an amino acid position corresponding to Y96 of SEQ ID NO: 53 (e.g., Y96A).
  • a subject ILT2 CIR includes a mutation at an amino acid position corresponding to Y96 of SEQ ID NO: 29 (e.g., Y96A).
  • a subject ILT4 CIR includes a mutation at an amino acid position corresponding to Y394 of SEQ ID NO: 53 (e.g., Y394A) (also see SEQ ID NOs: 60-61).
  • a subject ILT2 CIR includes a mutation at an amino acid position corresponding to Y395 of SEQ ID NO: 29 (e.g., Y395A).
  • a subject ILT4 CIR includes a mutation at an amino acid position corresponding to Y96 of SEQ ID NO: 53 (e.g., Y96A) and a mutation at an amino acid position corresponding to Y394 of SEQ ID NO: 53 (e.g., Y394A) (e.g., Y96A/Y394A).
  • a subject ILT2 CIR includes a mutation at an amino Atty Docket No.: NKLT-002WO acid position corresponding to Y96 of SEQ ID NO: 29 (e.g., Y96A) and a mutation at an amino acid position corresponding to Y395 of SEQ ID NO: 29 (e.g., Y395A) (e.g., Y96A/Y395A).
  • NKLT-002WO acid position corresponding to Y96 of SEQ ID NO: 29 e.g., Y96A
  • Y395A amino acid position corresponding to Y395 of SEQ ID NO: 29
  • Other embodiments along similar lines may limit interaction with classical HLA proteins or CD1 while retaining binding for HLA-G. These mutations may replace interacting sites with ⁇ 3 domains that are specific to the heavy chains of these HLAs or on a surface bound to ⁇ 2-M.
  • a CIR may include a single-pass or multiple-pass transmembrane sequence (e.g., at the N-terminus or C-terminus of the chimeric protein, or within the protein, e.g., connecting the extracellular targeting region to the intracellular domain).
  • Single pass transmembrane regions are found in certain CD molecules, tyrosine kinase receptors, serine/threonine kinase receptors, TGF ⁇ , BMP, activin and phosphatases.
  • Single pass transmembrane regions often include a signal peptide region and a transmembrane region of about 20 to about 25 amino acids, many of which are hydrophobic amino acids and can form an alpha helix.
  • a short track of positively charged amino acids often follows the transmembrane span to anchor the protein in the membrane.
  • Multiple pass proteins include ion pumps, ion channels, and transporters, and include two or more helices that span the membrane multiple times. All or substantially all of a multiple pass protein sometimes is incorporated in a chimeric protein. Sequences for single pass and multiple pass transmembrane regions are known and can be selected for incorporation into a chimeric protein molecule.
  • the transmembrane domain is fused to the extracellular domain of the CIR. In some embodiments, the transmembrane domain is fused to the extracellular region and the intracellular region, thereby connecting the extracellular and intracellular regions to one another. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CIR is used. In other embodiments, a transmembrane domain that is not naturally associated with one of the domains in the CIR is used.
  • the transmembrane domain can be selected or modified by amino acid substitution (e.g., typically changed to a hydrophobic residue) to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • amino acid substitution e.g., typically changed to a hydrophobic residue
  • Transmembrane (TM) domains may, for example, be derived from the alpha, beta, or zeta chain of the T cell receptor, CD3- ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD8 ⁇ , CD9, CD16, CD22, CD28, CD33, CD38, CD64, CD80, CD86, CD134, CD137, ILT2, HER2, ILT4 or CD154 - or transmembrane regions containing functional variants thereof such as Atty Docket No.: NKLT-002WO those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof can be used. See e.g., Kahlon et al.
  • the transmembrane domain may be synthesized de novo, comprising mostly hydrophobic residues, such as, for example, leucine, isoleucine, phenylalanine and valine.
  • hydrophobic residues such as, for example, leucine, isoleucine, phenylalanine and valine.
  • suitable CD8 stalk sequences, transmembrane sequences, and CD3 ⁇ sequences are disclosed herein.
  • the TM domain of a subject CIR is selected from: an ILT2 (see, e.g., SEQ ID NO: 39), ILT4 (see, e.g., SEQ ID NO: 59), CD28 (see, e.g., SEQ ID NOs: 47 and 104), and CD8 (see, e.g., SEQ ID NOs: 43 and 100) TM domain.
  • ILT2 see, e.g., SEQ ID NO: 39
  • ILT4 see, e.g., SEQ ID NO: 59
  • CD28 see, e.g., SEQ ID NOs: 47 and 104
  • CD8 see, e.g., SEQ ID NOs: 43 and 100
  • VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLL LLFLILRHRRQ (SEQ ID NO: 39), which includes an ILT2 stalk and TM domain; IYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR (SEQ ID NO: 100), which includes a CD8 TM domain; PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VITLYCNHRNRRRVCKCPR (SEQ ID NO: 43), which includes a CD8 stalk and TM domain; FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 104), which includes a CD28 TM domain; IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWV (SEQ ID NO: 47), which includes a CD28
  • the TM domain of a subject CIR includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the TM domain sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.
  • the TM domain of a subject CIR includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the TM domain sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.
  • the TM domain of a subject CIR includes the TM domain sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.
  • the stalk domain plus the TM domain (stalk/TM domain) of a subject CIR includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.
  • the stalk domain plus the TM domain (stalk/TM domain) of a subject CIR includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.
  • the stalk domain plus the TM domain (stalk/TM domain) of a subject CIR includes an amino acid sequence having the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.
  • the TM domain of a subject CIR includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the TM domain sequence set forth in any one of SEQ ID NOs: 100 and 104.
  • the TM domain of a subject CIR includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the TM domain sequence set forth in any one of SEQ ID NOs: 100 and 104.
  • the TM domain of a subject CIR includes the TM domain sequence set forth in any one of SEQ ID NOs: 100 and 104.
  • ICD Intracellular Domain
  • a subject chimeric ILT receptor includes an intracellular region (Intracellular domain or ICD) that replaces the natural intracellular portion of ILT2 or ILT4, which is inhibitory, with an ICD of a CAR, which is activating.
  • the ICD of a subject CIR includes a “signaling region”, which has at least one signaling domain that causes activation of the cell, and can optionally include a “costimulatory region”, which can include one or more costimulatory domains.
  • Signaling Region [00145] Activation of T cells is directed by engagement of the T cell receptor with a peptide- MHC complex and activation of the CD3 signaling complex. Cell signaling is triggered by phosphorylation ITAM motives present on the CD3 ⁇ , CD3d, CD3 ⁇ and CD3e components of this complex.
  • CD3 ⁇ contains three ITAMs that recruit the ZAP70 kinase that initiates a downstream signaling cascade to activate the T cell canonically through the activation of NF-AT.
  • NK cells also express CD3 ⁇ which acts as the intracellular signaling adaptor for the Natural cytotoxicity receptor (NCR) NKp46.
  • NCRs such as CD94/NKG2C engage with the ITAM-containing signaling adaptor DAP12 and NKG2D engages with the DAP10 signaling adaptor.
  • DAP12 contains a single ITAM motif that can be phosphorylated by Syk or ZAP70 to activate NK cell signaling for cytotoxicity.
  • the “signaling region” (or “intracellular signaling domain”), e.g., of a CIR, refers to the part that participates in transducing the signal from binding (e.g., CIR) binding to a target molecule (HLA-G in the case of a subject CIR) into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and/or cytotoxic activity, including the release of cytotoxic factors to the CIR-bound target cell, or other cellular responses elicited with target molecule binding to the extracellular CIR domain.
  • a target molecule HLA-G in the case of a subject CIR
  • signaling region refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of a full-length intracellular signaling domain as long as it transduces the effector function signal.
  • signaling region is meant to include any truncated portion of an intracellular signaling domain sufficient for transducing effector function signal. In some cases, the signaling region includes signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or “ITAMs”).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • the ITAM motives of CD3 ⁇ are contained on the intracellular domain of, e.g., a canonical CAR.
  • the signaling region does not include an ITAM.
  • the signaling region of a subject CIR includes an intracellular domain (e.g., CD3 ⁇ ) that includes an ITAM.
  • the signaling region of a subject CIR includes an intracellular domain (e.g., DAP10) that does not include an ITAM.
  • intracellular domain sequences that can be used in a signaling region of a subject CIR include those derived from an intracellular signaling domain of a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit, an IL-2 receptor subunit, CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, CD278(ICOS), FcsRl, DAP10, and DAP12. [00148] In some zeta (CD3 ⁇ ) .
  • the ICD (e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)) includes a signaling region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 33.
  • the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 33.
  • the signaling region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 33. In some embodiments, the signaling region includes the amino acid sequence set forth as SEQ ID NO: 33. [00149] In some embodiments, the signaling region (e.g., of a subject CIR) includes a DAP10 signaling domain (see, e.g., SEQ ID NO: 4), e.g., with or without a CD3 ⁇ domain.
  • the signaling region (e.g., of a subject CIR) includes a DAP12 signaling domain (see, e.g., SEQ ID NO: 5), e.g., with or without a CD3 ⁇ domain.
  • DAP10 of DAP12 e.g., SEQ ID NO: 4, SEQ ID NO: 5
  • DAP10 of DAP12 can supplement CD3 ⁇ signaling by fusion. Examples are described herein where DAP10 or DAP12 replace or supplement CD3 ⁇ alone or in combination with further signaling motives that drive coactivation of NK cells or costimulation of T cells.
  • the signaling region (e.g., of a subject CIR) includes a DAP10 signaling domain (see, e.g., SEQ ID NO: 4).
  • the ICD e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)
  • the ICD includes a signaling region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 4.
  • the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 4.
  • the signaling region includes an Atty Docket No.: NKLT-002WO amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 4.
  • the signaling region includes the amino acid sequence set forth as SEQ ID NO: 4.
  • the signaling region (e.g., of a subject CIR) includes a DAP12 signaling domain (see, e.g., SEQ ID NO: 23).
  • the ICD e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)
  • the ICD includes a signaling region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 23.
  • the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 23. In some embodiments, the signaling region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 23. In some embodiments, the signaling region includes the amino acid sequence set forth as SEQ ID NO: 23.
  • Costimulatory Region [00152] T cell activation is generated by antigen presenting cells (APC), typically dendritic cells that present peptide-MHC to receptive T cells.
  • costimulatory elements further stimulate T cells engaged with high affinity to presenting cells.
  • These elements are typically derived from B7-H1 and B7-H2 ligation with CD28 on T cells and further by 4-1BB or OX40 interaction with their ligands once T cells are partially activated.
  • the costimulation provided by CD28 or 4-1BB in combination with activation of ITAMs from the TCR/CD3 complex drives T cell differention into a fully activated state that supports proliferation and persistence to a memory cell state.
  • Signaling derived from receptors that drive T cell costimulation can be incorporated into CIR or CAR products to support differentiation to a fully activated and persistent cell state.
  • NK cells express 4-1BB, but not CD28 and are not typically stimulated by cell:cell contact with support cells but are activated directly by target cells.
  • Coactivation of NK cells is promoted by cytokines such as IL-12, IL-18, IL-21 and IL-1 within an inflammatory environment.
  • IL-18 and IL-1 signaling is mediated by the cytoplasmic signaling node MyD88.
  • Toll-like Receptors (TLR) activation by pathogenic ligands are also a potent NK cell activation mechanism employed by NK cells that is also directed downstream by MyD88.
  • the examples below include the incorporation of signaling elements that drive NK cell activation to a degree further than costimulatory elements currently employed in CAR- T cell products and CAR-NK products that are derived from constructs designed for incorporation in T cells.
  • the costimulatory polypeptide may comprise one or more costimulatory signaling regions such as a truncated MyD88, 4-1BB or HVEM or a combination of these or other costimulatory motives.
  • the costimulatory polypeptide may comprise one or more suitable costimulatory signaling regions that activate the signaling pathways activated by MyD88, 4-1BB or HVEM.
  • Costimulating polypeptides include any molecule or polypeptide that activates the NF- ⁇ B pathway, MyD88 pathway, STAT5 pathway, STAT1 pathway, Akt pathway, and/or p38 pathway of tumor necrosis factor receptor (TNFR) family (i.e., CD40, RANK/TRANCE-R, OX40, 4-1BB) and CD28 family members (CD28, ICOS). More than one costimulating polypeptide or costimulating polypeptide cytoplasmic region may be expressed in the modified cells [00156]
  • the ICD e.g., of a subject CIR
  • the ICD also includes a costimulatory region.
  • the costimulatory region includes at least one costimulatory domain (e.g., one, two, three, one or more, two or more, or three or more costimulatory domains).
  • costimulatory domains include, but are not limited to: CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, and ICOS costimulatory domains.
  • costimulatory domains include, but are not limited to: 4-1BB, OX40, ICOS, CD28, CD27, MyD88, IL-1R ⁇ , HVEM, TRANCE, IL-1R ⁇ , CD70, IL-18R ⁇ , CD40, IL-18R ⁇ , IL-33R ⁇ , CD30, and IL-33R ⁇ .
  • costimulatory domains include, but are not limited to: 4-1BB, OX40, ICOS, RANK, DAP10, DAP12, CD28, CD27, MyD88, IL-1R ⁇ , HVEM, TRANCE, IL-1R ⁇ , CD70, IL-18R ⁇ , CD40, IL-18R ⁇ , IL- 33R ⁇ , CD30, and IL-33R ⁇ .
  • the costimulatory region includes one or more (e.g., one, two, three, one or more, or two or more) costimulatory domains selected from the group consisting of: CD28 (see, e.g., SEQ ID NO: 49), 4-1BB (see, e.g., SEQ ID NO: 35), and OX40 – or any combination thereof.
  • CD28 see, e.g., SEQ ID NO: 49
  • 4-1BB see, e.g., SEQ ID NO: 35
  • OX40 – or any combination thereof e.g., a CD28 costimulatory domain is used.
  • a 4-1BB costimulatory domain is used.
  • both a CD28 costimulatory domain and a 4-1BB costimulatory domain is used (i.e., they are both used).
  • the costimulatory region includes a truncated MyD88 polypeptide fused with signaling domains of receptor mediators of costimulation, such as, for example, CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, or ICOS.
  • the costimulatory region includes a MyD88 polypeptide or a truncated MyD88 polypeptide and a costimulatory domain selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40, and DAP10.
  • the ICD (e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)) includes a costimulatory region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49.
  • the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49.
  • the signaling region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49. In some embodiments, the signaling region includes the amino acid sequence set forth as SEQ ID NO: 49.
  • the ICD (e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)) includes a costimulatory region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35 (4-1BB).
  • the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35.
  • the signaling region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35. In some embodiments, the signaling region includes the amino acid sequence set forth as SEQ ID NO: 35.
  • the ICD (e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)) includes a costimulatory region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35 and an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49.
  • sequence identity e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%
  • the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35 and . an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49.
  • the signaling region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35 and an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49.
  • the signaling region includes the amino acid sequence set forth as SEQ ID NO: 35 and the amino acid sequence set forth as SEQ ID NO: 49.
  • the signaling region (e.g., of a subject CIR) includes a CD3 zeta (CD3 ⁇ ) signaling domain and the costimulatory region includes a CD28 costimulatory domain.
  • the signaling region includes a CD3 zeta (CD3 ⁇ ) signaling domain and the costimulatory region includes a 4-1BB costimulatory domain.
  • the signaling region includes a CD3 zeta (CD3 ⁇ ) signaling domain and the costimulatory region includes a 4-1BB costimulatory domain and a CD28 costimulatory domain.
  • the signaling region includes a CD3 zeta (CD3 ⁇ ) signaling domain and the costimulatory region includes a CD28 costimulatory domain and an OX40 costimulatory domain.
  • the signaling region e.g., of a subject CIR
  • the costimulatory region includes a CD28 costimulatory domain.
  • the signaling region includes a DAP10 or DAP12 signaling domain (without a CD3 ⁇ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain.
  • the signaling region includes a DAP10 or DAP12 signaling domain (without a CD3 ⁇ signaling domain) and the costimulatory region includes a 4- 1BB costimulatory domain and a CD28 costimulatory domain.
  • the signaling region includes a DAP10 or DAP12 signaling domain (without a CD3 ⁇ signaling domain) and the costimulatory region includes a CD28 costimulatory domain and an OX40 costimulatory domain.
  • the signaling region includes a DAP12 signaling domain (without a CD3 ⁇ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain.
  • the signaling region includes a DAP10 signaling domain (without a CD3 ⁇ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain.
  • the signaling region e.g., of a subject CIR
  • the costimulatory region includes a CD28 costimulatory domain.
  • the signaling region includes a DAP10 or DAP12 signaling domain (fused with a CD3 ⁇ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain.
  • the signaling region includes a DAP10 or DAP12 signaling domain (fused with a CD3 ⁇ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain and a CD28 costimulatory domain. In some embodiments, the signaling region includes a DAP10 or DAP12 signaling domain (fused with a CD3 ⁇ signaling domain) and the costimulatory region includes a CD28 costimulatory domain and an OX40 costimulatory domain.
  • the Atty Docket No.: NKLT-002WO signaling region includes a DAP12 signaling domain (fused with a CD3 ⁇ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain.
  • the signaling region includes a DAP10 signaling domain (fused with a CD3 ⁇ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain.
  • Cells may include chimeric signaling polypeptides, including, for example, chimeric signaling polypeptides where a truncated MyD88 polypeptide has also been fused with signaling domains of receptor mediators of costimulation, such as, for example, 4- 1BB or HVEM.
  • the fusion may incorporate other signaling domains such as those from CD40, CD27, CD28, 4-1BB, OX40, or ICOS. More than one costimulating polypeptide or costimulating polypeptide cytoplasmic region may be expressed in the modified cells.
  • Cells may include chimeric signaling polypeptides, including, for example, chimeric signaling polypeptides where a truncated MyD88 polypeptide has also been fused with signaling domains of receptor mediators of costimulation, such as, for example, CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, or ICOS.
  • chimeric signaling polypeptides including, for example, chimeric signaling polypeptides where a truncated MyD88 polypeptide has also been fused with signaling domains of receptor mediators of costimulation, such as, for example, CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, or ICOS.
  • a chimeric signaling polypeptide comprises cytoplasmic signaling regions from two costimulatory polypeptides, such as, for example, 4-1BB and CD28, or one, or two or more costimulatory polypeptide cytoplasmic signaling regions selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, or OX40,.
  • the chimeric signaling polypeptide comprises a MyD88 polypeptide or a truncated MyD88 polypeptide and a costimulatory polypeptide cytoplasmic signaling region selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40.
  • Non-limiting examples of a 4-1BB, CD28, and OX40 costimulatory signaling domains can be found in U.S.20130266551, U.S. Pat. No.5,686,281; Geiger, T. L. et al., Blood 98: 2364-2371 (2001); Hombach A. et al., J Immunol 167: 6123-6131 (2001); Maher J. et al. Nat Biotechnol 20: 70-75 (2002); Haynes N. M. et al., J Immunol 169: 5780-5786 (2002); Haynes N. M. et al., Blood 100: 3155-3163 (2002); and in U.S.
  • Non-limiting examples of chimeric polypeptides useful for inducing cell activation, and related methods for inducing therapeutic cell activation including, for example, expression constructs, methods for constructing vectors, and assays for activity or function, may also be found in the following patents and patent applications: US2014- 0286987-A1; WO2014/151960; US2016/0046700; WO2015/123527; US2004/0209836; U.S. Patent No.7,404,950; WO2004/073641; US2011/0033388; U.S.
  • cell activation domains e.g., cell signaling and costimulatory domains.
  • cells are designed to provide constitutively active therapy.
  • genetically modified cells comprise a nucleic acid comprising a first polynucleotide encoding a Chimeric ILT2 or ILT4 Receptor (or CIR), and a second polynucleotide encoding a chimeric signaling polypeptide.
  • the second polynucleotide is positioned 5’ of the first polynucleotide. In some embodiments, the second polynucleotide is positioned 3’ of the first polynucleotide.
  • a third polynucleotide encoding a linker polypeptide is positioned between the first and second polynucleotides. Where the third polynucleotide is positioned 3’ of the first polynucleotide and 5’ of the second polynucleotide, the linker polypeptide, may remain intact following translation, or may separate the polypeptides encoded by the first and second polynucleotides during, or after translation. In some embodiments, the linker polypeptide is a 2A polypeptide (see elsewhere herein), which may separate the polypeptides encoded by the first and second polynucleotides during, or after translation.
  • High level costimulation is provided constitutively through an alternate mechanism in which a leaky 2A cotranslational sequence (see elsewhere herein). is used to separate the CAR from the chimeric signaling polypeptide.
  • a leaky 2A cotranslational sequence see elsewhere herein.
  • most of the expressed chimeric signaling polypeptide molecules are separated from the chimeric antigen receptor polypeptide and may remain cytosolic, and some portion or the chimeric signaling polypeptide molecules remain attached, or linked, to the CAR.
  • Constitutively active is meant that the chimeric stimulating polypeptide’s cell activation activity is active even in the absence of an inducer.
  • MyD88 encoded by myeloid differentiation primary response gene 88
  • TLRs Toll-like Receptors
  • MyD88 is also a principal mediator of signaling downstream of the Interleukin-1 family of receptors including the receptors for IL-1, IL-18 and IL-33.
  • MyD88 contains two domains that direct its activity – an amino-terminal Death Domain that directs oligomerization into a complex that further directs downstream signaling through the NK- ⁇ B pathway for induced cytokine Atty Docket No.: NKLT-002WO production and coactivation of signals from separate ITAM-directed signals, the AKT growth and survival pathway and the Interferon Response Factor pathway.
  • a carboxy terminal TIR domain directs recruitment to Toll-like Receptors and IL-1 family receptors through interaction with their TIR domains. Similar TIR-TIR interactions can recruit related signaling proteins that act as nodes, for example TRIF, to TLR3 and TLR4.
  • a TIR domain from an IL1 family receptor or toll-like receptor can be used to recruit MyD88 or TRIF signaling to an activated chimeric receptor (e.g., a CAR, a CIR) by direct fusion of a the TIR to the receptor as a coactivation domain, e.g., a “costimulatory region” can include a TIR domain (e.g., from IL1 family receptors or TLRs).
  • the costimulatory region e.g., of a CIR or CAR
  • the costimulatory region includes a TIR domain from an IL1 family receptor or a TLR.
  • the costimulatory region includes a TIR from TLR2.
  • a costimulatory region includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 111.
  • a costimulatory region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 111.
  • a costimulatory region includes an amino acid an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 111.
  • a costimulatory region includes the amino acid sequence set forth as SEQ ID NO: 111. [00174] In some cases, the costimulatory region includes a TIR from TLR3.
  • a costimulatory region includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 113.
  • a costimulatory region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 113.
  • a costimulatory region includes an amino acid an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 113.
  • a costimulatory region includes the amino acid sequence set forth as SEQ ID NO: 113. [00175] In some cases, the costimulatory region includes a TIR from IL-18R1 (IL-18 receptor alpha).
  • a costimulatory region includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with Atty Docket No.: NKLT-002WO SEQ ID NO: 109.
  • a costimulatory region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 109.
  • a costimulatory region includes an amino acid an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 109. In some embodiments, a costimulatory region includes the amino acid sequence set forth as SEQ ID NO: 109. [00176] Any of the above TIR domains can be used in combination with any desired signaling domain, e.g. CD3 ⁇ , DAP10, DAP12. In some case, a signaling region includes a CD3 ⁇ signaling domain and a TIR from TLR2. In some case, a signaling region includes a CD3 ⁇ signaling domain and a TIR from TLR3.
  • a signaling region includes a CD3 ⁇ signaling domain and a TIR from IL-18R1. In some case, a signaling region includes a DAP10 signaling domain and a TIR from TLR2. In some case, a signaling region includes a DAP10 signaling domain and a TIR from TLR3. In some case, a signaling region includes a DAP10 signaling domain and a TIR from IL- 18R1. In some case, a signaling region includes a DAP12 signaling domain and a TIR from TLR2. In some case, a signaling region includes a DAP12 signaling domain and a TIR from TLR3.
  • a signaling region includes a DAP12 signaling domain and a TIR from IL-18R1.
  • Fusions of a truncated MyD88 polypeptide, lacking the TIR domain with the intracellular domain of CD40 to produce a chimeric polypeptide amplifies certain signals directed by MyD88.
  • T cells are transfected or transduced with nucleic acids that encode MC, in combination with a Chimeric Antigen Receptor (CAR), MC delivers potent costimulatory signals that enhance T and NK cell growth, persistence, and cytotoxic activity against cells specifically targeted by the CAR (Foster et al, Mol. Ther.25:2176 (2017), Duong et al., Mol. Ther.
  • CAR Chimeric Antigen Receptor
  • a truncated MyD88 polypeptide has also been fused with signaling domains of receptor mediators of costimulation, such as, for example, 4-1BB or HVEM.
  • signaling domains of receptor mediators of costimulation such as, for example, 4-1BB or HVEM.
  • chimeric truncated MyD88 polypeptides that, when expressed in, for example, CIR-NK cells, produce significantly fewer of certain toxic inflammatory cytokines such as TNF- ⁇ than CIR-NK cells that express an MyD88-CD40 chimeric Atty Docket No.: NKLT-002WO polypeptide, while retaining potent or even enhanced tumor cell killing.
  • modified chimeric receptors where the chimeric receptor polypeptide comprises truncated MyD88 polypeptides alone.
  • immune cells such as, for example, activated NK cells that express an chimeric signaling polypeptide.
  • the activated cells may be used to increase the immune response against a disease, or to treat cancer by, for example, reducing the size of a tumor.
  • Therapeutic courses of treatment using the activated NK cells and activated CIR-NK cells may be monitored by determining the size and vascularity of tumors by various imaging modalities (e.g. CT, bonescan, MRI, PET scans, Trofex scans), by various standard blood biomarkers (e.g. PSA, Circulating Tumor Cells), or by serum levels of various inflammatory, hypoxic cytokines, or other factors in the treated patient [00181]
  • a costimulatory region includes an MyD88 polypeptide (see, e.g., SEQ ID NO: 27).
  • a costimulatory region includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 27.
  • a costimulatory region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 27.
  • a costimulatory region includes an amino acid an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 27.
  • a costimulatory region includes the amino acid sequence set forth as SEQ ID NO: 27.
  • the MyD88 polypeptides of this paragraph can combined with other costimulatory molecules as part a costimulatory region. For example, in some cases with CD40. In some cases with 4-1BB. In some cases with HVEM (see, e.g., SEQ ID NO: 25).
  • a signaling region includes a CD3 ⁇ signaling domain and a MyD88, MyD88-CD40, MyD88-4-1BB, or MyD88-HVEM costimulatory region.
  • a signaling region includes a DAP10 signaling domain and a MyD88, MyD88-CD40, MyD88-4-1BB, or MyD88-HVEM costimulatory region.
  • a signaling region includes a DAP12 signaling domain and a MyD88, MyD88-CD40, MyD88-4- 1BB, or MyD88-HVEM costimulatory region. In some case, a signaling region includes a DAP12 signaling domain and a MyD88 costimulatory region. In some case, a signaling region (e.g., of a CIR) includes a CD3 ⁇ signaling domain and a MyD88-4- 1BB costimulatory region. Atty Docket No.: NKLT-002WO CIR Variations [00182] Provided are functional portions of the CIRs described herein.
  • the term “functional portion” when used in reference to a CIR refers to any part or fragment of the CIR, which part or fragment retains the biological activity of the CIR of which it is a part (the parent CIR).
  • Functional portions encompass, for example, those parts of a CIR that retain the ability to recognize the target (HLA-G) or target cells, or detect, treat, or prevent a disease, to a similar extent, the same extent, or to a higher extent, as the parent CIR.
  • the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent CIR.
  • ICD polypeptides are also provided.
  • the functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CIR.
  • the additional amino acids do not interfere with the biological function, e.g., recognize target cells, detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent CIR.
  • Included in the scope of the disclosure are functional variants or biological equivalent of the inventive CIRs disclosed herein.
  • a functional variant can, for example, comprise the amino acid sequence of the parent polypeptide with at least one conservative amino acid substitution.
  • the functional variants can comprise the amino acid sequence of the parent polypeptide with at least one non- conservative amino acid substitution.
  • the non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent polypeptide.
  • Such biological variant (including functional portions thereof) can comprise synthetic amino acids in place of one or more naturally-occurring amino acids.
  • Such biological variant can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.
  • Such biological variant can be obtained by methods known in the art.
  • the polypeptides may be made by any suitable method of making polypeptides or proteins.
  • polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods.
  • Safety switches [00188] Genetically modified cells that express a subject chimeric ILT receptor (CIR) may also express a safety switch, also known as an inducible suicide gene or suicide switch, which can be used to eradicate the therapeutic cells in vivo if desired e.g. if GvHD develops.
  • CIR chimeric ILT receptor
  • therapeutic cells may trigger an adverse event, such as off-target toxicity due to a CIR, or a patient might experience a negative symptom during therapy using modified cells, or there may be side effects due to non-specific attacks on healthy tissue; or, sometimes, the therapeutic cells may no longer be needed, or the therapy is intended for a specified amount of time, for example, the therapeutic cells may work to decrease the tumor cell, or tumor size, and may no longer be needed.
  • genetically modified cells can also inducibly express a polypeptide which causes the cells to die, such as an inducible caspase-9 polypeptide. If there is a need, for example, to reduce the number of therapeutic cells, the switch can be triggered.
  • a safety switch is based on a pro-apoptotic protein that can be triggered by administering a trigger molecule (also referred to as a ligand inducer) to a subject.
  • a trigger molecule also referred to as a ligand inducer
  • pro-apoptotic protein is fused to a polypeptide sequence which binds to the trigger molecule, delivery of this trigger molecule can bring two pro-apoptotic proteins into proximity such that they trigger apoptosis.
  • caspase-9 can be fused to a modified human FK-binding protein which can be induced to dimerize in response to the pharmacological agent rimiducid (AP1903).
  • a safety switch based on a human pro-apoptotic protein such as, for example, caspase-9 minimizes Atty Docket No.: NKLT-002WO the risk that cells expressing the switch will be recognized as foreign by a human subject’s immune system.
  • chimeric polypeptides useful for inducing cell death or apoptosis may be found in the following patents and patent applications, each of which is incorporated by reference herein in its entirety for all purposes.
  • caspase-9 It is latent in the absence of ligand but drives dimerization of the initiator caspase, caspase-9, from the intrinsic pathway for cell apoptosis. Dimerization leads to caspase-9 activation, cleavage and activation of the effector caspase, caspase-3, and rapid cell death by apoptosis. Inducible caspase-9 has particular utility as a safety switch in cell therapies to block toxic responses. [00193] Caspase-9 switches: Examples are described in Di Stasi et al. (2011) supra; see also Yagyu et al. (2015) Mol Ther 23(9):1475-85; Rossigloni et al.
  • the safety switch may comprise a modified Caspase-9 polypeptide having modified activity, such as, for example, reduced basal activity in the absence of the homodimerizer ligand.
  • Modified Caspase-9 polypeptides are discussed in, for example, US patent 9,913,882 and US2015/0328292, supra, and may include, for example, amino acid substitutions at position 330 (e.g., D330E or D330A) or, for example, amino acid substitutions at position 450 (e.g., N405Q), or combinations thereof, including, for example, D330E-N405Q and D330A-N405Q.
  • Caspase-9 polypeptide with lower basal activity have been described previously, e.g. in U.S. Patent Nos.9,434,935, 9,932,572 and 9,913,882, and U.S. Patent Application Nos.
  • the safety switch may be, for example, iCasp9 discussed in Di Stasi et al. (2011) supra, which consists of the sequence of the human FK506-binding protein (FKBP12) (GenBank AH002818) with an F36V mutation, connected through a SGGGS linker to a modified human caspase 9 (CASP9) which lacks its endogenous Atty Docket No.: NKLT-002WO caspase activation and recruitment domain.
  • FKBP12 human FK506-binding protein
  • FKBP12-allele specific binding by rimiducid Rimiducid binds with high affinity ( ⁇ 0.1 nM) to the valine-36 allele of FKBP12 but with low affinity ( ⁇ 500 nM) to the wild-type phenylalanine-36 FKBP12 allele. Rapamycin and rapalogs can bind to either FKBP allele.
  • Rimiducid has two identical, protein-binding surfaces arranged tail-to-tail, each with high affinity and specificity for the valine-36 form (known variously as FKBP12(F36V), FKBP12v36, FKBPV, FV36, or simply Fv). See Jemal et al., CA Cancer J. Clinic.58, 71-96 (2008); Scher & Kelly Journal of Clinical Oncology 11, 1566-72 (1993)). Two tandem copies of the protein may also be used in the construct so that higher-order oligomers are induced upon cross-linking by rimiducid.
  • FKBP12 variants may also be used. Variants may bind to rapamycin, or rapalogs, but with less affinity to rimiducid than, for example, FKBP12v36. Examples of FKBP12 variants include those from many species, including, for example, yeast. In one embodiment, the FKBP12 variant is FKBP12.6 (calstablin).
  • the suicide switch may be controlled by a pharmaceutical composition comprising a trigger molecule (such as a dimerizing or multimerizing ligand).
  • An effective amount of a pharmaceutical composition comprising the trigger molecule is an amount that achieves the desired result of killing the genetically-modified cells.
  • the degree of killing may be high (e.g. over 60%, 70%, 80%, 85%, 90%, 95%, or 97%) or complete; conversely, sometimes only partial removal will be desired (e.g. under 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the genetically modified cells are killed).
  • genetically-modified may display a range of sensitivities to a trigger molecule.
  • the trigger molecule may thus be used to eradicate only a portion of the cells (e.g. at least 10%) while permitting some of the cells (e.g. at least 10%) to survive.
  • the concentration of the trigger molecule can be selected according to the desired balance of cell death and survival e.g. a higher concentration will be delivered if a higher proportion of cell eradication (or complete eradication) is desired. [00198] These concentrations can be determined by simple dose-ranging experiments, monitoring levels of cell death in response to the trigger molecule. Any appropriate assay may be used to determine the percent of genetically modified cells that are killed. An assay may include the steps of obtaining a first sample from a subject before administration of the trigger molecule and obtaining a second sample from the subject after administration of the trigger molecule and comparing the number or concentration of therapeutic cells in the first and second samples to determine the Atty Docket No.: NKLT-002WO percent of therapeutic cells that are killed.
  • Chimeric antigen receptors are artificial receptors designed to convey antigen specificity to cells. They generally include an antigen-specific component, a transmembrane component, and an intracellular component selected to activate the cell. CAR-expressing cells may be used in various therapies, including cancer therapies.
  • a CAR is, for example, a chimeric polypeptide which comprises a polypeptide sequence that recognizes a target antigen (an antigen-recognition domain) linked to a transmembrane polypeptide and an intracellular domain polypeptide selected to activate the cell, and thereby provide specific immunity.
  • the antigen-recognition domain may be a single-chain variable fragment (scFv), or may, for example, be derived from other molecules such as, for example, a T cell receptor or camelid VhH domain.
  • the intracellular domain comprises at least one polypeptide which causes activation of the cell, such as, for example, but not limited to, CD3 zeta (CD3 ⁇ ), and, optionally, co-stimulatory molecules (for example, but not limited to, CD28, OX40 and 4-1BB).
  • cells are modified to express a CAR that comprises a single chain antibody variable fragment (scFv) fused with a transmembrane domain containing a linker region and an intracellular domain derived from the CD3 zeta component.
  • scFv single chain antibody variable fragment
  • signals from CD3zeta drive the initial activation of the T cell through signaling to the NF-ATc transcription factor. These signals drive targeted cell killing in cytotoxic T lymphocytes and synergize with costimulatory signaling pathways to drive the robust cell proliferation of T cell immune response.
  • the genetically modified cells may be modified by transduction or transfection with a nucleic acid that expresses the CAR and a nucleic acid (the same or different) that comprises a polynucleotide that encodes a chimeric signaling polypeptide (see below).
  • a CAR is expressed without also expressing a chimeric signaling polypeptide.
  • Chimeric antigen receptors can be expressed in NK cells, iNKT cells or in macrophages to generate antigen specific cytotoxicity.
  • CARs include chimeric receptors that are derived from antibodies, but also include chimeric T cell receptors.
  • chimeric T cell receptors may comprise a polypeptide sequence that recognizes a target antigen, where the recognition sequence may be, for example, but not limited to, the recognition sequence derived from a T cell receptor or a scFv.
  • the intracellular domain polypeptides are those that act to activate the T Atty Docket No.: NKLT-002WO cell. Chimeric T cell receptors are discussed in, for example, Gross & Eshar FASEB Journal (1992) 6:3370-3378, and Zhang et al., (2010) PLOS Pathogens 6:1-13. 8.
  • Linker polypeptides [00204] Where it is desired to encode two polypeptides in a single gene, such that they are encoded on a single transcript, the two polypeptides can be joined by a linker polypeptide. For instance, these may be included between MyD88 and CD40 in a MyD88-CD40 chimeric polypeptide, or between the costimulatory polypeptide cytoplasmic signaling region and the CD3 ⁇ portion of a CAR or CIR. A linker can be positioned between any of the regions/domains described herein, where desired.
  • a linker is positioned: between the TM domain and the signaling region or costimulatory region, between the ILT2 or ILT4 targeting region (e.g., D1-D2 domain) and the stalk, between a signaling region and a costimulatory region, between two costimularoty domains, between a costimulatory or signaling region and a T2A sequence, or any combination thereof.
  • Linker polypeptides include cleavable and non-cleavable linker polypeptides. Examples of linkers include, but are not limited to: SGR, GS, VD, and PRGSG (SEQ ID NO: 67).
  • Linker polypeptides include those for example, consisting of about 2 to about 30 amino acids e.g. furin cleavage site, (GGGGS)n. In some embodiments, the linker polypeptide consists of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the linker polypeptide consists of about 18 to 22 amino acids. In some embodiments, the linker polypeptide consists of 20 amino acids. [00207] Cleavable linkers include linkers that are cleaved by an enzyme in the modified cells.
  • the enzyme may be exogenous to the cells, for example, an enzyme encoded by a polynucleotide that is introduced into the cells by transfection or transduction, either at the same time or a different time as the polynucleotide that encodes the linker.
  • cleavable linkers include linkers that are cleaved by an enzyme endogenous to the modified cells in the population, including, for example, enzymes that are naturally expressed in the cell, and enzymes encoded by polynucleotides native to the cell, such as, for example, lysozyme.
  • cleavable linker also extends to a linker which is cleaved by any means, including, for example, non- enzymatic means, such as peptide skipping.
  • a cleavable linker permits an essentially fixed stoichiometric ratio of expression of two polypeptides (a 1:1 ratio if two mature polypeptides are linked by a single cleavable linker).
  • NKLT-002WO Atty Docket No.: NKLT-002WO
  • the linker polypeptide may be a 2A-like sequence, which can be derived from many different viruses, including, for example, from the Thosea asigna insect virus.
  • sequences are sometimes also known as “peptide skipping sequences.”
  • this type of sequence When this type of sequence is placed within a cistron, between two polypeptides that are intended to be separated, the ribosome appears to skip a peptide bond, in the case of Thosea asigna sequence; the bond between the Gly and Pro amino acids at the carboxy terminal “P-G-P” is omitted.
  • This may leave two to three polypeptides, for example, an inducible chimeric pro-apoptotic polypeptide and a chimeric antigen receptor, or, for example, a marker polypeptide and an inducible chimeric pro- apoptotic polypeptide.
  • the polypeptide that is encoded 5’ of the 2A sequence may end up with additional amino acids at the carboxy terminus, including the Gly residue and any upstream residues in the 2A sequence.
  • the peptide that is encoded 3’ of the 2A sequence may end up with additional amino acids at the amino terminus, including the Pro residue and any downstream residues following the 2A sequence.
  • the cleavable linker is a 2A polypeptide derived from porcine teschovirus-1 (P2A).
  • the 2A cotranslational sequence is a 2A- like sequence.
  • the 2A cotranslational sequence is T2A (thosea asigna virus 2A), F2A (foot and mouth disease virus 2A), P2A (porcine teschovirus-1 2A), BmCPV 2A (cytoplasmic polyhedrosis virus 2A) BmIFV 2A (flacherie virus of B. mori 2A), or E2A (equine rhinitis A virus 2A).
  • the 2A cotranslational sequence is T2A-GSG, F2A-GSG, P2A-GSG, or E2A-GSG.
  • the 2A cotranslational sequence is selected from the group consisting of T2A, P2A and F2A.
  • a 2TA comprises (or consists of) a sequence disclosed herein. comprises (consists of) a sequence disclosed herein (e.g., a sequence disclosed in the Examples below).
  • 2A-like sequences are sometimes “leaky” in that some of the polypeptides are not separated during translation, and instead, remain as one long polypeptide following translation.
  • One theory as to the cause of the leaky linker, is that the short 2A sequence occasionally may not fold into the required structure that promotes ribosome skipping (a “2A fold”). In these instances, ribosomes may not miss the proline peptide bond, which then results in a fusion protein.
  • a GSG (or similar) linker may be added to the amino terminal side of the 2A polypeptide; the GSG linker blocks secondary structures of newly-translated polypeptides from spontaneously folding and disrupting the ‘2A fold’.
  • a leaky 2A sequence can be used, for example, so that the same encoded polypeptide can sometimes be directed to the cell surface but other times remain in the cytosol.
  • a 2A linker includes the amino acid sequence of SEQ ID NO: 11.
  • the 2A linker further includes a GSG amino acid sequence at the amino terminus of the polypeptide
  • the 2A linker includes a GSGPR (SEQ ID NO: 68) amino acid sequence at the amino terminus of the polypeptide.
  • a “2A” sequence the term may refer to a 2A sequence in an example described herein or may also refer to a 2A sequence as listed herein further comprising a GSG or GSGPR (SEQ ID NO: 68) sequence at the amino terminus of the linker.
  • the linker for example, the 2A linker
  • the linker is cleaved in about 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% of the translated polypeptides.
  • suitable linker polypeptides including T2A linkers
  • T2A linkers examples of suitable linker polypeptides (including T2A linkers) are disclosed herein.
  • the genetically modified cells may be any cells useful in cell therapy, e.g., immune cells.
  • the cells may be, for example, natural killer (NK) cells, iNK-T cells, NKT cells, T cells, B cells, macrophages, peripheral blood cells, hematopoietic progenitor cells, or bone marrow cells.
  • the modified cells are NK cells, natural killer T cells (NKT cells / NK-T cells), or T cells.
  • Cells which are genetically modified as disclosed herein are useful for administering to subjects who can benefit from receiving them e.g. who can benefit from donor lymphocyte administration. These subjects will typically be humans, so the methods will typically be performed using human cells.
  • Cells to be genetically modified may be autologous, syngeneic, or allogeneic. Allogeneic cells can be derived from any healthy donor, and syngeneic cells from any healthy donor who is appropriately related to the intended recipient. The donor will generally be an adult (at least 18 years old) but children are also suitable as cell donors (e.g. see Styczynski 2018, Transfus Apher Sci 57(3):323-330).
  • autologous means a cell derived from the same individual to which it is later administered.
  • allogeneic refers to HLA or MHC loci that are antigenically distinct between the host and donor cells. Thus, cells from the same species can be antigenically distinct.
  • the term “syngeneic” refers to cells that have genotypes that are identical or closely related enough to allow tissue transplant, or are immunologically compatible. For example, identical twins or close relatives can be syngeneic. Atty Docket No.: NKLT-002WO [00219]
  • the cells may be blood cells.
  • the source of the cells may be, for example, umbilical cord blood, bone marrow, or peripheral blood, and they may be peripheral blood mononuclear cells (PBMCs). These include lymphocytes (e.g. T cells, B cells, NK cells) or monocytes.
  • peripheral blood refers to cellular components of blood (e.g., red blood cells, white blood cells and platelets), which are obtained or prepared from the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver or bone marrow.
  • Umbilical cord blood is distinct from peripheral blood and blood sequestered within the lymphatic system, spleen, liver or bone marrow, and it refers to blood that remains in the placenta and in the attached umbilical cord after child birth.
  • Cord blood often contains stem cells including hematopoietic cells.
  • NK cells also known as natural killer cells or large granular lymphocytes (LGL), are cytotoxic lymphocytes critical to the innate immune system.
  • NK cells provide rapid responses to virus-infected cells and respond to tumor formation.
  • T cells rely on priming interactions between the T-cell receptor (TCR) and MHC- peptide complexes on target cells as a necessary first step in T-cell activation.
  • T cells can recognize a single antigen, and tumor cells may avoid T-cell recognition through mutations that significantly reduce antigen presentation.
  • NK cells are capable of recognizing a multitude of transformed and infected cells without being dependent on the presentation of a single antigen. Therefore, treatment with NK cells can bypass some of the resistance mechanisms to T-cell based therapy.
  • NK cells can secrete proinflammatory chemokines and cytokines to recruit and activate the body’s adaptive immune system, consisting of T and B cells, creating a second wave of durable antitumor response. Furthermore, NK cells are not associated with certain toxicities associated with CAR-T cell therapy such as cytokine release syndrome and central nervous system toxicity. [00224] NK cells can be useful as a source for antigen or receptor-based directed cell therapy because of their innate cytotoxic mechanisms. NK cells comprise approximately 10- 15% of the lymphocytes in peripheral blood of a typical donor and can be readily Atty Docket No.: NKLT-002WO purified, expanded and virally transduced.
  • an activated NK cell has alternative inate mechanisms to direct cytotoxic function including NKG2D, p46, p44, p30, DNAM and CD16 CD4+ and CD8+ T cells
  • a subject composition can include CD4+ and CD8+ T cells. Whereas the ratio of CD4+ cells to CD8+ cells in a leukopak is typically above 2, in some embodiments the ratio of genetically-modified CD4+ cells to genetically-modified CD8+ cells in a composition of the disclosure is less than 2 e.g. less than 1.5.
  • the ratio is less than 1 e.g. less than 0.9, less than 0.8, less than 0.7, less than 0.6, or preferably even less than 0.5.
  • an overall procedure starting from donor cells and producing genetically-modified T cells ideally enriches for CD8+ cells T cells relative to CD4+ T cells.
  • at least 60% of the genetically-modified T cells are CD8+ T cells, and more preferably at least 65%.
  • a preferred range for CD8+ T cells is between 55- 75% e.g. from 63-73%.
  • a population of genetically-modified T cells can include terminal effector memory T cells (defined as CD45RA+CD45RO-CCR7- cells; ‘TEMRA’), T-effector memory cells (defined as CD45RA-CD45RO+CCR7- cells; ‘EM’), T-central memory cells (defined as CD45RA-CD45RO+CCR7+ cells; ‘CM’), and na ⁇ ve T cells (defined as CD45RA+CD45RO-CCR7+ cells).
  • An average leukopak typically contains ⁇ 20% each of terminal effector and T-effector memory cells.
  • An overall procedure from donor cells to genetically-modified T cells may enrich for terminal effector memory T cells relative to T-effector memory cells.
  • less than 60% of the genetically-modified T cells are na ⁇ ve T cells e.g. less than 58%, preferably less than 55%, and more preferably less than 50%.
  • na ⁇ ve T cells Within the population of genetically-modified CD3+ T cells a preferred range for na ⁇ ve T cells is between 30-60%, more preferably 42-49%, and most preferably from 43-46%. This proportion of na ⁇ ve T cells has been seen to correlate with favourable Atty Docket No.: NKLT-002WO outcomes in T cell recipients. Na ⁇ ve EM cells can be assessed by flow cytometry using the CD45RA/RO and CCR7 markers. [00229] Within a population of genetically modified T cells, in addition to TEMRA, EM and na ⁇ ve T cells, the proportion of T-central memory cells is generally ⁇ 10%.
  • a population of genetically-modified T cells in a composition comprises about 10% to about 40% CD4+ T cells and about 60% to about 90% CD8+ T cells.
  • the population of genetically-modified CD3+ T cells can comprise about 15 % to about 40% CD4+ T cells and about 60% to about 85% CD8+ T cells, more preferably about 20% to about 40% CD4+ T cells and about 60% to about 80% CD8+ T cells.
  • Genetic modification of cells [00231] Cells are genetically modified by transferring an expression construct (e.g., encoding a subject chimeric ILT receptor) into them. Such transfer may employ viral or non-viral methods of gene transfer. This section provides a discussion of methods and compositions of gene transfer.
  • An expression vector can be introduced into a cell by various means.
  • the term "transfection” and “transduction” are interchangeable and refer to the process by which an exogenous nucleic acid sequence is introduced into a eukaryotic host cell. Transfection (or transduction) can be achieved by any one of a number of means including electroporation, microinjection, gene gun delivery, retroviral infection, lipofection, superfection and the like.
  • Any appropriate method may be used to transfect or transform the cells (e.g. the T cells, NKT cells, or NK cells). Certain non-limiting examples are presented herein.
  • the viral vector is an SFG-based viral vector, as discussed in Tey et al.
  • the cells can be transduced using a viral vector encoding polypeptides described herein. Suitable transduction techniques may involve fibronectin fragment CH-296.
  • cells can be transfected with any suitable method known in the art such as with DNA encoding the relevant polypeptides e.g. using calcium phosphate, cationic polymers (such as PEI), magnetic beads, electroporation and commercial lipid-based reagents such as LipofectamineTM and FugeneTM.
  • the viral vector used for transduction is the retroviral vector disclosed by Tey et al. (2007) Biol Blood Marrow Transpl 13:913-24 and by Di Stasi et Atty Docket No.: NKLT-002WO al. (2011) supra.
  • This vector is based on Gibbon ape leukemia virus (Gal-V) pseudotyped retrovirus encoding an iCasp9 suicide switch and a ⁇ CD19 cell surface transgene marker (see further below – and see SEQ ID NOs: 12-13).
  • the CIR may not be suitable for positive selection of desired cells, so in some embodiments, the genetically-modified cells should express a cell surface transgene marker of interest (see below).
  • Cells which express this surface marker can be selected e.g. using immunomagnetic techniques. For instance, paramagnetic beads conjugated to monoclonal antibodies which recognise the cell surface transgene marker of interest can be used, for example, using a CliniMACS system (available from Miltenyi Biotec).
  • genetically-modified cells are selected after a step of transduction, are cultured, and are then fed. Thus, the order of transduction, feeding, and selection can be varied.
  • the result of these procedures is a composition containing cells which have been genetically modified, and which can thus express the Chimeric ILT receptor (and any other desired polypeptides e.g. a costimulatory polypeptide, a suicide switch, a cell surface transgene marker, etc.).
  • These genetically-modified cells can be administered to a recipient, but they might first be preserved (e.g. cryopreserved), optionally after further expansion, before being administered.
  • Selectable Markers [00240] Cells may be modified to express polypeptides whose expression can be identified in vitro or in vivo, thereby permitting selection of genetically-modified cells e.g. to separate them from unmodified cells.
  • Such markers confer an identifiable change to the cell, permitting easy identification of cells containing the desired expression construct.
  • Inclusion of a drug selection marker aids in cloning and in the selection of transformants.
  • genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as Herpes Simplex Virus thymidine kinase (tk) are employed.
  • Immunologic surface markers containing the extracellular, non-signaling domains or various proteins (e.g.
  • CD34, CD19, LNGFR also can be employed, permitting a straightforward method for magnetic or fluorescence antibody-mediated sorting.
  • markers can be detected e.g. using a labelled antibody which binds to the protein.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a desired gene product e.g. a subject CIR.
  • the marker should ideally be a polypeptide which is not expressed by the initial (donor) cells, although difference in expression levels can be used in situations where the marker is indeed endogenous to the initial cells.
  • the marker is based on a human proteins as this minimises the risk that cells expressing the marker will be recognised as foreign by a human subject’s immune system (e.g. after they are administered therapeutically).
  • T cells are the desired type of cell
  • human CD proteins which are not naturally expressed by T cells can be used for this purpose.
  • the genetically modified cells provided herein may express a cell surface transgene marker, present on an expression vector that expresses a subject CIR, and/or, in some embodiments, present on an expression vector that encodes a protein other than the CIR, such as, for example a CAR, a pro-apoptotic polypeptide safety switch, or a costimulatory polypeptide.
  • the cell surface transgene marker is a truncated CD19 ( ⁇ CD19) polypeptide (Di Stasi et al. (2011) supra) that comprises a human CD19 truncated at amino acid 333 to remove most of the intracytoplasmic domain (see, e.g., SEQ ID NO: 12 (nucleotides) and SEQ ID NO: 13 (protein)).
  • the extracellular CD19 domain can still be recognised (e.g. in flow cytometry, FACS or MACS) but the potential to trigger intracellular signalling is minimised.
  • CD19 is normally expressed by B cells, rather than by T cells or NK cells, so selection of CD19+ cells permits genetically-modified cells (e.g.
  • T cells, NK cells or NKT cells to be separated from unmodified cells.
  • Another useful marker is CD34, which has a 16 amino acid minimal epitope that is useful as a marker.
  • CD34 which has a 16 amino acid minimal epitope that is useful as a marker.
  • nucleic acids that include a nucleotide sequence encoding a subject chimeric ILT receptor (CIR) (and optionally, other desired polypeptides such as chimeric antigen receptors, signaling polypeptides, safety switches, IL-15, etc.).
  • CIR chimeric ILT receptor
  • other desired polypeptides such as chimeric antigen receptors, signaling polypeptides, safety switches, IL-15, etc.
  • expression constructs for expressing the Chimeric ILT Receptor are provided herein.
  • one or more polypeptides is said to be “operably linked” to a promoter, which indicates that the promoter sequence is functionally linked to a second sequence, wherein the promoter sequence is in the correct location and orientation in relation to that second sequence to control RNA polymerase initiation and transcription of the DNA corresponding to the second sequence, whereby the resulting transcript encodes a polypeptide of interest.
  • a "promoter” is a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • the promoter is a developmentally regulated promoter i.e.
  • expression construct is any type of genetic construct containing a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • the transcript can be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding genes of interest.
  • Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism.
  • A“vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • target cells e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • vector construct e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes
  • expression vector e transfer vector
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • a polynucleotide coding for the CIR is included in the same vector, such as, for example, a viral or plasmid vector, as a polynucleotide coding for a second polypeptide.
  • This second polypeptide may be, for example (and as described elsewhere herein), a downregulator of endogenous proteins, a blocking antibody or Atty Docket No.: NKLT-002WO scFv for inhibitory receptors, a signaling polypeptide, an inducible suicide switch, or a marker polypeptide.
  • a construct may be designed with one promoter operably linked to a nucleic acid comprising a polynucleotide coding for a fusion protein of the polypeptides, linked by a linker polypeptide (e.g. a cleavable linker polypeptide, such as a 2A polypeptide).
  • linker polypeptide e.g. a cleavable linker polypeptide, such as a 2A polypeptide.
  • the two polypeptides may be expressed separately from the same vector, where each nucleic acid comprising a polynucleotide coding for one of the polypeptides is operably linked to a separate promoter.
  • one promoter may be operably linked to the two polynucleotides, directing the production of two separate RNA transcripts, and thus two polypeptides; in one example, the promoter may be bi-directional, and the coding regions may be in opposite directions 5’-3’. Therefore, expression constructs discussed herein may comprise at least one, or at least two promoters.
  • two polypeptides may be expressed in a cell using two separate vectors.
  • the cells may be co- transfected or co-transformed with the vectors, or the vectors may be introduced to the cells at different times.
  • Any combinations of these approaches may be used, in order to achieve expression of desired polypeptides in a genetically modified cell.
  • a nucleic acid construct is contained within a viral vector.
  • the viral vector is a retroviral vector.
  • the viral vector is an adenoviral vector or a lentiviral vector.
  • a cell is contacted with the viral vector ex vivo, and in some embodiments, the cell is contacted with the viral vector in vivo.
  • an expression construct may be inserted into a vector, for example a viral vector or plasmid.
  • the steps of the methods provided may be performed using any suitable method; these methods include, without limitation, methods of transducing, transforming, or otherwise providing nucleic acid to the cell, described herein.
  • the particular promoter employed to control the expression of a polynucleotide sequence of interest is generally not of particular importance, so long as it is capable of directing the expression of the polynucleotide in a desired cell.
  • the polynucleotide sequence-coding region may, for example, be placed adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either Atty Docket No.: NKLT-002WO a human or viral promoter. Promoters may be selected that are appropriate for the vector used to express the CIRs and other polypeptides provided herein.
  • the expression vector is a retrovirus
  • an example of an appropriate promoter is the Murine Moloney leukemia virus promoter.
  • the promoter may be, for example, the CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, ⁇ 2-microglobulin, ribosomal protein 31, phosphoglycerate kinase, EF1 ⁇ , ⁇ - actin, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest.
  • the use of other viral or mammalian cellular promoters which are well known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • the expression vector is a transposon such that the genetic elements encoding a CIR and associated marker proteins, coactivation proteins or inhibitors of endogenous factors or the tumor microenvironment a carried on a plasmid vector carrying elements recognized by a transiently coexpressed transposase.
  • the action of the transposase is to catalyse the fusion of the transgenes carried between repeated elements recognized by the transposase with the cells genome. Examples of transposon systems that can be used in these embodiments are the Sleeping Beauty system and the Piggyback system.
  • the promoter may be, for example the CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, ⁇ 2-microglobulin, ribosomal protein 31, phosphoglycerate kinase, EF1 ⁇ , ß-actin, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase.
  • the methods for introduction of the plasmids for the transposon and transposase to cells is transfection rather than viral transduction.
  • Promoters, and other regulatory elements are selected such that they are functional in the desired cells or tissue.
  • the order of the polynucleotides may vary and may be tested to determine the suitability of the construct for any particular method, thus, the nucleic acid may include the polynucleotides in varying orders, which also take into account a variation in the order of components.
  • the cell is transfected or transduced with the nucleic acid that encodes two of the polynucleotides, and the cell also comprises a nucleic acid Atty Docket No.: NKLT-002WO comprising a polynucleotide coding for a third polypeptide and/or the cell also comprises a nucleic acid comprising a polynucleotide coding for the fourth polypeptide.
  • the cell is transfected or transduced with the nucleic acid that encodes three of the polynucleotides, and the cell also comprises a nucleic acid comprising a polynucleotide coding for the fourth polypeptide.
  • a cell may comprise a nucleic acid comprising the first, second and third polynucleotides, and the cell may also comprise a nucleic acid comprising a polynucleotide coding for a chimeric Caspase-9 polypeptide.
  • a cell may comprise a nucleic acid comprising the first, second and fourth polynucleotides, and the cell may also comprise a nucleic acid comprising a polynucleotide coding for a Chimeric ILT receptor, an scFv modulator of natural ILT2 function or Interleukin-15.
  • Methods for treating a disease where administration of cells (e.g., cells expressing a subject CIR) by, for example, infusion, may be beneficial.
  • the cells may, for example, be used in regeneration, for example, to replace the function of diseased cells.
  • the genetically-modified cells described herein may be used for cell therapy.
  • the terms "treatment,” “treating,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • the terms "individual,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).
  • mammalian sport animals e.g., horses
  • mammalian farm animals e.g., sheep, goats, etc.
  • mammalian pets dogs, cats, etc.
  • rodents e.g., mice, rats, etc.
  • an "effective amount” or “sufficient amount” refers to an amount (e.g., an effective amount of cells) providing, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic agents such as a drug), treatments, protocols, or therapeutic regimens, a detectable response of any duration of time (long or short term), an expected or desired outcome in or a benefit to a subject of any Atty Docket No.: NKLT-002WO measurable or detectable degree or for any duration of time (e.g., for minutes, hours, days, months, years, or cured).
  • the doses of an "effective amount” or “sufficient amount” for treatment typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of a disease, one or more adverse symptoms, disorders, illnesses, pathologies, or complications, for example, caused by or associated with the disease, to a measurable extent, although decreasing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the disease is also a satisfactory outcome.
  • Genetically-modified cells provided herein i.e., cells expressing a subject CIR
  • the cells will usually be delivered to the recipient subject by infusion.
  • the genetically-modified cells may be T cells, iNKT cells, macrophage or NK cells.
  • a typical dose of T or NK cells for therapy in a subject is between 10 5 -10 7 cells/kg.
  • genetically modified T and NK cells of the disclosure can be used in the same manner as known donor leukocyte infusion (DLI), but they have the added benefit of the CIR.
  • a recipient may undergo lymphodepletive conditioning prior to receiving the genetically-modified lymphocytes (and prior to receiving an allograft).
  • the recipient may have a hematological cancer (such as a treatment-refractory hematological cancer) or an inherited blood disorder.
  • a hematological cancer such as a treatment-refractory hematological cancer
  • the recipient may have acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), severe combined immune-deficiency (SCID), Wiskott-Aldrich syndrome (WA), Fanconi Anemia, chronic myelogenous leukemia (CML), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), or multiple myeloma.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • SCID severe combined immune-deficiency
  • WA Wiskott-Aldrich syndrome
  • Fanconi Anemia chronic myelogenous leukemia
  • CML chronic myelogenous leukemia
  • NHL non-Hodgkin lymphoma
  • the recipient of CIR-expressing T cells or NK cells may have non-hematological cancer expressing HLA-G.
  • the recipient may have renal cell cancer (RCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer, neuroblastoma, hepatocellular cancer (HCC), ovarian cancer, endometrial cancer or prostate cancer.
  • the therapeutic cells may be, for example, immune cells such as, for example, T cells, natural killer cells (NK cells), NK-T cells, B cells, tumor infiltrating lymphocytes, or macrophages, or a combination thereof; the therapeutic cells may be, for example, peripheral blood cells, hematopoietic progenitor Atty Docket No.: NKLT-002WO cells, bone marrow cells, or tumor cells.
  • the treatment may be combined with one or more adjuvants (e.g., IL-12, checkpoint inhibitors, IDO inhibitors, etc.).
  • the cells may be delivered to treat a solid tumor, such as, for example, delivery of the cells to a tumor bed.
  • the cells may be delivered to treat a liquid tumor, such as, for example, delivery of the cells to treat a leukemia such as AML.
  • a liquid tumor such as, for example, delivery of the cells to treat a leukemia such as AML.
  • nucleic acids which may be administered to a subject, thereby transforming or transducing target cells in vivo to form the genetically- modified cells in situ.
  • An effective amount of genetically-modified cells is administered. To determine if an effective amount of ligand or modified cells is administered, any means of assaying or measuring the number of target cells, or amount of target antigen, or size of a tumor may be used to determine whether the number of target cells, amount of target antigen or size of a tumor has increased, decreased, or remained the same.
  • Samples, images, or other means of measurement taken before administration of the modified cells or ligand may be used to compare with samples, images, or other means of measurement taken after administration of the modified cells or ligand.
  • a first sample may be obtained from a subject before administration of the ligand or modified cells
  • a second sample may be obtained from a subject after administration of the ligand or modified cells.
  • the amount or concentration of cells expressing the target antigen in the first sample may be compared with the amount or concentration of cells expressing the target antigen in the second sample, in order to determine whether the amount or concentration of cells expressing the target antigen has increased, decreased, or remained the same following administration of the ligand or modified cell.
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, and/or the severity of the disease or condition. One can empirically determine the effective amount of a particular composition presented herein.
  • the administration of the pharmaceutical composition may precede, be concurrent with and/or follow the other agent(s) by intervals ranging from minutes to weeks.
  • NKLT-002WO embodiments where the pharmaceutical composition and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the pharmaceutical composition and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism.
  • one or more agents may be administered from substantially simultaneously, about 1 minute, to about 24 hours to about 7 days to about 1 to about 8 weeks or more, and any range derivable therein, prior to and/or after administering the expression vector. Yet further, various combination regimens of the pharmaceutical composition presented herein, and one or more agents may be employed. [00281] Diseases that may be treated or prevented include diseases caused by viruses, bacteria, yeast, parasites, protozoa, cancer cells and the like.
  • Exemplary diseases that can be treated and/or prevented include, but are not limited, to infections of viral etiology such as HIV, influenza, herpes, viral hepatitis, Epstein Barr, polio, viral encephalitis, measles, chicken pox, papillomavirus etc.; or infections of bacterial etiology such as pneumonia, tuberculosis, syphilis, etc.; or infections of parasitic etiology such as malaria, trypanosomiasis, leishmaniasis, trichomoniasis, amoebiasis, etc.
  • viral etiology such as HIV, influenza, herpes, viral hepatitis, Epstein Barr, polio, viral encephalitis, measles, chicken pox, papillomavirus etc.
  • infections of bacterial etiology such as pneumonia, tuberculosis, syphilis, etc.
  • infections of parasitic etiology such as malaria, try
  • Preneoplastic or hyperplastic states which may be treated or prevented using the pharmaceutical composition include but are not limited to preneoplastic or hyperplastic states such as colon polyps, Crohn's disease, ulcerative colitis, breast lesions and the like.
  • Cancers including solid tumors and/or liquid tumors, which may be treated using the cells include, but are not limited to primary or metastatic melanoma, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, leukemias (e.g., Chronic lymphocytic leukemia, Acute myeloid leukemia, Chronic myeloid leukemia, Acute lymphocytic leukemia), uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, NPC, bladder cancer, cervical cancer and the like.
  • leukemias e.g., Chronic lymphocytic leukemia, Acute myeloid leukemia, Chronic myeloid leukemia, Acute lymph
  • Other hyperproliferative diseases that may be treated using the therapeutic cells and other therapeutic cell activation system presented herein include, but are not limited to rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions (such as adenomatous hyperplasia and Atty Docket No.: NKLT-002WO prostatic intraepithelial neoplasia), carcinoma in situ, oral hairy leukoplakia, or psoriasis.
  • rheumatoid arthritis inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions (such as adenomatous hyperplasia
  • Solid tumors from any tissue or organ may be treated using the present methods, including, for example, any tumor expressing a target antigen, for example, HLA-G, in the vasculature, for example, solid tumors present in, for example, lungs, bone, liver, prostate, or brain, and also, for example, in breast, ovary, bowel, testes, colon, pancreas, kidney, bladder, neuroendocrine system, soft tissue, boney mass, and lymphatic system.
  • Other solid tumors that may be treated include, for example, glioblastoma, and malignant multiple myeloma.
  • Liquid tumors may be treated using the present methods, including, for example, any cancer (e.g., any leukemia or lymphoma) in which the cancer cells express a target antigen, for example, HLA-G.
  • any cancer e.g., any leukemia or lymphoma
  • Subjects may be given a zinc supplement to ensure that any zinc-dependent factors contained within a CIR or the cofactors expressed in a cell therapy product including a CIR have an adequate source of this ion to permit their full activity.
  • transfecting or transducing cells with a nucleic acid or expression vector of the present disclosure (e.g., one encoding for a subject CIR).
  • the term “transfection” is used to refer to the uptake of foreign DNA by a cell.
  • a cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3 rd edition, Cold Spring Harbor Laboratories, New York, Davis et al.
  • a cell of the present disclosure is produced by transducing the cell with a viral vector encoding a CIR.
  • the polypeptide includes a CIR and the cell is a T cell, such that provided are methods of producing a CIR T cell.
  • such methods include activating a population of T cells (e.g., T cells obtained from an individual to which a CIR T cell therapy will be administered), stimulating the population of T cells to proliferate, and transducing the T cell with a viral vector encoding the polypeptide including the CIR.
  • an immune cell e.g., T cells, NK cells, macrophages
  • NKLT-002WO vector e.g., a gamma retroviral vector, or lentiviral vector, or AAV encoding a CIR.
  • the immune cell T cells are transduced with a lentiviral vector encoding the polypeptide.
  • the polypeptide includes a CIR and the cell is an NK cell, such that provided are methods of producing a CIR NK cell (e.g., by using a viral vector such as an AAV, lentiviral, or retroviral vector).
  • a viral vector such as an AAV, lentiviral, or retroviral vector.
  • the word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition.
  • the term “between” with reference to two values includes those two values e.g. the range “between” 10 mg and 20 mg encompasses inter alia 10, 15, and 20 mg.
  • a method comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
  • sequence identity herein means the extent to which two nucleotide or amino acid sequences are invariant.
  • sequence alignment means the process of lining up two or more sequences to achieve maximal levels of identity for the purpose of assessing the degree of similarity.
  • sequences Atty Docket No.: NKLT-002WO of individual polypeptides provided in these examples, such as, for example, the truncated ILT4 D1-D2 polypeptides, co-stimulatory polypeptide cytoplasmic signaling regions, ITAM-containing cytotoxicity regions, IL-15 or safety switches may be used to construct other expression vectors that encode chimeric signaling polypeptides of the present embodiments.
  • a chimeric receptor protein comprising: (a) a targeting region, that targets HLA-G, comprising a D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region, comprising a transmembrane amino acid sequence; and (c) an intracellular domain (ICD), comprising a signaling region capable of transducing a signal, upon binding of said targeting region to HLA-G, into the interior of an immune effector cell to elicit effector cell function, wherein the signaling region comprises a costimulatory region comprising a MyD88 polypeptide.
  • Atty Docket No.: NKLT-002WO The chimeric receptor protein of any one of 1-4, wherein the signaling region comprises a CD3 ⁇ signaling domain, a DAP10 signaling domain, a DAP12 signaling domain, or any combination thereof.
  • the chimeric receptor protein of 4 wherein the signaling region comprises a CD3 ⁇ signaling domain.
  • the chimeric receptor protein of 1 or 2 wherein the signaling region comprises a DAP12 signaling domain.
  • An intracellular domain (ICD) polypeptide comprising a signaling region capable of transducing a signal in an immune effector cell to elicit effector cell function, wherein the signaling region comprises (i) a CD3 ⁇ signaling domain, a DAP10 signaling domain, or a DAP12 signaling domain, and (ii) a costimulatory region that comprises a Toll/Interleukin-1 Receptor/Resistance Protein (TIR) domain.
  • TIR Toll/Interleukin-1 Receptor/Resistance Protein
  • the ICD polypeptide of 10 wherein the TIR domain comprises an amino acid sequence having 85% or more sequence identity with the TLR2 TIR domain of SEQ ID NO: 111.
  • a targeting region that targets HLA-G, comprising a D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4)
  • TM transmembrane
  • a chimeric receptor protein comprising: (a) a targeting region, that targets HLA-G, comprising a D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region, comprising a transmembrane amino acid sequence; and (c) an intracellular domain (ICD), comprising a signaling region capable of transducing a signal, upon binding of said targeting region to HLA-G, into the interior of an immune effector cell to elicit effector cell function, wherein the signaling region comprises a DAP10 signaling domain or a DAP12 signaling domain.
  • a targeting region that targets HLA-G, comprising a D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4)
  • TM transmembrane
  • ICD intracellular domain
  • the chimeric receptor of any one of 17-19, wherein the signaling region does not include a CD3 ⁇ signaling domain.
  • the chimeric receptor of any one of 17-21, wherein the signaling region further comprises a CD40, 4-1BB, or HVEM costimulatory domain.
  • NKLT-002WO The chimeric receptor protein of any one of 1-25, wherein the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain.
  • the chimeric receptor protein of any one of 1-25 wherein: the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain, the extracellular domain lacks an ILT2 D3-D4 extracellular domain, the chimeric receptor protein comprises a CD8 ⁇ stalk domain, and the TM region is a CD8 ⁇ TM.
  • a nucleic acid comprising a nucleotide sequence encoding the chimeric receptor protein of any one of 1-35.
  • the nucleic acid of 36 wherein said nucleotide sequence is operably linked to a constitutive promoter.
  • the nucleic acid of 36, wherein said nucleotide sequence is operably linked to an inducible promoter.
  • a genetically modified cell expressing the chimeric receptor protein of any one of 1-35.
  • the genetically modified cell of 42, wherein the immune cell is a natural killer (NK) cell, an NK-T cell, a T cell, an iNKT cell, or a macrophage.
  • the genetically modified cell of 42, wherein the immune cell is a natural killer (NK) cell.
  • a method of treatment comprising administering the genetically modified cell of any one of 41-44 to an individual in need.
  • the method of 45 wherein the genetically modified cell is autologous to the individual.
  • the method of 45 wherein the genetically modified cell is allogeneic to the individual.
  • the method of any one of 45-47 wherein the individual has cancer.
  • the method of 48 wherein the individual has a solid tumor.
  • the method of 48 wherein the individual has a liquid tumor.
  • a method of producing a genetically modified cell the method comprising: introducing the nucleic acid of any one of 36-40 into a cell, thus producing a genetically modified cell.
  • the method of 51 wherein the genetically modified cell is an immune cell.
  • the method of 52 wherein the immune cell is a natural killer (NK) cell, an NK- T cell, a T cell, an iNKT cell, or a macrophage.
  • NK natural killer
  • iNKT iNKT
  • macrophage a natural killer
  • DNA encoding a CIR was cloned 5’ to a gene encoding Interleukin-15 (IL-15), a growth factor important for sustained NK cell growth and survival that is provided in an autocrine fashion to support CIR-NK cells in culture.
  • IL-15 Interleukin-15
  • the cistrons encoding the ILT4 CIRs and IL-15 and CD19 were separated by the T2A cotranslational cleavage site derived from Thosea asigna virus or the P2A sequence derived from porcine teschovirus to permit separate protein expressing from individual mRNA molecules.
  • ⁇ -Retroviruses were produced from these DNA constructs by transfection into HEK293 cells together with helper plasmids encoding reverse transcriptase and viral capsid and envelope proteins. These retroviral vectors were used to transduce primary human NK cells selected for CD56 expression from peripheral blood mononuclear cells (PBMCs) derived from 2 healthy donors. All NK cells used in comparative experiments were derived from the same donors. The efficiency of transduction was marked by expression of ILT4 present on the CIR, but not normally expressed by NK cells as detected by flow cytometry. Stability of transgene expression was monitored weekly.
  • PBMCs peripheral blood mononuclear cells
  • ANTI-TUMOR EFFICACY WITH CIR-NK CELLS WITH ALTERED ITAM- CONTAINING CYTOTOXICITY DOMAINS [00378]
  • AML Acute Myeloid Leukemia
  • KG1-GFPffluc cells did not express HLA-G protein detectable by flow cytometry or HLA-G1, HLA-G2 mRNA that encodes the most common membrane bound HLA-G isoforms determined by quantitative PCR.
  • Molm13-GFPffluc and Kasumi1- GFPffluc cells are AML lines that express low, but measurable levels of HLA-G1 protein and HLA-G1 and HLA-G5 mRNA. These cells were used to engage and activate CIR-NK cells.
  • NKLT-002WO primary human NK cells from two healthy donors Atty Docket No.: NKLT-002WO were transduced with ⁇ -retroviruses encoding the extracellular and stalk/transmembrane domain described in Example 1 and a CD3 ⁇ , DAP10 or DAP12 intracellular domain. It is notable that DAP10 does not contain a canonical ITAM domain but instead signals through mechanisms similar to CD28 in T cells. No coactivation domain was present in the expressed CIR. A separate ⁇ -retrovirus encoding Red Fluorescent Protein (RFP) was co-transduced to assess NK cell growth during coculture experiments.
  • RFP Red Fluorescent Protein
  • Mock transduced (cells that were manipulated identically as transduced cells, but without virus) and cells transduced with RFP without a CIR retrovirus were produced as negative controls.
  • 2 x 105 CIR-NK cells were cultured without a target to assess the degree of tonic NK cell activity that may be induced by signaling domains present on a CIR construct.
  • 2 x 103 of the same NK cells were cocultered with HLA-G+ Kasumi1 cells at an effector to target (E:T) ratio of 1:5.
  • E:T effector to target
  • Production of the cytokines Tumor Necrosis Factor- ⁇ (TNF- ⁇ ) and Interferon- ⁇ was determined to assess tonic and stimulated NK cell activity ( Figures 3 and 4, respectively).
  • Results were normalized to a cell number of 2 x 105 for comparison and assessment of NK cell stimulation.
  • Tonic secretion of TNF- ⁇ and IFN- ⁇ was elevated in CIR-NK cells relative to NK cells lacking CIR expression. Following stimulation with Kasumi1 target cells, overall cytokine secretion was elevated in mock or RFP-transduced NK cells indicating innate targeting of Kasumi1 cells.
  • CIR-NK cells were not further stimulated for TNF- ⁇ production, but ILT4.DAP12 CIR-NK cells were markedly elevated for IFN- ⁇ production following Kasumi1 stimulation relative to CIR-NK cells expressing DAP10 or CD3 ⁇ or control NK cells.
  • IFN- ⁇ production is a commonly used surrogate marker of NK cell activation.
  • DAP12 may be a superior signaling moiety for the promotion of inflammation at a tumor site.
  • NK cells expressing RFP Control cultures with NK cells expressing RFP but no CIR did not control KG1 cell outgrowth relative to cultures lacking NK cells (Tumor only) and innate activity of CIR-NK cells was low.
  • CIR-NK cells containing DAP12 were capable of enhanced control of Molm13 cell outgrowth relative to CIR-NK cells expressing DAP10 or CD3 ⁇ .
  • NK cell proliferation over seven days was measured in the Incucyte by imaging of the RFP marker expressed in the NK cells.
  • Enhanced CIR-NK cell proliferation was found with each of the CIR activation domains relative to NK cells expressing only RFP.
  • NKLT-002WO results provided further indication that DAP12 provided superior NK cell performance relative to CD3 ⁇ or DAP10 when expressed without a coactivation signaling moiety.
  • constructs were prepared that included both DAP10 and CD3 ⁇ or DAP12 and CD3 ⁇ in the intracellular domain CIR-NK cell performance was compared with CIR-NK cells expressing each signaling element alone. Tonic and Kasumi1 target-stimulated cytokine production was compared and addition of CD3 ⁇ signaling with DAP10 or DAP12 did not augment the production of TNF- ⁇ ( Figure 7) or IFN- ⁇ ( Figure 8).
  • 4-1BB (TNFRS9) and HVEM (TNFRS14) are members of the TNF Receptor superfamily and each can signal through the NF- ⁇ B and other pathways to promote cytokine gene transcription and cytokine release. These cooperate with ITAM-directed signaling pathways to promote the growth and survival of immune cells. These properties are termed costimulation in T cells and coactivation in NK cells. Retrovirus vectors were prepared that contain 4-1BB or HVEM together with the CD3 ⁇ cytotoxicity domain and CIR-NK cells transduced with these constructs were evaluated relative to CIR-NK cells expressing CD3 ⁇ signaling elements alone.
  • Tonic NK cell signaling to production of TNF- ⁇ was not impacted by inclusion of 4-1BB or HVEM, but signaling to drive IFN- ⁇ production was enhanced when 4-1BB was included but not when HVEM was included ( Figure 12 and Figure 13).
  • Production of TNF- ⁇ and IFN- ⁇ by CIR-NK cells activated by coculture with Kasumi1 targets was not impacted by inclusion of 4-1BB or HVEM.
  • Coactivation by 4-1BB reduced tonic production of TNF- ⁇ and IFN- ⁇ when combined with DAP12 signaling with or without further CD3 ⁇ signaling while DAP10 signaling to cytokine production was relatively unaffected by combination with 4-1BB ( Figure 15, Figure 16).
  • Target-specific cytokine production by DAP12-containing CIR-NK cells in cocultures with Kasumi1 cells was markedly enhanced by inclusion of 4-1BB with or without the further inclusion of CD3 ⁇ .
  • Cytotoxicity against HLA-G- KG1 cells was unaffected by 4-1BB inclusion with DAP10, DAP12 or CD3 ⁇ alone or in combination ( Figure 17).
  • Target-specific cytotoxicity against Molm13 cells was improved by addition of 4-1BB with DAP10 and CD3 ⁇ (ILT4.4- 1BB.DAP10.CD3 ⁇ ), but other combinations of DAP10 and DAP12 with 4-1BB did not improve cytotoxicity against Molm13 ( Figure 18).
  • MyD88 is a cytoplasmic protein that is recruited to receptors including IL-1 type receptor and Toll-like receptors.
  • Retroviral constructs encoding CIRs were created that added only the Death Domain signaling elements of MyD88 but not the receptor-engaging TIR domain as an element to replace 4-1BB coactivation together with CD3 ⁇ , DAP12 or DAP12/CD3 ⁇ . Tonic production of TNF- ⁇ was only impacted by MyD88 inclusion in the combination of DAP12, and MyD88 signaling had little effect of target-specific TNF- ⁇ production (Figure 19).
  • Tonic IFN- ⁇ production by CIR-NK cells was higher when MyD88 was included with CD3 ⁇ , DAP12 or DAP12/CD3 ⁇ ( Figure 20).
  • MyD88 coactivation did not lead to enhanced IFN- ⁇ or TNF- ⁇ production when CIR-NK cells were cocultured with Kasumi1 targets.
  • Cytotoxicity against HLA-G-negative KG1 cells was not observed with or without MyD88 signaling, but enhanced NK cell proliferation was observed when MyD88 was included with each ITAM-containing cytotoxicity domain (ILT4.MyD88.CD3 ⁇ , ILT4.MyD88.DAP12, ILT4.MyD88.DAP12.CD3 ⁇ ) ( Figure 21).
  • Retroviral constructs were created that combine MyD88 with 4-1BB, HVEM or CD40 together with the CD3 ⁇ cytotoxicity element in the intracellular signaling domain of the ILT4 CIR.
  • CIR-NK cells expressing these so-called 3rd generation CIR constructs (ILT4.MyD88.4-1BB.CD3 ⁇ , ILT4.MyD88.HVEM.CD3 ⁇ , ILT4.MyD88.CD40.CD3 ⁇ ) were compared with CIR-NK cells with intracellular domains containing CD3 ⁇ only, MyD88.CD3 ⁇ , 4-1BB.CD3 ⁇ and HVEM.CD3 ⁇ .
  • Tonic cytokine production was enhanced in all CIR-NK cells containing MyD88 coactivation elements.
  • Native MyD88 is recruited to IL-1 family receptors and TLRs by interaction between TIR domains on the receptor and a TIR domain on MyD88. Downstream signaling is directed by the separate MyD88 Death Domain that was incorporated in the CIRs described in Example 4.
  • constructs were Atty Docket No.: NKLT-002WO created in which CD3 ⁇ cytotoxicity domains were fused with the TIR domain of the Interleukin 18-Receptor 1 (or ⁇ ) chain (ILT4.IL18R1.CD3 ⁇ ). Further constructs were created that recruit MyD88 indirectly.
  • TLR2 The intracellular TIR domain of TLR2 interacts with the TIR domain of MAL which further interacts in a ternary complex with MyD88.
  • a CIR construct was created that fused the TLR2 CIR with CD3 ⁇ .
  • a still further CIR construct fused the TIR domain of TLR3 with CD3 ⁇ .
  • the TIR domain of TLR3 does not interact with MyD88, but instead recruits TRIF through its TIR domain.
  • TRIF signaling has overlapping and distinct downstream signaling pathways to MyD88.
  • Tonic cytokine production was low in CIR-NK cells expressing TIR domains, much lower than the ILT4.MyD88.CD3 ⁇ direct CIR fusion, and TNF- ⁇ production in activated TIR-containing CIR-NK cell cocultures with Kasumi1 cells was not enhanced relative to direct ILT4.MyD88.CD3 ⁇ cocultures ( Figure 27).
  • IFN- ⁇ was dramatically stimulated by CIR engagement of each TIR domain-containing CIR-NK cell coculture with Kasumi1 cells ( Figure 28). Both innate killing activity (Figure 29) and target- specific killing of Molm13 cells (Figure 30) was not elevated by TIR domain-containing CIR-NK cells possibly due to insufficient expression to support cytotoxic signaling.
  • MyD88 signaling is activated by oligomerization of the signaling death domains.
  • the low tonic signaling for IFN- ⁇ production and high inducibility by CIR-target engagement supports the hypothesis that TIR domain receptors are engaged by dimeric HLA-G and themselves dimerize. This creates CIR-NK cells that have low tonic activity and high inducible potential.
  • EXAMPLE 6 COMPARISON OF ILT4 AND ILT2 BINDERS WITH ALTERNATE SIGNALING DOMAINS. [00392] CIR constructs described in the previous examples that demonstrated the highest levels of enhanced potency when expressed in NK cells were selected and the ILT4 binder replaced with ILT2. The signaling domains described in this example are listed in Figure 31.
  • Each construct contained a CIR and IL-15 separated by a 2A sequence and RFP encoding cistron to mark the cells and assess transduction efficiency. These constructs were transduced into NK cells to produce CIR-NK cells and the CIR expression level, viability of NK cells with different signaling capabilities, capacity to produce cytokines in the presence and absence of target and cytotoxicity against a range of targets expressing HLA-G was determined. [00393] Viability of NK cells at day 8 and day 14 was determined by staining with Actinomycin D and propidium iodide such that the integrity of the cell membrane to exclude dye measured individual cell viability (Figure 32).
  • the growth potential of transduced cells was examined by counting populations at day 5, day 8 and day 14 and comparing the expanded level of cells at day 8 or day 14 relative to that at day 5 ( Figure 33).
  • All CIR- NK cells expressing an ILT4 binder expanded well through to day 14, but CIR-NK cells with an ILT2 binder and only a first-generation (1G) CAR (ILT2.CD3 ⁇ ), a 4-1BB domain or MyD88 domain linked to DAP10 and CD3 ⁇ failed to expand significantly between days 8 and 14.
  • ILT2 CIR-NK cells expanded readily.
  • the mean levels of ILT4 CIR expression were determined in CIR-NK cells with each alternative signaling domain at day 8 and day 14 by flow cytometry with an antibody specific for the ILT4 D1/D2 domain.
  • CIR expression was high at day 8 (five days post- transduction) and generally stabilized to an appreciable mean fluorescence intensity (MFI) at day 14 ( Figure 34).
  • MFI mean fluorescence intensity
  • CIR-NK cells with MyD88 linked to all domains readily produced tonic IFN- ⁇ which is a good marker for signaling transduced through the NF- ⁇ B pathway following expansion to day 14 (Figure 35).
  • Levels were elevated relative to a first-generation CAR (1G) lacking coactivation and comparable to constructs expressing 4-1BB, DAP12, DAP10 and recruiting endogenous MyD88 through fusion with the TIR domain of TLR2 in various combinations.
  • CIR-NK cells expressing MyD88 demonstrated more potent production of IFN- ⁇ than CIR-NK cells not containing MyD88 though all CIR-NK with coactivation domains demonstrated enhanced cytokine production relative to 1G CIR-NK cells.
  • levels of cytokine production upon CIR stimulation were enhanced relative to tonic levels ( Figure 35).
  • ILT2 and ILT4 CIR-NK cells containing enhanced activation domains were potently cytotoxic against a range of HLA-G expressing target cells, but not against HLA-G- target cells.
  • NKLT-002WO observed when the same CIR-NK cell groups were cocultured with Kasumi1 cells that also express HLA-G but are somewhat harder to kill ( Figures 41 and 42).
  • CIR-NK cells also demonstrated potent cytotoxicity and selectivity against target cell lines derived from solid tumors.
  • Levels of HLA-G RNA were measured in HCT-116 cells derived from a human colon carcinoma and HT-1376 cells derived from a human bladder carcinoma. HCT-116 expressed low to no HLA-G RNA while HT-1376 expressed the HLA-G1, HLA-G2 and HLA-G5 isoforms.
  • HLA-G was determined by flow cytometry with the MEMG/9 antibody and HLA-G was readily detected in HT-1376 cells, but not HCT-116 cells ( Figure 43). CIR-NK cell cytotoxicity was readily observed with HT-1376 cells ( Figure 44) but not with HCT-116 cells ( Figure 45).
  • SU8686 cells are derived from a pancreatic ductal adenocarcinoma (PDAC) and were previously noted to be particularly hard to kill with PSCA-targeted CAR-T cells despite robust expression of that target.
  • PDAC pancreatic ductal adenocarcinoma
  • SU8686 cells robustly express HLA-G1 on the cell surface ( Figure 46) and isoform-specific QPCR detected abundant HLA-G1 (9359 copies/cell) and HLA-G2 (77,668 copies/cell) expression in SU8686 cells. While speculative, it is possible that the resistance of this cell line to targeted cell therapy was due to immunosuppression by HLA-G through ILT2. In cocultures of mock- transduced or CIR-NK cells with SU8686 cells little innate cytotoxicity was observed with the mock-transduced cells lacking CIR expression while ILT2 and ILT4 CIR-NK cells could recognize and target SU8686 cells effectively ( Figures 47 and 48).
  • CIR-NK cells are potent at high effector to target ratios of up to 1:40 demonstrating that many or most CIR-NK cells have so-called ‘serial killing’ ability.
  • CIR-NK cells are also effective to control the outgrowth of target cells derived from solid tumors indicating that this technology has the potential for development as a therapy against a wide range of leukemia and solid tumor indications.
  • NK cells have a limited functional life span in a natural setting, typically about 2 weeks, and are constantly replenished by hematopoiesis. As a cell therapy product, it is advantageous to maximize persistence of NK cell potency to treat a high tumor burden. To examine the persistence of CIR-NK cell functionality and evaluate the capacity of different activation domains to extend this potency, cocultures of ILT4 CIR- NK cells grown for a standard 14 days or extended for 26 days were prepared with Molm13 target cells at and E:T of 1:20.
  • Activation domains contained the cytotoxicity domains of CD3 ⁇ , DAP10 and DAP12 in varying combinations and were compared with the same combinations with 4-1BB coactivation or the TIR domain of TLR2 in varying amino-to-carboxy orientations of TLR2 relative to the cytotoxicity domains ( Figure 49).
  • Each of the CIR-NK cells was highly cytotoxic against Molm13 targets when expanded for 14 days. This potency was reduced in coculture containing ‘older’ first generation CIR-NK cells grown for 26 days and in several of the CIR-NK cells with alternative activation domains.
  • CIR-NK cells containing 4-1BB in combination with DAP10 or DAP12 maintained potency at 26 days as did combinations of 4-1BB with DAP10 and TLR2 (BB.DAP10.TLR2).
  • BB.DAP10.TLR2 TLR2
  • mock-transduced or CIR-NK cells were repeatedly challenged with Molm13 tumor target cells up to 4 times over a 9-day culture period ( Figure 50). Innate cytotoxicity of mock-transduced NK cells was relatively robust against this target line and was exhibited when Molm13 were cultured only one time with NK cells and quickly exhausted if further Molm13 targets were added at day 2.
  • First generation CIR-NK cells had reduced potency with 3 or more serial additions of tumor target.
  • Enhanced signaling elements were differentiated with 3 or 4 additions of Molm13 target and it was evident that the BB.DAP10 and particularly the BB.DAP10.TLR2 combination of signaling elements with either an ILT2 or ILT4 ‘binder’ maintained NK cell potency with repeated tumor challenge.
  • This finding was confirmed and extended in a similar experiment with a refined cohort of candidate CIR-NK cells.
  • the combination of BB.DAP10.TLR2 on ILT2 and ILT4 CIRs exhibited the most effective maintenance of NK cell cytotoxicity (Figure 51) and proliferation (Figure 52) relative to other CIRs and NK cells transduced only with the RFP marker.
  • OE19 cells are derived from an esophageal tumor and express relatively low levels of HLA-G1 (2080 RNA copies/cell) and HLA-G2 (899 copies/cell). Despite relatively low levels of HLA-G expression, ILT4 CIR-NK cells specifically target OE19 cells with CIR-NK cells harboring a BB.DAP10.TLR2 activation domain particularly effective at an E:T ratio of 1:10 ( Figure 57).
  • NK cells were cocultured with the same or different tumor targets expressing varying levels of HLA-G expression.
  • CIR-NK cells containing BB.DAP10 or BB.DAP10.TLR2 were most effective for tumor control ( Figures 60 and 61).
  • Donor variation was wide and somewhat expected in this model system. NK cell expansion in coculture was most evident with the BB.DAP10 combination while expansion of BB.DAP10.TLR2 was relatively poor after 26 days of tumor exposure.
  • NSG mice are immunodeficient due to the Non-obese diabetic (NOD) MHC haplotype, mutation at the Severe Combined Immunodeficiency (Scid) locus and nullizygosity for the IL-2 receptor ⁇ -chain.
  • NOD Non-obese diabetic
  • Scid Severe Combined Immunodeficiency locus
  • nullizygosity for the IL-2 receptor ⁇ -chain This mouse model is severely limited in murine T, B and NK cell numbers and supports the engraftment of human cells.
  • mice were euthanized by day 17 and blood, spleen and bone marrow collected from select groups including two control populations (Mock and first generation CIR-NK cell) and the groups of ILT2 and ILT4 CIR-NK cells with BB.DAP10.TLR2.
  • Molm13-GFPffluc cells were widely present in the femoral bone marrow of mice with mock-transduced or first generation CIR-NK cells but were absent in CIR-NK cells with enhanced BB.DAP10.TLR2 signaling ( Figure 66). Further examination by flow cytometry revealed that mock transduced NK cells did not effectively engraft and traffic to bone marrow ( Figure 67). First generation (CD3 ⁇ ) CIR-NK cells were abundant in the bone Atty Docket No.: NKLT-002WO marrow but failed to control Molm13. Conversely, ILT2 and ILT4 effectively trafficked to and had sufficient cytotoxicity to control the expansion of Molm13 in the bone marrow.
  • Molm13 cells are derived from an acute myeloid leukemia (AML), a tumor known to populate the bone marrow of patients and devastate hematopoiesis.
  • AML acute myeloid leukemia
  • CIR-NK cells with enhanced activation to traffic to bone marrow and target HLA-G expressing AML indicates that CIR technology in combination with these activation domains can offer an effective NK cell therapeutic option for AML patients.
  • ⁇ 112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for” or the exact phrase “step for” is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. ⁇ 112 (f) or 35 U.S.C. ⁇ 112(6) is not invoked.

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Abstract

L'invention concerne des récepteurs ILT chimériques (CIR) qui comportent une région de ciblage d'ILT2 ou d'ILT4, un domaine transmembranaire et un domaine intracellulaire (ICD). L'ICD comporte une région de signalisation (p. ex., CD3 zêta (CD3ζ), DAP10, DAP12) et éventuellement une région costimulatrice (p. ex., MyD88, un domaine TIR et analogues). L'invention concerne en outre des acides nucléiques (p. ex., vecteurs d'expression) codant un CIR sujet, et des cellules génétiquement modifiées (p. ex., des cellules immunitaires telles que des cellules NK, des cellules NK-T, des cellules T, des cellules iNKT, des macrophages et analogues) exprimant un CIR sujet.
PCT/US2024/039649 2023-07-28 2024-07-25 Compositions et méthodes de récepteur ilt chimérique Pending WO2025029597A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210147507A1 (en) * 2017-05-09 2021-05-20 Bellicum Pharmaceuticals, Inc. Methods to augment or alter signal transduction
US20210252058A1 (en) * 2018-05-17 2021-08-19 St. Jude Children's Research Hospital, Inc. Chimeric antigen receptors with myd88 and cd40 costimulatory domains
WO2022178367A2 (fr) * 2021-02-19 2022-08-25 University Of Southern California Récepteurs d'antigènes synthétiques à chaîne unique et à chaînes multiples pour diverses cellules immunitaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210147507A1 (en) * 2017-05-09 2021-05-20 Bellicum Pharmaceuticals, Inc. Methods to augment or alter signal transduction
US20210252058A1 (en) * 2018-05-17 2021-08-19 St. Jude Children's Research Hospital, Inc. Chimeric antigen receptors with myd88 and cd40 costimulatory domains
WO2022178367A2 (fr) * 2021-02-19 2022-08-25 University Of Southern California Récepteurs d'antigènes synthétiques à chaîne unique et à chaînes multiples pour diverses cellules immunitaires

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