[go: up one dir, main page]

WO2024215987A1 - CELLULES IMMUNITAIRES POUR LE TRAITEMENT DU CANCER EN COMBINAISON AVEC DES CONJUGUÉS IL-15/IL-15Rα - Google Patents

CELLULES IMMUNITAIRES POUR LE TRAITEMENT DU CANCER EN COMBINAISON AVEC DES CONJUGUÉS IL-15/IL-15Rα Download PDF

Info

Publication number
WO2024215987A1
WO2024215987A1 PCT/US2024/024213 US2024024213W WO2024215987A1 WO 2024215987 A1 WO2024215987 A1 WO 2024215987A1 US 2024024213 W US2024024213 W US 2024024213W WO 2024215987 A1 WO2024215987 A1 WO 2024215987A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
domain
polypeptide
immune
cell population
Prior art date
Application number
PCT/US2024/024213
Other languages
English (en)
Inventor
Amy JENSEN
Emily KUIPER
Kathleen WHITEMAN
Irena ADKINS
Original Assignee
Sotio Biotech Inc.
Sotio Biotech Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sotio Biotech Inc., Sotio Biotech Ag filed Critical Sotio Biotech Inc.
Publication of WO2024215987A1 publication Critical patent/WO2024215987A1/fr

Links

Classifications

    • 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
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] 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
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4244—Enzymes
    • A61K40/4251—Kinases, e.g. Raf or Src
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • Cancer immunotherapy including cell-based therapy, is used to provoke immune responses attacking tumor cells while sparing normal tissues. It is a promising option for treating various types of cancer because of its potential to evade genetic and cellular mechanisms of drug resistance, and to target tumor cells while sparing normal tissues.
  • Cell-based therapy may involve various immune cells including NK cells and cytotoxic T cells having reactivity skewed toward cancer cells (Eshhar et al.1993, Geiger et al.1999, Brentjens et al.2003, Cooper et al.2003, Imai et al.2004).
  • TEE tumor microenvironment
  • an IL-15/IL-15R ⁇ sushi fusion protein i.e., an exemplary covalent conjugate of IL-15 fused with a fragment of the IL-15R ⁇ receptor comprising its sushi domain
  • an immune cell population comprising immune effector cells responsive to an IL-15/IL-15R ⁇ conjugate, i.e., cells expressing IL-2/IL-15R ⁇
  • the therapeutic effect was further Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC increased by the co-expression of a metabolism modulating polypeptide in the immune cells of the immune cell population.
  • This allows for novel combination treatments combining immune cell populations and IL-15/IL-15R ⁇ conjugates for use in the treatment of cancer in patients, especially immune cell populations comprising ⁇ T cells, a ⁇ T cells, natural killer (NK) cells and NKT cells.
  • immune cells e.g., CAR-T cells or BOXR-CAR-T cells as disclosed herein
  • an IL-15/IL-15R ⁇ fragment conjugate e.g., a fusion polypeptide
  • the present disclosure provides a method for treating cancer in a subject that involves a combination of (a) an effective amount of an immune cell population and (b) an effective amount of a conjugate (e.g., a fusion polypeptide) comprising an IL-15 polypeptide (a polypeptide comprising the amino acid sequence of a native interleukin 15 (IL-15) or a derivative thereof) and a fragment of an interleukin 15-receptor alpha (IL-15R ⁇ ) polypeptide (a native IL-15R ⁇ or a derivative thereof), the fragment comprising the sushi domain of the IL- 15R ⁇ polypeptide.
  • a conjugate e.g., a fusion polypeptide
  • an IL-15 polypeptide a polypeptide comprising the amino acid sequence of a native interleukin 15 (IL-15) or a derivative thereof
  • IL-15R ⁇ interleukin 15-receptor alpha
  • the method provided herein comprises administering to a subject in need of the treatment an effective amount of the immune cell population of (a) and an effective amount of the conjugate (e.g., fusion polypeptide) of (b).
  • the method provided herein comprises administering to a subject in need of the treatment an effective amount of the immune cell population of (a), wherein the subject has received treatment or is on treatment of the conjugate of (b).
  • the method provided herein comprises administering to a subject in need of the treatment an effective amount of the conjugate of (b), wherein the subject has received treatment or is on treatment of the immune cell population.
  • the immune cell population may comprise immune cells selected from B cells, ⁇ T cells, ⁇ T cells, natural killer (NK) cells, or NKT cells.
  • the immune cells may comprise a combination of any of B cells, ⁇ T cells, ⁇ T cells, natural killer (NK) cells, and NKT cells.
  • the immune cells are genetically unmodified (e.g., naturally-occurring immune cells). In other instances, the immune cells are genetically modified. In some instances, the immune cells are autologous to the subject. Alternatively, the immune cells are allogenic to the subject.
  • the immune cells are expanded ex vivo prior to administration.
  • the immune cells can be activated ex vivo in the presence of one or more of 4- 1BB ligand, anti-4-1BB antibody, anti-CD3 antibody, anti-CD28 antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL-7, IL-12, IL-21 and K562 cells, and an engineered artificial stimulatory cell or particle prior to administration.
  • the immune cells are derived from one or more cell lines.
  • the immune cells are derived from peripheral blood mononuclear cells (PBMC), hematopoietic stem cells (HSCs), cord blood stem cells or induced pluripotent stem cells (iPSCs).
  • the immune cells are from tumor tissue, for example, comprising tumor-infiltrating lymphocytes (TILs).
  • TILs tumor-infiltrating lymphocytes
  • the immune cells comprise genetically engineered immune cells expressing a chimeric receptor polypeptide.
  • the chimeric receptor polypeptide comprises: (a) an extracellular target binding domain; (b) a transmembrane domain; and (c) a cytoplasmic signaling domain.
  • the chimeric receptor polypeptide is a chimeric antigen receptor (CAR) polypeptide or a T-Cell Receptor (TCR) polypeptide.
  • the CAR polypeptide may comprise an extracellular antigen binding domain, which may bind to a tumor antigen.
  • the extracellular antigen binding domain may be a single chain variable fragment (scFv).
  • the extracellular antigen binding may be a single domain antibody.
  • the transmembrane domain (b) of any of the chimeric receptor polypeptide provided herein may be of a membrane protein.
  • the cytoplasmic signaling domain of (c) of any of the chimeric receptor polypeptides provided herein may comprise a cytoplasmic domain of CD3 ⁇ . Alternatively, it may comprise a cytoplasmic domain of Fc ⁇ R1 ⁇ . Any of the chimeric receptor polypeptides provided herein may further comprise a signal peptide at its N-terminus.
  • the chimeric receptor polypeptide may further comprise a hinge domain, which may be located at the C-terminus of (a) and the N-terminus of (b). Alternatively, the chimeric receptor polypeptide may be free of a hinge domain. In some examples, the chimeric receptor polypeptide may further comprise at least one co-stimulatory signaling domain.
  • Examples include, but are not limited to, an co-stimulatory domain from CD8 ⁇ , CD28, 4-1BB, 2B4, OX40, OX40L, ICOS, CD27, GITR, HVEM, TIM1, LFA1, CD2, DAP10, DAP12, DNAM-1, NKG2D, NKp30, NKp44, NKp46 and JAMAL, or any functional Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC variant thereof.
  • the chimeric receptor polypeptide may be free of a co-stimulatory signaling domain.
  • the chimeric receptor polypeptide may comprise a cytoplasmic signaling domain that comprises an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • the immune cells disclosed herein may be genetically engineered to further express or overexpress a metabolism-regulating polypeptide. Such a polypeptide may (a’) improve glucose uptake activity; (b’) modulate the Krebs cycle; (c’) enhance lactate concentration; or (d’) divert or redirect glucose metabolites out of a glycolysis pathway.
  • the metabolism-regulating polypeptide may be encoded by an exogenous nucleic acid introduced into the immune cells.
  • the genetically engineered immune cells disclosed herein may express/overexpress the metabolism-regulating polypeptide disclosed herein and any of the chimeric receptor polypeptides as also disclosed herein.
  • the metabolism-regulating polypeptide improves glucose uptake activity. Examples include a glucose transporter (GLUT) or a sodium-glucose cotransporter (SGLT).
  • GLUT glucose transporter
  • SGLT sodium-glucose cotransporter
  • the glucose importation polypeptide may be GLUT1, GLUT3, GLUT1 S226D, SGLT1, SGLT2, GLUT8, GLUT8 L12A L13A, GLUT11, GLUT7, or GLUT4.
  • the metabolism-regulating polypeptide modulates Krebs cycle.
  • Examples include an enzyme that catalyzes a reaction in the Krebs cycle (e.g., isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), or phosphoglycerate dehydrogenase (PHGDH)), an enzyme that uses a Krebs cycle metabolite as a substrate (e.g., glutamic- oxaloacetic transaminase (GOT) or phosphoenolpyruvate carboxykinase 1 (PCK1)), or an enzyme that converts a precursor to a Krebs cycle metabolite (e.g., phosphoserine aminotransferase (PSAT1), glutamate dehydrogenase (GDH1), glutamate-pyruvate transaminase 1 (GPT1), or glutaminase (GLS)).
  • IDH isocitrate dehydrogenase
  • MDH malate dehydrogenase
  • PDGDH phospho
  • the metabolism-regulating polypeptide enhances lactate concentration.
  • examples include monocarboxylate transporter (MCT) (e.g., MCT1, MCT2, or MCT4), an enzyme involved in lactate synthesis (e.g., lactate dehydrogenase A (LDHA)), or a polypeptide that inhibits a pathway that competes for lactate-synthesis substrates (e.g., pyruvate dehydrogenase kinase 1 (PDK1)).
  • MCT monocarboxylate transporter
  • LDHA lactate dehydrogenase A
  • PDK1 pyruvate dehydrogenase kinase 1
  • the metabolism-regulating polypeptide diverts or redirects glucose metabolites out of a glycolysis pathway.
  • any of the immune cell populations disclosed herein is administered to the subject at a dose of at least about 1 x 10 4 immune cells/kg.
  • the dose is from about 1 x 10 4 to about 1 x 10 12 cells/kg.
  • the conjugate provided herein may comprise the amino acid sequence of human IL-15 or a derivative thereof.
  • the IL-15 derivative has an amino acid sequence at least 92.5 % identical with the amino acid of SEQ ID NO: 69.
  • the conjugate may comprise the sushi domain of the human IL-15R ⁇ or a derivative thereof, which may have an amino acid sequence at least 92% identical with the amino acid sequence of SEQ ID NO: 71.
  • the human IL-15R ⁇ derivative comprises the amino acid sequence of SEQ ID NO: 72.
  • the conjugate is a fusion polypeptide comprising the human IL-15 moiety and human IL-15R ⁇ moiety disclosed herein.
  • the amino acid sequence of the IL-15 or derivatives thereof and the amino acid sequence of the sushi domain of the IL-15R ⁇ or derivatives are separated by a linker amino acid sequence.
  • the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 74.
  • the conjugate provided herein (e.g., a fusion polypeptide) comprises a first polypeptide comprising the amino acid sequence of an IL-15 derivative having at least 0.1% of the activity of human IL-15 on the proliferation induction of kit225 cell line, and a second polypeptide comprising the amino acid sequence of the sushi domain of IL-15R ⁇ or derivatives thereof having at least 10% of the binding activity of the sushi domain of human IL- 15R ⁇ to human IL-15.
  • the IL-15 derivative has at least 25% (e.g., at least 50%) of the activity of human interleukin-15 on the proliferation induction of kit225 cell line.
  • Such an IL-15 derivative may have an amino acid sequence at least 92.5% (e.g., at least 96% or at least 98.5%) identical with SEQ ID NO: 69.
  • the IL-15 derivative comprises the amino acid sequence of SEQ ID NO: 69.
  • the derivative of the sushi domain of IL-15R ⁇ has at least 25% (e.g., at least 50%) of the binding activity of the sushi domain of human IL-15R ⁇ .
  • Such a derivative may comprise an amino acid sequence at least 92.5% (e.g., at least 96% or at least 98%) identical with SEQ ID NO: 71 or SEQ ID NO: 72.
  • the IL-15R ⁇ derivative comprises the amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 72.
  • an effective dose of the immune cell population and an effective dose of the conjugate are administered sequentially.
  • the conjugate e.g., a fusion polypeptide as provided herein
  • the cyclical administration regimen may comprise: (a) a first period of “x” days during which the conjugate is administered at a daily dose on “y” consecutive days at the beginning of the first period followed by “x-y” days without administration of the conjugate, wherein “x “is 7, 14, or 21 days, preferably 7 days and “y” is 1, 2, 3 or 4 consecutive days; (b) repeating the first period at least once; and (c) a second period of “z” days without administration of the conjugate wherein z is 7, 14 or 21 days, preferably 7 or 14 days.
  • the conjugate can be administered at a daily dose of 4.4 pmol/kg to 2200 pmol/kg, preferably 22 pmol/kg to 660 pmol/kg, and more preferably 220 pmol/kg to 530 pmol/kg.
  • the administration of the conjugate provided herein may results in one or more of the following effects: (a) an increase of the % of Ki-67 + NK of total NK cells in comparison to no administration of the conjugate, and wherein the administration of the conjugate after at least one repetition of the first period (step (b)) results in a Ki-67 + NK cell level that is at least 70% of the of the Ki-67 + NK cells; (b) maintenance of NK cell numbers or preferably an increase of NK cell numbers to at least 110% as compared to no administration of conjugate after at least one repetition of the first period (step (b)); and/or (c) NK cell numbers of at least 1.1 x 10 3 NK cells/ ⁇ l after at least one repetition of the first period (step (b)).
  • the present disclosure provides a method for treating cancer, involving the combination of a viral particle capable of transducing immune cells such as T cells as disclosed herein and any of the conjugates (e.g., fusion polypeptides) provided herein.
  • the viral particle comprises a vector genome comprising a polynucleotide sequence encoding any of the chimeric receptor polypeptides provided herein.
  • the vector genome may further Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC comprise a polynucleotide sequence encoding any of the metabolism-regulating polypeptides also provided herein.
  • the viral particle is a retroviral particle.
  • the viral particle can be a lentiviral particle.
  • the method provided above comprises administering to a subject in need of the treatment an effective amount of the viral particle and an effective amount of the conjugate (e.g., fusion polypeptide).
  • the viral particle and the conjugate are administered sequentially.
  • the method provided herein comprises administering to a subject in need of the treatment an effective amount of the viral particle, wherein the subject has received treatment or is on treatment of the conjugate.
  • the method provided herein comprises administering to a subject in need of the treatment an effective amount of the conjugate of, wherein the subject has received treatment or is on treatment of the viral particle.
  • the viral particle is for intravenous, intraperitoneal, subcutaneous or intranodal injection.
  • the present disclosure provides a method for treating cancer, involving a combination of a nucleic acid encoding a chimeric receptor polypeptide as disclosed herein and any of the conjugates (e.g., fusion polypeptides) as also disclosed herein.
  • the nucleic acid can be a vector.
  • the nucleic acid e.g., the vector
  • the nucleic acid or the non-viral transduction system and the conjugate may be administered to a subject in need of the treatment sequentially.
  • Any of the cancer treatment methods disclosed herein may further comprise one or more of the following: (a) a lymphocyte reduction treatment, which preferably comprises cyclophosphamide and/or fludarabine; (b) a therapeutic agent; and/or (c) a further anti-tumor treatment.
  • the subject to be treated by any of the cancer treatment methods provided herein can be a human patient suffering from a cancer.
  • the human patient may have a solid tumor (e.g., carcinoma, lymphoma, sarcoma and blastoma).
  • the human patient may have a liquid tumor.
  • the human patient may have a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC glioma, glioblastoma, liver cancer, and thyroid cancer.
  • the cancer of B-cell origin is Hodgkin lymphoma or non-Hodgkin lymphoma.
  • the tumor has a low-glucose tumor microenvironment.
  • methods for inhibiting and/or killing tumor cells in a subject using any of the combinations of (a) immune cells, viral particles, and/or nucleic acid, and (b) any of the conjugates provided herein.
  • Other exemplary embodiments provided herein include: A method for inhibiting and/or killing tumor cells in a subject, the method comprising administering to a subject in need thereof an immune cell population in combination with any of the conjugates disclosed herein and the immune cell population as also disclosed herein; wherein a therapeutically effective dose of the immune cell population and a therapeutically effective dose of the conjugate are administered sequentially.
  • a method for inhibiting and/or killing tumor cells in a subject comprising administering to a subject in need thereof a therapeutically effective amount of viral particles, a nucleic acid, a vector, or a non-viral transduction system as disclosed herein in combination with a conjugate disclosed herein; wherein the viral particles, the nucleic acid, the vector or the non- viral transduction system are suitable for transducing immune cells in vivo.
  • a therapeutically effective dose of the viral particles, the nucleic acid, the vector or the non-viral transduction system, and a therapeutically effective dose of the conjugate are administered sequentially.
  • FIGs.1A-1F include diagrams showing SOT101 concentration dependent proliferation of activated T cell products.
  • FIGs.1A-1C show proliferation of activated T cell products in the presence of SOT101. T cells were seeded in 96-well U bottom plate and treated with SOT101.
  • FIG.1A Untransduced T cells (UTD).
  • FIG.1B CAR T cells directed to ROR-1 (CAR).
  • FIG.1C GOT2 expressing CAR T cells directed to ROR-1(BOXR).
  • FIGs. 1D-1F show proliferation of activated T cell products in the presence of SOT 101 in comparison with 0.2 nM IL-2.
  • FIG.1D Untransduced T cells.
  • FIG.1E CAR-T cells.
  • FIG.1F BOXR T cells. Cells were gated on CD8 + , mean and SEM are of 3 donors.
  • FIGs.2A-2N include diagrams showing analysis of T cell memory subsets after SOT101 treatment.
  • FIGs.3A-3F include diagrams showing cytotoxicity of T cell products with SOT101 or IL-2 pre-treatment.
  • Indicated T cells were treated with 0.1 nM SOT101 or IL-2 for 3 days then applied to A549 ROR-1 positive tumor cells (FIGs.3A-3C) or Hep3b ROR-1 negative tumor cells (FIGs.3D-3F).
  • UTD for untransduced T cells CAR for T cells transduced with the CAR construct, GOT2 BOXR or BOXR for T cells transduced with CAR and GOT2.
  • Tumor cells Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC were counted with time.
  • FIGs.4A-4D include diagrams showing memory phenotype of T cell products post tumor cell exposure. T cells were treated with 0.1 nM SOT101 or IL-2 for 3 days then co- cultured with A549 ROR-1 + positive tumor cells or Hep3b ROR-1- tumor cells for 4 days after which cells were analyzed for T cell differentiation markers as outlined for FIG.2.
  • % of CAR + /CD8 + T cells according to the TCM, TSCM, TemRA or TEM phenotype (see gating strategy) after no stimulation (No) or stimulation with 0.1 nM SOT101 or IL-2 for co-culturing with ROR-1 + A549 tumor cells (FIG.4A), or co-culturing with ROR-1 – Hep3b tumor cells (FIG. 4B).
  • % of CAR + CD8 + TEM population CD45RA – , CCR7 –
  • FIG. 4D GOT2 BOXR T cells
  • FIGs.5A-5G include diagrams showing that SOT101 improves CAR T cell or BOXR CAR T cell in vivo efficacy in a human renal cell carcinoma mouse model. Mice implanted with CAKI-1 cell line xenografts were untreated (control) or treated with 3 x 10 6 CAR T cells or BOXR CAR T cells. Dashed vertical lines indicate SOT101 dosage.
  • FIG.5A CAR T cells or CAR T cells in combination with 15 ⁇ g/kg per dose of SOT101 at day 7, 8, 14, and 15 post T cell infusion.
  • FIG.5B BOXR CAR T cells or BOXR CAR T cells in combination with 15 ⁇ g/kg per dose SOT101 at day 7, 8, 14, and 15.
  • GOT2 is the BOXR transgene within the BOXR CAR construct.
  • FIG.5C Tumor volume curves of individual mice within the untreated group at day 7, 8, 14, and 15 post T cell infusion.
  • FIG.5D Tumor volume curves of individual mice within the treated group of CAR T cells at day 7, 8, 14, and 15 post T cell infusion.
  • FIG.5E Tumor volume curves of individual mice within the treated group of CAR T cells and SOT101 at day 7, 8, 14, and 15 post T cell infusion.
  • FIG.5F Tumor volume curves of individual mice within the treated group of BOXR CAR T cells at day 7, 8, 14, and 1.
  • FIG.5G Tumor volume curves of individual mice within the treated group of BOXR T cells in combination with SOT101 at day 7, 8, 14, and 15.
  • FIGs.6A-6C include diagrams showing peripheral blood T cell expansion upon SOT101 treatment. Enumeration of T cells from retro-orbital bleeds. GOT2 is the BOXR transgene within the BOXR CAR construct.
  • FIG.6A T cell counts from CAR T cell and BOXR CAR T cell treated mice with no SOT101 treatment at day 7, 8, 14, and 15 post T cell infusion.
  • FIG.6B T cell counts from mice treated with CAR T cells and SOT101 at day 7, 8, 14, and 15 post T cell infusion.
  • FIG.6C T cell counts from mice treated with BOXR CAR T cells in combination with SOT101 at day 7, 8, 14, and 15. SOT101 improves T cell peripheral expansion, with maximum expansion observed at Day 21.
  • FIGs.7A-7B include diagrams showing that SOT101 improves CAR T cell or BOXR CAR T cell efficacy in a human non-small cell lung cancer mouse model. Mice implanted with NCI-H1975 cell line xenografts were untreated or treated with 3 x 10 6 CAR T cells or BOXR CAR T cells. Dashed vertical lines indicate SOT101 dosage.
  • FIG.7A CAR T cells or CAR T cells in combination with SOT101 at day 7, 8, 14, and 15 post T cell infusion at day 7, 8, 14, and 15.
  • GOT2 is the BOXR transgene within the BOXR CAR construct.
  • FIG.7B BOXR CAR T cells, or BOXR CAR T cells in combination with SOT101 at day 7, 8, 14, and 15.
  • GOT2 is the BOXR transgene within the BOXR CAR construct.
  • Definitions, Abbreviations and Acronyms “Interleukin-15”, “IL-15” or “IL15” refers to the human cytokine as described by NCBI Reference Sequence NP_000576.1 or UniProt ID P40933 (SEQ ID NO: 68).
  • IL-15 derivative or “derivative of IL-15” refers to a protein having a percentage of identity of at least 92%, preferably of at least 96%, more preferably of at least 98%, and most preferably of at least 99% with the amino acid sequence of the mature human IL-15 (114 aa) (SEQ ID NO: 69). In one embodiment, an IL-15 derivative has at least 0.01% activity of human IL-15.
  • an IL-15 derivative has at least 0.1% of the activity of human IL-15, preferably at least 1%, more preferably at least 10%, more preferably at least 25%, even more preferably at least 50%, and most preferably at least 80%.
  • Interleukins are extremely potent but short-lived molecules, even such low activities as 0.01% or 0.1% of human IL-15 may still be sufficiently potent, especially if dosed higher or if an extended half-life compensates for the loss of activity.
  • a reduced binding of a mutant IL-15 or a mutant IL-15/IL-15R ⁇ conjugate to the IL- 2/IL-15R ⁇ receptor results in a lower target-mediated drug deposition and therefore to a longer in vivo half-life.
  • BXRCYT-2302-PC for reducing binding to the IL-15R ⁇ (WO 2016/142314A1); N65K and L69R for abrogating the binding of IL-15R ⁇ (WO 2014/207173A1); Q101D and Q108D for inhibiting the function of IL- 15 (WO 2006/020849A2); S7Y, S7A, K10A, K11A for reducing IL-15R ⁇ binding (Ring et al.
  • IL-15 muteins can be generated by standard genetic engineering methods and are well known in the art, e.g., from WO 2005/085282, US 2006/0057680, WO 2008/143794, WO 2009/135031, WO 2014/207173, WO 2016/142314, WO 2016/060996, WO 2017/046200, WO 2018/071918, WO 2018/071919, US 2018/0118805.
  • IL-15 derivatives may further be generated by chemical modification as known in the art, e.g., by PEGylation or other posttranslational modifications (see WO 2017/112528A2, WO 2009/135031).
  • the IL-15 derivative may have at least 0.01%, preferably at least 0.1% of the activity of human IL-15, more preferably at least 1%, even more preferably at least 10%, even more preferably at least 25%, even more preferably at least 50%, and most preferably at least 80%.
  • the activity of IL-15 derivatives and human IL-15 can be determined by induction of proliferation of kit225 cells as described by Hori et al. (1987). Preferably, methods such as colorimetry or fluorescence are used to determine proliferation activation due to IL-2 or IL-15 stimulation, as for example described by Soman et al. using CTLL-2 cells (Soman et al.2009).
  • PBMCs peripheral blood mononuclear cells
  • buffy coats can be used as an alternative to cell lines such as the kit225 cells.
  • a preferred bioassay to determine the activity of IL-2 or IL-15 is the IL-2/IL-15 Bioassay Kit using STAT5-RE CTLL-2 cells (Promega Catalog number CS2018B03/B07/B05).
  • IL-15R ⁇ refers to the human IL-15 receptor ⁇ or CD215 as described by NCBI Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC Reference Sequence AAI21142.1 or UniProt ID Q13261 (SEQ ID NO: 70).
  • IL-15R ⁇ sushi domain (or IL-15R ⁇ sushi, SEQ ID NO: 71) is the domain of IL-15R ⁇ which is essential for binding to IL-15 (Wei et al.2001).
  • the sushi+ fragment (SEQ ID NO: 72) comprising the sushi domain and part of the hinge region, defined as the fourteen amino acids which are located after the sushi domain of this IL-15R ⁇ , in a C-terminal position relative to said sushi domain, i.e., said IL-15R ⁇ hinge region begins at the first amino acid after said (C4) cysteine residue, and ends at the fourteenth amino acid (counting in the standard “from N-terminal to C-terminal” orientation).
  • the sushi+ fragment reconstitutes full binding activity to IL-15 (WO 2007/046006).
  • IL-15R ⁇ derivative refers to a polypeptide comprising an amino acid sequence having a percentage of identity of at least 92%, preferably of at least 96%, more preferably of at least 98%, and even more preferably of at least 99%, and most preferably 100% identical with the amino acid sequence of the sushi domain of human IL-15R ⁇ (SEQ ID NO: 71) and, preferably of the sushi+ domain of human IL-15R ⁇ (SEQ ID NO: 72).
  • the IL-15R ⁇ derivative is a N- and C-terminally truncated polypeptide, whereas the signal peptide (amino acids 1-30 of SEQ ID NO: 70) is deleted and the transmembrane domain and the intracytoplasmic part of IL- 15R ⁇ is deleted (amino acids 210 to 267 of SEQ ID NO: 70).
  • preferred IL-15R ⁇ derivatives comprise at least the sushi domain (aa 33-93 but do not extend beyond the extracellular part of the mature IL-15R ⁇ being amino acids 31- 209 of SEQ ID NO: 70.
  • IL-15R ⁇ derivatives are the sushi domain of IL-15R ⁇ (SEQ ID NO: 71), the sushi+ domain of IL-15R ⁇ (SEQ ID NO: 72) and a soluble form of IL-15R ⁇ (from amino acids 31 to either of amino acids 172, 197, 198, 199, 200, 201, 202, 203, 204 or 205 of SEQ ID NO: 70, see WO 2014/066527, (Giron-Michel et al.2005)).
  • the IL-15R ⁇ derivative may include natural occurring or introduced mutations. Natural variants and alternative sequences are e.g., described in the UniProtKB entry Q13261 (uniprot.org/uniprot/Q13261).
  • an IL-15R ⁇ derivative has at least 10% of the binding activity of the human sushi domain to human IL-15, e.g. as determined in (Wei et al.2001), more preferably at least 25%, even more preferably at least 50%, and most preferably at least 80%.
  • “RLI-15” refers to an IL-15/IL-15R ⁇ complex being a receptor-linker-interleukin (from N- to C-terminus; “RLI”) fusion protein of the human IL-15R ⁇ sushi+ fragment with the human IL-15.
  • Suitable linkers are flexible with low immunogenicity, examples are described in WO 2007/046006 and WO 2012/175222.
  • the sushi domain or fragment of human IL-15R ⁇ has the sequence as described by SEQ ID NO: 71 from the first to the fourth conserved cysteine, optionally extended N-terminally by T or IT and C-terminally by I.
  • the sushi+ fragment of human IL-15R ⁇ has the sequence as described by SEQ ID NO: 72, which additionally comprises part of the hinge region.
  • SOT101 also “nanrilkefusp alfa”, “RLI2” or “SO-C101” refers to an IL-15/IL-15R ⁇ complex being a receptor-linker-interleukin fusion protein of the human IL-15R ⁇ sushi+ fragment with the human IL-15 represented by (SEQ ID NO: 74) using the linker with the SEQ ID NO: 73.
  • Percentage of identity between two amino acids sequences means the percentage of identical amino acids, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the amino acid sequences.
  • “best alignment” or “optimal alignment” means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two amino acids sequences are usually realized by comparing these sequences that have been previously aligned according to the best alignment; this comparison is realized on segments of comparison in order to identify and compare the local regions of similarity.
  • the best sequences alignment to perform comparison can be realized, beside by a manual way, by using the global homology algorithm developed by Smith and Waterman (1981), by using the local homology algorithm developed by Needleman and Wunsch (1970), by using the method of similarities developed by Pearson and Lipman (1988), by using computer software using such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, WI USA), by using the MUSCLE multiple alignment algorithms (Edgar 2004), or by using CLUSTAL (Goujon et al. 2010).
  • To get the best local alignment one can preferably use the BLAST software with the BLOSUM 62 matrix.
  • the identity percentage between two sequences of amino acids is Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC determined by comparing these two sequences optimally aligned, the amino acids sequences being able to encompass additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences.
  • the percentage of identity is calculated by determining the number of identical positions between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.
  • Conservative amino acid substitutions refer to a substation of an amino acid, where an aliphatic amino acid (i.e.
  • Glycine Alanine, Valine, Leucine, Isoleucine
  • a hydroxyl or sulfur/selenium-containing amino acid i.e. Serine, Cysteine, Selenocysteine, Threonine, Methionine
  • an aromatic amino acid i.e. Phenylalanine, Tyrosine, Tryptophan
  • a basic amino acid i.e.
  • In vivo half-life refers to the time required for a quantity of a drug to be reduced to half of its initial amount in vivo.
  • the in vivo half-life of a particular drug can be determined in any mammal. For example, the in vivo half-life can be determined in humans, primates or mice.
  • the in vivo half-life determined in humans may considerably differ from the in vivo half- life in mice, i.e., the in vivo half-life in mice for a certain drug is commonly shorter than the in vivo half-life determined for the same drug in humans, such in vivo half-life determined in mice still gives an indication for a certain in vivo half-life in humans.
  • the in vivo half-life of the drug in humans can be extrapolated. This is particularly important since the direct determination of the in vivo half-life of a certain drug in humans is rarely possible due to prohibitions of experiments for merely scientific purposes involving humans.
  • the half-life can be determined in primates (e.g., cynomolgus monkeys) which is more similar to the half-life in humans. More specifically, the “in vivo half-life”, (terminal) plasma half-life or T1 ⁇ 2 is the half-life of elimination or half-life of the terminal phase, i.e. following administration, the in vivo half-life is the time required for plasma/blood concentration to decrease by 50% after pseudo-equilibrium of distribution has been reached (Toutain and Bousquet-Melou 2004).
  • the determination of the drug, here the IL- 2/IL-15 ⁇ agonist being a polypeptide, in the blood/plasma is typically done through a polypeptide-specific ELISA.
  • the term “genetically engineered” means in the context of this invention the artificial Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC manipulation, modification, and recombination of DNA or other nucleic acid molecules in order to modify a cell typically by introducing a vector into the immune cell comprising the respective transgene under control of a constitutive or inducible promoter.
  • a vector my remain extrachromosomal or may integrate into the genome of the immune cell at a site different to the location of the endogenous gene (if present).
  • Transient genetically engineering may also be achieved through transfection of mRNA encoding the transgene.
  • treating refers to the application or administration of a composition including one or more active agents to a subject, who has cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or the symptoms of the disease.
  • An effective amount refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and co-usage with other active agents.
  • compositions of the invention are administered in an effective amount that alone, or together with further doses, produces the desired response, e.g., increases anti-cancer effects (e.g., via increasing viability of the immune cell population disclosed herein and/or enhancing anti-tumor effects of the immune cell population in the tumor microenvironment).
  • the desired response includes inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods or can be monitored according to diagnostic methods of the invention discussed herein.
  • the desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC psychological reasons or for virtually any other reasons.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
  • “about” can mean within an acceptable standard deviation, per the practice in the art.
  • “about” can mean a range of up to ⁇ 20%, preferably up to ⁇ 10%, more preferably up to ⁇ 5%, and most preferably still up to ⁇ 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 2-fold, of a value.
  • references to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as “and/or” as defined above.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • IL-15 and IL-15 agonists have been combined with various cellular immune therapies, e.g., co-expressing IL-15 or IL-15/IL-15R ⁇ complexes in CAR T cells leading to constitutive, membrane-tethered IL-15 (WO-2022/192439), combining CAR T cell therapy with Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC pegylated IL-15 therapy in hematological malignancies (clinical trial NCT04136756), or ALT- 803 for treating Burkitt Lymphoma (Chu et al.2015) for quite some time, no real success has been obtained so far.
  • various cellular immune therapies e.g., co-expressing IL-15 or IL-15/IL-15R ⁇ complexes in CAR T cells leading to constitutive, membrane-tethered IL-15 (WO-2022/192439), combining CAR T cell therapy with Attorney Docket No.: 063642-526
  • one embodiment of the present invention is an immune cell population for use in treatment of cancer in a subject, wherein the immune cell population is administered in combination with a conjugate comprising: (i) a polypeptide comprising the amino acid sequence of the interleukin 15 (IL-15) or a derivative thereof, and (ii) a polypeptide comprising the amino acid sequence of the sushi domain of an interleukin 15-receptor alpha (IL-15R ⁇ ) or a derivative thereof.
  • IL-15 interleukin 15
  • IL-15R ⁇ interleukin 15-receptor alpha
  • Immune cell populations may be derived from peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis from each individual patient (i.e., autologous approach) or from a patient or healthy volunteer (i.e., allogeneic approach).
  • PBMCs peripheral blood mononuclear cells
  • PBMCs contain immune cells that are responsive to an IL-15/IL-15R ⁇ conjugate, i.e., cells expressing the IL-2/IL-15R ⁇ , which are able to exert strong anti-tumor activity, both through the innate immune response and the adaptive immune response.
  • the immune cell population of this invention comprises immune cells expressing the IL-2/IL-15R ⁇ receptor.
  • Another embodiment of the present invention is a conjugate comprising a polypeptide comprising the amino acid sequence of the interleukin 15 (IL-15) or a derivative thereof, and a polypeptide comprising the amino acid sequence of the sushi domain of an interleukin 15- receptor alpha (IL-15R ⁇ ) or a derivative thereof for use in treatment of cancer in a subject in combination with immune cell population.
  • the immune cell population comprises immune cells selected from B cells, ⁇ T cells, ⁇ T cells, natural killer (NK) cells or NKT cells.
  • BXRCYT-2302-PC results of clinical trials with infusions of chimeric receptor-expressing autologous T lymphocytes ( ⁇ T cells) provided compelling evidence of their clinical potential.
  • ⁇ T cells autologous T lymphocytes
  • NK cells natural killer cells due to their unique advantages such as low risk of on-target/off-tumor toxicity in normal tissues, cytokine release syndrome and neurotoxicity. They also exhibit natural cytotoxicity against tumor cells.
  • GVHD graft versus host disease
  • an off-the-shelf cell therapy product i.e., allogeneic
  • NK cells are preferred immune cells.
  • ⁇ T cells Another subtype of T cells, i.e., ⁇ T cells, has immense potential in cell therapy sharing similar advantages of that of NK cells additionally displaying immune regulatory functions (Sievers et al.2020, Park and Lee 2021), which makes them another preferred embodiment.
  • NK cells additionally displaying immune regulatory functions
  • persistent activation of these ⁇ T cells due to antigen dependent or independent CAR activation can lead to exhaustion and reduced bioactivity, which is again an advantage of the innate cells such as NK and ⁇ T cells.
  • both NK and ⁇ T cells expressing a chimeric receptor polypeptide may have increased effector functions such as increased inflammatory cytokine production, antigen acquisition and presentation or ability to activate adaptive immune responses.
  • the immune cells are genetically unmodified.
  • the immune cells are genetically modified/engineered.
  • NK cells suitable for therapy may be haploidentical NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, NK cell lines, adaptive NK cells, or cytokine-induced memory-like NK cells. They also may be genetically modified to express a chimeric antigen receptor. Cytokines or cytokine-based molecules including N-803 (ALT-803) are being explored to augment the cytotoxicity and longevity of NK cells, but also NK cell-engager molecules and immune-checkpoint inhibitors (Du et al.2021, Myers and Miller 2021).
  • TILs tumor- infiltrating lymphocytes
  • tumor tissues are Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC digested into small fragments or a single cell suspension and then expanded in culture with IL-2. Reactive TILs may then be enriched/selected by their recognition of autologous tumor cells.
  • the “young TIL” approach the selection stop is ommited.
  • the TIL culture is finally expanded to a clinically relevant level and infused back into the patient (Wang et al. 2021).
  • lifileucel a first TIL-based therapy has been approved by FDA for the treatment of patients with unresectable or metastatic melanoma.
  • the genetically modified T cell therapies especially T cells genetically engineered to express a CAR (Sterner and Sterner 2021) and optionally further transgenes as a safety switch, a cytokine, a metabolism modulating transgene etc.
  • the immune cells comprise one or more of the following features: (I) the immune cells are autologous; (II) the immune cells are allogeneic; (III) the immune cells are activated, expanded, or both ex vivo, and (IV) the immune cells are activated ex vivo in the presence of one or more of 4-1BB ligand, anti-4-1BB antibody, anti-CD3 antibody, anti-CD28 antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL-7, IL-12, IL-21 and K562 cells, and an engineered artificial stimulatory cell or particle prior to administration.
  • the immune cells may be autologous to the subject or patient, i.e., the immune cells are obtained from the subject in need of the treatment, optionally genetically engineered, and then administered to the same subject.
  • the autologous immune cells prior to re-introduction into the subject, are activated and/or expanded ex vivo. Administration of autologous cells to a subject may result in reduced rejection of the immune cells as compared to administration of non-autologous cells.
  • the immune cells are allogeneic cells, i.e., the cells are obtained from a first subject, optionally genetically engineered, and administered to a second subject that is different from the first subject but of the same species.
  • allogeneic immune cells may be derived from a human donor and administered to a human recipient who is different from the donor.
  • NK cells and ⁇ T cells are preferably used to generate off-the-shelf cell therapies.
  • T cells are allogeneic T cells in which the expression of the endogenous T cell receptor has been inhibited or eliminated.
  • the allogeneic immune cells prior to introduction into the subject, are activated and/or expanded ex vivo.
  • NK and T cells can be activated by any method known in the art, e.g., in the presence of one or more agents selected from the group consisting of 4-1BB ligand, anti- 4-1BB antibody, anti-CD3 antibody, anti-CD28 antibody, IL-15, anti-IL-15 receptor antibody, Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC IL-2, IL-7, IL-12, IL-21 and K562 cells, and/or engineered artificial stimulatory cells or particles. See, e.g., US 7,435,596 and US 8,026,097 for the description of useful methods for expanding NK cells.
  • NK cells used in the compositions or methods of the disclosure may be preferentially expanded by exposure to cells that lack or poorly express major histocompatibility complex I and/or II molecules and which have been genetically modified to express membrane bound IL-15 and 4-1BB ligand (CD137L).
  • Such cell lines include, but are not necessarily limited to, K562 [ATCC, CCL 243; (Lozzio and Lozzio 1975, Klein et al.1976)], and the Wilms tumor cell line HFWT (Fehniger and Caligiuri 2001, Harada et al.2004), the uterine endometrium tumor cell line HHUA, the melanoma cell line HMV-II, the hepatoblastoma cell line HuH-6, the lung small cell carcinoma cell lines Lu-130 and Lu-134-A, the neuroblastoma cell lines NB19 and N1369, the embryonal carcinoma cell line from testis NEC 14, the cervix carcinoma cell line TCO-2, and the bone marrow-metastasized neuroblastoma cell line TNB 1 (Harada et al.2002).
  • the cell line used lacks or poorly expresses both MHC I and II molecules, such as the K562 and HFWT cell lines.
  • a solid support may be used instead of a cell line.
  • Such support should preferably have attached on its surface at least one molecule capable of binding to NK cells and inducing a primary activation event and/or a proliferative response or capable of binding a molecule having such an affect thereby acting as a scaffold.
  • the support may have attached to its surface the CD137 ligand protein, a CD137 antibody, the IL-15 protein or an IL-15 receptor antibody.
  • the support will have IL-15 receptor antibody and CD137 antibody bound on its surface.
  • T lymphocytes can be activated by any method known in the art, e.g., in the presence of anti-CD3/CD28, IL-2, IL-15, phytohemoagglutinin, engineered artificial stimulatory cells or particles, or a combination thereof.
  • the method further comprises cryopreserving the population of immune cells for storage and/or transport to clinical sites.
  • the immune cell may be derived from one or more cell lines, from peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells (HSCs), cord blood stem cells or induced pluripotent stem cells (iPSCs), or from tumor tissue.
  • PBMCs peripheral blood mononuclear cells
  • HSCs hematopoietic stem cells
  • iPSCs induced pluripotent stem cells
  • the immune cells described herein can be derived from a cell line, e.g., selected from NK-92, NK-92MI, YTS, and KHYG-1. On one example, the immune cells are derived from NK-92 cells. In other embodiments, the immune cells described herein can be derived from peripheral blood mononuclear cells (PBMC), hematopoietic stem cells (HSCs), cord blood stem cells (CBSCs) or induced pluripotent stem cells (iPSCs). In other embodiments, the immune cell population described herein can be obtained from other sources, Attorney Docket No.: 063642-526001WO Client Ref.
  • PBMC peripheral blood mononuclear cells
  • HSCs hematopoietic stem cells
  • CBSCs cord blood stem cells
  • iPSCs induced pluripotent stem cells
  • the starting population of NK or T cells is obtained from isolating mononuclear cells using ficoll-paque density gradient.
  • the method further comprises depleting the mononuclear cells of CD3, CD14, and/or CD19 cells to obtain the starting population of NK cells.
  • the method further comprises depleting the mononuclear cells CD3, CD14, and CD19 cells to obtain the starting population of NK cells.
  • depleting comprises performing magnetic sorting.
  • NK cells could be positively selected using sorting, magnetic bead selection or other methods to obtain the starting populations of NK cells.
  • specifically lymphocytes are obtained from tumor tissue, i.e., TILs.
  • TILs tumor tissue
  • a source suitable for obtaining the type of immune cells desired would be evident to one of skill in the art.
  • the immune cells can be a mixture of different types of T cells and/or NK cells as known in the art.
  • the immune cells can be a population of immune cells isolated from a suitable donor (e.g., a healthy volunteer for allogeneic approaches or patient).
  • the population of immune cells is derived from PBMCs, which may be obtained from the patient, who is in need for the treatment described herein (autologous approach).
  • the type of immune cells desired e.g., T cells or NK cells
  • T cells or NK cells may be expanded within the population of cells obtained by co-incubating the cells with stimulatory molecules as described supra.
  • immune cells such as NK cells are derived from cord blood stem cells or induced pluripotent stem cells (iPSCs) providing from “off-the shelf” source for immunotherapy (Li et al.2018, Liu et al.2018, Morgan et al.2020, Wrona et al.2021).
  • the starting population of NK cells is obtained from cord blood.
  • the cord blood has previously been frozen.
  • cells are derived from cell lines (e.g., NK-92 and V ⁇ 9V ⁇ 2 T cell).
  • the immune cells derived from tumor tissue are TILs. Methods for obtaining are known in the art. (Wang et al.2021). Chimeric Receptor Polypeptides
  • the immune cell population comprises immune cells, which are genetically engineered to express a chimeric receptor polypeptide, wherein the chimeric receptor polypeptide comprises an extracellular target binding domain, a transmembrane domain; and a cytoplasmic signaling domain.
  • a chimeric receptor polypeptide refers to a non-naturally occurring molecule that can be expressed on the Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC surface of an immune cell and comprises an extracellular target binding domain, a transmembrane domain and at least one cytoplasmic signaling domain.
  • the extracellular target binding domain targets an antigen of interest (e.g., an antigen associated with a disease such as cancer). It may bind to an antigen of interest directly (e.g., an extracellular antigen binding domain in a CAR polypeptide as disclosed herein), or may bind to the antigen of interest via an intermediate, for example, an Fc-containing agent such as an antibody.
  • the transmembrane domain ankers the chimeric receptor polypeptide within the cellular membrane of an immune cell.
  • the chimeric receptor polypeptides are configured such that, when expressed in an immune cell, the extracellular target binding domain is located extracellularly for binding to a target antigen, directly or indirectly, whereas the cytoplasmic signaling domain is located intracellularly to allow for signaling into the cell upon binding of the target binding domain to the target.
  • a chimeric receptor polypeptide may further comprise a hinge domain, one or more co-stimulatory domains, or a combination thereof.
  • Chimeric Antigen Receptor (CAR) In one embodiment, the chimeric receptor polypeptide is a chimeric antigen receptor (CAR) polypeptide.
  • a CAR polypeptide refers to a non-naturally occurring molecule that can be expressed on the surface of an immune cell and comprises an extracellular antigen binding domain, together with the transmembrane domain and the cytoplasmic signaling domain.
  • the CAR polypeptides described herein may further include at least one co-stimulatory signaling domain.
  • the extracellular antigen binding domain re-directs the specificity of immune cells (e.g., NK, ⁇ T or ⁇ T cells) expressing the CAR polypeptide to the target tumor cells.
  • an extracellular antigen binding domain refers to a peptide or polypeptide having binding specificity to a target antigen of interest, which can be a naturally occurring antigen.
  • a target antigen may be any molecule that is associated with a disease or condition, including, but are not limited to, tumor antigens.
  • the target antigen binding domain targets a native tumor antigen protein.
  • the target antigen binding domain targets a variant (e.g., mutation) of a tumor antigen protein.
  • Some examples include EGFRvIII scFv recognizes the tumor specific variant of EGFR (Wang et al. 2020).
  • the target antigens are pathogenic antigens, e.g., bacterial, fungal, or viral antigens, or antigens present on diseased cells, such as those described herein.
  • the immune cell population of this invention is used in one embodiment for Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC use in the treatment of infectious diseases caused by bacteria, fungi or viruses.
  • Non-limiting examples of the extracellular antigen binding domains are tumor antigens, pathogenic antigens and immune cells specific to an autoantigen (Gubin et al.2015, Linnemann et al.2015). Respective diseases and/or conditions to be treated include tumors, inflammatory conditions and auto-immune disorders.
  • the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues.
  • the extracellular antigen binding domain binds to a tumor antigen, which is associated with a hematologic or solid tumor.
  • Non-limiting examples of solid tumor extracellular binding domains are domains of GD2, GPC3, FOLR (e.g., FOLR1 or FOLR2), HER2, EphA2, EGFRVIII, IL13RA2, VEGFR2, ROR1, NKG2D, EpCAM, CEA, Mesothelin, MUC1, CLDN18.2, CD171, CD133, PSCA, cMET, EGFR, PSMA, FAP, CD70, MUC16, L1-CAM, B7H3, GUCY2C, Nectin4, LRRC15, PSMA and CAIX.
  • tumor antigens are derived from cancers that are characterized by tumor-associated antigen expression, such as HER2 expression.
  • the extracellular antigen binding domain of any CAR polypeptides described herein is a peptide or polypeptide capable of binding to a cell surface antigen (e.g., a native and mutated tumor antigen), and may be presented on the cell surface of an antigen- presenting cell.
  • the extracellular antigen binding domain is a single-chain variable fragment (scFv).
  • extracellular antigen binding domain is a single domain antibody.
  • the scFv or single domain antibody binds to a tumor antigen, a pathogenic antigen, or an immune cell specific to an autoantigen. These may be Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC derived from an antibody that binds the target cell surface antigen with a high binding affinity.
  • Table 1 lists exemplary cell-surface target antigens and exemplary antibodies binding to such.
  • Table 1 Exemplary Cell Surface Target Antigen and Exemplary Antibodies Binding to Such Exemplary target Exemplary Exemplary Exemplary target Exemplary nti n ntib di nti n antibodies and Fc- s Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC Exemplary target Exemplary Exemplary target Exemplary antigens antibodies antigens antibodies and Fc- Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC Exemplary target Exemplary Exemplary target Exemplary antigens antibodies antigens antibodies and Fc- Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC Exemplary target Exemplary Exemplary target Exemplary antigens antibodies antigens antibodies and Fc- 7 Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC Exemplary target Exemplary Exemplary target Exemplary antigens antibodies antigens antibodies and Fc- s Attorney Docket No.: 063642-526001WO Client Ref.
  • the extracellular antigen binding domain may comprise an antigen binding fragment (e.g., an scFv) derived from any of the antibodies listed in Table 1 depending upon the target antigen of interest.
  • the antigen binding fragment e.g., an scFv
  • the antigen binding fragment may comprise the same heavy chain and light chain complementarity determining regions (CDRs) as the antibodies listed in Table 1 depending upon the target antigen of interest.
  • the antigen binding fragment (e.g., an scFv) may comprise the same heavy chain variable region (VH) and light chain variable region VL as the antibodies listed in Table 1 depending upon the target antigen of interest.
  • the extracellular antigen binding domain of any of the CAR polypeptides described herein may be specific to a pathogenic antigen, such as a bacterial antigen, a viral antigen, or a fungal antigen.
  • influenza virus neuraminidase hemagglutinin, or M2 protein
  • human respiratory syncytial virus (RSV) F glycoprotein or G glycoprotein herpes simplex virus glycoprotein gB, gC, gD, or gE, Chlamydia MOMP or PorB protein
  • Dengue virus core protein matrix protein, or glycoprotein E
  • measles virus hemagglutinin herpes simplex virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoproteins of HIV 1, hepatitis B core antigen or surface antigen, diptheria toxin, Streptococcus 24M epitope, Gonococcal pilin, pseudorabies virus g50 (gpD), pseudorabies virus II (gpB), pseudorabies virus III (gpC), pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, coronavirus polypeptides, transmissible gastroenteritis glycoprotein 195, transmiss
  • RSV human respiratory
  • the extracellular antigen binding domain of the CAR polypeptide described herein may be specific to a tag conjugated to a therapeutic agent, which targets an antigen associated with a disease or disorder (e.g., a tumor antigen or a pathogenic antigen as described herein).
  • the tag conjugated to the therapeutic agent can be antigenic and the extracellular antigen binding domain of the CAR polypeptide can be an antigen-binding fragment (e.g., scFv) of an antibody having high binding affinity and/or specificity to the antigenic tag.
  • Exemplary antigenic tags include, but are not limited to, biotin, avidin, a fluorescent molecule (e.g., GFP, YRP, luciferase, or RFP), Myc, Flag, His (e.g., poly His such as 6xHis), HA (hemagglutinin), GST, MBP (maltose binding protein), KLH (keyhole limpet hemocyanins), trx, T7, HSV, VSV (e.g., VSV-G), Glu-Glu, V5, e-tag, S-tag, KT3, E2, Au1, Au5, and/or thioredoxin.
  • biotin avidin
  • a fluorescent molecule e.g., GFP, YRP, luciferase, or RFP
  • Myc Flag
  • His e.g., poly His such as 6xHis
  • HA hemagglutinin
  • GST hemagglutinin
  • MBP maltose binding
  • the tag conjugated to the therapeutic agent is a member of a ligand- receptor pair and the extracellular antigen binding domain comprises the other member of the Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC ligand-receptor pair or a fragment thereof that binds the tag.
  • the tag conjugated to the therapeutic agent can be biotin and the extracellular antigen binding domain of the CAR polypeptide can comprise a biotin-binding fragment of avidin. See, e.g., (Urbanska et al.2012, Lohmueller et al.2017).
  • anti-Tag CAR in which the extracellular antigen binding domain is a scFv fragment specific to a protein tag, such as FITC (Tamada et al. 2012, Kim et al.2015, Cao et al.2016, Ma et al.2016), PNE (Ma et al.2016), La-SS-B (Cartellieri et al.2016), Biotin (Lohmueller et al.2017) and Leucine-Zipper (Cho et al.2018). Selection of the antigen binding domain for use in the CAR polypeptides described herein will be apparent to one of skill in the art.
  • binding affinity refers to the apparent association constant or KA or the KD.
  • the KA is the reciprocal of the dissociation constant (KD).
  • the extracellular antigen binding domain for use in the CAR polypeptides described herein may have a binding affinity (KD) of at least 10 -5 , 10 -6 , 10 7 , 10 -8 , 10 -9 , 10 -10 M, or lower for the target antigen or antigenic epitope.
  • KD binding affinity
  • An increased binding affinity corresponds to a decreased KD.
  • Higher affinity binding of an extracellular antigen binding domain for a first antigen relative to a second antigen can be indicated by a higher K A (or a smaller numerical value KD) for binding the first antigen than the KA (or numerical value KD) for binding the second antigen.
  • the extracellular antigen binding domain has specificity for the first antigen (e.g., a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein).
  • Differences in binding affinity can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 100,000-fold.
  • Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay).
  • Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration.
  • the concentration of bound binding protein [Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation: Attorney Docket No.: 063642-526001WO Client Ref.
  • the chimeric receptor polypeptide is a T-Cell Receptor (TCR) polypeptide.
  • TCR T-Cell Receptor
  • the TCR polypeptide comprising an extracellular domain, or portion thereof, of a TCR ⁇ chain, a TCR ⁇ chain, a TCR ⁇ chain, a TCR ⁇ chain, a CD3 ⁇ TCR subunit, a CD3 ⁇ TCR subunit, a CD3 ⁇ TCR subunit or a CD3 ⁇ TCR subunit.
  • the TCR is further modified (e.g., express multiple TCRs against same or different target antigens or same or different epitopes on the same antigen or increasing affinity for the target or amino acid substitutions to increase preferential TCR chain pairing).
  • TCR-expressing immune cells e.g., TCR-expressing T cells, also called “TCR T cells”
  • they may enhance toxicity against cancer cells targeted by the TCR-CD3 complex via their binding to the peptide-MHC complex (Wieczorek et al.2017, Shafer et al.2022).
  • Exemplary TCR polypeptides for use with the methods and compositions described herein are affinity matured TCRs or recombinant TCR-like molecules, but also naturally occurring TCRs. Typically they are, or are introduced as, ⁇ and ⁇ chains that dimerize within the cell and complex with endogenous CD3 components to form a functional TCR that redirects T cell specificity towards an antigen of interest as reviewed by Shafer et al. (2022).
  • Respective CD3 components provided by the transfected immune cell are the CD3 chains ⁇ , ⁇ , ⁇ and ⁇ forming together with the transfected ⁇ and ⁇ chains the T cell receptor that redirects the T cell towards an antigenic peptide presented at the cell surface by MHC molecules.
  • antigen-presenting MHC alleles are broadly classified as HLA class I (A, B, or C), presenting predominantly cytosolic peptides, or HLA class II (DR, DP, or DQ), which predominantly present extracellular derived peptides.
  • TCR modifications may be introduced to prevent mispairing and maximize surface expression.
  • Various modifications to TCRs are known in the art (Shafer et al.2022).
  • TCRs according to the invention may be murinized TCRs, where the human TCR constant regions is replaced Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC with those of a mouse TCR constant region.
  • TCR constant region stabilizes interchain binding affinity of engineered TCR ⁇ / ⁇ chains while reducing their binding affinity with endogenous TCR ⁇ / ⁇ chains.
  • the stability of the engineered TCR ⁇ chain can be increased through select hydrophobic substitutions in its transmembrane region.
  • Domain swapped TCRs also exist, where large or specific segments of the engineered TCR ⁇ / ⁇ constant regions are inverted, which reduces propensity of engineered TCRs to mispair.
  • scTCR single-chain TCRs
  • the chimeric receptor polypeptide is an antibody-coupled T cell Receptor (ACTR) protein, which can be expressed in an immune cell (e.g., an NK cell or a T cell).
  • an immune cell e.g., an NK cell or a T cell.
  • the ACTR protein containing an extracellular Fc-binding domain.
  • the chimeric receptor polypeptides may also be antibody-coupled T cell receptor (ACTR) polypeptides that comprise an Fc binding domain.
  • the ACTR polypeptides described herein comprise an extracellular target binding domain that is an Fc binding domain, i.e., capable of binding to the Fc portion of an immunoglobulin (e.g., IgG, IgA, IgM, or IgE) of a suitable mammal (e.g., human, mouse, rat, goat, sheep, or monkey).
  • Suitable Fc binding domains may be derived from naturally occurring proteins such as mammalian Fc receptors or certain bacterial proteins (e.g., protein A, protein G).
  • Fc binding domains may be synthetic polypeptides engineered specifically to bind the Fc portion of any of the antibodies described herein with high affinity and specificity.
  • an Fc binding domain can be an antibody or an antigen-binding fragment thereof that specifically binds the Fc portion of an immunoglobulin. Examples include, but are not limited to, a single-chain variable fragment (scFv), a domain antibody, or single domain antibodies (e.g., nanobodies).
  • an Fc binding domain can be a synthetic peptide that specifically binds the Fc portion, such as a Kunitz domain, a small modular immune pharmaceutical (SMIP), an adnectin, an avimer, an affibody, a designed ankyrin repeat proteins (DARPin), or an anticalin, which may be identified by screening a peptide combinatory library Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC for binding activities to Fc.
  • the Fc binding domain is an extracellular ligand-binding domain of a mammalian Fc receptor.
  • an “Fc receptor” is a cell surface bound receptor that is expressed on the surface of many immune cells (including B cells, T cells and NK cells and exhibits binding specificity to the Fc domain of an antibody.
  • Fc receptors are typically comprised of at least two immunoglobulin (Ig)-like domains with binding specificity to an Fc (fragment crystallizable) portion of an antibody.
  • Ig immunoglobulin
  • binding of an Fc receptor to an Fc portion of the antibody may trigger antibody dependent cell-mediated cytotoxicity (ADCC) effects.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • the Fc receptor used for constructing an ACTR polypeptide as described herein may be a naturally occurring polymorphism variant (e.g., the CD16 V158 variant), which may have increased or decreased affinity to Fc as compared to a wild-type counterpart.
  • the Fc receptor may be a functional variant of a wild-type counterpart, which carry one or more mutations (e.g., up to 10 amino acid residue substitutions including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations) that alter the binding affinity to the Fc portion of an Ig molecule.
  • the mutation may alter the glycosylation pattern of the Fc receptor and thus the binding affinity to Fc.
  • Transmembrane domain refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane.
  • transmembrane domains may also or alternatively be classified based on the transmembrane domain topology, including the number of passes that the transmembrane domain makes across the membrane and the orientation of the protein.
  • single-pass membrane proteins cross the cell membrane once
  • multi- Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC pass membrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times).
  • Membrane proteins may be defined as Type I, Type II or Type III depending upon the topology of their termini and membrane-passing segment(s) relative to the inside and outside of the cell.
  • Type I membrane proteins have a single membrane-spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is present on the cytoplasmic side.
  • Type II membrane proteins also have a single membrane-spanning region but are oriented such that the C-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the N- terminus of the protein is present on the cytoplasmic side.
  • Type III membrane proteins have multiple membrane-spanning segments and may be further sub-classified based on the number of transmembrane segments and the location of N- and C-terminus.
  • the transmembrane domain of the chimeric receptor polypeptide described herein is derived from a Type I single-pass membrane protein.
  • the transmembrane domain is of a membrane protein selected from the group consisting of CD8 ⁇ , CD8 ⁇ , 4-1BB/CD137, CD27, CD28, CD34, CD4, Fc ⁇ RI ⁇ , CD16 ⁇ , OX40/CD134, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD32, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, FGFR2B, CD2, IL-15R, DNAM-1, 2B4, NKG2D, NKp44 and NKp46.
  • the transmembrane domain is from a membrane protein selected from the group consisting of CD8 ⁇ , CD8 ⁇ , 4-1BB, CD28, CD34, CD4, Fc ⁇ RI ⁇ , CD16 ⁇ , OX40, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , CD32, CD64, VEGFR2, FAS, NKG2D, NKp44, NKp46 and FGFR2B.
  • the transmembrane domain is of CD8 ⁇ (e.g., the transmembrane domain is of CD8 ⁇ ).
  • the transmembrane domain is of 4-1BB/CD137.
  • the transmembrane domain is of CD28.
  • the transmembrane domain is of NKG2D, NKp44 or NKp46. In other examples, the transmembrane domain is of CD34. In yet other examples, the transmembrane domain is not derived from human CD8 ⁇ . In some embodiments, the transmembrane domain of the chimeric receptor polypeptide is a single-pass alpha helix.
  • the amino acid sequences of exemplary transmembrane domains are provided in Table 2. Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC Table 2: Exemplary Transmembrane Domains Transmembrane Sequence SEQ domain ID NO.
  • Transmembrane domains from multi-pass membrane proteins may also be compatible for use in the chimeric receptor polypeptides described herein.
  • Multi-pass membrane proteins may comprise a complex alpha helical structure (e.g., at least 2, 3, 4, 5, 6, 7 or more alpha helices) or a beta sheet structure.
  • the N-terminus and the C-terminus of a multi-pass membrane protein are present on opposing sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side.
  • the reverse orientation of such a native transmembrane protein may be constructed for efficient orientation of the chimeric receptor Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC polypeptide (e.g., CAR) within the immune cell membrane.
  • BXRCYT-2302-PC polypeptide e.g., CAR
  • Either one or multiple helices passes from a multi-pass membrane protein can be used for constructing the chimeric receptor polypeptide described herein.
  • Transmembrane domains for use in the chimeric receptor polypeptides described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment.
  • the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet.
  • the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids.
  • Examples of synthetic transmembrane domains are known in the art, for example in US 7,052,906B1 and WO 2000/032776A2, the relevant disclosures of each of which are incorporated by reference herein.
  • the amino acid sequence of the transmembrane domain does not comprise cysteine residues.
  • the amino acid sequence of the transmembrane domain comprises one cysteine residue.
  • the amino acid sequence of the transmembrane domain comprises two cysteine residues.
  • the amino acid sequence of the transmembrane domain comprises more than two cysteine residues (e.g., 3, 4, 5, or more).
  • the transmembrane domain may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain.
  • the cytoplasmic region of the transmembrane domain may comprise three or more amino acids and, in some embodiments, helps to orient the transmembrane domain in the lipid bilayer.
  • one or more cysteine residues are present in the transmembrane region of the transmembrane domain.
  • one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain.
  • the cytoplasmic region of the transmembrane domain comprises positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine. In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic.
  • the transmembrane region comprises a poly- leucine-alanine sequence.
  • the hydropathy, hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed by any method known in the art including, for example, the Kyte and Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC Doolittle hydropathy analysis.
  • Cytoplasmic signaling domain Any cytoplasmic signaling domain can be used to create the chimeric receptor polypeptides described herein (e.g., CAR polypeptides, TCR, or ACTR polypeptides).
  • Such a cytoplasmic domain may be any signaling domain involved in triggering cell signaling (primary signaling) that leads to immune cell proliferation and/or activation.
  • the cytoplasmic signaling domain as described herein is not a co-stimulatory signaling domain, which, as known in the art, relays a co-stimulatory or secondary signal for fully activating immune cells (e.g., CAR-T).
  • the amino acid sequence of one exemplary cytoplasmic signaling domain of human CD3 ⁇ is: cytoplasmic Sequence SEQ ID NO. signaling domain
  • the cytoplasmic signaling domain is of CD3 ⁇ or Fc ⁇ R1 ⁇ .
  • the cytoplasmic signaling domain is of CD3 ⁇ .
  • cytoplasmic signaling domain is not derived from human CD3 ⁇ .
  • the cytoplasmic signaling domain is not derived from an FcR, when the extracellular Fc-binding domain of the same chimeric receptor polypeptide is derived from CD16A.
  • several signaling domains can be fused together for additive or synergistic effect.
  • useful additional signaling domains include part or all of one or more of TCR ⁇ chain, CD28, OX40/CD134, 4-1BB/CD137, Fc ⁇ RI ⁇ , ICOS/CD278, IL2R ⁇ /CD122, IL-2R ⁇ /CD132, and CD40.
  • the chimeric receptor polypeptide comprises one or more of the following features: (i) the chimeric receptor polypeptide further comprises a signal peptide at its N-terminus; (ii) the chimeric receptor polypeptide further comprises a hinge domain, which is located at the C-terminus of the extracellular target binding domain and the N-terminus of transmembrane domain; (iii) the chimeric receptor polypeptide is free of a hinge domain; (iv) the chimeric receptor polypeptide further comprises at least one co-stimulatory signaling domain; (v) the chimeric receptor polypeptide is free of a co-stimulatory signaling domain; and (vi) the Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC cytoplasmic signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • the chimeric receptor polypeptide e.g., CAR, TCR or ACTR polypeptide
  • the nucleic acid encoding the chimeric receptor polypeptide is also encoding a signal peptide, whereas in the mature polypeptide the signal peptide has been cleaved off.
  • signal sequences are peptide sequences that target a polypeptide to the desired site in a cell.
  • the signal sequence targets the chimeric receptor polypeptide to the secretory pathway of the cell and will allow for integration and anchoring of the chimeric receptor polypeptide into the lipid bilayer.
  • Signal sequences including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences that are compatible for use in the chimeric receptor polypeptides described herein will be evident to one of skill in the art.
  • the signal sequence is from CD8 ⁇ (24).
  • the signal sequence is from CD28 (25).
  • the signal sequence is from the murine kappa chain (26).
  • the signal sequence is from CD16. See Table 3. Table 3. Exemplary Signal Peptides Signal Peptide Sequence SEQ ID NO.
  • the chimeric receptor polypeptides (e.g., CAR, TCR, or ACTR polypeptide) described herein further comprise a hinge domain that is located between the extracellular ligand-binding domain and the transmembrane domain.
  • a hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular ligand- binding domain relative to the transmembrane domain of the chimeric receptor polypeptide can be used.
  • chimeric receptor polypeptide comprises a hinge domain, which is a hinge domain selected from the list of CD28, CD16A, CD8, IgG, murine CD8 ⁇ , and DAP12.
  • the hinge domain is of CD8 ⁇ (e.g., the hinge domain is of CD8 ⁇ ).
  • the hinge domain is a portion of the hinge domain of CD8, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8.
  • the hinge domain stems from CD28.
  • the hinge domain is a portion of the hinge domain of CD28, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD28.
  • the hinge domain and/or the transmembrane domain may be linked to additional amino acids (e.g., 15 aa, 10-aa, 8-aa, 6-aa, or 4-aa) at the N-terminal portion, at the C-terminal portion, or both. Examples can be found, e.g., in Ying et al. (2019).
  • the hinge domain is of a CD16A receptor, for example, the whole hinge domain of a CD16A receptor or a portion thereof, which may consist of up to 40 consecutive amino acid residues of the CD16A receptor (e.g., 20, 25, 30, 35, or 40).
  • Such a chimeric receptor polypeptide may contain no hinge domain from a different receptor (a non-CD16A receptor).
  • the chimeric receptor polypeptide described herein may be free of a hinge domain from any non-CD16A receptor.
  • such a chimeric receptor polypeptide may be free of any hinge domain.
  • Hinge domains of IgG antibodies such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the chimeric receptor polypeptides described herein.
  • the hinge domain joins the constant domains CH1 and CH2 of an antibody.
  • the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody.
  • the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody, preferably IgG1 and IgG4.
  • the hinge region comprises the hinge region and the CH2 and CH3 Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC constant regions of an IgG1 antibody.
  • the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.
  • Non-naturally occurring peptides may also be used as hinge domains for the chimeric receptor polypeptides described herein.
  • the hinge domain between the C- terminus of the extracellular target-binding domain and the N-terminus of the transmembrane domain is a peptide linker, such as a (Gly x Ser) n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.
  • the hinge domain is (Gly 4 Ser) n , wherein n can be an integer between 3 and 60, including 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In certain embodiments, n can be an integer greater than 60. In some embodiments, the hinge domain is (Gly4Ser)3. In some embodiments, the hinge domain is (Gly4Ser)6. In some embodiments, the hinge domain is (Gly4Ser)9.
  • the hinge domain is (Gly4Ser)12. In some embodiments, the hinge domain is (Gly4Ser)15. In some embodiments, the hinge domain is (Gly4Ser)30. In some embodiments, the hinge domain is (Gly4Ser)45. In some embodiments, the hinge domain is (Gly4Ser)60. In other embodiments, the hinge domain is an extended recombinant polypeptide (XTEN), which is an unstructured polypeptide consisting of hydrophilic residues of varying lengths (e.g., 10-80 amino acid residues).
  • XTEN extended recombinant polypeptide
  • the hinge domain is an XTEN peptide and comprises 60 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 30 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 45 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 15 amino acids. Any of the hinge domains used for making the chimeric receptor polypeptide as described herein may contain up to 250 amino acid residues.
  • the chimeric receptor polypeptide may contain a relatively long hinge domain, for example, containing 150- 250 amino acid residues (e.g., 150-180 amino acid residues, 180-200 amino acid residues, or 200-250 amino acid residues).
  • the chimeric receptor polypeptide may contain a medium sized hinge domain, which may contain 60-150 amino acid residues (e.g., 60-80, 80- 100, 100-120, or 120-150 amino acid residues).
  • the hinge domain may be a flexible linker consisting of glycine and serine amino acids having a length between 15 and 60 amino acids, preferably composed of Gly 4 Ser units, especially one of the linkers of 38 to 40.
  • the chimeric receptor polypeptide may contain a short hinge domain, which may contain less than 60 amino acid residues (e.g., 1-30 amino acids or 31-60 amino acids).
  • a chimeric receptor polypeptide (e.g., an ACTR polypeptide) described herein contains no hinge domain or no hinge domain from a non-CD16A receptor.
  • the amino acid sequences of exemplary hinge domains are provided in Table 4: Table 4: Exemplary Hinge Domains Hinge domain Sequence SEQ ID NO.
  • chimeric receptor polypeptides described herein may further Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC comprise a hinge domain, which may be located at the C-terminus of the extracellular target binding domain and the N-terminus of the transmembrane domain.
  • the hinge domain may be of any suitable length.
  • the chimeric receptor polypeptide described herein may have a shortened hinge domain (e.g., including up to 25 amino acid residues).
  • the chimeric receptor polypeptide described herein is free of a hinge domain, i.e., does not have a hinge domain.
  • Co-stimulatory signaling domain For many immune cells (e.g., NK or T cells) it is beneficial to include a co-stimulatory signaling domain for stimulation of an antigen-specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell.
  • co- stimulatory signaling domain refers to at least a fragment of a co-stimulatory signaling protein that mediates signal transduction within a cell to induce an immune response such as an effector function (a secondary signal).
  • T cells such as T cells often require two signals: (1) the antigen specific signal (primary signal) triggered by the engagement of T cell receptor (TCR) and antigenic peptide/MHC complexes presented by antigen presenting cells, which typically is driven by CD3 ⁇ as a component of the TCR complex; and (ii) a co-stimulatory signal (secondary signal) triggered by the interaction between a co-stimulatory receptor and its ligand.
  • a co-stimulatory receptor transduces a co- stimulatory signal (secondary signal) as an addition to the TCR-triggered signaling and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.
  • Activation of a co-stimulatory signaling domain in an immune cell may induce the cell to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity.
  • the co-stimulatory signaling domain of any co-stimulatory molecule may be compatible for use in the chimeric receptor polypeptides described herein.
  • the type(s) of co-stimulatory signaling domain is selected based on factors such as the type of the immune cells in which the chimeric receptor polypeptides would be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC).
  • the chimeric receptor polypeptide of the genetically engineered immune cell comprises at least one co- stimulatory signaling domain.
  • co-stimulatory signaling domains for use in the chimeric receptor polypeptides may be the cytoplasmic signaling domain of co-stimulatory proteins, including, without limitation, members of the B7/CD28 family (e.g., B7-1/CD80, B7- Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC 2/CD86 B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, and PDCD6); members of the TNF superfamily (e.g.,4-1BB/TNFRSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF
  • the chimeric receptor polypeptides may contain a CD28 co-stimulatory signaling domain or a 4-1BB (CD137) co-stimulatory signaling domain.
  • at least one co-stimulatory signaling domain is selected from the group consisting of CD8 ⁇ , CD28, 4-1BB, 2B4, OX40, OX40L, ICOS, CD27, GITR, HVEM, TIM1, LFA1, CD2, DAP10, DAP12, DNAM-1, NKG2D, NKp30, NKp44, NKp46 and JAMAL, or any functional variant thereof.
  • the co-stimulatory signaling domains comprise up to 10 amino acid residue mutations (e.g., 1, 2, 3, 4, 5, or 8) such as amino acid substitutions, deletions, or additions as compared to a wild-type counterpart.
  • Such co-stimulatory signaling domains comprising one or more amino acid variations may be referred to as variants.
  • Mutation of amino acid residues of the co-stimulatory signaling domain may result in an increase in signaling transduction and enhanced stimulation of immune responses relative to co- stimulatory signaling domains that do not comprise the mutation. Mutation of amino acid residues of the co-stimulatory signaling domain may result in a decrease in signaling transduction and reduced stimulation of immune responses relative to co-stimulatory signaling Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC domains that do not comprise the mutation. For example, mutation of residues 186 and 187 of the native CD28 amino acid sequence may result in an increase in co-stimulatory activity and induction of immune responses by the co-stimulatory domain of the chimeric receptor polypeptide.
  • the mutations are substitution of a lysine at each of positions 186 and 187 with a glycine residue of the CD28 co-stimulatory domain, referred to as a CD28 LL ⁇ GG variant. Therefore, a suitable variant of CD28 is the CD28 LL ⁇ GG variant. Additional mutations can be made in co-stimulatory signaling domains that may enhance or reduce co-stimulatory activity of the domain will be evident to one of ordinary skill in the art. In some embodiments, the co-stimulatory signaling domain is selected from the group of 4-1BB, CD28, OX40, and CD28 LL ⁇ GG variant.
  • the chimeric receptor polypeptides may contain a single co- stimulatory domain such as, for example, a CD27 co-stimulatory domain, a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, an ICOS co-stimulatory domain, an OX40 co- stimulatory domain, an OX40L co-stimulatory domain, a 2B4 co-stimulatory domain, a GITR co-stimulatory domain, a NKG2D co-stimulatory domain, a NKp30 co-stimulatory domain, a NKp44co-stimulatory domain, a NKp46 co-stimulatory domain, a DAP10 co-stimulatory domain, a DAP12 co-stimulatory domain, a DNAM1 co-stimulatory domain, a LFA-1 co- stimulatory domain, a HVEM co-stimulatory domain or a JAMAL co-stimulatory domain.
  • a single co- stimulatory domain such as, for example
  • the at least one co-stimulatory signaling domain is a CD28 co-stimulatory signaling domain or a 4-1BB co-stimulatory signaling domain. Selection of the type(s) of co-stimulatory signaling domains may be based on factors such as the type of immune cells (e.g., ⁇ T, ⁇ T or NK cells) to be used with the chimeric receptor polypeptides and the desired immune effector function.
  • the chimeric receptor polypeptides may comprise more than one co-stimulatory signaling domain (e.g., 2, 3, or more).
  • the chimeric receptor polypeptide comprises at least two co-stimulatory signaling domains.
  • the chimeric receptor polypeptide comprises two co-stimulatory signaling domains. In some embodiments, the chimeric receptor polypeptide comprises two or more of the same co- stimulatory signaling domains, for example, two copies of the co-stimulatory signaling domain of CD28. In some embodiments, the chimeric receptor polypeptide comprises two or more co- stimulatory signaling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein.
  • the chimeric receptor polypeptide may comprise two or more co-stimulatory signaling domains from different co- stimulatory receptors, such as any two or more co-stimulatory receptors described herein, for Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC example, CD28 and 4-1BB, CD28 and CD27, CD28 and ICOS, CD28 LL ⁇ GG variant and 4-1BB, CD28 and OX40, or CD28 LL ⁇ GG variant and OX40.
  • the two co- stimulatory signaling domains are CD28 and 4-1BB.
  • the two co- stimulatory signaling domains are CD28 LL ⁇ GG variant and 4-1BB.
  • the two co-stimulatory signaling domains are CD28 and OX40. In some embodiments, the two co- stimulatory signaling domains are CD28 LL ⁇ GG variant and OX40. In some embodiments, the chimeric receptor polypeptides described herein may contain a combination of a CD28 and ICOSL. In some embodiments, the chimeric receptor polypeptide described herein may contain a combination of CD28 and CD27. In certain embodiments, the 4-1BB co-stimulatory domain is located N-terminal to the CD28 or CD28 LL ⁇ GG variant co-stimulatory signaling domain.
  • one of the co-stimulatory signaling domains is a CD28 co- stimulatory signaling domain and the other co-stimulatory domain is selected from the group consisting of a CD8 ⁇ , 4-1BB, 2B4, OX40, OX40L, ICOS, CD27, GITR, HVEM, TIM1, LFA1, CD2, DAP10, DAP12, DNAM-1, NKG2D, NKp30, NKp44, NKp46 and JAMAL co-stimulatory signaling domain.
  • one of the co-stimulatory signaling domains is a CD8 ⁇ co-stimulatory signaling domain and the other co-stimulatory domain is selected from the group consisting of a CD28, 4-1BB, 2B4, OX40, OX40L, ICOS, CD27, GITR, HVEM, TIM1, LFA1, CD2, DAP10, DAP12, DNAM-1, NKG2D, NKp30, NKp44, NKp46 and JAMAL co-stimulatory signaling domain.
  • one of the co-stimulatory signaling domains is a 4- 1BB co-stimulatory signaling domain and the other co-stimulatory domain is selected from the group consisting of a CD8 ⁇ , CD28, 2B4, OX40, OX40L, ICOS, CD27, GITR, HVEM, TIM1, LFA1, CD2, DAP10, DAP12, DNAM-1, NKG2D, NKp30, NKp44, NKp46 and JAMAL co- stimulatory signaling domain.
  • the amino acid sequences of exemplary co-stimulatory signaling domains are provided in Table 5.
  • Table 5 Exemplary Co-Stimulatory Signaling Domains Co-stimulatory Sequence SEQ O.
  • chimeric receptor polypeptide is free of any co-stimulatory signaling domain.
  • the cytoplasmic signaling domain described herein may comprise an immunoreceptor tyrosine-based activation motif (ITAM) domain (e.g., at least one ITAM domain, at least two ITAM domains, or at least three ITAM domains).
  • ITAM immunoreceptor tyrosine-based activation motif
  • An “ITAM,” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells.
  • the motif may comprise two repeats of the amino acid sequence YxxL/I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL/Ix (6-8) YxxL/I.
  • ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule.
  • ITAMs may also function as docking sites for other proteins involved in signaling pathways.
  • Examples of ITAMs for use in the chimeric receptor polypeptides comprised within the cytoplasmic signaling domain may be: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , each containing a single ITAM motif while each ⁇ chain contains 3 distinct ITAM domains ( ⁇ a, ⁇ b and ⁇ c).
  • the number and ITAM sequences are also important in the design of CARs (Bettini et al.2017, Jayaraman et al.2020).
  • cytoplasmic signaling domain examples include, but are not limited to, the cytoplasmic signaling domain of Jak/STAT, Toll-interleukin receptor (TIR), and tyrosine kinase.
  • TIR Toll-interleukin receptor
  • tyrosine kinase examples include, but are not limited to, the cytoplasmic signaling domain of Jak/STAT, Toll-interleukin receptor (TIR), and tyrosine kinase.
  • TIR Toll-interleukin receptor
  • tyrosine kinase examples include, but are not limited to, the cytoplasmic signaling domain of Jak/STAT, Toll-interleukin receptor (TIR), and tyrosine kinase.
  • the cytoplasmic signaling domain described herein is free of the ITAM motif.
  • a chimeric receptor polypeptide as described herein may comprise, from N-terminus to C-terminus, the extracellular target binding
  • a chimeric receptor polypeptide as described herein comprises, from N-terminus to C-terminus, the extracellular target binding domain, the transmembrane domain, at least one co-stimulatory Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC signaling domain, and the cytoplasmic signaling domain.
  • a chimeric receptor polypeptide as described herein comprises, from N-terminus to C-terminus, the extracellular target binding domain, the transmembrane domain, the cytoplasmic signaling domains, and at least one co-stimulatory signaling domain.
  • the CAR comprises, from N- to C-terminus, a CD8 ⁇ signal peptide, an scFv against a tumor target of choice, an IgG4 hinge domain, a CD28 transmembrane domain, a 4-1BB co-stimulatory signaling domain and a CD3 ⁇ cytoplasmic signaling domain.
  • a CAR polypeptide described herein may comprise (i) a CD28 co- stimulatory domain or a 4-1BB co-stimulatory domain; and (ii) a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof.
  • a CAR polypeptide described herein may comprise (i) a CD28 co-stimulatory domain or a 4-1BB co- stimulatory domain, (ii) a CD8 ⁇ transmembrane domain, a CD8 ⁇ hinge domain, or a combination thereof.
  • the CAR polypeptides may further include one or more co-stimulatory signaling domains, one of which may be a CD28 co-stimulatory signaling domain or a 4-1BB co-stimulatory signaling domain.
  • a CAR polypeptide described herein may comprise (i) a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain, (ii) a CD28 transmembrane domain, a CD8 ⁇ hinge domain, or a combination thereof.
  • the CAR polypeptide comprises (i) a CD8 ⁇ hinge domain (ii) a CD8 ⁇ transmembrane domain, (iii) a CD28 co-stimulatory domain or a 4-1BB co- stimulatory domain, (iv) a CD3 ⁇ cytoplasmic signaling domain or a combination thereof.
  • the CAR polypeptide comprising two co-stimulatory domains further comprises (i) a CD8 ⁇ or CD28 hinge domain (ii) a CD8 ⁇ or CD28 transmembrane domain, (iii) a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain, (iv) a OX40L co-stimulatory domain, a 2B4 co-stimulatory domain, a DAP10 co-stimulatory domain, a DNAM-1 co- stimulatory domain, a NKG2D co-stimulatory domain, a NKp30 co-stimulatory domain, a NKp44 co-stimulatory domain, a NKp46 co-stimulatory domain or a JAMAL co-stimulatory domain, or (v) a CD3 ⁇ cytoplasmic signaling domain or a combination thereof.
  • the CAR polypeptide comprising two co-stimulatory domains further comprises (i) a CD8 ⁇ hinge domain (ii) a CD28 transmembrane domain, (iii) a CD28 co- stimulatory domain or a 4-1BB co-stimulatory domain, (iv) a OX40L co-stimulatory domain, a 2B4 co-stimulatory domain, a DAP10 co-stimulatory domain, a DNAM-1 co-stimulatory or a JAMAL co-stimulatory domain, or (v) a CD3 ⁇ cytoplasmic signaling domain or a combination thereof.
  • the CAR polypeptide comprising two co-stimulatory domains further comprises (i) a CD8 ⁇ hinge domain (ii) a CD28 transmembrane domain, a NKp44 transmembrane domain, a NKG2D transmembrane domain or a NKp46 transmembrane domain, (iii) a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, a 2B4 co- stimulatory domain or a DAP10 co-stimulatory, (iv) an OX40L co-stimulatory domain, a 2B4 co-stimulatory domain, a DAP10 co-stimulatory domain, a DAP12 co-stimulatory domain, a DNAM-1 co-stimulatory domain or a JAMAL co-stimulatory domain, or (v) a CD3 ⁇ cytoplasmic signaling domain, a DAP12 cytoplasmic signaling domain
  • the CAR polypeptide comprises (i) a CD8 ⁇ hinge domain (ii) a CD28 transmembrane domain, (iii) a CD28 co- stimulatory domain or a 4-1BB co-stimulatory domain, (iv) an OX40L co-stimulatory domain or an OX40 co-stimulatory domain, (v) a CD3 ⁇ cytoplasmic signaling domain or a combination thereof.
  • the CAR polypeptide may comprise an amino acid sequence selected from SEQ ID NO: 84 or SEQ ID NO: 85 provided below.
  • the CAR polypeptides are configured such that, when expressed on an immune cell (e.g., T or NK cell), the extracellular antigen-binding domain is located extracellularly for binding to a target molecule (e.g., a tumor antigen) and the CD3 ⁇ cytoplasmic signaling domain is located intracellularly for signaling into the cell.
  • a target molecule e.g., a tumor antigen
  • the co-stimulatory signaling domain may be located in the cytoplasm for triggering activation and/or effector signaling.
  • a CAR polypeptide as described herein may comprise, from N-terminus to C-terminus, the extracellular antigen binding domain, the transmembrane domain, the optional one or more co-stimulatory domains (e.g., a CD28 co-stimulatory domain, a 4-1BB co- stimulatory signaling domain, an OX40L co-stimulatory signaling domain, an OX40 co- stimulatory signaling domain, a CD27 co-stimulatory signaling domain, a 2B4 co-stimulatory signaling domain or an ICOS co-stimulatory signaling domain), and the CD3 ⁇ cytoplasmic signaling domain.
  • co-stimulatory domains e.g., a CD28 co-stimulatory domain, a 4-1BB co- stimulatory signaling domain, an OX40L co-stimulatory signaling domain, an OX40 co- stimulatory signaling domain, a CD27 co-stimulatory signaling domain, a 2B4 co-stimulatory signaling domain or
  • the CAR polypeptides described herein may contain two or more co-stimulatory signaling domains, which may link to each other or be separated by the cytoplasmic signaling domain.
  • the extracellular antigen binding domain, transmembrane domain, optional co-stimulatory signaling domain(s), and cytoplasmic signaling domain in a CAR polypeptide may be linked to each other directly, or via a peptide linker.
  • any of the CAR polypeptides described herein may comprise a signal sequence at the N-terminus.
  • a protein X transmembrane domain refers to any portion of a given protein, i.e., transmembrane-spanning Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC protein X, that is thermodynamically stable in a membrane.
  • a protein X cytoplasmic signaling domain for example, a CD3 ⁇ cytoplasmic signaling domain, refers to any portion of a protein (protein X) that interacts with the interior of a cell or organelle and is capable of relaying a primary signal as known in the art, which leads to immune cell proliferation and/or activation.
  • the cytoplasmic signaling domain as described herein differs from a co-stimulatory signaling domain, which relays a secondary signal for fully activating immune cells.
  • a protein X co-stimulatory signaling domain refers to the portion of a given co-stimulatory protein (protein X, such as CD28, 4-1BB, OX40, CD27, or ICOS) that can transduce co-stimulatory signals (secondary signals) into immune cells (such as T cells), leading to fully activation of the immune cells.
  • protein X such as CD28, 4-1BB, OX40, CD27, or ICOS
  • another embodiment of the present invention is an immune cell population comprising an immune cell, wherein the immune cell are genetically engineered to further expresses or overexpresses a polypeptide that (a’) improves glucose uptake activity; (b’) modulates the Krebs cycle; (c’) enhances lactate concentration; or (d’) diverts or redirects glucose metabolites out of a glycolysis pathway, referred herein as metabolism modulating polypeptides.
  • the expression or overexpression is mediated by genetic engineering of the immune cell. Typically, this is achieved by introducing a vector or vector genome into the immune cell comprising the respective transgene under control of a constitutive or inducible promoter.
  • Such vector my remain extrachromosomal or may integrate into the genome of the immune cell at a site different to the location of the endogenous gene (if present).
  • Suitable vectors are retroviral or lentiviral vectors that are routinely used also for introducing CARs into the immune cells.
  • adenovirus oder andeno-associated virus vectors may be used.
  • the conding sequence for such polypeptide is by the same viral vector under in the same expression cassette as the chimeric receptor polypeptide separated, for example, by a P2A ribosomal skip sequence.
  • BXRCYT-2302-PC “Expressed” is used for metabolism modulating polypeptides, respectively their genes, which are also referred to as bolt-on genes or BOXR genes, introduced into a immune cell which does not express such gene, whereas “overexpressed” is used for metabolism modulating polypeptides, respectively their genes, that are naturally expressed in the immune cell from its endogenous allels, but which introduction of a further copy leads to more of the respective polypeptide in the immune cells, which can be measured by increased RNA expression (e.g., by quantitative PCR), protein expression (e.g, Western Blot) or by measuring the resepective enzyme activity.
  • RNA expression e.g., by quantitative PCR
  • protein expression e.g, Western Blot
  • the metabolism modulating polypeptide is present on a vector within the cell or is integrated at a non-endogenous site of the genome within the cell. In one embodiment, the metabolism modulating polypeptide is present in the cell at a copy number that is higher than the endogenous copy number of said metabolism modulating polypeptide.
  • the metabolism modulating polypeptide may be present at a copy number of at least 3 copies, at least 5 copies or at least 7 copies per cell. The copy number refers to the number of genomic copies of the metabolism modulating polypeptide present within a cell.
  • the polypeptide that improves glucose uptake activity is a glucose importation polypeptide.
  • the glucose importation polypeptide is a glucose transporter (GLUT) or a sodium-glucose cotransporter (SGLT). Examples include GLUT1, GLUT1 S226D variant, GLUT3, GLUT8, GLUT8 L12A L13A variant, GLUT11, GLUT7, GLUT4, SGLT1 or SGLT2.
  • GLUT1 S226D variant GLUT3, GLUT8, GLUT8 L12A L13A variant, GLUT11, GLUT7, GLUT4, SGLT1 or SGLT2.
  • GLUT1, GLUT1 S226D variant, GLUT3, GLUT8, GLUT8 L12A L13A variant, GLUT11, GLUT7, GLUT4, SGLT1 or SGLT2 are disclosed therein as SEQ ID NOs: 81-90. Further included are functional vairants thereof as dislcosed in WO 2020/010110.
  • the preferred glucose importation polypeptide is GLUT1.
  • the polypeptide that modulates the Krebs cycle is: • an enzyme that catalyzes a reaction in the Krebs cycle, for example, isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), or phosphoglycerate dehydrogenase Attorney Docket No.: 063642-526001WO Client Ref.
  • IDH isocitrate dehydrogenase
  • MDH malate dehydrogenase
  • phosphoglycerate dehydrogenase Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC PEGDH
  • PSAT1 glutamic-oxaloacetic transaminase
  • GDH1 glutamate dehydrogenase
  • GGPT1 glutamate- pyruvate transaminase 1
  • GLS glutaminase
  • PCK1, GOT1, GOT2, GPT1, GLS, PSAT1 and GDH1 are disclosed therein as SEQ ID NOs: 86-92. Further included are functional vairants thereof as dislcosed in WO 2020/037066.
  • Preferred Krebs cycle modulating genes are GOT2 and GFPT1.
  • the polypeptide that enhances lactate concentration is: • monocarboxylate transporter (MCT), for example, MCT1, MCT2, or MCT4, • an enzyme involved in lactate synthesis, for example, lactate dehydrogenase A (LDHA), or • a polypeptide that inhibits a pathway that competes for lactate-synthesis substrates, for example, pyruvate dehydrogenase kinase 1 (PDK1).
  • MCT monocarboxylate transporter
  • LDHA lactate dehydrogenase A
  • PDK1 pyruvate dehydrogenase kinase 1
  • the polypeptide that diverts or redirects glucose metabolites is pyruvate kinase muscle isozyme M2 (PKM2), glutamine-fructose-6-phosphate aminotransferase 1 (GFPT1), or TP53-inducible glycolysis and apoptosis regulator (TIGAR).
  • PKM2 pyruvate kinase muscle isozyme M2
  • GFPT1 glutamine-fructose-6-phosphate aminotransferase 1
  • TIGAR TP53-inducible glycolysis and apoptosis regulator
  • the polypeptide that diverts or redirects glucose metabolism is TIGAR. Attorney Docket No.: 063642-526001WO Client Ref.
  • the expression or overexpression of the polypeptide comprising the amino acid sequence of any one of (a’) – (d’) or derivatives thereof, i.e., a polypeptide that improves glucose uptake activity, modulates the Krebs cycle, enhances lactate concentration, or diverts or redirects glucose metabolites out of a glycolysis pathway, or derivatives thereof lead to an at least 70% increase of proliferation of the immune cell population when compared to the native immune cell population of the same type not expressing or overexpressing such polypeptide.
  • the increase in proliferation of an immune cell population can be determined by the assay described in example 10.
  • the assay may be conducted at different glucose conditions, whereas 1.25 mM glucose reflects more typical energy-deprived conditions in the tumor/tumor microenvironment and 10 mM glucose reflects glucose levels in the peripheral blood.
  • glucose level preferably the expression or overexpression of the polypeptide lead to an at least 100% increase of proliferation of the immune cell population when compared to the native immune cell population of the same type not expressing or overexpressing such polypeptide.
  • two metabolism modulating polypeptides are transfected into immune cells.
  • the genetically engineered immune cells described herein, co-expressing at least two metabolism modulating polypeptides and a CAR polypeptide can be used in immune therapy such as T-cell therapy (both ⁇ and ⁇ T cells), NK-cell therapy or NKT-cell therapy for inhibiting and/or killing diseased cells expressing an antigen to which a CAR or TCR polypeptide targets, directly or indirectly (e.g., via a therapeutic agent conjugated to a tag to which the CAR polypeptide binds).
  • Examples of the at least two metabolism modulating polypeptides include: (a) GOT2 and TIGAR; (b) GOT2 and GLUT1; (c) GOT2 and PDK1; (d) TIGAR and GLUT1; (e) PDK1 and Cystathionine gamma-lyase (CTH), CTH; (f) CTH and Phosphoserine phosphatase (PSPH); (g) GLUT1 and Argininosuccinate synthase 1 (ASS1); and (h) GLUT1 and PSPH.
  • nucleic acid or nucleic acid set encoding the metabolism modulating polypeptide and/or a chimeric receptor polypeptide is comprised within one or more Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC viral vectors The nucleic acids and the vector(s) may be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of the nucleic acid encoding at least two metabolism modulating polypeptides and/or the chimeric receptor polypeptides.
  • the synthetic linkers may contain nucleic acid sequences that correspond to a particular restriction site in the vector.
  • An exemplary embodiment is a method of modifying the metabolism of immune cells, comprising transfecting immune cells transiently or stably with the vector or vector set and collecting immune cells transfected with the vector or vector set.
  • the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene or the kanamycin gene for selection of stable or transient transfectants in immune cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; intron sequences from the human EF1-alpha gene, transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 or polyomavirus origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESs), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase or an inducible caspase such as iCasp9), and reporter gene(s) for assessing expression of the metabolism modulating poly
  • such vectors also include a suicide gene.
  • suicide gene refers to a gene that causes the cell expressing the suicide gene to die.
  • the suicide gene can be a gene (e.g., HSV thymidine kinase) that confers sensitivity to an agent, e.g., a drug (e.g., ganciclovir for HSV thymidine kinase), upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent.
  • agent e.g., a drug for HSV thymidine kinase
  • Suicide genes are known in the art (see, for example, Springer, C. J.
  • HSV Herpes Simplex Virus
  • TK thymidine kinase
  • cytosine deaminase purine nucleoside phosphorylase
  • nitroreductase and caspases such as caspase 8 or caspase-9 (iCasp9).
  • Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Examples of the preparation of vectors for expression of metabolism Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC modulating polypeptides and/or chimeric receptor polypeptides can be found, for example, in US 2014/0106449, herein incorporated in its entirety by reference.
  • Any of the vectors comprising a nucleic acid sequence that encodes the metabolism modulating polypeptides and/or a chimeric receptor polypeptide described herein is also within the scope of the present invention.
  • Such a vector, or the sequence encoding such metabolism modulating polypeptides and/or a chimeric receptor polypeptide contained therein may be delivered into immune cells such as immune cells by any suitable method.
  • Methods of delivering vectors to immune cells are well known in the art and may include DNA electroporation, RNA electroporation, transfection using reagents such as liposomes, or viral transduction (e.g., retroviral transduction such as lentiviral or gamma-retroviral transduction).
  • viral transduction e.g., retroviral transduction such as lentiviral or gamma-retroviral transduction.
  • the vectors for expression of the at least two metabolism modulating polypeptides and/or the chimeric receptor polypeptides are delivered to immune cells by viral transduction (e.g., retroviral transduction such as lentiviral or gamma-retroviral transduction).
  • Exemplary viral methods for delivery include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; and WO 91/02805; US 5,219,740 and US 4,777,127; GB 2,200,651; and EP 0345242), alphavirus-based vectors, and adeno-associated virus (AAV) vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984; and WO 95/00655).
  • retroviruses see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230;
  • the vectors for expression of the at least two metabolism modulating polypeptides and/or the chimeric receptor polypeptides are retroviruses.
  • the vectors are lentiviruses. Examples of references describing retroviral transduction include Anderson et al., US 5,399,346; (Mann et al.1983); US 4,650,764; US 4,980,289; (Markowitz et al.1988); US 5,124,263; WO 95/07358 and (Kuo et al.1993).
  • WO 95/07358 describes high efficiency transduction of primary B lymphocytes.
  • viral vectors or viral particles that capable of infecting the immune cells may be produced by any method known in the art and can be found, for example in WO 91/02805A2, WO 98/09271A1, and US 6,194,191.
  • the viral particles are harvested from the cell culture supernatant and may be isolated and/or purified prior to contacting the viral particles with the immune cells.
  • RNA molecules encoding the at least two metabolism modulating polypeptides and/or the chimeric receptor polypeptides as described herein may be prepared by a Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC conventional method (e.g., in vitro transcription) and then introduced into suitable immune cells, e.g., those described herein, via known methods, e.g., Rabinovich, Komarovskaya et al. (2006).
  • the nucleic acid encoding the metabolism modulating polypeptides and/or the nucleic acid encoding a suitable chimeric receptor polypeptide may be cloned into separate expression vectors, which may be introduced into suitable immune cells concurrently or sequentially.
  • an expression vector (or an RNA molecule) for expressing the metabolism modulating polypeptides may be introduced into immune cells first and the transfected immune cells expressing the metabolism modulating polypeptides may be isolated and cultured in vitro.
  • an expression vector (or an RNA molecule) expressing a suitable chimeric receptor polypeptide can be introduced into the immune cells expressing the metabolism modulating polypeptide(s).
  • the nucleic acid(s) encoding the metabolism modulating polypeptides and/or the chimeric receptor polypeptide may be delivered into immune cells via transposons (e.g., piggybac).
  • the encoding nucleic acid(s) may be delivered into immune cells via gene editing, for example, by CRISPR, TALEN, zinc-finger nuclease (ZFN), or meganucleases.
  • the nucleic acid encoding the metabolism modulating polypeptides and the nucleic acid encoding the chimeric receptor polypeptide may be cloned into the same expression vector.
  • Polynucleotides for expression of the chimeric receptor polypeptide and the metabolism modulating polypeptides are also within the scope of the present disclosure.
  • useful vectors of the disclosure include viral vectors such as, e.g., retroviral vectors including gamma retroviral vectors and lentiviral vectors, adeno- associated virus vectors (AAV vectors) and adenoviruses.
  • the nucleic acid described herein may comprise two or three coding sequences, one encoding a chimeric receptor polypeptide as described herein, and the other one or two encoding metabolism modulating polypeptides.
  • the nucleic acid comprising the coding sequences described herein may be configured such that the coding sequences encoding the metabolism modulating polypeptides can be expressed as independent (and physically separate) polypeptides.
  • the nucleic acid described herein may contain a fourth, optionally fifth or sixth, nucleotide sequence located between the coding sequences for the metabolism modulating polypeptides.
  • This fourth, fifth and/or sixth nucleotide sequence may, for example, encode a ribosomal skip site.
  • a ribosomal skip site is a sequence that impairs normal peptide bond formation. This mechanism results in the translation of additional open reading frames from one messenger RNA.
  • This fourth, fifth and/or sixth nucleotide sequence Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC may, for example, encode a P2A, T2A, or F2A peptide (see, for example, Kim et al. (2011). See Table 6 below.
  • Table 6 Exemplary Ribosomal Skip Site Ribosomal Skip Sequence SEQ ID NO Site bosome . d- independent manner, also permitting the translation of additional open reading frames from one messenger RNA.
  • the fourth nucleotide sequence may encode a promoter controlling the expression of the second polypeptide and/or the third polypeptide.
  • the fourth nucleotide sequence may also encode more than one ribosomal skipping sequence, IRES sequence, additional promoter sequence, or a combination thereof.
  • IRES sequence is provided below (SEQ ID NO: 67): GAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTC TCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCT TCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGG CGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCAC AACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCA AGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGA TCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAA
  • the additional coding sequences may be separated from other coding sequences for a polypeptide by one or more nucleotide sequences encoding one or more ribosomal skipping sequences, IRES sequences, or additional promoter sequences.
  • the nucleic acid e.g., an expression vector or an RNA molecule as described herein
  • the nucleic acid may comprise coding sequences for at least two metabolism modulation polypeptides and a suitable chimeric receptor polypeptide, the coding sequences (for example, Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC three), in any order, being separated by a fourth nucleotide sequence coding for a P2A peptide.
  • three separate polypeptides, at least two metabolism modulating polypeptides and the chimeric receptor can be produced from such a nucleic acid, wherein at least one P2A skip site is linked C-terminally to the upstream polypeptide (encoded by the upstream coding sequence) and the C-terminal residue P from the P2A peptide is linked to the downstream polypeptide (encoded by the downstream coding sequence).
  • the chimeric receptor polypeptide is the upstream one and the metabolism modulating polypeptide(s) is/are downstream.
  • the metabolism modulating polypeptide(s) is/are upstream and the chimeric receptor polypeptide is downstream.
  • the nucleic acid may comprise coding sequences of at least two metabolism modulating polypeptides (e.g., those described herein) and a suitable ACTR or CAR polypeptide, the coding sequences, in any order, being separated by a fourth nucleotide sequence coding for a P2A peptide.
  • nucleic acid described above may further encode a linker (e.g., a GSG linker) between two segments of the encoded sequences, for example, between the upstream polypeptide and the P2A peptide.
  • a linker e.g., a GSG linker
  • RNA molecules can be prepared by in vitro transcription or by chemical synthesis.
  • the RNA molecules can then be introduced into suitable immune cells (e.g., ⁇ or ⁇ T or NK cells) by, e.g., electroporation.
  • suitable immune cells e.g., ⁇ or ⁇ T or NK cells
  • electroporation e.g., electroporation.
  • RNA molecules can be synthesized and introduced into immune cells following the methods described in Rabinovich, Komarovskaya et al. (2006) and WO 2013/040557.
  • a vector(s) or RNA molecule(s) comprising at least two metabolism modulating polypeptides and/or the chimeric receptor polypeptide may be introduced to the immune cells in vivo.
  • this may be accomplished by administering a vector or RNA molecule described herein directly to the subject (e.g., through intravenous administration).
  • the IL-15/IL-15R ⁇ conjugates (e.g., fusion polypeptides) according to the invention comprise an IL-15 or derivative thereof and at least the sushi domain of the IL-15R ⁇ or derivative thereof.
  • IL-2/IL-15R ⁇ i.e., the receptor consisting of the IL-2/IL-15R ⁇ and the ⁇ c subunits, which is expressed on NK cells, CD8 + T cells, NKT cells and ⁇ T cells.
  • IL-2/IL-15R ⁇ the receptor consisting of the IL-2/IL-15R ⁇ and the ⁇ c subunits, which is expressed on NK cells, CD8 + T cells, NKT cells and ⁇ T cells.
  • the complex comprises a human IL-15 (SEQ ID NO: 69) or a derivative thereof, and the sushi domain of the human IL-15R ⁇ (SEQ ID NO: 71), the sushi+ fragment/domain of the human IL-15R ⁇ (SEQ ID NO: 72) or a soluble form of the human IL- 15R ⁇ (from amino acids 31 to either of amino acids 172, 197, 198, 199, 200, 201, 202, 203, 204 or 205 of SEQ ID NO: 70, see WO 2014/066527, (Giron-Michel et al.2005)).
  • the IL-15/IL-15R ⁇ conjugate comprises the polypeptides (i) comprising the amino acid sequence of human IL-15 or a derivative thereof, wherein the IL-15 derivative has an amino acid sequence having a percentage of identity of at least 92.5 % with the amino acid of SEQ ID NO: 69 (mature human IL-15), and the polypeptide (ii) comprising the sushi domain of the human IL-15R ⁇ or a derivative thereof, wherein the sushi domain derivative has an amino acid sequence having a percentage of identity of at least 92% with the amino acid sequence of SEQ ID NO: 71 (sushi domain of human IL-15R ⁇ ).
  • the sushi domain comprises an amino acid sequence at least 92% identical with SEQ ID NO: 72 (sushi+ fragment of human IL-15R ⁇ ).
  • SEQ ID NO: 72 ushi+ fragment of human IL-15R ⁇
  • One specific example is an IL-15/IL-15R ⁇ conjugate comprising an IL-15 derivative with an amino acid sequence having a percentage of identity of at least 92.5 % with the amino acid of SEQ ID NO: 69 and comprising the sushi domain of the human IL-15R ⁇ with the amino acid of SEQ ID NO: 71.
  • the conjugate comprises the amino acid sequence of the IL-15 or derivatives thereof in a C-terminal position relative to the amino acid sequence of the sushi domain of the IL-15R ⁇ or derivatives thereof.
  • the amino acid sequence of the IL-15 or derivatives thereof and the amino acid sequence of the sushi domain of the IL-15R ⁇ or derivatives are separated by a linker amino acid sequence.
  • the linker is preferably flexible, preferably has a length of 2-30 amino acids, more preferably of 10-30 amino acids, more preferably of 15-30 amino acids, still more preferably of 15-25 amino acids, most preferably of 18-22 amino acids. G and S are preferred amino acids for the flexible linker.
  • a suitable linker is provided as SEQ ID NO: 73.
  • the human IL-15 preferably comprises the sequence of SEQ ID NO: 69 and the human IL-15R ⁇ Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC sushi domain comprises the sequence of SEQ ID NO: 71, more preferably comprising the sushi+ fragment/domain of SEQ ID NO: 72.
  • Such fusion protein is preferably in the order (from N- to C-terminus) IL-15R ⁇ -linker-IL-15 (RLI-15).
  • An especially preferred IL-15/IL-15R ⁇ conjugate is the fusion protein designated SOT101 (RLI2) having the sequence of SEQ ID NO: 74.
  • Other formats of IL-15/IL-15R ⁇ conjugates are also encompassed by the invention.
  • the IL-15 and the IL-15R ⁇ sushi domain-containing polypeptide may be covalently linked through an introduced disulfide bond, e.g., by introduced L52C substation in IL-15 and S40C substation in the sushi domain.
  • the IL-15 and the IL-15R ⁇ may be independently fused to a carrier protein/dimerizing carrier proteins, e.g., a heterodimeric Fc fusion protein as described in US 2018/0118805 (para.0161 to 0228), where IL-15 is bound to the N-terminus of one Fc chain and the IL-15R ⁇ sushi domain is bound to the N-terminus of a second Fc chain to form a heterodimeric conjugate through the disulfide bonds of the Fc domains.
  • a carrier protein/dimerizing carrier proteins e.g., a heterodimeric Fc fusion protein as described in US 2018/0118805 (para.0161 to 0228)
  • P-22339 are XmAb24306.
  • P-22339 refers to an IL-15/IL-15R ⁇ sushi conjugate, where IL-15 is bound to the N-terminus of one Fc chain and the IL-15R ⁇ sushi domain is bound to the N-terminus of a second Fc chain as described in WO 2016/095642 and Hu et al. (2016) with the L52C substitution on the IL-15 polypeptide (SEQ ID NO: 78) and the S40C substitution on the IL-15R ⁇ sushi+ polypeptide (SEQ ID NO: 79) forming a disulfide bond.
  • XmAb24306 refers to an IL-15/IL-15R ⁇ sushi conjugate, where a mutant IL-15 is bound to the N-terminus of one Fc chain and the IL-15R ⁇ sushi domain is bound to the N-terminus of a second Fc chain as described in as described in US 2018/0118805 (see XENP024306 in Fig.94C, SEQ ID NO: 80 and SEQ ID NO: 81).
  • the IL-15/IL-15R ⁇ conjugates is a half-life extended conjugate. Half-life extension is partly leading to the improved stimulation by the IL-15/IL-15R ⁇ conjugates according to this invention, as the prolonged half-life leads to a stronger activation of NK and CD8 + T cells.
  • the IL-15/IL-15R ⁇ conjugate comprises (i) a polypeptide comprising the amino acid sequence of an IL-15 derivatives having at least 0.1% of the activity of human IL-15 on the proliferation induction of kit225 cells, and (ii) a polypeptide comprising the amino acid sequence of the sushi domain of IL-15R ⁇ or derivatives thereof having at least 10% of the binding activity of the sushi domain of human IL-15R ⁇ to human IL-15.
  • the IL-15 derivative has only at least 0.01 of the activity of human IL-15 on the Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC proliferation induction of kit225 cells.
  • the (in vitro) activity of IL-15 can be determined by induction of proliferation of kit225 cells as described by Hori et al. (1987). Kit225 cells (Hori et al.1987) are passaged in kit225 base medium and used for the potency assay at passage 4-7.
  • kit225 cells are cultivated in kit225 base medium without IL-2 for 24h (starvation period).1x10 4 kit225 cells are plated in 96-well plate and a serial dilution of test IL- 15 compounds added to cells. Cells are incubated at 37°C, 5% CO 2 for 72 ⁇ 3h. Following the incubation, 10 ⁇ l (10% of the volume in the well) of Alamar Blue is added to each well and, after 6 h, absorbance is measured at 560 nm with a 620 nm reference using a Tecan Spark absorbance microplate reader (set mixing before detection for 15 s).
  • kits225 cells when lower potency of IL-15 mutants is tested, the incubation with kit225 cells should be prolonged from 3 days (72h ⁇ 3h) to 5 days.
  • methods such as colorimetry or fluorescence are used to determine proliferation activation due to IL-15 stimulation, as for example described by Soman et al. using CTLL-2 cells (Soman et al.2009).
  • PBMCs peripheral blood mononuclear cells
  • buffy coats can be used.
  • a preferred bioassay to determine the activity of IL-15 is the IL-2/IL-15 Bioassay Kit using STAT5-RE CTLL-2 cells (Promega Catalog number CS2018B03/B07/B05).
  • an IL-15 derivative has at least 0.1% of the activity of human IL-15, preferably 1%, more preferably at least 10%, more preferably at least 25%, even more preferably at least 50%, and most preferably at least 80%.
  • Interleukins are extremely potent but short-lived molecules, even such low activities as 0.1% of human IL-15 may still be sufficiently potent, especially if dosed higher or if an extended half-life compensates for the loss of activity.
  • a reduced binding of a mutant IL-15 or a mutant IL-15/IL-15R ⁇ conjugate to the IL-2/IL-15R ⁇ receptor results in a lower target-mediated drug deposition and therefore to a longer in vivo half- life.
  • said interleukin 15 derivative has at least 25% of the activity of human interleukin-15 on the proliferation induction of kit225 cell line, more preferably at least 50%, and has an amino acid sequence having a percentage of identity of at least 92.5% with an amino acid sequence of SEQ ID NO: 69, preferably of at least 96%, and more preferably of at least 98.5% or of at least 99% identiy.
  • said derivative of the sushi domain of IL-15R ⁇ has at least 25%, more preferably at least 50% of the binding activity of the sushi domain of human IL-15R ⁇ , and has an amino acid sequence having a percentage of identity of at least 92.5% with an amino acid Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC sequence selected in the group comprising SEQ ID NO: 71 or SEQ ID NO: 72, preferably of at least 96%, and more preferably of at least 98% identity.
  • Administration The immune cell population of the invention and the IL-15/IL-15R ⁇ conjugate of the invention are manufactured as separate pharmaceutical compositions, each being formulated with respective buffers and excipients.
  • an effective dose of the immune cell population and an effective dose of the conjugate are administered sequentially, more preferably the conjugate is administered after the immune cell population. In one embodiment, the conjugate is administered at least about 5 days, but not more than 21 days after the immune cell population.
  • the immune cell population comprising immune effector cells especially for the treatment of solid tumors over a couple of days lose (part of) their tumoricidal activity due to one or more of the factors of upregulation of PD-1, entering/staying in the immune-suppressive tumor microenvironment, suffering from energy deprivation in the tumor microenvironment, and getting exhausted.
  • the immune cell population is administered to the subject at a dose of at least about 1 x 10 4 immune cells/kg, preferably from about 1 x 10 4 to about 1 x 10 12 cells/kg.
  • patients can be treated by infusing therapeutically effective doses of the immune cell population as described here in the range of about 10 5 to 10 10 or more cells per kilogram of body weight (cells/kg).
  • the infusion can be repeated as often and as many times as the patient can tolerate until the desired response is achieved, to be aligned with the dosing schedule of the IL-15/IL-15R ⁇ conjugate as disclosed below.
  • the immune cell population is administered at day 1, the conjugate is then administered at days 8, 9, 15, and 16 (i.e., twice per week at two following days for two weeks starting one week after the immune cell administration, followed by one week of treatment break).
  • the next cycle starting again with the administration of the immune cells begins at day 29 corresponding to day 1 of the first cycle.
  • the appropriate infusion dose and schedule will vary from patient to patient, but can be determined by the treating physician for a particular patient. Typically, initial doses of approximately 10 6 cells/kg will be infused, escalating to 10 8 or more cells/kg.
  • the administration of an IL-15/IL-15R ⁇ conjugate which Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC also at low concentration in the tumor remains undissociated as a fusion protein, is then capable of reactivating such immune effector cells and thereby boosting their activity.
  • the conjugate is administered about 7 days after the administration of the immune cell population, preferably the conjugate is administered in treatment cycles starting at about 7 days after the administration of the immune cell population.
  • Treatment cycles for IL-15/IL-15R ⁇ conjugates typically comprise the administration for one or multiple times within a treatment period of two to three weeks, e.g., once or twice per week for 2 or 3 weeks, followed by a period of, e.g., 1 or 2 weeks without conjugate administration for resetting the immune system. Then a new cycle can begin. Treatment cycles are continued over several months or until disease progression.
  • the conjugate and the immune cell population are administered sequentially, wherein the conjugate is administered using a cyclical administration regimen, wherein the cyclical administration regimen comprises: (a) a first period of “x” days during which the conjugate is administered at a daily dose on “y” consecutive days at the beginning of the first period followed by “x-y” days without administration of the conjugate, wherein “x “is 7, 14, or 21 days, preferably 7 days and “y” is 1, 2, 3 or 4 consecutive days, preferably 1 or 2 days; (b) repeating the first period at least once; and (c) a second period of “z” days without administration of the conjugate wherein z is 7, 14, or 21 days, preferbaly 7 or 14 days.
  • the cyclical administration regimen comprises: (a) a first period of “x” days during which the conjugate is administered at a daily dose on “y” consecutive days at the beginning of the first period followed by “x-y” days without administration of the conjugate, wherein “x “is 7, 14, or 21 days, preferably 7
  • this cyclic administration regimen begins at least 5 days, preferably 7 days after the administration of the immune cell population.
  • the cycle is typically repeated at least two times, preferably at least three times, more preferably until disease progression. Repeated administration is especially preferred for NK cell therapies.
  • this cyclic administration of the conjugate is merged with the repeated administration of the immune cell population, so that prior to each start of conjugate cycle there is one administration of the immune cell population.
  • the conjugate is administered at a daily dose of 4.4 pmol/kg to 2200 pmol/kg, preferably 22 pmol/kg to 660 pmol/kg, and more preferably 220 pmol/kg to 530 pmol/kg.
  • the conjugate is administered at a daily dose of 0,1 ⁇ g/kg to 50 ⁇ g/kg, preferably 0,5 ⁇ g/kg to 15 ⁇ g/kg, and more preferably 5 ⁇ g/kg to 12 ⁇ g/kg.
  • Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC The pharmacodynamic effect of the administration can be easily determined by the activation of proliferation of immune cells, e.g., NK cells in blood.
  • the administration of the conjugate results in: (a) an increase of the % of Ki-67 + NK of total NK cells in comparison to no administration of the conjugate, and wherein the administration of the conjugate after at least one repetition of the first period (step(b)) results in a Ki-67 + NK cell level that is at least 70% of the of the Ki-67 + NK cells; (b) maintenance of NK cell numbers or preferably an increase of NK cell numbers to at least 110% as compared to no administration of conjugate after at least one repetition of the first period (step(b)); and/or (c) NK cell numbers of at least 1.1 x 10 3 NK cells/ ⁇ l after at least one repetition of the first period (step(b)).
  • the treatment of tumor patients further comprises administering: (a) a lymphocyte reduction treatment, preferably selected from cyclophosphamide and fludarabine; (b) a further therapeutic agent; and/or (c) a further anti-tumor treatment.
  • Lymphocyte reduction treatment or lymphodepletion, is routinely applied prior to the administration of the immune cell population in order to reduce the number of circulating immune cells to allow expansion of the administered immune cells. In some cases, there is no lymphodepletion treatment.
  • Further therapeutic agents may be agents that patients receive to treat comorbidities, pain and a like. In tumor therapy, typically different anti-tumor treatments are combined.
  • the subject is a human patient suffering from a cancer which is a solid tumor or liquid tumor.
  • a solid tumor may be selected from the group consisting of carcinoma, lymphoma, sarcoma and blastoma.
  • BXRCYT-2302-PC Cancer may be selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer; optionally wherein the cancer of B-cell origin is selected from the group consisting of Hodgkin lymphoma and non-Hodgkin lymphoma.
  • the patient is suffering from a solid tumor.
  • the solid tumor has a tumor microenvironment having a low glucose level.
  • the cancer is not a hematologic malignancy, i.e., blood cancers, which impact the normal production and function of blood cells, including the three categories of leukemias, lymphomas, and multiple myeloma.
  • a viral particle suitable for transducing an immune cell as defined supra comprising a vector genome comprising a polynucleotide sequence encoding a chimeric receptor polypeptide as defined supra for use in treatment of cancer in a subject in combination with an IL-15/IL-15R ⁇ conjugate as defined supra.
  • transducing the immune cells in vivo with an off-the-shelf viral particle product carrying the polynucleotides encoding the chimeric receptor polypeptide overcomes these shortcomings.
  • the viral particles are retroviral particles or lentiviral particles. Respective viral particles are disclosed in WO 2019/200056A2, WO 2020/106992A1, WO 2022/164935A1, WO 2022/183072A1, and WO 2023/015217A1.
  • the immune cell is a T cell.
  • WO 2023/015217A1 discloses lentiviral vectors comprising a CD4 binding agent to specifically target CD4 + T cells.
  • WO 2022/183072A1 discloses lentiviral vectors further encoding heterologous lymphocyte targeting protein specifically targeting T cells or NK cells. Examples for heterologous lymphocyte Attorney Docket No.: 063642-526001WO Client Ref.
  • BXRCYT-2302-PC targeting proteins are: (a) a T cell marker selected from CD3, CD28, CD80, 4-1BB, AhR, CD3, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, PD- 1, TCRa/b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, TIGIT, LAG-3, TIM-3, and IL6ST, or any combination thereof; (b) a ⁇ T cell marker selected from ⁇ TCR, Vdelta1, Vdelta2, and NKG2D (KLRK1, CD314); or (c) an ⁇ T
  • the polynucleotide sequence further encodes a metabolism modulating polypeptide as defined supra.
  • a metabolism modulating polypeptide as defined supra.
  • the same advantages of the expression or overexpression of a polypeptide that (a’) improves glucose uptake activity; (b’) modulates the Krebs cycle; (c’) enhances lactate concentration; or (d’) diverts or redirects glucose metabolites out of a glycolysis pathway applies to the immune cells transduced in vivo, and therefore, the combination of expressing or overexpressing metabolism modulating polypeptides with co- treatment with IL-15/IL-15R ⁇ conjugates are equally beneficial.
  • the viral particle for in vivo transduction may be administered transdermally, intravenously, intratumorally, intraperitoneally, intramuscularly, intranodally or subcutaneously.
  • the viral particle is for intravenous, intraperitoneal, subcutaneous or intranodal injection.
  • the viral particles are preferably formulated for such administration with conventional buffers and/or excipients as known to a person skilled in the art.
  • non-viral in vivo transduction methods are envisaged, as these have the potential to make cellular therapies more cost-effective and avoid risks associated with viral transduction such as undesired immune responses, pre- existing immunity or dysregulation of oncogenes by viral integration.
  • LNPs typically comprise ionizable and cationic lipids, cholesterol, phospholipids and PEG lipids having a size of 50 to 100 nm in diameter.
  • the helper lipids e.g., phospholipids and cholesterol
  • Polymeric nanoparticles are produced from natural polymers, e.g. dextran, chitosan and/or cyclodextrins, or synthetic polymers, e.g.
  • poly-L-lysine Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC polyethyleneimine, polyamidoamine or poly(lactic-co-glycolic acid) in the form of nanocapsules or nanospheres.
  • the cationic polymers e.g. polyethyleneimine, chitosan or biodegradable polyesters
  • Liposomes are based on phospholipids with polar head groups and non-polar tails (e.g.
  • DOTMA N-[1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOTAP 1,2-dioleoyl-3- trimethylammonium propane
  • DOPE 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine
  • stabilizers e.g. cholesterol
  • inorganic nanoparticles such as gold nanoparticles, silica nanoparticles or iron oxide nanoparticles may be used.
  • a further embodiment of the invention is a nucleic acid or a nucleic acid set comprising a polynucleotide sequence encoding a chimeric receptor polypeptide according to the invention for use in treatment of cancer in a subject in combination with a conjugate of the invention.
  • the nucleic acid is a vector suitable for transient or stable transduction of an immune cell as described in the section “Vectors” above.
  • the nucleic acid, the set of nucleic acids or the vector according to the invention may be formulated for non-viral transduction according to non-viral transduction methods/delivery systems known in the art.
  • Exemplary non- viral transduction systems/formulations are LNPs, liposomes, polymeric nanoparticles and inorganic nanoparticles.
  • the formulation comprises the nucleic acid, the set of nucleic acids or the vectors of the invention, and ionizable and cationic lipids, cholesterol, phospholipids and PEG lipids.
  • the formulation comprises the set of nucleic acids or the vectors of the invention, and phospholipids with polar head groups and non-polar tails, e.g., N-[1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoyl-3- trimethylammonium propane (DOTAP) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) and a stabilizer, e.g. cholesterol.
  • DOTMA N-[1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOTAP 1,2-dioleoyl-3- trimethylammonium propane
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • the formulation comprises the set of nucleic acids or the vectors of the invention, and a natural polymer, e.g., dextran, chitosan and/or cyclodextrins, or a synthetic polymer, e.g. poly-L-lysine, polyethyleneimine, polyamidoamine or poly(lactic-co-glycolic acid).
  • a natural polymer e.g., dextran, chitosan and/or cyclodextrins
  • a synthetic polymer e.g. poly-L-lysine, polyethyleneimine, polyamidoamine or poly(lactic-co-glycolic acid).
  • the invention further relates to a method for inhibiting and/or killing tumor cells in a subject, the method comprising administering to a subject in need thereof an immune cell population in combination with an IL-15/IL-15R ⁇ conjugate as defined herein, wherein an effective dose of the immune cell population and an effective dose of the conjugate are Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC administered sequentially.
  • the invention further relates to a method for increasing the viability of a previously administered immune cell population in the tumor microenvironment by subsequently administering an IL-15/IL-15R ⁇ conjugate as defined herein.
  • the invention further relates to a method for increasing the anti-tumor cytotoxicity of a previously administered immune cell population in the tumor microenvironment by subsequently administering an IL-15/IL-15R ⁇ conjugate as defined herein.
  • the invention further relates to a method for inhibiting and/or killing tumor cells in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of viral particles as defined supra, a nucleic acid as defined supra, a vector as defined supra, or a non-viral transduction system as defined supra, wherein the viral particles, the nucleic acid, the vector or the non-viral transduction system are suitable for transducing immune cells in vivo, in combination with an IL-15/IL-15R ⁇ conjugate as defined supra, wherein a therapeutically effective dose of the viral particles, the nucleic acid, the vector or the non-viral transduction system and a therapeutically effective dose of the conjugate are administered sequentially.
  • the invention further relates to a method for increasing the viability of in vivo transduced immune cells in the tumor microenvironment after administration of viral particles, the nucleic acid, the vector or the non-viral transduction system as defined in supra in a subject by subsequently administering an IL-15/IL-15R ⁇ conjugate as defined supra.
  • the invention further relates to a method for increasing the anti-tumor cytotoxicity of in vivo transduced immune cells in the tumor microenvironment after administration of viral particles, the nucleic acid, the vector or the non-viral transduction system as supra in a subject by subsequently administering an IL-15/IL-15R ⁇ conjugate as defined supra.
  • the efficacy of the combined treatments or methods described herein may be assessed by any method known in the art and would be evident to a skilled medical professional.
  • the efficacy of the compositions or methods described herein may be assessed by survival of the subject or cancer or pathogen burden in the subject or tissue or sample thereof.
  • the compositions and methods described herein may be assessed based on the safety or toxicity of the therapy (e.g., administration of the immune cells expressing the metabolism modulating polypeptides that redirect glucose metabolites and the CAR polypeptides) in the subject, for example, by the overall health of the subject and/or the presence of adverse events or severe adverse events.
  • BXRCYT-2302-PC SEQ ID NO: 75 - Leader peptide of (IL-15N72D)2:IL-15R ⁇ sushi-Fc: METDTLLLWV LLLWVPGSTG SEQ ID NO: 76 - IL-15R ⁇ sushi (65aa)-Fc (IgG1 CH2-CH3): 1 ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS 61 LKCIREPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED 120 PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 180 PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN 240 YKTTPPV
  • BXRCYT-2302-PC SEQ ID NO: 87 – GFPT1 MCGIFAYLNYHVPRTRREILETLIKGLQRLEYRGYDSAGVGFDGGNDKDWEANACKIQLIKKKGKVKALDEEVHKQ QDMDLDIEFDVHLGIAHTRWATHGEPSPVNSHPQRSDKNNEFIVIHNGIITNYKDLKKFLESKGYDFESETDTETI AKLVKYMYDNRESQDTSFTTLVERVIQQLEGAFALVFKSVHFPGQAVGTRRGSPLLIGVRSEHKLSTDHIPILYRT ARTQIGSKFTRWGSQGERGKDKKGSCNLSRVDSTTCLFPVEEKAVEYYFASDASAVIEHTNRVIFLEDDDVAAVVD GRLSIHRIKRTAGDHPGRAVQTLQMELQIMKGNFSSFMQKEIFEQPESVVNTMRGRVNFDDYTVNLGGLKDHIKE IQRCRRLIA
  • Example 1 Production of Retroviral Particles and Viral Titer Determination 15 x 10 6 low passage HEK293 cells were plated in 15 cm coated tissue culture plates using DMEM (+10% FBS) media so that the following day they were approximately 80% confluent. The next day, 1.5 ml of transfection mixture containing 10 ⁇ g of GAG/Pol, 6.6 mg of GALV helper plasmids, 20 ⁇ g of transfer plasmid and Opti-MEM (Cat # 31985062; Thermo Fisher:) were mixed with 1.5 ml of Opti-MEM, and 75 ⁇ l of PEIPro transfection reagent (PolyPlus: 115-010) were added to the plates.
  • DMEM +10% FBS
  • Example 2 PBMC Initiation and Viral Transduction Approximately 100 x 10 6 PBMCs per donor were initiated by stimulation with anti-CD3 (Cat # 130-093-387, Miltenyi), anti-CD28 (Cat #130-093-375; Miltenyi), and IL-2 (100 UI/ml; Cat # 1020-500; CellGenix) in X Vivo 15 (Cat # BEBP04-744Q; Lonza) in 100 ml on Day 0 in T175 flaks. On Day 2, activated PBMC were harvested and 1 ml containing 1 x 10 6 cells were plated in a 24 well plate.
  • transduction 1 ml of viral supernatant was added to each well. Plates were centrifuged 1200 g for 45 min and allowed to rest overnight in an incubator at 37°C. One day after transduction, cells were transferred to a GREX vessel (Cat # 80192M; Wilson Wolf) filled with X-VIVO + 100 IU/ml IL-2. The cells were maintained for an additional week with supplementation of IL-2 every 48 h. On Day 10, CAR T or BOXR CAR T cells were harvested and frozen until further analysis. The transduced immune cells were assessed for the transgene expression by immunoblotting.
  • the transduced cells e.g., T cells or NK-92
  • the transduced cells were harvested by centrifuging at 1500 rpm for 5 min at RT. The supernatant was removed, and the cell pellet washed twice in 1XPBS before flash freezing in liquid nitrogen and stored at -80°C until further use.
  • Cell pellets were subsequently lysed in 200 ⁇ l of SDS Lysis buffer (Cat # NP0008; Novex) containing 1x HALT Protease Inhibitor Cocktail (Cat# 78430; Thermo Fisher) followed by sonication. The suspension was centrifuged at 15,000 rpm for 15 min at RT and the supernatant containing total protein was collected.
  • the total protein concentration was measured using Pierce 660 nm Protein Assay (Cat# 1861426; Thermo Scientific) followed by immunoblotting.10 ⁇ g total protein was loaded in each lane of a NovexTM 4 to 12 % Tris- Glycine Plus, 1.0 mm, 20-well Midi Protein Gel (Invitrogen), transferred onto PVDF membrane using Transblot Turbo (Biorad) and blocked for 1 h at RT using LICOR Blocking buffer.
  • the membrane was probed for transgenes (e.g., GOT2, TIGAR) using mouse ⁇ -Actin (3700S, CST; dilution 1:2000), Rabbit ⁇ -TIGAR (14751S CST; dilution 1:1000) and Rabbit ⁇ - Got2 (NBP232241, Novus; dilution 1:2000) antibodies overnight (in 0.1% Tween 20 + LICOR Blocking buffer) at 4°C.
  • membranes are washed thrice with 1x TBS containing 0.1% Tween20 detergent (w/v) for 5 min each.
  • Membranes are subsequently incubated with standard rabbit or mouse secondary antibodies (LICOR; dilution 1:10,000) for 1 h.
  • Cells were stained in a 1:1000 dilution of live dead dye in PBS (Zombie Aqua; Cat # 423102; Biolegend) for 10 min at room temperature in the dark. Cells were washed in FACS buffer (PBS +2% FBS). Cells were stained for CAR with ROR1-Fc-biotin protein, streptavidin-AF647 and cells surface markers (CD3, CD4, CD8, CD45RA, CCR7), see Table 7 below. For Ki67 staining, cells were further processed and washed in PBS +2% FBS then fixed and permeabilized for 30 minutes at 4 ⁇ C following manufacturer’s protocol (Cat # 00-5523-00; ebiosciences).
  • Example 5 Analysis of T cell Memory Subsets After SOT101 Treatment CAR-expressing T cells from Example 4 above stimulated with 0.1 mM SOT1010 or 0.2 mM IL-2 were further analyzed by FACS to group them into the subtypes of memory T cell being (i) CD45RA + , CCR7 + stem cell like T cell (Tscm); (ii) CD45RA – , CCR7 + na ⁇ ve central memory T cell (TCM), (iii) CD45RA – ,CCR7 – effector memory T cell (TEM), or (iv) CD45RA + , CCR7 – effector memory cells re-expressing CD45RA (T emRA ).
  • T cell differentiation goes from Tscm to TCM to TEM to TemRA.
  • T cell memory subsets were comparable for CAR T cells expressing only the anti-ROR-1 CAR or the anti-ROR-1 CAR and GOT2. Comparing increasing doses of SOT101, a slight dose-dependent increase of TCM was observed at day 5 and day 7 (although still lower than in absence of SOT101). Overall, a slight shift in T cell memory populations was observed with increasing SOT101 doses.
  • FIGs.2A- 2C and FIGs.2D-2E From day 3 to day 7, we observed slight shifts in T cell memory populations towards T EM , with corresponding decreases in T scm and T CM , resulting in a more differentiated (T EM ) phenotype. Comparing SOT101 and IL-2, at day 7 this trend was more pronounced for SOT101 compared to IL-2 treatment. Further, at day 3, T scm and T emRA populations were reduced with SOT101 compared to IL-2 and we observed a reduction for both Tscm and TCM and an increase in T EM at day 7.
  • CAR or GOT2 BOXR T cells were analyzed on day 4 by FACS for T cell differentiation markers.
  • cytokine treatment with both SOT101 and IL-2 resulted in an increase in the TEM population (CD45RA – , CCR7 – ) for both CAR T cells (FIGS.4A and 4C) and GOT2 BOXR T cells (FIGS.4A and 4D), whereas no significant change was observed for antigen negative tumor cells (FIG.4B).
  • Example 7 In vivo Efficacy Using a Caki-1 (Human Renal Cell Carcinoma) Xenograft Mouse Model
  • 5-6 week-old NOD scid gamma (NSG) mice were implanted with 5 million Caki-1 cells suspended in 50% RPMI 1640:matrigel via s.c. injection into the right flank.
  • NSG NOD scid gamma
  • mice were implanted with 5 million anti-ROR-1 CAR + T cells via i.v. tail vein injection (Day 0).
  • SOT101 was administered s.c. at doses of 15 ⁇ g/mouse on days 7, 8, 14 and 15. Tumor growth and body weight were monitored 2 times per week.
  • Tumor volume was measured using calipers and calculated using the formula (LxWxH)/2 mm3.
  • anti-ROR-1 CAR + T cells had a minor effect on tumor growth (measured as mean tumor volume), whereas the addition of SOT101 substantially reduced the tumor volume (FIG.5A).
  • anti-ROR-1 GOT2 expressing BOXR T cells showed a stronger anti-tumor effect compared to the CAR + T cells. This antitumor effect was strongly enlarged by the additional treatment with SOT101 (FIG. 5B). This synergistic effect can be further observed looking at the individual mouse data for the groups in FIGs.5C to 5G.
  • the combination of the CAR T cell therapy or the BOXR CAR T cell therapy, here expressing the GOT2 metabolism modulating polypeptide, with an IL-15/IL- 15R ⁇ conjugate, here the IL-15/IL-15R ⁇ sushi+ fusion protein SOT101 was well tolerated and lead to likely additive to even synergistic treatment improvements in the CAKI-1 xenograft model in vivo.
  • SOT101 clearly improved the efficacy of CAR-expressing T cells, and this effect was even stronger when the BOXR T cells co-expressed an exogenous copy of a gene optimizing the metabolism of the T cell, here the GOT2 gene.
  • Example 8 Pharmacokinetics Blood from retro-orbital bleeds of mice from Example 7 above was collected on study days 7, 14, 21 and 28, and 20 ⁇ l of blood were frozen with 80 ⁇ l of Bambanker (Cat # BB01; Bulldog bio) and stored at -80°C. Samples were thawed, washed with PBS and lysed with 150 ⁇ l of FACS lysing solution (Cat # 349202; BD Biosciences) for 5 min. Cells were washed with PBS and stained with 1:1000 dilution of Live/Dead in PBS for 10 min.
  • Bambanker Cat # BB01; Bulldog bio
  • FACS lysing solution Cat # 349202; BD Biosciences
  • Example 9 In vivo Efficacy Using NCI-H1975 (Human Non-Small Cell Lung Carcinoma) Xenograft Mouse Model Female, 5-6 week-old NOD scid gamma (NSG) mice were implanted with 5 million NCI-H1975 tumor cells suspended in 50% RPMI 1640:matrigel via s.c. injection into the right flank.
  • NCI-H1975 Human Non-Small Cell Lung Carcinoma
  • mice per group were dosed with 3 million anti- ROR-1 CAR + T cells via i.v. tail vein injection (Day 0).
  • SOT101 was administered s.c. at doses Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC of 15 ⁇ g/mouse on days 7, 8, 14 and 15. Tumor growth and body weight were monitored 2 times per week. Tumor volume was measured using calipers and calculated using the formula (LxWxH)/2 mm3.
  • Example 10 Impact of Expressing Metabolism Modulating Polypeptide on T cell Function
  • Gamma-retrovirus encoding the test antigen-targeting CAR polypeptide expression construct may be generated via recombinant technology and for use to infect primary human T- cells to generate cells expressing the test antigen-targeting CAR polypeptide on their cell surface.
  • gamma-retroviruses encoding the test antigen-targeting CAR polypeptide and a test metabolism modulating polypeptide are to be generated via recombinant technology and used to infect primary human T-cells to generate cells that express the test antigen-targeting CAR polypeptide and the test metabolism modulating polypeptide. It is preferred that both transgenes are encoded on one vector to ensure that cells do not get transfected with only one vector. Accordingly, transfected cells expressing the CAR, which can easily be determined, e.g., by FACS analysis, also contain the metabolism modulating polypeptide.
  • a test metabolism modulating polypeptide e.g., GOT2, GLUT1, TIGAR or other metabolism modulating polypeptides of interest
  • the two polypeptides may be separated by a P2A ribosomal skip sequence.
  • an IRES sequence or a second expression cassette/second promoter may be used to express two transgenes from one vector.
  • a six-day flow-based proliferation assay can then be used to test the functionality of the test antigen-targeting CAR expressing cells compared to control.
  • the co-culture is incubated at typical conditions for the tumor cell line, e.g., 37° C in a 5% CO2, for six days in the presence of 1.25 mM glucose (tumor-relevant) or 10 mM glucose (approximate peripheral blood levels). At the end of six days, co-cultures are harvested and stained with an anti-CD3 antibody.
  • the analogous assay may be conducted by replacing the CAR polypeptide by the respective chimeric receptor polypeptide, e.g., by a TCR or an ACTR polypeptide, and ensuring that the matching antigen is expressed by the tumor cell line and/or that, in case of an ACTR polypeptide, the respective anti-tumor antibody is added to the assay, to allow for specific binding of the immune cell to tumor cell.
  • the assay is to be conducted by only comparing respective untransduced immune cells with immune cells transduced with the gene encoding the test metabolism modulating polypeptide.
  • T cells For measuring T cells, the number of CD3 + cells is evaluated by flow cytometry as a measure of T cell proliferation.
  • T cells expressing the test metabolism modulating polypeptides in addition to the CAR polypeptide relative to T cells expressing the CAR construct alone are measured to determine the induction of proliferation of the immune cell population. This enhanced proliferation also occurs at tumor-relevant low glucose concentrations.
  • CD56 and CD16 are used to define the main subsets of NK cell, CD56 bright CD16 lo/ ⁇ and CD56 dim CD16 + , often simplified as CD56 bright and CD56 dim , respectively.
  • ⁇ T cells can be measured through FACS by gating for CD3 + and ⁇ -TCR + gating.
  • Example 11 Expression of GLUT1, GOT2, and TIGAR Elevated Glucose Uptake and Lactate Production Healthy donor PBMCs are stimulated with anti-CD3 and anti-CD28 until day 2 followed by transduction with transgene packaged into a lentiviral vector.
  • Examples for metabolism modulating polypeptides are GLUT1 (SEQ ID NO: 88), GOT2 (SEQ ID NO: 86), or TIGAR (SEQ ID NO: 89).
  • the transduced cells are supplemented with fresh IL-2 each day until day10.10,000 cells/well (384-well plate) are resuspended in PBS and assayed for glucose uptake.
  • the luminescence read-out is evaluated as a fold change for each transgene and compared to null (non-transduced control; baseline as fold change 1) T cells under the same Attorney Docket No.: 063642-526001WO Client Ref. No.: BXRCYT-2302-PC condition. Cells showing elevated levels of glucose uptake are indicative of higher metabolic activity.
  • PBMCs are stimulated with anti-CD3 and anti-CD28 until day 2 followed by transduction with transgene (described above) packaged into lentiviral vectors.
  • the transduced cells are supplemented with fresh IL-2 each day until day 9.
  • a subset of T cells is stimulated with PMA and Ionomycin for 24 h.10,000 harvested cells/well (384- well plate) are resuspended in RPMI without FBS and incubated at 37 °C for 2 h to remove residual lactate from the media and assayed for lactate production.
  • the luminescence read-out is evaluated as a fold change for each transgene compared to null (non-transduced control; baseline as fold change 1) T cells under the same condition.
  • Stimulated cells showing elevated levels of lactate production are indicative of higher metabolic adaptability in nutrient deficient environments.
  • Previous data show that T cells transduced with GLUT1, GOT2, or TIGAR showed enhanced metabolic activity and adaptability in nutrient deficient environment.
  • therapeutic T cells e.g., T cells expressing an ACTR or CAR polypeptide as disclosed herein
  • co-expressing for e.g., GLUT1, GOT2, or TIGAR would be better adapted to tumor microenvironment (which could be deficient in nutrient) and exhibit better therapeutic activity as compared with counterpart T cells that are not transduced with the transgene (e.g., GLUT1, GOT2, or TIGAR).
  • inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Chemical & Material Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Population de cellules immunitaires destinée à être utilisée dans le traitement du cancer chez un sujet en combinaison avec un conjugué, qui comprend : (i) un polypeptide comprenant la séquence d'acides aminés de l'interleukine 15 (IL-15) ou un dérivé de celle-ci ; et (ii) un polypeptide comprenant la séquence d'acides aminés du domaine sushi d'un récepteur alpha de l'interleukine 15 (IL-15Rα) ou d'un dérivé de celui-ci.
PCT/US2024/024213 2023-04-14 2024-04-12 CELLULES IMMUNITAIRES POUR LE TRAITEMENT DU CANCER EN COMBINAISON AVEC DES CONJUGUÉS IL-15/IL-15Rα WO2024215987A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363496222P 2023-04-14 2023-04-14
US63/496,222 2023-04-14
EP23173531.7 2023-05-16
EP23173531 2023-05-16

Publications (1)

Publication Number Publication Date
WO2024215987A1 true WO2024215987A1 (fr) 2024-10-17

Family

ID=91022564

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/024213 WO2024215987A1 (fr) 2023-04-14 2024-04-12 CELLULES IMMUNITAIRES POUR LE TRAITEMENT DU CANCER EN COMBINAISON AVEC DES CONJUGUÉS IL-15/IL-15Rα

Country Status (1)

Country Link
WO (1) WO2024215987A1 (fr)

Citations (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4650764A (en) 1983-04-12 1987-03-17 Wisconsin Alumni Research Foundation Helper cell
GB2200651A (en) 1987-02-07 1988-08-10 Al Sumidaie Ayad Mohamed Khala A method of obtaining a retrovirus-containing fraction from retrovirus-containing cells
US4777127A (en) 1985-09-30 1988-10-11 Labsystems Oy Human retrovirus-related products and methods of diagnosing and treating conditions associated with said retrovirus
EP0345242A2 (fr) 1988-06-03 1989-12-06 Smithkline Biologicals S.A. Expression de protéines gag de rétrovirus dans les cellules eucaryotes
WO1990007936A1 (fr) 1989-01-23 1990-07-26 Chiron Corporation Therapies de recombinaison pour infections et troubles hyperproliferatifs
US4980289A (en) 1987-04-27 1990-12-25 Wisconsin Alumni Research Foundation Promoter deficient retroviral vector
WO1991002805A2 (fr) 1989-08-18 1991-03-07 Viagene, Inc. Retrovirus de recombinaison apportant des constructions de vecteur a des cellules cibles
US5124263A (en) 1989-01-12 1992-06-23 Wisconsin Alumni Research Foundation Recombination resistant retroviral helper cell and products produced thereby
WO1993003769A1 (fr) 1991-08-20 1993-03-04 THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Transfert induit par adenovirus de genes vers la voie gastro-intestinale
WO1993010218A1 (fr) 1991-11-14 1993-05-27 The United States Government As Represented By The Secretary Of The Department Of Health And Human Services Vecteurs comprenant des genes etrangers et des marqueurs selectifs negatifs
WO1993011230A1 (fr) 1991-12-02 1993-06-10 Dynal As Cellule souche modifiee de mammifere bloquant la replication virale
US5219740A (en) 1987-02-13 1993-06-15 Fred Hutchinson Cancer Research Center Retroviral gene transfer into diploid fibroblasts for gene therapy
WO1993019191A1 (fr) 1992-03-16 1993-09-30 Centre National De La Recherche Scientifique Adenovirus recombinants defectifs exprimant des cytokines pour traitement antitumoral
WO1993025234A1 (fr) 1992-06-08 1993-12-23 The Regents Of The University Of California Procedes et compositions permettant de cibler des tissus specifiques
WO1993025698A1 (fr) 1992-06-10 1993-12-23 The United States Government As Represented By The Particules vecteurs resistantes a l'inactivation par le serum humain
WO1994003622A1 (fr) 1992-07-31 1994-02-17 Imperial College Of Science, Technology & Medicine Vecteurs retroviraux du type d, bases sur le virus du singe mason-pfizer
WO1994012649A2 (fr) 1992-12-03 1994-06-09 Genzyme Corporation Therapie genique de la fibrose kystique
WO1994028938A1 (fr) 1993-06-07 1994-12-22 The Regents Of The University Of Michigan Vecteurs d'adenovirus pour therapie genique
WO1995000655A1 (fr) 1993-06-24 1995-01-05 Mc Master University Vecteurs a base d'adenovirus destines a la therapie genique
WO1995007358A1 (fr) 1993-07-30 1995-03-16 University Of Medicine & Dentistry Of New Jersey Transfert efficace de genes dans des lymphocytes primaires
US5399346A (en) 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
WO1995011984A2 (fr) 1993-10-25 1995-05-04 Canji, Inc. Vecteur recombinant d'adenovirus et procedes d'utilisation
WO1998009271A1 (fr) 1996-08-28 1998-03-05 Burgett, Inc. Procede et appareil pour actionner des solenoides dans un piano mecanique
WO2000032776A2 (fr) 1998-12-01 2000-06-08 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques les codant
US6194191B1 (en) 1996-11-20 2001-02-27 Introgen Therapeutics, Inc. Method for the production and purification of adenoviral vectors
WO2005085282A1 (fr) 2004-02-27 2005-09-15 Inserm (Institut National De La Sante Et De La Recherche Medicale) Site de liaison pour le recepteur alpha de l'il 15 et mutants specifiques de l'il-15 presentant une activite agoniste/antagoniste
WO2006020849A2 (fr) 2004-08-11 2006-02-23 Beth Israel Deaconess Medical Center, Inc. Polypeptides d'interleukine-15 mutants
US7052906B1 (en) 1999-04-16 2006-05-30 Celltech R & D Limited Synthetic transmembrane components
WO2007046006A2 (fr) 2005-10-20 2007-04-26 Institut National De La Sante Et De La Recherche Medicale (Inserm) Domaine sushi de l'il-15ralpha utilise en tant que renforçateur puissant de l'action de l'il-15 par l'intermediaire de l'il-15rbeta/gamma, et proteines de fusion hyperagonistes (ilralpha sushi-il 15)
US7435596B2 (en) 2004-11-04 2008-10-14 St. Jude Children's Research Hospital, Inc. Modified cell line and method for expansion of NK cell
WO2008143794A1 (fr) 2007-05-11 2008-11-27 Altor Bioscience Corporation Molécules de fusion et variantes de il-15
WO2009135031A1 (fr) 2008-04-30 2009-11-05 The United States Of America, As Represented By The Secretary, Department Of Health And Human Servic Il-15 substitué
WO2012175222A1 (fr) 2011-06-24 2012-12-27 Cytune Immunocytokines à base d'il-15 et domaine sushi d'il-15rα
WO2013040557A2 (fr) 2011-09-16 2013-03-21 The Trustees Of The University Of Pennsylvania Lymphocytes t à arn modifié pour le traitement du cancer
US8673860B2 (en) 2009-02-03 2014-03-18 Amunix Operating Inc. Extended recombinant polypeptides and compositions comprising same
US20140106449A1 (en) 2010-12-09 2014-04-17 The Trustees Of The University Of Pennsylvania Use of Chimeric Antigen Receptor-Modified T Cells to Treat Cancer
WO2014066527A2 (fr) 2012-10-24 2014-05-01 Admune Therapeutics Llc Formes d'il-15r alpha, cellules exprimant des formes d'il-15r alpha, et utilisations thérapeutiques d'il-15r alpha et de complexes il-15/il-15r alpha
WO2014207173A1 (fr) 2013-06-27 2014-12-31 INSERM (Institut National de la Santé et de la Recherche Médicale) Antagonistes de l'interleukine 15 (il-15) et leurs utilisations pour le traitement de maladies auto-immunes et de maladies inflammatoires
EP2884999A1 (fr) 2012-08-20 2015-06-24 Fred Hutchinson Cancer Research Center Procédé et compositions pour l'immunothérapie cellulaire
WO2016040441A1 (fr) 2014-09-09 2016-03-17 Unum Therapeutics Récepteurs chimériques et utilisations de ceux-ci en thérapie immunitaire
WO2016060996A2 (fr) 2014-10-14 2016-04-21 Armo Biosciences, Inc. Compositions d'interleukine-15 et leurs utilisations
WO2016095642A1 (fr) 2014-12-19 2016-06-23 江苏恒瑞医药股份有限公司 Complexe protéine-interleukine 15 et utilisation associée
WO2016142314A1 (fr) 2015-03-06 2016-09-15 Deutsches Krebsforschungszentrum Protéines de fusion comprenant une protéine de liaison et un polypeptide interleukine-15 ayant une affinité réduite pour il15ra, et leurs usages thérapeutiques
WO2017046200A1 (fr) 2015-09-16 2017-03-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Polypeptide antagoniste spécifique de l'interleukine-15 (il-15) et ses utilisations pour le traitement de maladies inflammatoires et auto-immunes
WO2017112528A2 (fr) 2015-12-21 2017-06-29 Armo Biosciences, Inc. Compositions d'interleukine-15 et leurs utilisations
WO2018071919A1 (fr) 2016-10-14 2018-04-19 Xencor, Inc. Protéines de fusion fc hétérodimères il15/il15rα
WO2019160956A1 (fr) * 2018-02-13 2019-08-22 Novartis Ag Thérapie par récepteur antigénique chimérique en combinaison avec il-15 r et il15
WO2019200056A2 (fr) 2018-04-12 2019-10-17 Vivo-Til Therapeutics Inc. Vecteurs viraux et lignées d'encapsidation
WO2020010110A1 (fr) 2018-07-03 2020-01-09 Unum Therapeutics Inc. Récepteurs chimériques en association avec des molécules de métabolisme trans améliorant l'importation de glucose et leurs utilisations thérapeutiques
EP3593812A2 (fr) * 2014-03-15 2020-01-15 Novartis AG Traitement du cancer à l'aide d'un récepteur d'antigène chimérique
WO2020037066A1 (fr) 2018-08-14 2020-02-20 Unum Therapeutics Inc. Récepteurs d'antigène chimériques en association avec des molécules de trans-métabolisme modulant le cycle de krebs et leurs utilisations thérapeutiques
WO2020051493A1 (fr) 2018-09-07 2020-03-12 Unum Therapeutics Inc. Polypeptides récepteurs chimériques en association avec des molécules métaboliques trans modulant des concentrations intracellulaires en lactate et utilisations thérapeutiques associées
WO2020106992A1 (fr) 2018-11-21 2020-05-28 Umoja Biopharma, Inc. Vecteur multicistronique pour ingénierie de surface de particules lentivirales
WO2020206395A1 (fr) * 2019-04-05 2020-10-08 Nektar Therapeutics Procédé d'amélioration de l'immunothérapie cellulaire
WO2022164935A1 (fr) 2021-01-27 2022-08-04 Umoja Biopharma, Inc. Lentivirus permettant de générer des cellules exprimant un récepteur antigénique chimérique anti-cd19
WO2022183072A1 (fr) 2021-02-26 2022-09-01 Kelonia Therapeutics, Inc. Vecteurs lentiviraux ciblant les lymphocytes
WO2022192439A1 (fr) 2021-03-11 2022-09-15 Kite Pharma, Inc. Amélioration de la fonction de cellules immunitaires
WO2023015217A1 (fr) 2021-08-04 2023-02-09 Sana Biotechnology, Inc. Utilisation de vecteurs viraux ciblant cd4
WO2023049933A1 (fr) 2021-09-27 2023-03-30 Sotio Biotech Inc. Polypeptides récepteurs chimériques en combinaison avec des molécules de métabolisme trans qui réorientent des métabolites de glucose hors de la voie de glycolyse et leurs utilisations thérapeutiques

Patent Citations (63)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4650764A (en) 1983-04-12 1987-03-17 Wisconsin Alumni Research Foundation Helper cell
US4777127A (en) 1985-09-30 1988-10-11 Labsystems Oy Human retrovirus-related products and methods of diagnosing and treating conditions associated with said retrovirus
GB2200651A (en) 1987-02-07 1988-08-10 Al Sumidaie Ayad Mohamed Khala A method of obtaining a retrovirus-containing fraction from retrovirus-containing cells
US5219740A (en) 1987-02-13 1993-06-15 Fred Hutchinson Cancer Research Center Retroviral gene transfer into diploid fibroblasts for gene therapy
US4980289A (en) 1987-04-27 1990-12-25 Wisconsin Alumni Research Foundation Promoter deficient retroviral vector
EP0345242A2 (fr) 1988-06-03 1989-12-06 Smithkline Biologicals S.A. Expression de protéines gag de rétrovirus dans les cellules eucaryotes
US5124263A (en) 1989-01-12 1992-06-23 Wisconsin Alumni Research Foundation Recombination resistant retroviral helper cell and products produced thereby
WO1990007936A1 (fr) 1989-01-23 1990-07-26 Chiron Corporation Therapies de recombinaison pour infections et troubles hyperproliferatifs
US5399346A (en) 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
WO1991002805A2 (fr) 1989-08-18 1991-03-07 Viagene, Inc. Retrovirus de recombinaison apportant des constructions de vecteur a des cellules cibles
WO1993003769A1 (fr) 1991-08-20 1993-03-04 THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Transfert induit par adenovirus de genes vers la voie gastro-intestinale
WO1993010218A1 (fr) 1991-11-14 1993-05-27 The United States Government As Represented By The Secretary Of The Department Of Health And Human Services Vecteurs comprenant des genes etrangers et des marqueurs selectifs negatifs
WO1993011230A1 (fr) 1991-12-02 1993-06-10 Dynal As Cellule souche modifiee de mammifere bloquant la replication virale
WO1993019191A1 (fr) 1992-03-16 1993-09-30 Centre National De La Recherche Scientifique Adenovirus recombinants defectifs exprimant des cytokines pour traitement antitumoral
WO1993025234A1 (fr) 1992-06-08 1993-12-23 The Regents Of The University Of California Procedes et compositions permettant de cibler des tissus specifiques
WO1993025698A1 (fr) 1992-06-10 1993-12-23 The United States Government As Represented By The Particules vecteurs resistantes a l'inactivation par le serum humain
WO1994003622A1 (fr) 1992-07-31 1994-02-17 Imperial College Of Science, Technology & Medicine Vecteurs retroviraux du type d, bases sur le virus du singe mason-pfizer
WO1994012649A2 (fr) 1992-12-03 1994-06-09 Genzyme Corporation Therapie genique de la fibrose kystique
WO1994028938A1 (fr) 1993-06-07 1994-12-22 The Regents Of The University Of Michigan Vecteurs d'adenovirus pour therapie genique
WO1995000655A1 (fr) 1993-06-24 1995-01-05 Mc Master University Vecteurs a base d'adenovirus destines a la therapie genique
WO1995007358A1 (fr) 1993-07-30 1995-03-16 University Of Medicine & Dentistry Of New Jersey Transfert efficace de genes dans des lymphocytes primaires
WO1995011984A2 (fr) 1993-10-25 1995-05-04 Canji, Inc. Vecteur recombinant d'adenovirus et procedes d'utilisation
WO1998009271A1 (fr) 1996-08-28 1998-03-05 Burgett, Inc. Procede et appareil pour actionner des solenoides dans un piano mecanique
US6194191B1 (en) 1996-11-20 2001-02-27 Introgen Therapeutics, Inc. Method for the production and purification of adenoviral vectors
WO2000032776A2 (fr) 1998-12-01 2000-06-08 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques les codant
US7052906B1 (en) 1999-04-16 2006-05-30 Celltech R & D Limited Synthetic transmembrane components
WO2005085282A1 (fr) 2004-02-27 2005-09-15 Inserm (Institut National De La Sante Et De La Recherche Medicale) Site de liaison pour le recepteur alpha de l'il 15 et mutants specifiques de l'il-15 presentant une activite agoniste/antagoniste
WO2006020849A2 (fr) 2004-08-11 2006-02-23 Beth Israel Deaconess Medical Center, Inc. Polypeptides d'interleukine-15 mutants
US20060057680A1 (en) 2004-08-11 2006-03-16 Zheng Xin X Mutant interleukin-15 polypeptides
US8026097B2 (en) 2004-11-04 2011-09-27 St. Jude Children's Research Hospital Expansion of NK cells and therapeutic uses thereof
US7435596B2 (en) 2004-11-04 2008-10-14 St. Jude Children's Research Hospital, Inc. Modified cell line and method for expansion of NK cell
WO2007046006A2 (fr) 2005-10-20 2007-04-26 Institut National De La Sante Et De La Recherche Medicale (Inserm) Domaine sushi de l'il-15ralpha utilise en tant que renforçateur puissant de l'action de l'il-15 par l'intermediaire de l'il-15rbeta/gamma, et proteines de fusion hyperagonistes (ilralpha sushi-il 15)
WO2008143794A1 (fr) 2007-05-11 2008-11-27 Altor Bioscience Corporation Molécules de fusion et variantes de il-15
WO2009135031A1 (fr) 2008-04-30 2009-11-05 The United States Of America, As Represented By The Secretary, Department Of Health And Human Servic Il-15 substitué
US8673860B2 (en) 2009-02-03 2014-03-18 Amunix Operating Inc. Extended recombinant polypeptides and compositions comprising same
US20140106449A1 (en) 2010-12-09 2014-04-17 The Trustees Of The University Of Pennsylvania Use of Chimeric Antigen Receptor-Modified T Cells to Treat Cancer
WO2012175222A1 (fr) 2011-06-24 2012-12-27 Cytune Immunocytokines à base d'il-15 et domaine sushi d'il-15rα
WO2013040557A2 (fr) 2011-09-16 2013-03-21 The Trustees Of The University Of Pennsylvania Lymphocytes t à arn modifié pour le traitement du cancer
EP2884999A1 (fr) 2012-08-20 2015-06-24 Fred Hutchinson Cancer Research Center Procédé et compositions pour l'immunothérapie cellulaire
WO2014066527A2 (fr) 2012-10-24 2014-05-01 Admune Therapeutics Llc Formes d'il-15r alpha, cellules exprimant des formes d'il-15r alpha, et utilisations thérapeutiques d'il-15r alpha et de complexes il-15/il-15r alpha
WO2014207173A1 (fr) 2013-06-27 2014-12-31 INSERM (Institut National de la Santé et de la Recherche Médicale) Antagonistes de l'interleukine 15 (il-15) et leurs utilisations pour le traitement de maladies auto-immunes et de maladies inflammatoires
EP3593812A2 (fr) * 2014-03-15 2020-01-15 Novartis AG Traitement du cancer à l'aide d'un récepteur d'antigène chimérique
WO2016040441A1 (fr) 2014-09-09 2016-03-17 Unum Therapeutics Récepteurs chimériques et utilisations de ceux-ci en thérapie immunitaire
WO2016060996A2 (fr) 2014-10-14 2016-04-21 Armo Biosciences, Inc. Compositions d'interleukine-15 et leurs utilisations
WO2016095642A1 (fr) 2014-12-19 2016-06-23 江苏恒瑞医药股份有限公司 Complexe protéine-interleukine 15 et utilisation associée
WO2016142314A1 (fr) 2015-03-06 2016-09-15 Deutsches Krebsforschungszentrum Protéines de fusion comprenant une protéine de liaison et un polypeptide interleukine-15 ayant une affinité réduite pour il15ra, et leurs usages thérapeutiques
WO2017046200A1 (fr) 2015-09-16 2017-03-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Polypeptide antagoniste spécifique de l'interleukine-15 (il-15) et ses utilisations pour le traitement de maladies inflammatoires et auto-immunes
WO2017112528A2 (fr) 2015-12-21 2017-06-29 Armo Biosciences, Inc. Compositions d'interleukine-15 et leurs utilisations
WO2018071919A1 (fr) 2016-10-14 2018-04-19 Xencor, Inc. Protéines de fusion fc hétérodimères il15/il15rα
WO2018071918A1 (fr) 2016-10-14 2018-04-19 Xencor, Inc. Protéines de fusion hétérodimères bispécifiques contenant des protéines de fusion fc il -15/il -15 rαlpha et des fragments d'anticorps pd -1
US20180118805A1 (en) 2016-10-14 2018-05-03 Xencor, Inc. IL15/IL15Ralpha HETERODIMERIC Fc-FUSION PROTEINS
WO2019160956A1 (fr) * 2018-02-13 2019-08-22 Novartis Ag Thérapie par récepteur antigénique chimérique en combinaison avec il-15 r et il15
WO2019200056A2 (fr) 2018-04-12 2019-10-17 Vivo-Til Therapeutics Inc. Vecteurs viraux et lignées d'encapsidation
WO2020010110A1 (fr) 2018-07-03 2020-01-09 Unum Therapeutics Inc. Récepteurs chimériques en association avec des molécules de métabolisme trans améliorant l'importation de glucose et leurs utilisations thérapeutiques
WO2020037066A1 (fr) 2018-08-14 2020-02-20 Unum Therapeutics Inc. Récepteurs d'antigène chimériques en association avec des molécules de trans-métabolisme modulant le cycle de krebs et leurs utilisations thérapeutiques
WO2020051493A1 (fr) 2018-09-07 2020-03-12 Unum Therapeutics Inc. Polypeptides récepteurs chimériques en association avec des molécules métaboliques trans modulant des concentrations intracellulaires en lactate et utilisations thérapeutiques associées
WO2020106992A1 (fr) 2018-11-21 2020-05-28 Umoja Biopharma, Inc. Vecteur multicistronique pour ingénierie de surface de particules lentivirales
WO2020206395A1 (fr) * 2019-04-05 2020-10-08 Nektar Therapeutics Procédé d'amélioration de l'immunothérapie cellulaire
WO2022164935A1 (fr) 2021-01-27 2022-08-04 Umoja Biopharma, Inc. Lentivirus permettant de générer des cellules exprimant un récepteur antigénique chimérique anti-cd19
WO2022183072A1 (fr) 2021-02-26 2022-09-01 Kelonia Therapeutics, Inc. Vecteurs lentiviraux ciblant les lymphocytes
WO2022192439A1 (fr) 2021-03-11 2022-09-15 Kite Pharma, Inc. Amélioration de la fonction de cellules immunitaires
WO2023015217A1 (fr) 2021-08-04 2023-02-09 Sana Biotechnology, Inc. Utilisation de vecteurs viraux ciblant cd4
WO2023049933A1 (fr) 2021-09-27 2023-03-30 Sotio Biotech Inc. Polypeptides récepteurs chimériques en combinaison avec des molécules de métabolisme trans qui réorientent des métabolites de glucose hors de la voie de glycolyse et leurs utilisations thérapeutiques

Non-Patent Citations (79)

* Cited by examiner, † Cited by third party
Title
"Cell and Tissue Culture: Laboratory Procedures", August 1993, J. WILEY AND SONS
"Current Protocols in Immunology", 1991
"DNA Cloning: A practical Approach", vol. 1,2, 1985
"GenBank", Database accession no. HQ401283.1
"Handbook of Experimental Immunology", 1987, HUMANA PRESS, article "Gene Transfer Vectors for Mammalian Cells"
"Immobilized Cells and Enzymes", 1986, LRL PRESS
"Monoclonal antibodies: a practical approach", 2000, OXFORD UNIVERSITY PRESS
"NCBI", Database accession no. NP_000576.1
"PCR: The Polymerase Chain Reaction", 1994
"The Antibodies", 1995, HARWOOD ACADEMIC PUBLISHERS
"UniProt", Database accession no. Q13261
B. PERBAL ET AL.: "A practical Guide To Molecular Cloning", 1984
BETTINI, M. L. ET AL., J IMMUNOL, vol. 199, no. 5, 2017, pages 1555 - 1560
BRENTJENS, R. J. ET AL., BLOOD, vol. 118, no. 18, 2011, pages 4817 - 4828
BRENTJENS, R. J. ET AL., NAT MED, vol. 9, no. 3, 2003, pages 279 - 286
BRENTJENS, R. J. ET AL., SCI TRANSL MED, vol. 5, no. 177, 2013, pages 177 - 138
C. A. JANEWAYP. TRAVERS: "Immunobiology", 1997
CAO, Y. ET AL., ANGEW CHEM INT ED ENGL, vol. 55, no. 26, 2016, pages 7520 - 7524
CARTELLIERI, M. ET AL., BLOOD CANCER J, vol. 6, no. 8, 2016, pages e458
CHO, J. H. ET AL., CELL, vol. 173, no. 6, 2018, pages 1426 - 1438
CHU, Y. ET AL., BLOOD, vol. 126, no. 23, 2015, pages 3085 - 3085
COOPER, L. J. ET AL., BLOOD, vol. 101, no. 4, 2003, pages 1637 - 1644
DU, N. ET AL., CANCERS, vol. 13, no. 16, 2021, pages 4129
EDGAR, R. C., NUCLEIC ACIDS RES, vol. 32, no. 5, 2004, pages 1792 - 1797
ESHHAR, Z. ET AL., PROC NATL ACAD SCI U S A, vol. 90, no. 2, 1993, pages 720 - 724
FEHNIGER, T. A.M. A. CALIGIURI, INT REV IMMUNOL, vol. 20, no. 3-4, 2001, pages 503 - 534
GEIGER, T. L. ET AL., THE JOURNAL OF IMMUNOLOGY, vol. 162, no. 10, 1999, pages 5931 - 5939
GIRON-MICHEL, J. ET AL., BLOOD, vol. 106, no. 7, 2005, pages 2302 - 2310
GONG, Y. ET AL., J HEMATOL ONCOL, vol. 14, no. 1, 2021, pages 73
GOUJON, M. ET AL., NUCLEIC ACIDS RES, vol. 38, 2010, pages W695 - 699
GUBIN, M. M. ET AL., J CLIN INVEST, vol. 125, no. 9, 2015, pages 3413 - 3421
HARADA, H. ET AL., EXP HEMATOL, vol. 32, no. 7, 2004, pages 614 - 621
HARADA, H. ET AL., JPN J CANCER RES, vol. 93, no. 3, 2002, pages 313 - 319
HORI, T. ET AL., BLOOD, vol. 70, no. 4, 1987, pages 1069 - 1072
HU, Q. ET AL., SCI REP, vol. 8, no. 1, 2018, pages 7675
IMAI, C. ET AL., LEUKEMIA, vol. 18, no. 4, 2004, pages 676 - 684
J. P. MATHERP. E. ROBERTS: "Introduction to Cell and Tissue Culture", 1998, PLENUM PRESS
JAYARAMAN, J. ET AL., EBIOMEDICINE, vol. 59, 2020, pages 102975
KIM, J. H. ET AL., PLOS ONE, vol. 6, no. 4, 2011, pages e18556
KIM, M. S. ET AL., J AM CHEM SOC, vol. 137, no. 8, 2015, pages 2832 - 2835
KLEIN, E. ET AL., INT J CANCER, vol. 18, no. 4, 1976, pages 421 - 431
KNUDSON K M ET AL: "Rationale for IL-15 superagonists in cancer immunotherapy", EXPERT OPINION ON BIOLOGICAL THERAPY, vol. 20, no. 7, 11 March 2020 (2020-03-11), pages 705 - 709, XP093030394, ISSN: 1471-2598, DOI: 10.1080/14712598.2020.1738379 *
KOCHENDERFER, J. N. ET AL., BLOOD, vol. 119, no. 17, 2012, pages 3940 - 3950
KUDO, K. ET AL., CANCER RES, vol. 74, no. 1, 2014, pages 93 - 103
KUO, M. L. ET AL., BLOOD, vol. 82, no. 3, 1993, pages 845 - 852
LI, Y. ET AL., CELL STEM CELL, vol. 23, no. 2, 2018, pages 181 - 192
LINNEMANN, C. ET AL., NAT MED, vol. 21, no. 1, 2015, pages 81 - 85
LIU, E. ET AL., LEUKEMIA, vol. 32, no. 2, 2018, pages 520 - 531
LOHMUELLER, J. J. ET AL., ONCOIMMUNOLOGY, vol. 7, no. 1, 2017, pages el368604
LOZZIO, C. B.B. B. LOZZIO, BLOOD, vol. 45, no. 3, 1975, pages 321 - 334
MA, J. S. ET AL., PROC NATL ACAD SCI U S A, vol. 113, no. 4, 2016, pages E450 - 458
MANN, R. ET AL., CELL, vol. 33, no. 1, 1983, pages 153 - 159
MARKOWITZ, D. ET AL., J VIROL, vol. 62, no. 4, 1988, pages 1120 - 1124
MENDES, B. B. ET AL., NATURE REVIEWS METHODS PRIMERS, vol. 2, no. 1, 2022, pages 24
MORGAN, M. A. ET AL., FRONT IMMUNOL, vol. 11, 2020, pages 611163
MYERS, J. A.J. S. MILLER, NATURE REVIEWS CLINICAL ONCOLOGY, vol. 18, no. 2, 2021, pages 85 - 100
NEEDLEMAN, S. B.C. D. WUNSCH, J MOL BIOL, vol. 48, no. 3, 1970, pages 443 - 453
NUSSBAUMER, O.M. KOSLOWSKI, IMMUNOONCOL TECHNOL, vol. 1, 2019, pages 3 - 10
PARK, J. H.H. K. LEE, EXP MOL MED, vol. 53, no. 3, 2021, pages 318 - 327
PEARSON, W. R.D. J. LIPMAN, PROC NATL ACAD SCI U S A, vol. 85, no. 8, 1988, pages 2444 - 2448
PORTER, D. L. ET AL., NEW ENGLAND JOURNAL OF MEDICINE, vol. 365, no. 8, 2011, pages 725 - 733
PULE, M. A. ET AL., NAT MED, vol. 14, no. 11, 2008, pages 1264 - 1270
RABINOVICH, P. M. ET AL., HUMAN GENE THERAPY, vol. 17, no. 10, 2006, pages 1027 - 1035
RING, A. M. ET AL., NAT IMMUNOL, vol. 13, no. 12, 2012, pages 1187 - 1195
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR PRESS
SHAFER, P. ET AL., FRONT IMMUNOL, vol. 13, 2022, pages 835762
SIEVERS, N. M. ET AL., INT J MOL SCI, vol. 21, no. 10, 2020
SMITH, T. F.M. S. WATERMAN, ADVANCES IN APPLIED MATHEMATICS, vol. 2, no. 4, 1981, pages 482 - 489
SOMAN, G. ET AL., J IMMUNOL METHODS, vol. 348, no. 1-2, 2009, pages 83 - 94
STERNER, R. C.R. M. STERNER, BLOOD CANCER JOURNAL, vol. 11, no. 4, 2021, pages 69
TAMADA, K. ET AL., CLIN CANCER RES, vol. 18, no. 23, 2012, pages 6436 - 6445
TOUTAIN, P. L.A. BOUSQUET-MELOU, J VET PHARMACOL THER, vol. 27, no. 6, 2004, pages 427 - 439
URBANSKA, K. ET AL., CANCER RES, vol. 72, no. 7, 2012, pages 1844 - 1852
WANG, S. ET AL., BMC MEDICINE, vol. 19, no. 1, 2021, pages 140
WANG, W. ET AL., CANCER LETT, vol. 472, 2020, pages 175 - 180
WEI, X. ET AL., J IMMUNOL, vol. 167, no. 1, 2001, pages 277 - 282
WIECZOREK, M. ET AL., FRONT IMMUNOL, vol. 8, 2017, pages 292
WRONA, E. ET AL., INT J MOL SCI, vol. 22, no. 11, 2021
YING, Z. ET AL., NAT MED, vol. 25, no. 6, 2019, pages 947 - 953

Similar Documents

Publication Publication Date Title
US20240376442A1 (en) Chimeric antigen receptor polypeptides in combination with trans metabolism molecules modulating krebs cycle and therapeutic uses thereof
US20220160760A1 (en) Receptors providing targeted costimulation for adoptive cell therapy
US12187778B2 (en) Receptors providing targeted costimulation for adoptive cell therapy
US20210340219A1 (en) Chimeric receptor polypeptides in combination with trans metabolism molecules modulating intracellular lactate concentrations and therapeutic uses thereof
US20220054545A1 (en) ANTI-GPC3 CHIMERIC ANTIGEN RECEPTORS (CARs) IN COMBINATION WITH TRANS CO-STIMULATORY MOLECULES AND THERAPEUTIC USES THEREOF
JP2024102286A (ja) グルコース輸入を向上させるトランス代謝分子と組み合わせたキメラ受容体及びその治療的使用
US20240390495A1 (en) Chimeric receptor polypeptides in combination with trans metabolism molecules that re-direct glucose metabolites out of the glycolysis pathway and therapeutic uses thereof
US20210261646A1 (en) Chimeric receptors in combination with trans metabolism molecules enhancing glucose import and therapeutic uses thereof
US20230055694A1 (en) Receptors providing targeted costimulation for adoptive cell therapy
US20230227576A1 (en) Receptors providing targeted costimulation for adoptive cell therapy
WO2022098750A1 (fr) Tcr restreints au hla de classe ii contre la mutation activant kras g12>v
CN116041518A (zh) 靶向b7-h3的抗体或抗体片段、以及其在嵌合抗原受体免疫细胞疗法领域的应用
WO2024215987A1 (fr) CELLULES IMMUNITAIRES POUR LE TRAITEMENT DU CANCER EN COMBINAISON AVEC DES CONJUGUÉS IL-15/IL-15Rα
WO2024215989A1 (fr) CELLULES IMMUNITAIRES MODIFIÉES POUR LE TRAITEMENT D'UN CANCER EN COMBINAISON AVEC DES AGONISTES βγ DU RÉCEPTEUR IL-2/IL-15
WO2024040208A1 (fr) Cellules immunitaires génétiquement modifiées avec des polypeptides récepteurs chimériques en combinaison avec de multiples molécules du métabolisme trans et leurs utilisations thérapeutiques
WO2024040207A1 (fr) Cellules tueuses naturelles (nk) génétiquement modifiées avec des polypeptides récepteurs chimériques en combinaison avec des molécules de trans-métabolisme et leurs utilisations thérapeutiques
CN118234848A (zh) 嵌合受体多肽与将葡萄糖代谢物重定向出糖酵解通路的反式代谢分子的组合和其治疗用途
KR20250049550A (ko) 개선된 car t 세포 활성화를 위한 길이-조절된 키메라 항원 수용체 및 길이-조절된 보조 수용체

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24724060

Country of ref document: EP

Kind code of ref document: A1