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WO2025193475A1 - Amélioration des réponses innées et adaptatives de cellules immunitaires - Google Patents

Amélioration des réponses innées et adaptatives de cellules immunitaires

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Publication number
WO2025193475A1
WO2025193475A1 PCT/US2025/018370 US2025018370W WO2025193475A1 WO 2025193475 A1 WO2025193475 A1 WO 2025193475A1 US 2025018370 W US2025018370 W US 2025018370W WO 2025193475 A1 WO2025193475 A1 WO 2025193475A1
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cells
cell
recombinant polypeptide
domain
polypeptide
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Avishai SHEMESH
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Publication of WO2025193475A1 publication Critical patent/WO2025193475A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K40/40—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
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • 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
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K14/70521—CD28, CD152
    • 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
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • 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
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • 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

  • NK cells are highly cytotoxic immune effectors, killing their targets in a non-specific manner. NK cells lack the potential to cause graft-versus-host disease (GVHD) and thus open opportunities to produce an off-the-shelf allogeneic product that could be readily available for immediate clinical use. Thus, the inherent qualities of NK cells make them promising candidates for immunotherapy. Strategies that improve the innate and/or adaptive anti-tumor activity of NK cells and T cells are of great clinical value.
  • GVHD graft-versus-host disease
  • a recombinant polypeptide comprising: (i) the transmembrane domain of Fc ⁇ R1 ⁇ or CD3 ⁇ , or a variant of the transmembrane domain that binds to NKp30, NKp46 and/or CD16, (ii) a co-stimulatory domain, and (iii) one or more immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • NKp30 which are natural NK cell activating receptors
  • CD16 which is the Fc receptor and mediates antibody-dependent cellular cytotoxicity (ADCC)
  • transmembrane domains of these proteins are mediated by the transmembrane domains of these proteins and, as such, are expected to occur in recombinant polypeptides that comprise a transmembrane domain of an adapter protein selected from Fc ⁇ R1 ⁇ and CD3 ⁇ , or a variant of the transmembrane domain that retains the ability to bind to NKp30, NKp46 and/or CD16.
  • the Fc ⁇ R1 ⁇ and CD3 ⁇ adapter proteins do not have an extracellular binding domain in their wild-type form and, as such, in several embodiments the recombinant peptide may not have an extracellular binding domain.
  • the NK cells or T cells may be activated via endogenous signaling pathways (which may increase the expression and activation of the NKp30, NKp46, and/or CD16 receptors). It has also been found that adding an extracellular binding domain to these recombinant polypeptides provides a potent way to activate NK cells or T cells when they bind to a specific antigen on a cancer cell.
  • the recombinant protein may comprise an extracellular binding domain, e.g., a scFv or nanobody, that recognizes a cancer antigen.
  • FIGS. 1A-1F Fc ⁇ R1 ⁇ 41BB increases NK cell NKp30 and NKp46 expression.
  • FIG. 1A Schematic representation of the Fc ⁇ R1 ⁇ 41BB (FCRG 41BB ) design relative to Fc ⁇ R1 ⁇ (FCRG) native form.
  • FIG. 1B Fc ⁇ R1 ⁇ constructs were overexpressed in the Fc ⁇ R1 ⁇ -negative CD16- negative human natural killer cell line, NK92.
  • FIG. 1A Schematic representation of the Fc ⁇ R1 ⁇ 41BB (FCRG 41BB ) design relative to Fc ⁇ R1 ⁇ (FCRG) native form.
  • FIG. 1B Fc ⁇ R1 ⁇ constructs were overexpressed in the Fc ⁇ R1 ⁇ -negative CD16- negative human natural killer cell line, NK92.
  • FIG. 1C Flow-cytometry histograms of Fc ⁇ R1 ⁇ SF2024-070-2 or CD3 ⁇ intracellular protein expression in the transduced NK92 cells (Parental: bottom, FCRG middle, or FCRG 41BB : top).
  • FIG. 1D Flow-cytometry histograms of NKp30 or NKp46 protein surface expression in the transduced NK92 cells (Parental: bottom, FCRG: middle, or FCRG 41BB : top).
  • FIG. 1E Flow-cytometry histograms of NKp30 or NKp46 protein surface expression in the transduced FCRG 41BB positive NK92 cells following FCER1G gene CRISPR-knockout.
  • FIG. 1F Flow-cytometry histograms of the inhibitory receptor NKG2A, the activating receptor NKG2D or NKp44, or the activation markers 41BB or TRAIL surface protein expression between parental (bottom), FCRG (middle), or FCRG 41BB (top) NK92 cells.
  • FIGS. 2A-2C Fc ⁇ R1 ⁇ 41BB increases NK cell NKp30 function.
  • FIG. 2A Top panels: schematic representation of the pro-B murine cell line, Ba/F3 (left), engineered to express B7H6 (activating NKp30 ligand) (middle) or both B7H6 and HLA-E HLA-G (inhibitory NKG2A ligand) (right). Lower panels: flow-cytometry dot-plot panels of the expression of B7H6 vs. HLA-E for cell lines corresponding to top panels.
  • FIG. 2B 5 hours lysis assay: NK92 cells were co-cultured with Ba/F3 cells at an effector to target (E:T) ratio of 1:1.
  • FIG. 2C 24 hours lysis assay: NK92 cells were co-cultured with Ba/F3 cells at an effector to target (E:T) ratio of 1:4, 1:8, or 1:16. (left to right: Ba/F3, Ba/F3-B7H6+, Ba/F3-B7H6+HLA-E+). Mean+/ SEM, unpaired t-test, one-tailed, *p ⁇ 0.05, **p ⁇ 0.001, ***p ⁇ 0.001.
  • FIGS. 3A-3D 24 hours lysis assay: NK92 cells were co-cultured with Ba/F3 cells at an effector to target (E:T) ratio of 1:4, 1:8, or 1:16. (left to right: Ba/F3, Ba/F3-B7H6+, Ba/F3-B7H6+HLA-E+). Mean+/ SEM, unpaired t-test, one-tailed, *p ⁇ 0.05, **p ⁇ 0.001, ***p ⁇ 0.00
  • FIG. 3A Median binding values of NK cell receptors- human Fc fusions to the human cell line, K562. Mean ⁇ SEM.
  • FIG. 3B Flow-cytometry histograms of B7H6 surface expression in Ba/F3 cells or K562 cells relative to FMO controls. K562 cells express lower B7H6 levels.
  • FIG. 3C 24 hours lysis assay: parental, FCRG, or FCRG 41BB NK92 cells were co-cultured with K562 cells at an effector to target (E:T) ratio of 2:1 to 1:16.
  • E:T effector to target
  • FIG. 4A SF2024-070-2 CD16 was overexpressed in the CD16-negative NK92 cell line.
  • Right panel Flow-cytometry histograms of CD16 surface expression in NK92 cells.
  • CD107a assay CD16+ NK92 cells or NK92 cells were stimulated with anti-NKp30, anti-NKp46, anti-CD16, or anti- NKG2D mIgG1 coated beads relative to mIgG1 control. Mean ⁇ SEM, unpaired t-test, one- tailed, ***p ⁇ 0.001.
  • FIG. 4C Flow-cytometry histograms of NKp30 or CD16 in NK92 cells (parental: bottom, CD16+: second from bottom, CD16+ FCRG: second from top, or CD16+ FCRG 41BB : top).
  • FIG. 4D Left panel: 5 hours CD107a assay where CD16+, CD16+ FCRG, or CD16+ FCRG 41BB NK92 cells were stimulated with anti-CD16 beads. Right panel: negative control: stimulation of CD16 negative cells with anti-CD16 beads. Mean ⁇ SEM, unpaired t-test, one-tailed, **p ⁇ 0.001, ***p ⁇ 0.001.
  • FIG. 4E Flow-cytometry histograms of human CD20 expression in Ba/F3 cells (top left). Schematic representation of lysis assay by ADCC using Rituximab (anti-hCD20, top right).
  • FIGS. 5A, 5B CD19 ScFv-CD8hinge fused to Fc ⁇ R1 ⁇ 41BB increases NKp30 and CD16 expression.
  • FIG. 5A Schematic representation of CD19 ScFv CD8 Hinge fused to Fc ⁇ R1 ⁇ 41BB (FCRG10; X1 ITAM) design relative to Fc ⁇ R1 ⁇ 41BB (FCRG 41BB, X1 ITAM) or CD19CAR 41BB- CD3 ⁇ (X3 ITAMs) design.
  • a Myc-tag was integrated into the N-terminal of FCRG10 or CD19CAR 41BB-CD3 ⁇ for detection of surface expression.
  • FIGS. 6A-6F CD19 ScFv- Fc ⁇ RI ⁇ 41BB CAR improves NK cell function against HLA-E + target cells.
  • FIG. 6A Top row: schematic representation of Ba/F3 cells engineered to co- express human CD19, B7H6 and HLA-E.
  • FIG. 6B Flow-cytometry histograms of human CD19 surface expression in Ba/F3 cells.
  • FIGS. 6C-6F 5 hours lysis assay: NK92 cells were co- cultured with Ba/F3 cells at an effector to target (E:T) ratio of 1:1.
  • E:T effector to target
  • FIGS. 7A, 7B CD19 ScFv- Fc ⁇ RI ⁇ 41BB CAR is functional in primary human NK cells.
  • FIG. 7A Flow-cytometry histograms of CD19 ScFv- Fc ⁇ RI ⁇ 41BB CAR surface expression in primary human NK cells.
  • FIG. 7B 5 hours lysis assay: CD19 ScFv- Fc ⁇ RI ⁇ 41BB CAR positive or non-transduced primary human NK cells were co-cultured with CD19+ Ba/F3 cells at an effector to target (E:T) ratio of 1:5.
  • FIG. 8A illustrates certain exemplary fusion proteins of the present disclosure.
  • FIG. 8B shows sequence alignments of the transmembrane domains of various proteins. D ETAILED D ESCRIPTION
  • the 8A is a recombinant polypeptide comprising: (i) the transmembrane domain of Fc ⁇ R1 ⁇ of CD3 ⁇ , or a variant of the transmembrane domain that retains the ability to bind to NKp30, NKp46 and/or CD16, (ii) a co-stimulatory domain, and (iii) one or more immunoreceptor tyrosine-based activation motifs (ITAMs).
  • the co-stimulatory domain and the one or more immunoreceptor tyrosine-based activation motifs (ITAMs) are intracellular domains and, in these embodiments, may be in either order.
  • the full-length sequence of the wild-type Fc ⁇ R1 ⁇ (including the signal sequence, which is not part of the mature protein) is deposited at Genbank as accession numbers NM_004106.2 and EAW52623.1 (which deposit is incorporated by reference herein).
  • Fc ⁇ R1 ⁇ has a single ITAM.
  • the wild type Fc ⁇ R1 ⁇ transmembrane domain has the following amino acid sequence: LCYILDAILFLYGIVLTLLYCRLK; SEQ ID NO: 1.
  • the full sequence of the wild-type CD3 zeta chain (referred to herein as CD3 ⁇ ) is deposited at Genbank as accession number XP_011508446.1 (which deposit is incorporated by reference herein).
  • CD3 ⁇ has three ITAMs.
  • the wild type CD3 ⁇ transmembrane domain has the following amino acid sequence: LCYLLDGILFIYGVILTALFLRVK (SEQ ID NO: 2). While many embodiments of the recombinant polypeptide comprise the wild-type transmembrane domain of Fc ⁇ R1 ⁇ (i.e., SEQ ID NO: 1) or CD3 ⁇ (i.e., SEQ ID NO: 2) in certain instances the recombinant polypeptide may comprise a variant of the wild-type transmembrane domain of Fc ⁇ R1 ⁇ or CD3 ⁇ defined by the following consensus sequence, which was generated by comparing the Fc ⁇ R1 ⁇ , or CD3 ⁇ transmembrane domains (which bind to the same or overlapping natural cytotoxicity receptors or CD16) and to the orthologs from other species (see FIG.
  • a variant sequence may comprise the conserved Cys at position 2, the conserved Asp at position 6, and up to 5 amino acid substitutions (e.g., 1, 2, 3, 4, or 5 substitutions) relative to SEQ ID NO: 1 or 2. For example, since this is a transmembrane domain, up to five of the hydrophobic residues can be substituted with another hydrophobic amino acid.
  • the recombinant polypeptide may lack an extracellular binding domain and, as such, the mature form of the recombinant polypeptide may have the same N- terminus as a wild-type adapter protein (which also lacks an extracellular binding domain).
  • the protein may have less than 50, less than 20, less than 10 or less than 5 amino acids that are extracellular (N-terminal to the transmembrane domain) and those amino acids do not form an extracellular binding domain.
  • the recombinant polypeptide may comprise an extracellular binding domain, e.g., a scFv or nanobody, that recognizes a cancer antigen.
  • the recombinant polypeptide may follow the general design of a chimeric antigen receptor (CAR).
  • CARs can be designed in several ways (see, generally, e.g., Guedan et al, Methods and Clinical Development 201912: 145-156).
  • CARs generally include an extracellular domain that contains an antigen binding domain such as a scFv or nanobody, a hinge, a transmembrane region, an ITAM and a co-stimulatory domain.
  • the recombinant polypeptide may comprise, in order from N- to C-, the extracellular binding domain, the transmembrane domain, the co-stimulatory domain and the one or more ITAMs.
  • the co-stimulatory domain may be the co-stimulatory domain of CD28, ICOS, CD27, 4 ⁇ 1BB, OX40, CD40L, DNAM1 or 2B4 (as reviewed in Weinkove Clin Transl Immunology. 2019; 8: e1049, among others). These domains have been proposed for chimeric antigen receptors and can be readily used herein.
  • the co- stimulatory domain may be the 4-1BB co-stimulatory domain, as illustrated in the examples section of the present application.
  • the present recombinant polypeptide may contain a single co-stimulatory domain.
  • the recombinant SF2024-070-2 polypeptide may contain multiple co-stimulatory domains (e.g., 2, 3, 4 or 5 co-stimulatory domains).
  • the recombinant polypeptide may contain one or more other signaling domains (e.g., STAT3, STAT4, STAT5 signaling domains), gene regulatory domains such an NFAT regulatory domain, or sites for recruiting signaling proteins protein such as phospholipase C gamma 1 (PLC ⁇ 1), TNF receptor-associated factor associated factors (TRAFs), growth factor receptorbound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (GADS), Src homology region 2 domain-containing phosphatase (SHP-1), vav guanine nucleotide exchange factor 1 (Vav1), phosphatidylinositol-3-kinase (PI), phospholipase C gamma 1 (PLC ⁇ 1), TNF receptor-associated factor associated factors (TRAFs
  • the recombinant polypeptide could contain a truncated cytoplasmic domain from IL-2R ⁇ and/or a STAT3-binding tyrosine-X-X- glutamine motif, among many others.
  • the recombinant polypeptide may contain 1, 2, 3, 4, or 5 or more ITAM motifs, wherein an ITAM conforms to the consensus sequence (YX1X2L/I)(X3)n(YX1X2L/I), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid (see, e.g., Chong et al Biochim Biophys Acta Gen Subj. 20221866: 130221).
  • the recombinant polypeptide may comprise two ITAMs or three ITAMs.
  • the one or more ITAMs may be independently selected from the ITAMs of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TYROBP (DAP12), DAP10, Fc ⁇ RI, Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, Dectin-1, CLEC-1, CD28, and CD72.
  • the one or more ITAMs may comprise the ITAM of Fc ⁇ RI ⁇ .
  • any of these domains may be a variation of a wild-type sequence, e.g., a sequence that is at least 90%, 95%, or 98% identical to a sequence described in WO2014127261, for example.
  • the recombinant polypeptide may have a signaling domain from CD3 ⁇ in which two of the three ITAM motifs (the second and third ITAM motifs) have been altered to be non-functional. More specifically, both tyrosine (Y) phosphorylation sites in the second and third ITAMs may be substituted by phenylalanine, thereby rendering those sites incapable of being phosphorylated. This altered CD3 ⁇ signaling domain is described in Feucht et al (Nat Med. 201925: 82-88).
  • the recombinant polypeptide may further comprise one or more other signaling motifs and/or protein binding motifs that are not already present in the adapter protein.
  • nucleic acid and cells also provided is a nucleic acid encoding the recombinant polypeptide.
  • the nucleic acid may be in a plasmid or viral vector such as a lentiviral or retroviral vector, and it may be operably linked to suitable promoter and, if necessary, terminator.
  • the coding sequence for the recombinant polypeptide may additionally comprise a signal sequence for targeting the protein to the plasma membrane.
  • the NK cell or T cell may be mammalian (e.g., mouse, human, primate). Human cells are often used, however.
  • the NK cell or T cell may be a primary NK or T cell, i.e., a cell obtained from peripheral blood, or a progenitor of the same (e.g., cell made by culturing primary NK cells or T cells in proliferation or expansion medium) and in any embodiment, the NK cell or T cell may be genetically modified to be allogeneic in a human host.
  • NK cells or T cells may be obtained from any suitable source. For example, NK cells or T cells may be differentiated in vitro from a hematopoietic stem cell population, or NK cells or T cells may be obtained from a subject.
  • NK cells or T cells may be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMCs peripheral blood mononuclear cells
  • NK cells or T cells may be derived from one or more NK cell or T cell lines available in the art.
  • NK cells or T cells may also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLLTM separation and/or apheresis.
  • the cells that are expanded can be primary NK cells or T cells.
  • NK cells or T cells may be genetically modified, made from iPSCs, obtained from umbilical cord blood, etc.
  • a method comprising genetically modifying an NK cell or T cell to express the recombinant polypeptide is also provided. As would be apparent, this method may be done ex vivo (on primary NK cells or T cells or their progenitors) or a cell line, etc.
  • a nucleic acid encoding the recombinant polypeptide may be introduced into the NK cell or T cell.
  • a nucleic acid encoding the co-stimulatory domain may be knocked into an existing gene.
  • SF2024-070-2 Populations of NK cells or T cells A population of the NK cells or T cells is also provided.
  • these cells may be present in vitro and may be progenitors of primary NK cells or T cells that have been genetically modified to express the recombinant protein.
  • NK cells or T cells may be genetically modified to be allogeneic in a human host, although this may be unnecessary in many cases.
  • the cells may be frozen.
  • the population may comprise any number of the NK cells or T cells (e.g. 100,000-1 Bn cells).
  • the harvested cells may be cryopreserved, where the term “cryopreserved” refers to cells that have been preserved or maintained by cooling to low sub- zero temperatures, such as 77 K or -196 deg. C. (the boiling point of liquid nitrogen).
  • cryopreservation and thawing cryopreserved cells include but are not limited to e.g., those described in U.S. Patent Nos. 10370638; 10159244; 9078430; 7604929; 6136525; and 5795711, the disclosures of which are incorporated herein by reference in their entirety.
  • freshness may refer to cells that have not been cryopreserved and, e.g., may have been directly obtained and/or used (e.g., transplanted, cultured, etc.) following collection from a subject or organ thereof.
  • Harvested therapeutic cell populations produced by the methods as described herein and therapeutic or pharmaceutical compositions thereof may be present in any suitable container (e.g., a culture vessel, tube, flask, vial, cryovial, cryo-bag, etc.) and may be employed (e.g., administered to a subject) using any suitable delivery method and/or device.
  • Such populations of cells and pharmaceutical compositions may be prepared and/or used fresh or may be cryopreserved.
  • populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a “ready-to-use” format, including e.g., where the therapeutic cells are present in a suitable diluent and/or at a desired delivery concentration (e.g., in unit dosage form) or a concentration that can be readily diluted to a desired delivery concentration (e.g., with a suitable diluent or media).
  • a desired delivery concentration e.g., in unit dosage form
  • a concentration that can be readily diluted to a desired delivery concentration e.g., with a suitable diluent or media.
  • populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a delivery device or a device compatible with a desired delivery mechanism or the desired route of delivery, such as but not limited to e.g., a syringe, an infusion bag, or the like.
  • the present disclosure provides one or a plurality of cell therapy doses, e.g., each contained in suitable container.
  • Cell therapy doses may be generated through a variety of methods. Aliquoting expanded populations of therapeutic cells into cell therapy doses may be performed by a variety of means.
  • the compositions may include the therapeutic cells present in a liquid medium.
  • the liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like.
  • One or more additives such as a salt (e.g., NaCl, MgCl 2 , KCl, MgSO 4 ), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N- tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.
  • a population may include a therapeutically effective amount of the cells.
  • therapeutically effective amount it is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to affect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease or disorder associated, e.g., with the target cell or a population thereof, as compared to a control.
  • An effective amount can be administered in one or more administrations.
  • a “therapeutically effective amount” of such cells may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the cells to elicit a desired response in the subject.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects.
  • the term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient).
  • a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • a therapeutically effective amount of NK cells or T cells may be 100,000-200M of the NK cells or T cells.
  • SF2024-070-2 The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration.
  • the cells of the present disclosure can be formulated for administration by combination with appropriate excipients, diluents and/or the like.
  • Formulations of the cells suitable for administration to a patient are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.
  • the cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.
  • An aqueous formulation of the cells may be prepared in a pH-buffered solution, e.g., at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5.
  • buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers.
  • the buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.
  • a tonicity agent may be included in the formulation to modulate the tonicity of the formulation.
  • Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof.
  • the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable.
  • the term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum.
  • Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.
  • a composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m- cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations SF2024-070-2 thereof.
  • a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).
  • Natural killer or T cell therapies can be manufactured from a variety of sources: these include peripheral blood, either steady-state or taking advantage of apheresis performed to collect hematopoietic stem and progenitor cells mobilized with growth factors such as granulocyte colony-stimulating factor (G-CSF), bone marrow, cord blood, ES cells, iPS cells and NK cell lines or T cell lines.
  • G-CSF granulocyte colony-stimulating factor
  • ES cells iPS cells
  • NK cell lines or T cell lines T cell lines.
  • the method may comprise expanding the NK cells or T cells ex vivo to produce expanded NK or T cells and harvesting the expanded NK or T cells to produce an NK or T cell population, where the term “harvesting” is intended to refer to a step in which the cells are removed from the container(s)/bioreactor(s) in which the cells were expanded.
  • the cells may be concentrated if desired, e.g., by centrifugation, a suitable cell separation technique (e.g., magnetic beads), and/or the like.
  • a cell population manufactured according to this method is also provided.
  • Methods of treatment A method of treatment is also provided. This method may comprise administering an effective amount of a population of the NK or T cells to a patient in need thereof where, in some embodiments, an effective amount may be 100,000-2 Bn cells.
  • the NK or T cells may be autologous/autogeneic (“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic).
  • “Autologous” as used herein refers to cells obtained from the subject to whom the therapeutic cells are later administered. “Allogeneic” as used herein refers to cells obtained from a donor other than the subject to whom the therapeutic cells are administered. In some embodiments, the cells are obtained from a mammalian subject. In certain embodiments, the mammalian subject is a primate. In some embodiments, the cells are obtained from a human. Cells are typically infused into the patient, although routes of administration can be used.
  • the patient may be a cancer patient where the treatment may result in least an amelioration of one or more symptoms associated with the condition of the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated.
  • amelioration also includes situations where the condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition.
  • the cancer being treated may be a blood cancer or a solid tumor, e.g., a carcinoma or a sarcoma.
  • the carcinoma is a basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma.
  • Cancers that can be treated with a method disclosed herein include any cancer that can be targeted by NK cells or T cells, including, but not limited to carcinomas, sarcomas, melanoma, leukemias, lymphomas and multiple myeloma.
  • Cancers that can be treated with a method disclosed herein include solid tumors, and cancers that begin in blood-forming tissue, i.e., hematological cancers such as leukemias, lymphomas and multiple myeloma.
  • Carcinomas that can be treated with a method disclosed herein include metastatic cancers.
  • Carcinomas that can treated by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma,
  • Sarcomas that can be treated by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, SF2024-070-2 lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.
  • solid tumors that can be treated by a method disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
  • the recombinant polypeptide comprises a binding domain, then the choice of binding domain may be determined by the type of cancer being treated.
  • the binding domain may target CD19, CD20 or BCMA for B-cell malignancies, including acute myeloid leukemia (AML), multiple myeloma (MM), B-cell acute lymphoblastic leukemia (B-ALL), lymphoma, etc.
  • Breast cancer antigens include MUC1, HER2, mesothelin, CEA, CAIX, FR- ⁇ , CD171, GD2, EGFRvIII, FAP, and vascular endothelial growth factor receptor2 (VEGF-R2).
  • Ovarian cancer antigens include CA125, MUC16, HER2, hepatocyte growth factor receptor (c-Met), mesothelin, folate receptor alpha (FR ⁇ ), and cancer/testis antigen 1B.
  • Lung cancer antigens include mesothelin (MSLN), EGFR, PSCA, MUC1, CEA, CD80/CD86, programmed death-ligand 1 (PD-L1), inactive tyrosine-protein kinase transmembrane receptor (ROR1), and HER2.
  • Colorectal cancer antigens include CEA, EGFR, MUC1, NKG2DL, HER2, and CD133.
  • Pancreatic cancer antigens include mesothelin, CD133, PSCA, claudin 6, claudin 18.2, EGFR, CEA, MUC1, and HER2.
  • Glioblastoma antigens include interleukin-13 receptor alpha 2 (IL-13 ⁇ R2), epidermal growth factor receptor variant III (EGFRvIII), and HER2.
  • Neuroblastoma antigens include GD2 and L1-CAM (CD171).
  • Melanoma antigens include HER2, ganglioside GD2 and c-MET.
  • Hepatocellular carcinoma antigens include CEA, MUC-1, and GPC-3.
  • Gastric cancer antigens include folate receptor 1 (FOLR1) and NKG2D.
  • Prostate cancer antigens include prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), PSCA, T-cell receptor gamma alternate reading frame protein (TARP), transient receptor potential (trp)-p8, and PSMA.
  • PSA prostate-specific antigen
  • PAP prostatic acid phosphatase
  • PSCA PSCA
  • trp transient receptor potential
  • SF2024-070-2 Renal cell carcinoma antigens include VEGFR2, CAIX, CCT301-38, CCT301-59, and CD70.
  • Conventional and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, intraventricular, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration.
  • the NK or T cells may be administered along with at least one additional therapeutic agent or therapeutic treatment (together or sequentially).
  • additional therapeutic agents include, but are not limited to, antibodies that stimulate ADCC, NK/T cell engagers, a small molecule cancer chemotherapeutic agent, and an immune checkpoint inhibitor.
  • Suitable additional therapeutic treatments include, e.g., radiation, surgery (e.g., surgical resection of a tumor), and the like.
  • a treatment method of the present disclosure can comprise co-administration of the NK or T cells and at least one additional therapeutic agent.
  • both the NK cells or T cells and at least one additional therapeutic agent are administered to an individual, although not necessarily at the same time, in order to achieve a therapeutic effect that is the result of having administered both the NK cells or T cells and the at least one additional therapeutic agent.
  • the administration of the NK or T cells and the at least one additional therapeutic agent can be substantially simultaneous, e.g., the polypeptide can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of administration of the at least one additional therapeutic agent.
  • the NK or T cells of the present disclosure are administered to an individual who is undergoing treatment with, or who has undergone treatment with, the at least one additional therapeutic agent.
  • the administration of the NK or T cells can occur at different times and/or at different frequencies.
  • the subject may additionally receive a monoclonal antibody that stimulates ADCC, or an NK cell engager (NKCE), such as a BiKE (bispecific killer cell engager) or TriKE SF2024-070-2 (trispecific killer cell engager).
  • NKCE NK cell engager
  • BiKEs and TriKEs are reviewed in Felices et al (Methods Mol Biol. 2016; 1441: 333–346).
  • BiKEs and TriKEs are molecules that contain a single variable portion of an antibody linked to one (BiKE) or two (TriKE) variable portions from other antibodies of different specificity. See, e.g., Shanshal et al (Cancers (Basel). 202315: 2824). Similar types of antibodies are available for T cells (e.g., BiKEs and TriKEs, etc.) Multispecific antibodies can be in a variety of different formats, including, but not limited to IgG-like antibody formats (including an Fc domain) and non-IgG-like antibody formats (without an Fc domain).
  • Multispecific antibodies with IgG-like antibody formats can be in a variety of different formats, including, but not limited to knob-into-hole (KIH), TrioMab, Duobody, ⁇ body, CrossMab, common light chain, strand exchange engineered domain bodies (SEEDBodies), Azymetric heterodimeric Fc, dual action Fab (DAF), dual-variable-domain immunoglobulin (DVD-Ig), IgG-scFv, Fab-Fab-Fc, DutaMab, and DutaFab.
  • Non-IgG-like antibody formats may lack an Fc region entirely.
  • Fab, Fv and VHH antibody regions may be genetically engineered and combined in various orientations and pairings.
  • Multispecific antibodies in non-IgG-like formats can be in a variety of different formats, including, but not limited to bivalent dual-affinity re-targeting protein (DART), tetravalent DART, half-life extended bispecific T-cell engager (HLE-BiTE), bispecific T-cell engager (BiTE), immune mobilizing monoclonal T-cell receptor (ImmTAC), tandem diabody (TandAb), bispecific killer cell engager (BiKE), trispecific killer cell engager (TRiKE), multispecific scFV single-chain variable fragment, trispecific T-cell activation construct (TriTAC), bispecific nanobody, and cross-over dual variable region (CODV).
  • DART bivalent dual-affinity re-targeting protein
  • HLE-BiTE half-life extended bispecific T-cell engager
  • BiTE bispecific T-cell engager
  • ImmTAC immune mobilizing monoclonal T-cell receptor
  • TandAb bispecific killer cell engager
  • TRiKE trispecific
  • immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2.
  • an immune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CT
  • the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122 and CD137.
  • the immune checkpoint polypeptide is an inhibitory checkpoint molecule SF2024-070-2 selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT and VISTA.
  • Co-therapies include for example, (a) anthracycline therapy (e.g., by administering daunomycin, doxorubicin, or mitoxantrone), (b) alkylating agent therapy (e.g., by administering mechlorethane, cyclophosphamide, ifosfamide, melphalan, cisplatin, carboplatin, nitrosourea, dacarbazine,procarbazine or busulfan), (c) topoisomerase II inhibitor therapy (e.g., by administering etoposide or teniposide), (d) bleomycin therapy, (e) anti-metabolite therapy (e.g., by administering methotrexate, 5-fluorocil, cytarabine, 6-mercaptopurine or 6-thioguanine), (f) vinca alkyloid therapy (e.g., by administering vincristine or vinblastine), (g) steroid therapy
  • Alternative therapies include targeted therapies and non-targeted chemotherapies, where targeted therapy includes treatment with erlotinib (Tarceva), afatinib (Gilotrif), gefitinib (Iressa) or osimertinib (Tagrisso) which may be administered to patients having an activating mutation in EGFR, crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa) or brigatinib (Alunbrig) which may be administered to patients having an ALK fusion, crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF- 06463922), ropotrectinib (TPX-0005), DS-6051b, ce
  • the therapy may be, for example, a platinum-based doublet chemotherapy (in which the platinum-based doublet chemotherapy may comprise a platinum-based agent selected from cisplatin (CDDP), carboplatin (CBDCA), and nedaplatin (CDGP)) and one third-generation agent (selected from docetaxel (DTX), paclitaxel (PTX), vinorelbine (VNR), gemcitabine (GEM), irinotecan (CPT-11), pemetrexed (PEM), and tegafur gimeracil oteracil (S1)).
  • DTX docetaxel
  • PTX paclitaxel
  • VNR vinorelbine
  • GEM gemcitabine
  • CPT-11 pemetrexed
  • PEM pemetrexed
  • S1 tegafur gimeracil oteracil
  • the present NK cell or T cell therapy may be combined with FOLFOX therapy (i.e., a chemotherapy regimen that comprises administering folinic acid, fluorouracil and oxaliplatin (FOLFOX) to the patient), e.g., for the treatment of colorectal and other cancers.
  • FOLFOX therapy i.e., a chemotherapy regimen that comprises administering folinic acid, fluorouracil and oxaliplatin (FOLFOX) to the patient
  • FOLFOX therapy i.e., a chemotherapy regimen that comprises administering folinic acid, fluorouracil and oxaliplatin (FOLFOX) to the patient
  • FOLFOX therapy i.e., a chemotherapy regimen that comprises administering folinic acid, fluorouracil and oxaliplatin (FOLFOX) to the patient
  • FOLFOX fluorouracil and oxaliplatin
  • the recombinant polypeptide may alternatively comprise the trans
  • the full-length sequence of the wild-type DAP12 is deposited at Genbank as accession number NP_001166986.1 (which deposit is incorporated by reference herein). DAP12 has a single ITAM.
  • the wild type DAP12 transmembrane domain has the following amino acid sequence: CSCSTVSPGVLAGIVMGDLVLTVLIALAV; SEQ ID NO: 4. See Lanier (Immunol Rev. 2009227: 150–160).
  • the full-length sequence of the wild-type DAP10 is deposited at Genbank as accession number AAD47911.1 (which deposit is incorporated by reference herein). DAP10 has a single YINM sequence .
  • the wild type DAP10 transmembrane domain has the following amino acid sequence: LAGLVAADAVASLLIVGAVFLC; SEQ ID NO: 5. See Lanier (Immunol Rev. 2009 227: 150–160). While many embodiments of the alternative recombinant polypeptide may comprise the wild-type transmembrane domain of DAP12 (i.e., SEQ ID NO: 4) or DAP10 (i.e., SEQ ID NO: 5) in certain instances the recombinant polypeptide may comprise a variant of the wild-type transmembrane domain of DAP12 or DAP10 comprising up to 5 amino acid substitutions (e.g., 1, 2, 3, 4, or 5 substitutions) relative to SEQ ID NO: 4 or 5.
  • this is a transmembrane domain
  • up to five of the hydrophobic residues can be substituted with another hydrophobic amino acid.
  • the other components of these alternative polypeptides e.g., the types of co-stimulatory domain, the types and number of ITAMs, whether the recombinant polypeptide contains or lack an extracellular binding domain, etc.
  • methods of use are the same for the embodiments that employ the transmembrane domain of Fc ⁇ RI ⁇ of CD3 ⁇ , or a variant of the transmembrane domain that retains the ability to bind to NKp30, NKp46 and/or CD16, as described above.
  • the following examples are offered by way of illustration and not by way of limitation.
  • SF2024-070-2 EXPERIMENTAL Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
  • the transmembrane adaptor protein Fc ⁇ R1 ⁇ is required for the expression and signaling of the activating receptors NKp30, NKp46, and CD16. These receptors are naturally expressed by human NK cells and a subset of human T cells.
  • Fc ⁇ R1 ⁇ -41BB Fc ⁇ R1 ⁇ -41BB in this disclosure.
  • addition of an extracellular binding domain in this example a binding domain that binds to CD19
  • Fc ⁇ R1 ⁇ -41BB retains and/or enhances human NK cell activity in the presence of cancer cells that express an antigen that is recognized by the binding domain.
  • fusion of the Fc ⁇ R1 ⁇ -41BB protein with the anti-human CD19 chimeric antigen receptor (CD19CAR) resulted in a new anti-CD19CAR-Fc ⁇ R1 ⁇ TM-41BB-Fc ⁇ R1 ⁇ - ITAM product that can enhance human CAR NK cell activity, which is believed to be mediated by NKp30, NKp46, and/or CD16 expression and downstream signaling. Therefore, these fusion proteins have the potential to provide a better human NK cell cancer immunotherapy treatment. It is noted that the experimental section of this application provides data for NK cells.
  • T cells express NKp30, NKp46, or CD16 and the mechanism for NKp30, NKp46, or CD16 activation is the same (see, e.g., Correia et a, Proc. Natl. Acad. Sci. 115 (26) E5980- E5989). As such, the present findings can be extrapolated to T cells.
  • FCRG2 contained nucleic acids 1-273 of human FCER1G (Sequence ID: NM_004106.2).
  • FCRG8 contained nucleic acids 1-273 of human FCER1G and a human 41BB co stimulatory motif (e.g., KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL ; SEQ ID NO:6) inserted between exon 3 and exon 4 of FCER1G cDNA sequence.
  • a human 41BB co stimulatory motif e.g., KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL ; SEQ ID NO:6
  • FCRG10 contained nucleic acids 1- 954 of CD19 ScFv -CD8 Hinge -41BB-CD3 ⁇ chimeric antigen receptor (CAR) ectodomain (e.g., CD8 signal peptide, Myc-tag, CD19 ScFv , and CD8 hinge ) fused to transmembrane and intracellular domains of FCRG8 at amino acid # 17 (e.g., FCER1G exon 2).
  • DNA fragments were obtained from Integrated DNA Technologies (e.g., IDT).
  • Lentivirus preparation was done by using the pMD2.G and pCMV dr8.91 packaging vectors and transfection of the Lenti-XTM 293T cell Line (TAKARA, cat.
  • NK92 cells (CRL-2407) were obtained from “The American Type Culture Collection” (ATCC). NK92 cell culture media: Minimum Essential Medium Eagle (Sigma Aldrich, cat. M0200-500ML) + 20% horse serum (Sigma Aldrich, cat.
  • mouse pro-B cell line Ba/F3 pre- engineered to express mouse IL-3 or human K562 cells were generously provided by Dr. L. Lanier.
  • Ba/F3 or K562 culture media complete RPMI-1640 + 10% heat-inactivated FCS.
  • lentiviral transductions 50,000 cells were mixed with concentrated lentiviral particles in 96-well round-bottom plate (200 ⁇ l of culture media) and were centrifuged at 1000 RCF for 1 hour at room temperature. After centrifugation, cells were incubated at 37°C, 5% CO2) for 24 hours. Cells were then transferred to T25 flask for expansion.
  • SF2024-070-2 Anti-Fc ⁇ R1 ⁇ (Milipore, cat. FCABS440F), anti-CD3 ⁇ (BioLegend, cat. 644103), anti-NKp30 (BioLegend, cat. 325208, cat. 325210), anti-NKp46 (BioLegend, cat. 331908, cat. 331936), anti- NKG2A (Miltenyi Biotech, act. 130-113-563), anti-NKG2D (R&D, cat. FAB139P), anti-NKp44 (BioLegend, cat.
  • proteins were incubated with the cells for 60 minutes at 4 o C, washed off and then cells were incubated with anti-human IgG for 30 minutes at 4 o C.
  • cells were incubated for 20 min at 4°C with 100 ⁇ l/well Cytofix/Cytoperm buffer (51-2090KZ; Becton Dickinson). Following incubation, cells were washed twice using Perm Wash buffer (cat. 421002; BioLegend) diluted 1:10 in PBS and then the antibodies were incubated with the cells for 60 minutes at room temperature.
  • Protein expression, or co-expression was detected by flow cytometry LSR-II; Becton Dickinson Immunocytometry Systems.
  • Engineered FCRG2, FCRG8, FCRG10, or CD19CAR NK92 cells were sorted by the increased expression or co-expression of NKp30, NKp46, CD16, or Myc-tag.
  • Ba/F3 cells were sorted by the expression or co-expression of B7H6, HLA-E, CD20, CD19 using FACSAria: Becton Dickinson Immunocytometry Systems. Dead cells were excluded by using Zombie NIRTM Fixable Viability Kit (BioLegend, cat. 423106) diluted 1:500 in PBS + 2% FCS.
  • CRISPR-Cas9 knockout of FCER1G or NCR3LG1 (B7H6): Fc ⁇ R1 ⁇ 41BB positive NK92 cells or K562 cells were edited using the non-viral CRISPR-Cas9 system.
  • crRNA was resuspended in IDT duplex buffer at a final concentration of 160 ⁇ M.
  • crRNA and tracrRNA, obtained from IDT were mixed at a 1:1 volume ratio and were incubated at 37 °C for 30 min to form gRNA solution.
  • gRNA solution and Cas9-NLS (Berkeley QB3 MacroLab) were mixed at a 1:1 volume ratio and were incubated at 37 °C for 15–30 min.
  • Electroporation codes NK92 cells; CM137, K562 cells; FF120.
  • FCER1G crRNA-1; GCCCAAGATGATTCCAGCAG (SEQ ID NO:7), crRNA-2: CAGCTCTGCTATATCCTGGA (SEQ ID NO:8).
  • NCR3LG1 crRNA; AAGTAGAGATGATGGCAGGG (SEQ ID NO:9), ATGGTGACATTGTCATTCAG (SEQ ID NO:10).
  • Antibody-conjugated beads and cell stimulation Antibody-conjugated beads were prepared according to the company’s protocol (Invitrogen Dynabeads Antibody Coupling Kit, cat. 14311D) at 10 ⁇ g antibody per 1 mg beads. Following conjugation, beads were resuspended in sterile PBS at an antibody concentration of 0.1 ⁇ g/ ⁇ l. Antibody conjugation was evaluated by flow cytometry with APC-conjugated anti-mouse or rat IgG. BioLegend: anti-CD16 (cat. 302002, IgG1k), anti-NKp30 (cat. 325204, IgG1k), (cat. 325102, IgG1k), anti-NKp46 (cat.
  • Antibody-conjugated beads were kept at 4°C until further use. The assays were performed in 96 well round-bottom plates. Antibody-coated beads were diluted at 1:1,000 or as indicated in NK92 cell culture media and used by adding 50 ⁇ l/well. NK92 cells (5 ⁇ 10 4 cells/well) were added at 150 ⁇ l/well in NK92 cell culture media. Alexa Fluor 647 anti- human CD107a antibody (BioLegend, cat.
  • NK92 cells were incubated at 37°C with 5% CO 2 for the duration of the assay. Cells were analyzed for the detection of CD107a by flow cytometry (LSR-II; Becton Dickinson Immunocytometry Systems). Dead cells were excluded by using propidium iodide (1 mg/ml, 1:500). NK92 cells incubated without beads were used for CD107a background control and the increase in CD107a signal was calculated relative to each cell line.
  • NK cell lysis assay and ADCC NK92 cells were labeled with cell trace violet (CTV) according to the company’s protocol (Invitrogen, cat.
  • NK92 cells were resuspended in NK92 cell culture at a concentration of 1 ⁇ 10 5 cells/100 ⁇ l / well, and were diluted 1:21:4 or as indicated in NK92 cell culture media.
  • Target cells were resuspended in complete RPMI-1640 + 10% heat-inactivated FCS, at a concentration of 5 ⁇ 10 4 cells/ 100 ⁇ l / well.
  • Fc ⁇ R1 ⁇ 41BB overexpression leads to NKp30 and NKp46 upregulation
  • Fc ⁇ R1 ⁇ is a transmembrane adaptor protein with one immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • Fc ⁇ R1 ⁇ protein expression is reported to regulate the expression and function of the activating receptors NKp30, NKp46, and CD16.
  • Loss of Fc ⁇ R1 ⁇ protein expression in human adaptive NK cells, g-NK cells, or following FCER1G CRISPR knockout is associated with loss of NKp30 and NKp46 surface protein expression.
  • Fc ⁇ R1 ⁇ protein expression leads to a mild decrease in CD16 protein expression but increases CD16 function due to transmembrane interaction with CD3 ⁇ homodimerize.
  • engineering Fc ⁇ R1 ⁇ with a co-stimulatory motif, such as 41BB may increase function and protein expression of NKp30, NKp46, and CD16 in human NK cells.
  • Fc ⁇ R1 ⁇ FCRG
  • Fc ⁇ R1 ⁇ 41BB FCRG 41BB
  • Fc ⁇ R1 ⁇ or Fc ⁇ R1 ⁇ 41BB in NK cells led to increased expression of Fc ⁇ R1 ⁇ without affecting the expression levels of CD3 ⁇ (FIG. 1C). Additionally, Fc ⁇ R1 ⁇ or Fc ⁇ R1 ⁇ 41BB overexpression increased NKp30 or NKp46 surface protein expression in NK cells (FIG. 1D), while FCER1G CRISPR knockout led to decreased NKp30 or NKp46 expression (FIG. 1E). Fc ⁇ R1 ⁇ or Fc ⁇ R1 ⁇ 41BB overexpression did not lead to changes in surface protein expression of the inhibitory receptor NKG2A, the activating receptors NKG2D or NKp44, and 41BB or TRAIL surface expression (FIG. 1F).
  • Fc ⁇ R1 ⁇ modification with an intracellular 41BB motif does not prevent the upregulation of NKp30 and NKp46 by Fc ⁇ R1 ⁇ .
  • Fc ⁇ R1 ⁇ or Fc ⁇ R1 ⁇ 41BB overexpression does not lead to alterations in the expression of main activating or inhibitory surface membrane receptors or to non-specific NK cell activation.
  • Example 2 SF2024-070-2 Fc ⁇ R1 ⁇ 41BB increase NKp30 function against B7H6 positive cells
  • the NKp30-B7H6 interaction was investigated.
  • the mouse pro-B cell line, Ba/F3 was engineered to express B7H6.
  • B7H6+ Ba/F3 cells were engineered to co-express the NKG2A inhibitory HLA-E HLA-G , as NK cell activation by activating receptors is further regulated by signaling from inhibitory receptors such as NKG2A (FIG. 2A). Challenging the Fc ⁇ R1 ⁇ or Fc ⁇ R1 ⁇ 41BB positive NK cells with parental Ba/F3 cells did not show unspecific target cell lysis relative to parental (not transduced) NK cells or no effector control (FIGS. 2B, 2C).
  • Fc ⁇ R1 ⁇ 41BB positive NK cells displayed an increased target cell lysis of B7H6 + or B7H6 + HLA-E + Ba/F3 cells relative to Fc ⁇ R1 ⁇ positive or parental NK cells (FIGS. 2B, 2C). This shows that Fc ⁇ R1 ⁇ 41BB expression leads to a higher NKp30-dependent (B7H6) target cell lysis even in the presence of high levels of an inhibitory ligand such as HLA- E.
  • CML chronic myelogenous leukemia
  • Activating receptor-human Fc fusion protein binding assay confirmed the binding of NKp30-hFc and NKG2D-hFc to K562 cells (FIG. 3A).
  • K562 cells express lower levels of surface B7H6 (FIG. 3B).
  • Fc ⁇ R1 ⁇ 41BB positive NK cells displayed an increased target cell lysis of K562 cells relative to parental or Fc ⁇ R1 ⁇ positive NK cells (FIG. 3C), while B7H6 knockout in K562 cells resulted in no target cell lysis (FIG.3D).
  • Fc ⁇ R1 ⁇ 41BB increases NKp30-dependent NK cell function against B7H6 positive cells.
  • Example 3 Fc ⁇ R1 ⁇ 41BB increases CD16 function and ADCC
  • NK92 cells were engineered to express human CD16 (FIG. 4A).
  • a CD107a assay using mouse IgG1 antibody-coated beads against activating receptors confirmed CD16 function in NK92 cells, while CD16 expression did not impact the function of NKp30, NKp46, or NKG2D (FIG. 4B).
  • Fc ⁇ R1 ⁇ or Fc ⁇ R1 ⁇ 41BB expression increased CD16 expression relative to parental NK cells (FIG. 4C).
  • Fc ⁇ R1 ⁇ SF2024-070-2 modification with an intracellular 41BB motif does not prevent the upregulation of CD16.
  • a CD107a assay was performed using anti-human CD16 mouse IgG1 coated beads. Following CD16 cross-linking, Fc ⁇ R1 ⁇ 41BB positive NK cells displayed a significant increase in CD107a surface expression relative to Fc ⁇ R1 ⁇ positive or parental cells (FIG. 4D).
  • Fc ⁇ R1 ⁇ 41BB positive NK cells displayed a significant increase in ADCC relative to Fc ⁇ R1 ⁇ positive or parental NK cells. Therefore, Fc ⁇ R1 ⁇ modification with an intracellular 41BB motif increases CD16 function in NK cells.
  • Example 4 CD19 ScFv -CD8 hinge fused to Fc ⁇ R1 ⁇ 41BB increases NKp30 and CD16 expression Chimeric antigen receptors (CARs) are used in cancer immunotherapy.
  • CD19CAR positive NK cells did not display upregulation of NKp30 or CD16 expression (FIG. 5B, top row).
  • the increase in NKp30 or CD16 expressions in FCRG10 positive NK cells corresponded to the expression of Myc-tag, located in the N-terminal of the CAR (FIG. 5B, bottom row). This shows that Fc ⁇ R1 ⁇ modification with an intracellular 41BB motif and an extracellular CD19 ScFv -CD8 hinge does not prevent the upregulation of NKp30 and CD16 by Fc ⁇ R1 ⁇ .
  • CD19 ScFv -CD8 hinge -Fc ⁇ R1 ⁇ 41BB CAR improves NK cell function against HLA-E + target cells
  • FCRG10 CD19 ScFv -CD8 hinge -Fc ⁇ R1 ⁇ 41BB CAR
  • Ba/F3, Ba/F3- B7H6+, or Ba/F3-B7H6+HLA-E+ were engineered to express human CD19 (FIGS. 6A, 6B).
  • a lysis assay against parental Ba/F3 cells did not show unspecific target cell lysis by FCRG10 positive NK cells relative to parental NK cells or no effector control (FIG. 6C).
  • FCRG10 or CD19CAR positive NK cells displayed increase target cell lysis, indicating the CD19 ScFv -CD8 hinge -Fc ⁇ R1 ⁇ 41BB CAR is functional. Still, FCRG10 positive NK cells displayed a higher target cell lysis, which may be due to the transmembrane dimerization of Fc ⁇ R1 ⁇ with CD3 ⁇ (FIG. 6D).
  • FCRG10 positive NK cells displayed increased target cell lysis relative to Fc ⁇ R1 ⁇ 41BB positive NK cells, indicating that the recognition of two activating ligands increases NK cell mediated lysis (FIG.
  • FCRG10 positive NK cells displayed a significant increase in target cell lysis relative to CD19CAR positive NK cells (FIG. 6F).
  • the results indicate that CD19 ScFv- CD8 hinge -Fc ⁇ R1 ⁇ 41BB CAR (X1 ITAM) leads to a similar NK cell mediated lysis as a CD19CAR 41BB-CD3 ⁇ (X3 ITAMs) against CD19 + cells (single activating ligand).
  • CD19 ScFv- CD8 hinge -Fc ⁇ R1 ⁇ 41BB CAR improves NK cell function relative to a CD19CAR 41BB-CD3 ⁇ (X3 ITAMs).
  • CD19 ScFv -CD8 hinge -Fc ⁇ R1 ⁇ 41BB CAR is functional in primary human NK cells
  • FCRG10 CD19 ScFv -CD8 hinge -Fc ⁇ R1 ⁇ 41BB CAR

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Abstract

L'invention concerne un polypeptide recombinant comprenant : un domaine transmembranaire de FcεR1γ ou CD3ζ, ou un variant du domaine transmembranaire qui conserve la capacité de se lier à NKp30, NKp46 et/ou CD16, un domaine de co-stimulation, et un ou plusieurs motifs d'activation à base de tyrosine d'immunorécepteur (ITAM). L'invention concerne également des cellules l'exprimant et des méthodes de traitement.
PCT/US2025/018370 2024-03-14 2025-03-04 Amélioration des réponses innées et adaptatives de cellules immunitaires Pending WO2025193475A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016054520A2 (fr) * 2014-10-03 2016-04-07 The California Institute For Biomedical Research Proteines de surface cellulaire génétiquement modifiées et leurs utilisations
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
US20230322894A1 (en) * 2020-08-26 2023-10-12 The Regents Of The University Of California Methods and compositions for treating glioblastoma

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016054520A2 (fr) * 2014-10-03 2016-04-07 The California Institute For Biomedical Research Proteines de surface cellulaire génétiquement modifiées et leurs utilisations
US20230322894A1 (en) * 2020-08-26 2023-10-12 The Regents Of The University Of California Methods and compositions for treating glioblastoma
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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