WO2023102109A1 - Stratégies d'amorçage par cytokines ex vivo pour la préparation de cellules immunitaires activées à forte expression de cd26 et utilisations dans les thérapies anticancéreuses - Google Patents

Stratégies d'amorçage par cytokines ex vivo pour la préparation de cellules immunitaires activées à forte expression de cd26 et utilisations dans les thérapies anticancéreuses Download PDF

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WO2023102109A1
WO2023102109A1 PCT/US2022/051528 US2022051528W WO2023102109A1 WO 2023102109 A1 WO2023102109 A1 WO 2023102109A1 US 2022051528 W US2022051528 W US 2022051528W WO 2023102109 A1 WO2023102109 A1 WO 2023102109A1
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cells
binds
expression
binding agent
specific binding
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Michael Brandon WARE
Megan Meek WYATT
Chrystal Mary PAULOS
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Emory University
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2312—Interleukin-12 (IL-12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2318—Interleukin-18 (IL-18)

Definitions

  • T cells can be isolated from the blood of a patient and genetically altered to express chimeric antigen receptors (CARs) that specifically target proteins expressed on the surface of cancerous cells and stimulate an immune response. When put back into the patient, the cells attack the cancerous cells.
  • CARs chimeric antigen receptors
  • Brentjens et al. report that T cells altered to bind CD19 can induce remissions of cancer in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med, 2013, 5(177): 177ra38.
  • CAR base therapies are clinically approved for treating certain hematological cancers. However, CAR therapies are reported to be less effective at treating solid tumors. Thus, there is a need to identify improvements.
  • CD4 and CD8 are molecule found on the outer membrane T cells which exist naturally in different ratios.
  • naive CD4+ T cells differentiate into one of several T helper (Th) subsets. These subsets are commonly identified by their ability to secrete IFN-y, IL-4 or IL-17A and are termed Thl, Th2 and Thl7 cells, respectively. Each subset has been reported to enhance immune responses against cancer.
  • This disclosure relates enriching and cytokine priming CD26 high T cells using exogenous cytokines ex vivo.
  • methods further comprise isolation of cytokine primed CD26 positive cells.
  • methods comprise isolation and enrichment of cytokine primed CD26 high T cells from human blood for use in cancer treatments.
  • this disclosure relates to methods of preparing purified activated T cells with high CD26 expression comprising separating cells from a blood product that express CD4 and CD26 providing isolated CD4 and CD26 T cells; contacting the isolated CD4 and CD26 T cells with IL- 18, IL-12, and IL-7 providing purified activated T cells with high CD26 expression.
  • this disclosure relates to methods of preparing purified activated T cells with high CD26 expression comprising separating cells from a blood product that express CD4 and CD26 providing isolated CD4 and CD26 T cells; contacting the isolated CD4 and CD26 T cells with IL-18, IL-12, IL-7, or combinations thereof providing purified activated T cells with high CD26 expression.
  • the purified activated T cells with high CD26 expression are further contacted with a specific binding agent that binds CD3, a specific binding agent that binds CD28, a specific binding agent that binds inducible T cell costimulator (ICOS/CD278), or combinations thereof.
  • the specific binding agent that binds CD3 and the specific binding agent that binds CD28 are beads coated with anti-CD3 and anti-CD28 antibodies or wherein the specific binding agent that binds CD3 and the specific binding agent that binds ICOS are beads coated with anti-CD3 and anti-ICOS antibodies.
  • the purified activated T cells with high CD26 expression are obtained without exposure to a specific binding agent that binds CD3, a specific binding agent that binds CD28, or a specific binding agent that binds CD3 inducible T cell costimulator (ICOS/CD278).
  • the separating cells from a blood product that express CD4 and CD26 providing isolated CD4 and CD26 T cells is by contacting the blood product with a specific binding agent that binds CD26 and optionally CD4 providing a CD4 and CD26 specific agent bound T cells; and purifying the CD4 and CD26 specific agent bound T cells from cells that lack CD26 providing purified T cells with high CD26 expression.
  • the purifying is by fluorescent activated cell sorting.
  • this disclosure relates to in vitro cell culture compositions comprising cells made by the methods reported herein.
  • the cell culture further comprises anti-CD3 antibodies, anti-CD28 antibodies and/or anti-ICOS antibodies optionally immobilized on a bead, in the form of a tetramer, or conjugated to a solid surface.
  • this disclosure relates to methods of expanding cells comprising contacting cells made by the method reported herein with an in vitro cell culture providing replicated activated T cells with high CD26 expression.
  • this disclosure relates to methods further comprising contacting the T cells having high CD26 expression made by methods disclosed herein with a vector having a nucleic acid encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a cancer targeting sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a signal-transduction component of a T-cell antigen receptor domain, under conditions such that the cells express the chimeric antigen receptor on the surface of the cells.
  • this disclosure relates to methods of treating cancer or a chronic infection comprising administering an effective amount of T cells having high CD26 expression made by the methods reported herein to a subject in need thereof.
  • the cancer is a hematological malignancy, tumor, or tissue-based cancer.
  • the T cells having high CD26 expression are administered in combination with another anticancer agent.
  • FIG. 1 A shows data from experiments.
  • FIG. 1C shows data from experiments.
  • FIG. 1A shows data from experiments.
  • Figure 2B illustrates methods of isolating contacting cells with cytokines.
  • Figure 3 A illustrates cellular changes.
  • Figure 3B illustrates methods of isolating cells.
  • Figure 3C shows data from experiments. DETAILED DISCUSSION
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of immunology, medicine, organic chemistry, biochemistry, molecular biology, pharmacology, physiology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
  • cell culture or “growth medium” or “media” refers to a composition that contains components that facilitate cell maintenance and growth through protein biosynthesis, such as vitamins, amino acids, inorganic salts, a buffer, and a fuel, e.g., acetate, succinate, a saccharide and/or optionally nucleotides.
  • a fuel e.g., acetate, succinate, a saccharide and/or optionally nucleotides.
  • Typical components in a growth medium include amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine and others); vitamins such as retinol, carotene, thiamine, riboflavin, niacin, biotin, folate, and ascorbic acid; carbohydrate such as glucose, galactose, fructose, or maltose; inorganic salts such as sodium, calcium, iron, potassium, magnesium, zinc; serum; and buffering agents. Additionally, a growth media may contain phenol red as a pH indication.
  • Components in the growth medium may be derived from blood serum or the growth medium may be serum-free.
  • the growth medium may optionally be supplemented with albumin, lipids, insulin and/or zinc, transferrin or iron, selenium, ascorbic acid, and an antioxidant such as glutathione, 2-mercaptoethanol or 1 -thioglycerol.
  • Other contemplated components contemplated in a growth medium include ammonium metavanadate, cupric sulfate, manganous chloride, ethanolamine, and sodium pyruvate.
  • Minimal Essential Medium is a term of art referring to a growth medium that contains calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium phosphate and sodium bicarbonate), essential amino acids, and vitamins: thiamine (vitamin Bl), riboflavin (vitamin B2), nicotinamide (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), folic acid (vitamin B9), choline, and myo-inositol (originally known as vitamin B8).
  • Various growth mediums are known in the art.
  • Dulbecco's modified Eagle's medium (DMEM) is a growth medium which contains additional components such as glycine, serine, and ferric nitrate with increased amounts of vitamins and amino acids.
  • Subject refers any animal, preferably a human patient, livestock, or domestic pet.
  • the terms “treat” and “treating” are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression. As used herein, the terms “prevent” and “preventing” include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.
  • the term "combination with” when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.
  • nucleic acid refers to a polymer of nucleotides, or a polynucleotide, e.g., RNA, DNA, or a combination thereof. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids may be single stranded or double stranded and may include coding regions and regions of various control elements.
  • encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • a "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are joined together in a manner that does not naturally occur in the same organism or any natural state.
  • nucleic acid molecule when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques provided that the entire nucleic acid sequence does not occurring in nature, i.e., there is at least one mutation in the overall sequence such that the entire sequence is not naturally occurring even though separately segments may occur in nature. The segments may be joined in an altered arrangement such that the entire nucleic acid sequence from start to finish does not naturally occur.
  • recombinant when made in reference to a protein or a peptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.
  • vector refers to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free expression systems.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • this disclosure contemplates a vector encoding a peptide disclosed herein in operable combination with a heterologous promoter.
  • a "chimeric antigen receptor” or “CAR” refers to a protein receptor, which introduces an antigen specificity, via an antigen binding domain, onto cells (immune cells) to which it is expressed (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof) thus combining the antigen binding properties of the antigen binding domain with the T cell activity (e.g. lytic capacity).
  • a CAR typically includes an extracellular antigen-binding domain (ectodomain), a transmembrane domain and an intracellular signaling domain.
  • the intracellular signaling domain generally contains at least one immunoreceptor tyrosine-based activation motif (ITAM) signaling domain, e.g., derived from CD3zeta, and optionally at least one costimulatory signaling domain, e.g., derived from CD28 or 4-1BB.
  • ITAM immunoreceptor tyrosine-based activation motif
  • T cells can be isolated from the blood of a patient and modified with a recombinant vector to express chimeric antigen receptors (CARs) that specifically target proteins expressed on the surface of cancerous cells and stimulate an immune response. When put back into the patient, the cells attack the cancerous cells.
  • CARs chimeric antigen receptors
  • the cells attack the cancerous cells.
  • Brentjens et al. report that T cells altered to bind CD19 can induce remissions of cancer in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med, 2013, 5(177): 177ra38.
  • the targeting sequence in a chimeric antigen receptor refers to any variety of polypeptide sequences capable of selectively binding to a targeted associated molecule.
  • the targeting sequences may be derived from variable binding regions of antibodies, single chain antibodies, and antibody mimetics.
  • targeting sequence is a single-chain variable fragment (scFv) derived from an antibody.
  • the targeting sequence is typically connected to intracellular domains by a hinge/transmembrane region, commonly derived from CD8 or IgG4.
  • the intracellular domains may contain co-stimulatory domains such as CD80, CD86, 4-1BBL, IL- 2Rbeta, OX40L and CD70 and/or CD28 linked to the cytoplasmic signaling domain of CD3zeta.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • leukocytes e.g., PBMCs
  • specific cell subsets e.g., isolate specific cells directly by using flow cytometry, depleting red blood cells, centrifugation, and/or apheresis.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs may be isolated by leukapheresis.
  • T cells can be enriched by mononuclear cells counter-flow elutriation and expanded by addition of anti-CD3/CD28 antibody coated paramagnetic beads for activation of T cells.
  • Cells may be expanded, harvested, and cryopreserved in infusible medium sometime after the subject has had an allogeneic stem-cell transplantation.
  • Cells may be obtained by isolation from peripheral blood and optionally purified by fluorescent activated cells sorting e.g., mixing cells with fluorescent antibodies or other fluorescent agents (molecular beacons) and separating the cells by flow cytometry based fluorescent sorting.
  • CD3 is expressed on T cells as it is associated with the T cells receptor (TCR).
  • TCR T cells receptor
  • the majority of TCR are made up of alpha beta chains (alpha beta T-cells).
  • Alpha beta T-cells typically become double-positive intermediates (CD4+CD8+) which mature into single-positive (CD4+CD8-) T helper cells or (CD4-CD8+) cytotoxic T cells.
  • T helper cells interact with antigen presenting dendritic cells and B cells. Upon activation with cognate antigen by dendritic cells, antigen specific CD4 T cells can differentiate to become various types of effector CD4 T cells with specific roles in promoting immune responses.
  • T cells may be isolated and separated from a human sample (blood or PBMCs or bone marrow) based on the expression of alpha beta T cells receptor (TCR), gamma delta T cells receptor, CD2, CD3, CD4, CD8, CD4 and CD8, NK1.1, CD4 and CD25 and other combinations based on positive or negative selection.
  • TCR alpha beta T cells receptor
  • CD2, CD3, CD4, CD8, CD4 and CD8, NK1.1, CD4 and CD25 and other combinations based on positive or negative selection.
  • T cells are purified and isolated from blood or bone marrow.
  • T cells are collected via apheresis, a process that withdraws blood from the body and removes one or more blood components (such as plasma, platelets, or other white blood cells).
  • the cells are exposed to a recombinant vector, such as a lentiviral vector, that infects the cells in a way that a chimeric antigen receptor (CAR) protein is produced and presented in the cell membrane.
  • a recombinant vector such as a lentiviral vector
  • separating refers to purify the cells from other cells or impurities that do not contain or contain less of a target molecule on the surface.
  • One method of selecting proteins that are on the outside of a cell is to provide a specific binding agent, such as a primary antibody, and further trap the primarily antibody bound to the cell using a secondary antibody that is conjugated to magnetic beads.
  • the magnetic beads can be captured by a magnetic field and separated from the rest of a solution.
  • secondary antibodies contain a fluorescent marker, and the cells can be separated using flow cytometry or fluorescence activated sorting.
  • the laser light would excite the fluorescent tag, or fluorochrome, which would emit photons of light at a higher wavelength (e.g. fluorescein isothiocyanate emits light at about 530nm when excited by a 488nm laser). This light can be collected and used to further categorize the cells.
  • fluorescence-activated sorting refers to a flow cytometry method of sorting a mixture of cells into two or more areas, typically one particle or cell at a time, based upon the fluorescent characteristics of each particle or cell. It is typically accomplished by applying an electrical charge and separating by movement through an electrostatic field. Typically, a vibrating mechanism causes a stream of cells to break into individual droplets. Just prior to droplet formation, a particle or cell in a fluid pass through an area for measuring fluorescence. An electrical charging mechanism is configured at the point where the stream breaks into droplets. Based on the fluorescence intensity measurement, a respective electrical charge is imposed on the droplet as it breaks from the stream.
  • CD4+ T cell and CD26high cells are distinct from T helper cell 17 (TH17) cells.
  • CD26 distinguishes three human CD4+ T cell subsets with varying degrees of responsiveness to tumors: one with regulatory properties (CD26neg), one with a naive/central memory phenotype (CD26int), and one with a durable stem memory profile (CD26high) (See Bailey et al. 2017).
  • CD26high T cells engineered with chimeric antigen receptors (CARs) persisted and regressed difficult-to-treat malignancies superior to CD26neg T cells and, unexpectedly, slightly better than naive CD26int T cells.
  • CD26high T cells secreted T helper cell 17 (TH17) cytokines, including interleukin-17A (IL-17A).
  • TH17 T helper cell 17
  • IL-17A interleukin-17A
  • CD26high T cells Given the abundance of IL- 17 produced by CD26high T cells and the high CD26 expression on TH17 cells, it was thought that CD26high T cells would have a similar cytokine profile as classic TH17 cells.
  • the level and type of cytokines produced by various CD4+ subsets were measured. These subsets were sorted from the peripheral blood of healthy individuals via extracellular markers. This sort yielded TH1 (CXCR3+CCR4-CCR6-), TH2 (CXCR3-CCR4+CCR6-), TH17 (CCR4+CCR6+CXCR3+/-), and CD26high T cells.
  • CD26high T cells also expressed high CXCR3 and CCR6 but nominal CCR4 on their surface.
  • CD26high T cells were not restricted to a TH17-like functional profile. Instead, CD26high T cells secreted comparable amounts of IL-17A and greater levels of interferon-gamma (IFN-gamma) and IL-22 than TH17 cells. CD26high T cells produced nearly as much IFN-alph as TH1 cells but less IL-4 than TH2 cells. This functional pattern was observed in CD26high T cells from several healthy individuals.
  • IFN-gamma interferon-gamma
  • CD4+ T cell populations were assayed for their capacity to secrete up to five cytokines at once, including IL-17A, IL-22, IFN-gamma, tumor necrosis factoralpha (TNF-alpha), and IL-2.
  • cytokines including IL-17A, IL-22, IFN-gamma, tumor necrosis factoralpha (TNF-alpha), and IL-2.
  • CD26high T cells secreted four to five cytokines simultaneously, while bulk CD4+, TH1, TH2, and TH17 cells, at most, cosecreted three cytokines.
  • CD26high T cells express more IL-23R mRNA than CD261ow or CD26int T cells.
  • IL-23 induce TH1 or TH17 cells to cosecrete elevated IL-17A and IFN-gamma to the levels mediated by CD26high T cells. While IL-23 did not provoke TH1 cells to cosecrete IL-17A and IFN-gamma, it did increase the coproduction of these two cytokines by TH17 cells to a less extent than CD26high T cells.
  • CD26high T cells have a functional profile distinct from other CD4+ T subsets and are more dynamic than classic TH17 cells.
  • CD4+ T helper subsets sorted from the peripheral blood via classic surface markers and engineered with a CAR was desired.
  • the impact of human Th 17 cells in the context of adoptive T cell transfer (ACT) therapy CD4+ T cells with high expression of the marker CD26 are cytotoxic cells with unique stem memory properties and dynamic polyfunctional capabilities.
  • CD26high T cells are distinct from Thl, Th2, or Thl7 cells. Most notably, CD26high T cells have tumor-killing capabilities in vivo. These cells can be obtained from healthy human donors. CD26high T cells express a multitude of cytokine and growth factor receptors.
  • IL- 18 receptor IL-18R
  • T cells expressing IL-18R are poised to secrete IFNgamma in response to IL- 18 without the need for T-cell Receptor (TCR) or mitogenic stimulation.
  • TCR T-cell Receptor
  • Co-stimulation with IL- 12 and IL-18 further enhances this effect.
  • IL- 18, IL-12 and IL-7 cytokines convert naive T cells into polyfunctional CD26high T cells without the need for TCR or mitogenic stimulation.
  • IL18R enriched CD4+ T cells are able to co-secrete IFNy, IL-2, IL- 21, IL17A, and TNFa. Surprisingly, this enrichment strategy bolsters T cell clustering in a manner which resembled conventional CD3/CD28 bead stimulation, but without logarithmic expansion.
  • IL-18R+ T cells induced by these cytokine priming strategies are referred to as Thl8R cells.
  • cytokine priming strategies which convert naive or CD26int cells into Thl8R cells with high CD26 expression, strong proliferative capacity, an enhanced polyfunctional cytokine profile and a superior cytotoxic phenotype.
  • Thl8R cells and CD26high T cells are useful in cellular therapeutics for the clinical treatment of cancer.
  • methods further comprise isolation of cytokine primed CD26 positive cells.
  • methods comprise isolation and enrichment of cytokine primed CD26 high T cells from human blood for used in cancer treatment.
  • this disclosure relates to methods of preparing purified activated T cells with high CD26 expression comprising separating cells from a blood product that express CD4 and CD26 providing isolated CD4 and CD26 T cells; contacting the isolated CD4 and CD26 T cells with IL-18, IL-12, IL-7, or combinations thereof providing purified activated T cells with high CD26 expression.
  • this disclosure relates to methods of preparing purified activated T cells with high CD26 expression comprising separating cells from a blood product that express CD4 and CD26 providing isolated CD4 and CD26 T cells; contacting the isolated CD4 and CD26 T cells with IL-18, IL-12, and IL-7 providing purified activated T cells with high CD26 expression.
  • the purified activated T cells with high CD26 expression are further contacted with a specific binding agent that binds CD3, a specific binding agent that binds CD28, a specific binding agent that binds inducible T cell costimulator (ICOS/CD278) or combinations thereof.
  • the specific binding agent that binds CD3 and the specific binding agent that binds CD28 are beads coated with anti-CD3 and anti-CD28 antibodies or wherein the specific binding agent that binds CD3 and the specific binding agent that binds ICOS are beads coated with anti-CD3 and anti-ICOS antibodies.
  • the purified activated T cells with high CD26 expression are obtained without exposure to a specific binding agent that binds CD3, a specific binding agent that binds CD28, or a specific binding agent that binds CD3 inducible T cell costimulator (ICOS/CD278).
  • the separating cells from a blood product that express CD4 and CD26 providing isolated CD4 and CD26 T cells is by contacting the blood product with a specific binding agent that binds CD26 and optionally CD4 providing a CD4 and CD26 specific agent bound T cells; and purifying the CD4 and CD26 specific agent bound T cells from cells that lack CD26 providing purified T cells with high CD26 expression.
  • the purifying is by fluorescent activated cell sorting.
  • this disclosure relates to in vitro cell culture compositions comprising cells made by the methods reported herein.
  • the cell culture further comprises anti-CD3 antibodies, anti-CD28 antibodies and/or anti-ICOS antibodies optionally immobilized on a bead, in the form of a tetramer, or conjugated to a solid surface.
  • this disclosure relates to methods of expanding cells comprising contacting cells made by the method reported herein with an in vitro cell culture providing replicated activated T cells with high CD26 expression.
  • this disclosure relates to methods further comprising contacting the T cells having high CD26 expression, e.g., Thl8R cells and CD26high T cells, with a vector having a nucleic acid encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a cancer targeting sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a signal-transduction component of a T-cell antigen receptor domain, under conditions such that the cells express the chimeric antigen receptor on the surface of the cells.
  • this disclosure relates to methods of treating cancer or a chronic infection comprising administering an effective amount of T cells having high CD26 expression made by the methods reported herein e.g., Thl8R cells and CD26high T cells, to a subject in need thereof.
  • the cancer is a hematological malignancy, tumor, or tissue-based cancer.
  • the T cells having high CD26 expression are administered in combination with another anticancer agent.
  • the T cells having high CD26 expression e.g., Thl8R cells and CD26high T cells
  • a chimeric antigen receptor on the surface of the cells such as those that specifically bind a cancer antigen, CD 138, CD 19, immunoglobulin kappa (Ig-Kappa), or B-cell maturation antigen (BCMA).
  • the chimeric antigen receptor specifically binds (EGFR) epidermal growth factor receptor, (HER2) human epidermal growth factor receptor 2, (MUC1) mucin 1, (MUC16) mucin 16, (EpCAM) epithelial cell adhesion molecule, (AFP) alpha-fetoprotein, (FAP) familial adenomatous polyposis, (CEA) carcinoembryonic antigen, (PSCA) prostate stem cell antigen, (PSMA) prostate-specific membrane antigen, (PSA) prostate-specific antigen, (AXL) AXL receptor tyrosine kinase, (DLL3) delta-like 3, (EPHA2) EPH receptor A2, (FRa) folate receptor alpha, (LMP1) Epstein-Barr virus latent membrane protein 1, (MAGE) melanoma antigen gene protein, MAGE-A1, MAGE- A3, MAGE-A4, (DR5) death receptor 5, (NKG2D) natural killer group 2 member D receptor, (CAI
  • T cells having high CD26 expression e.g., Thl8R cells and CD26high T cells, which are produced by methods disclosed herein, and which are used in methods for treating a subject diagnosed with cancer.
  • the cancer is a hematological malignancy such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia, acute monocytic leukemia (AMOL), chronic myeloid leukemia (CML), myeloproliferative neoplasms (MPNs), and lymphomas, Hodgkin's lymphomas, and non-Hodgkin's lymphomas such as Burkitt lymphoma, B-cell lymphoma.
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • SLL small
  • the cancer is a solid tumor, cellular malignancy, or hematological malignancy.
  • the cancer is lung cancer, non-small cell lung cancer, small cell lung cancer, bronchus cancer, mesothelioma, malignant pleural mesothelioma, lung adenocarcinoma, breast cancer, prostate cancer, colon cancer, rectum cancer, colorectal cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, cervical cancer, melanoma, kidney cancer, pancreatic cancer, pancreatic ductal adenocarcinoma (PDA), thyroid cancer, brain cancer, glioblastoma (GBM), medulloblastoma, glioma, neuroblastoma, liver cancer, bladder cancer, uterine cancer, bone cancer, osteosarcoma, sarcoma, rhabdomyosarcoma, Ewing's sarcoma, retinoblast
  • the T cells having high CD26 expression as reported herein are used for treating cancer and are administered in combination with another anticancer agent.
  • the anticancer agent is abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, afhbercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pami
  • the anticancer agent is an anti-PD-1, anti-PD-Ll anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti-PDl (e.g., nivolumab, pembrolizumab, cemiplimab) and anti-PD-Ll (e.g., atezolizumab, avelumab, durvalumab).
  • an anti-CTLA4 e.g., ipilimumab, tremelimumab
  • anti-PDl e.g., nivolumab, pembrolizumab, cemiplimab
  • anti-PD-Ll e.g., atezolizumab, avelumab, durvalumab.
  • the cells are administered to a subject with a lymphodepleted environment due to prior or concurrent administration of lymphodepleting agents such as cyclophosphamide and fludarabine).
  • lymphodepleting agents such as cyclophosphamide and fludarabine
  • Normal human donor peripheral blood cells may be obtained as a buffy coat or using leukapheresis.
  • Peripheral blood lymphocytes PBLs
  • CD4+ T cells were negatively isolated using magnetic bead separation and plated in culture medium with a low concentration of rhIL-2 [20 lU/ml] overnight.
  • CD8+ T cells were positively isolated before the enrichment of CD4+ T cells.
  • CD4+ T cells were stained using PE-CD26 (BA5b), AlexaFluor647-CXCR3 (G025H7), PECy7- CCR6 (G034E3), FITC-CCR4 (205410), and APCCy7-CD4 (OKT4).
  • Cells were sorted on the basis of the following gating strategies: bulk CD4:CD4+; THECD4+CCR6-CCR4-CXCR3+; TH2: CD4+CCR6-CCR4+CXCR3-; TH17:CD4+CCR6+CCR4+; and CD26: CD4+CD26high.
  • T cell subsets were expanded in RPMI 1640 culture medium supplemented with nonessential amino acids, 1-glutamine, sodium pyruvate, Hepes, penicillin/streptomycin, betamercaptoethanol, and fetal bovine serum. Cells were cultured at either a 1 : 1 or 1 : 10 bead-to-T cell ratio. Magnetic beads coated with antibodies to CD3 (OKT3) and/or ICOS (ISA-3) or CD28 (CD28.2) were produced in the laboratory according to the manufacturers’ protocols. rhIL-2 (100 lU/ml) was added on day 2, and media were replaced as needed.
  • alphaCD3/ICOS-activated, sorted CD4+ and bulk CD8+ T cells were transduced with a lentiviral vector encoding a chimeric anti-mesothelin singlechain variable fragment fusion protein containing the TCR signaling domain (first-generation meso-CAR) or a truncated CD3 nonsignaling domain.
  • CAR expression was determined using a flow cytometry antibody specific for the murine F(ab')2 fragment. Cells were normalized for CAR expression before adoptive transfer.

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Abstract

La présente divulgation concerne l'enrichissement et l'amorçage par cytokine des lymphocytes T à forte expresssion de CD26 à l'aide de cytokines exogènes ex vivo. Dans certains modes de réalisation, les lymphocytes T à forte expression de CD26 amorcés par des cytokines sont utilisés dans les traitements anticancéreux. Dans certains modes de réalisation, la présente divulgation concerne des procédés de préparation de lymphocytes T activés purifiés à forte expression de CD26, comprenant la séparation de cellules exprimant CD4 et CD26, fournissant des lymphocytes T CD4 et CD26 isolés, et la mise en contact des lymphocytes T CD4 et CD26 isolés avec IL-18, IL-12 et IL-7, fournissant des lymphocytes T activés purifiés à forte expression de CD26.
PCT/US2022/051528 2021-12-01 2022-12-01 Stratégies d'amorçage par cytokines ex vivo pour la préparation de cellules immunitaires activées à forte expression de cd26 et utilisations dans les thérapies anticancéreuses WO2023102109A1 (fr)

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WO2017189526A1 (fr) * 2016-04-25 2017-11-02 Musc Foundation For Research Development Cellules immunitaires à expression cd26 élevée et cellules immunes cd26-négatives activées et leurs utilisations
US20190247431A1 (en) * 2016-04-25 2019-08-15 Musc Foundation For Research Development Activated cd26-high immune cells and cd26-negative immune cells and uses thereof

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STEFANIE R. BAILEY, MICHELLE H. NELSON, KINGA MAJCHRZAK, JACOB S. BOWERS, MEGAN M. WYATT, AUBREY S. SMITH, LILLIAN R. NEAL, KEISUK: "Human CD26high T cells elicit tumor immunity against multiple malignancies via enhanced migration and persistence", NATURE COMMUNICATIONS, vol. 8, no. 1, 1 December 2017 (2017-12-01), XP055491138, DOI: 10.1038/s41467-017-01867-9 *
WRAGG KATHLEEN M., TAN HYON-XHI, KRISTENSEN ANNE B., NGUYEN-ROBERTSON CATRIONA V., KELLEHER ANTHONY D., PARSONS MATTHEW S., WHEATL: "High CD26 and Low CD94 Expression Identifies an IL-23 Responsive Vδ2+ T Cell Subset with a MAIT Cell-like Transcriptional Profile", CELL REPORTS, ELSEVIER INC, US, vol. 31, no. 11, 1 June 2020 (2020-06-01), US , pages 107773, XP093072172, ISSN: 2211-1247, DOI: 10.1016/j.celrep.2020.107773 *

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