WO2021006733A1 - Épuisement rapide in vitro de lymphocytes t - Google Patents

Épuisement rapide in vitro de lymphocytes t Download PDF

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WO2021006733A1
WO2021006733A1 PCT/NL2020/050446 NL2020050446W WO2021006733A1 WO 2021006733 A1 WO2021006733 A1 WO 2021006733A1 NL 2020050446 W NL2020050446 W NL 2020050446W WO 2021006733 A1 WO2021006733 A1 WO 2021006733A1
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cells
exhausted
peptide
exhaustion
cell
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Peter D. Katsikis
Yvonne M. MULLER
Manzhi ZHAO
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Erasmus University Medical Center Rotterdam
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T 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
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • 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]
    • C12N2501/2315—Interleukin-15 (IL-15)

Definitions

  • the present invention relates to a method for the rapid in vitro generation of viable, exhausted T cells and related cell cultures and applications of said method for drug discovery.
  • Exhausted T cells are effector T cells with decreased cytokine expression and effector function. Exhausted T cells are deemed to be resistant to reactivation and exhibit decreased proliferation. T cell exhaustion occurs naturally when the cells are chronically activated at sites of chronic inflammation, such as cancer, autoimmunity, and chronic infection. In a review by Crespo et al., T cell exhaustion was compared to T cell anergy, senescence and sternness in a tumor environment (Crespo et al., Curr Opin Immunol. 25(2): 214-221, 2013). Several markers and features have been identified that are required to distinguish T cell exhaustion from other T cell dysfunctions.
  • Exhausted T cells show a significant decrease in the cytokines IL-2, IFN Y , and TNFa expression (E. John Wherry et al., Immunity 27(4), 670-684, 2007), as well as cell cycle arrest.
  • a hallmark of exhaustion is the loss of poly-functionality which means that an individual cell looses the capacity to make multiple cytokines simultaneously (E. John Wherry et al, Immunity 27(4), 670-684., 2007).
  • Another critical feature of exhausted CD8 T cells is the sustained high expression of multiple inhibitory receptors, including PD-1, Tim-3a, Lag-3, CD160, CD244,TIGIT and others (Jin, H. T. et al. Proc. Natl Acad. Sci.
  • Characteristic of exhausted CD8 + T cells is also their deficient ability to proliferate. At the molecular level altered expression of transcription factors is a defining feature of exhausted CD8 + T cells (Kao, C. et al. Nat. Immunol. 12(7), 663- 671,2011), for example changes in expression of Tox and TCF1 can discriminate between fully exhausted T cells and those that retain proliferative capacity (B, C. Miller et al., Nat. Immunol. .20 (3), 326-336, 2019).
  • the weak IFN g production in these cells also does not support T cell exhaustion. Further, only 68% of cells express PD-1, the cardinal receptor for exhausted T cells, which argues for at best partial exhaustion. There is no evidence for co-expression of inhibitory receptors and no data on loss of polyfunctionality loss.
  • the induction of YY1 in these cultures suggests that differentiation of T cells is being affected and not exhaustion. YY1 regulates Th2 cell differentiation via the master regulator GATA3 and the Treg inducer Foxp3 so that it is likely instead of exhaustion the method of Balkhi et al. induces Th2 or Treg cells instead of exhaustion.
  • the present invention provides a method to rapidly generate viable, exhausted T cells in vitro.
  • the present inventors have found that this can be achieved through repeated antigen stimulation of T cells in the presence of IL-7 and/or IL-15.
  • the invention comprises a method for in vitro generation of large numbers of viable, exhausted T cells, comprising culturing T cells, especially CD8 + T cells, in a medium comprising at least IL-7 and/or IL-15; and antigen stimulation of said T cells to generate exhausted T cells.
  • the invention provides methods and means to induce exhausted T cells rapidly over a few days, for example 5 days, a significant improvement for intervention testing as exhaustion in vivo can take 30 days or more to be fully induced.
  • the approach taken allows to establish exhaustion as a result of T cell receptor stimulation without the presence of other factors that can influence gene expression or behavior of these T cells. It allows in vitro screening of compounds, drugs or other approaches to induce, prevent, accelerate or reverse T cell exhaustion.
  • treatment can include administration of a chemical compound, such as a small molecule, a peptide, a protein (including antibodies), a nucleotide and/or a carbohydrate.
  • Said antigen preferably is a peptide, more preferably a specific peptide is selected from the group of globular proteins and fragments thereof, preferably ovalbumin and fragments thereof, most preferably OVA257-264 peptide.
  • a total concentration of IL-7 and/or IL-15 together is 1- 100 ng/mL, preferably 5-50 ng/mL, most preferably 10-25 ng/mL.
  • Said antigen concentration preferably is 1 ng/mL to 10 mg/mL, preferably 5 ng/mL to 5 mg/mL, most preferably 10 ng/mL to 1 mg/mL.
  • Said T cells preferably are cultured for 2-15 days, more preferably for 3-10 days, most preferably for about 5 days.
  • the aforementioned animal preferably is a human or a rodent, most preferably a mouse.
  • a method for identifying molecular targets to prevent, restore, or accelerate T cell exhaustion comprising culturing T cells in a medium comprising at least IL-7 and/or IL-15; repeated antigen stimulation of said T cells to generate exhausted T cells; treating said T cells with a treatment that may prevent, restore, or accelerate T cell exhaustion; and comparing the treated T cells with untreated exhausted and non-exhausted T cells, to find relevant changes in said T cells.
  • a cell culture comprising viable exhausted T cells as obtained by a method according to the invention.
  • FIG. 1 In vitro exhaustion method induces loss of cytokine production and polyfuctionality.
  • Purified OT-I CD8+ T cells were cultured either without peptide (no peptide), stimulated one time for 2 days with OVA peptide (single peptide) or stimulated with OVA peptide daily (repeat peptide). On day 5 cells were harvested and re-stimulated with OVA peptide.
  • B) SPICE figures depicting the frequency of cells producing one, two or three cytokines in different combinations. Each symbol represents one animal (n ll-12), 9 independent experiments performed. Line depicts mean ⁇ SE.
  • FIG. 3 Repeat peptide stimulated CD8+ T cells have decreased cytotoxic function. On day 5, cells were harvested and re-stimulated with OVA peptide.
  • MFI Median fluorescence intensity
  • CD107a Median fluorescence intensity of the degranulation marker, CD107a, shown (left panel). Fold change of CD107a MFI induced by peptide re- stimulation depicted on the right panel.
  • Figure 4 In vitro exhaustion method induces the expression of inhibitory receptors.
  • A CD8 + T cells (CTL) submitted to in vitro exhaustion (multiple peptide) express multiple inhibitory receptors. Data are for inhibitory receptors PD-1, Lag3, Tim3a, CD 160, TIGIT and CD244.
  • FIG. 6 In vitro exhaustion method induces CTL with reduced capacity to proliferate in vivo.
  • OT-I CD8 + T cells CTL
  • OT-I CD8 + T cells CTL
  • In vitro exhausted (multiple peptide) expand much less indicating a defect in proliferative capacity.
  • a and b show percentage and absolute numbers of donor cells in lung.
  • transcriptome is enriched for gene sets associated with exhaustion.
  • RNAseq data from n 4- 5 mice and 4-5 experiments.
  • FIG. 8 Genome wide DNA methylation changes during T cell stimulation reveal Tcf7 promotor methylation. Sorted live CD8+ T cells were processed and whole genome methylated DNA sequencing (MeD-seq) was performed.
  • MeD-seq whole genome methylated DNA sequencing
  • A Hierarchical clustering on DMRs (differentially methylated regions) found between the three different peptide exposure conditions are shown.
  • B Boxplot of DNA methylation read count data in a 2kb window surrounding the TSS of mentioned genes are shown. The samples were collected from three independent experiments.
  • FIG. 10 Exposure to IL-2 does not alter the exhaustion phenotypes in repeat peptide stimulated cells and CD80 is upregulated on repeat stimulated cells.
  • CD8+ T cell exhaustion was induced in the presence of IL-2 (20U/ml) and IL-7/IL-15 (5ng/ml each). Cytokine production after OVA peptide restimulation for 6 hours (A) and inhibitory receptor expression on day 5 (B) are shown.
  • n 8 animals from 5-6 independent experiments depicted.
  • Figure 11 Anergy gene sets and pathways do not characterize repeat peptide exhausted T cells. Both upregulated and downregulated genes in anergic T cells are enriched in exhausted T cells.
  • A. GSEA of genes upregulated and B. downregulated in anergic T cells are both enriched in differentially expressed repeat peptide stimulated cells (repeat peptide versus no peptide cell).
  • C. GSEA of genes upregulated and D. downregulated in anergic T cells are both enriched in differentially expressed Day 30 gp33-specific CTL from LCMV clone 13 infected animals (Day 30 versus gp33- specific CTL naive CD8+ T cells; data from GSE41867).
  • T cell refers to lymphocytes developed in the thymus, and can be distinguished from other lymphocytes by the presence of a T- cell receptor on their surface.
  • exhaust refers to the dysfunctional state of T cells when the expression of cytokines and the effector function are decreased, and the T cells are resistant to reactivation and exhibit reduced proliferative capacity.
  • cell culture refers to an in vitro population of viable cells under cell cultivation conditions, i.e. under conditions wherein the cells are suspended in a culture medium that will allow their survival and preferably their growth.
  • the cell culture is usually comprised in a container holding the cell culture, referred to as a culture chamber, wherein sufficient exchange of gases such as oxygen and CO 2 is allowed between the cell culture and the atmosphere to support cell viability.
  • medium refers to a medium generally used in the culturing of mammalian T cells. Any available medium suitable for the culturing of mammalian T cells can be utilized, including a medium based on Dulbecco's Modified Eagle Medium (DMEM) or Ham’s F12 nutrient medium, or combinations of such basal media, optionally supplemented with compounds that prevent bacterial contamination, such as antibiotics, preferably in the form of a DMEM) or Ham’s F12 nutrient medium, or combinations of such basal media, optionally supplemented with compounds that prevent bacterial contamination, such as antibiotics, preferably in the form of a
  • DMEM Dulbecco's Modified Eagle Medium
  • Ham Ham’s F12 nutrient medium
  • the medium comprises Roswell Park Memorial Institute (RPMI) medium (commercially available from e.g. ThermoFisher Scientific, Paisley, UK).
  • RPMI Roswell Park Memorial Institute
  • antigen stimulation refers to the exposure of T cell receptors on the surface of T cells to their respective antigen or antigen fragments.
  • peerated antigen stimulation refers to antigen stimulation during multiple days, preferably at least 5 days, more preferably 5 to 10 days, more preferably 5 to 8 days, more preferably 5 to 7 days.
  • Antigen are for instance added to the culture medium once a day for the indicated number of days.
  • Antigens or antigen fragments should here be understood as molecules
  • the cells are culture in a medium comprising at least IL-7 and/or IL-15.
  • IL-7 and IL-15 refer to two cytokines.
  • IL-7, or interleukin- 7 is a growth factor for both T- and B-cells in an early lymphoid stage.
  • IL-15, or interleukin- 15 comprises multiple functions, including the proliferation of T cells.
  • IL-7 and IL-15 can be obtained from any source, preferably from mice. Most preferably, murine IL-7 and IL-15 can be commercially obtained from Peprotech (London, UK), catalogue numbers 210-07 and 210-15, respectively.
  • treatment “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • genetic modification refers to the direct manipulation of an organism’s genes. It comprises a subset of techniques, including gene delivery, mutagenesis and gene-editing.
  • Gene editing refers to the process of changing the genetic information present in the genome of a cell. This gene-editing may be performed by
  • Gene editing may e.g. be achieved by using CRISPR-Cas technology or zinc finger nuclease technology.
  • albumins refers to proteins with a spherical-like geometry and includes serum globulins and albumins.
  • Albumins and their fragments are very suitable as antigen- specific peptide.
  • Albumins are water- soluble proteins and are found in all animals, including humans.
  • the albumin or its fragment can be obtained from any source, preferably chicken. More preferably, a subset of albumins such as ovalbumins, or fragments thereof, are used.
  • the ovalbumin or its fragments can be obtained from any source, preferably from chickens. More preferably, an ovalbumin fragment is a peptide comprising an epitope recognizable by major histocompatibility (MHC) molecules, e.g.
  • MHC major histocompatibility
  • OVA257-264 peptide is a class I (Kb)-restricted peptide epitope of ovalbumin, and is presented by the class I MHC molecule, H-2Kb.
  • the OVA257-264 peptide has the following amino acid sequence: N-terminal-SIINFEKL, with a molecular mass of 963.2 kDa and may be commercially obtained from Eurogentec (Liege, Belgium).
  • fragment should be understood to comprise any subset of amino acids of a protein with a length of at least three amino acids, preferably at least five amino acids, which comprise an antigen function. It is also intended that a fragment according to the invention can include conservative or non-conservative amino acid substitutions that do not substantially alter its biologic activity.
  • the standard of exhaustion is set by restimulating T cells for 6h and determining their ability to produce cytokines and most importantly the ability of individual T cells to produce multiple cytokines simultaneously.
  • the upregulation of inhibitory receptors, alone, is not sufficient evidence for exhaustion as these receptors are upregulated by T cell activation in the absence of exhaustion.
  • M.Y. Balvic et al. have failed to provide critical evidence of exhaustion.
  • the in vitro generated T cells are not described as viable, i.e. preventing them from being cleared from the medium, thus reducing their applicability in in vitro experiments. It can thus not be concluded that partial or complete T cell exhaustion has been reached in the study of M.Y. Balkhi et al..
  • the current inventors now found that chronic antigen stimulation alone in the presence of the cytokines IL-7 and/or IL-15 results in the generation of in vitro viable, exhausted T cells.
  • the invention thus relates to a method for generating in vitro viable, exhausted T cells.
  • T cells that could be subject to T cell exhaustion include CD4 + T cells and naive T cells, or any other T cell known to the person skilled in the art.
  • the T cells are CD8 + T cells.
  • the cells may be cultured in precence of antigen presenting cells.
  • CD8 + T cells do not need antigen presenting cells to present peptides as they recognize peptides presented by major histocompatibility complex (MHC) I molecules which are found on the cell surface of all nucleated cells, including CD8 + T cells itself.
  • MHC major histocompatibility complex
  • the T cells are CD4 + T cells.
  • the repeated antigen stimulation is preferably in the presence of antigen presenting cells.
  • antigen presenting cells are macrophages, dendritic cells, langerhans cells, and B- lymphocytes but any type of cells known in the art to be capable of presenting antigens can be used, as well as combinations of different antigen presenting cells.
  • the antigen presenting cells and antigen can be added separately to the T cell culture. Alternatively, the antigen presenting cells are first incubated with the antigen and the antigen presenting cells are
  • One of the embodiments is the in vitro generation of exhausted cells, comprising culturing of T cells in a medium comprising at least IL-7 and/or IL-15, and repeated antigen stimulation of said T cells to exhaustion.
  • the concentration of T cells in culture can be varied.
  • an optimal cell density or concentration of T cells is 10 ⁇ 5 cells/mL to 2-3 *10 ⁇ 6 cells/mL, more preferably 0.5* 10 ⁇ 6 cells/mL to 10 ⁇ 6 cells/mL (see tools. thermofisher. com/content/sfs/manuals/t-cell-activation-in-vitro. pdf).
  • T cells can be harvested from the spleen, lymph nodes or blood of any mammal such as a human, more preferably from a human or a rodent, most preferably a mouse.
  • the cells are not limited to primary T cells but also cell lines are suitable, for example human or rodent, e.g. mouse, T cell lines.
  • T cells of any mouse for which a T cell antigen is available can be harvested.
  • an OT-I mouse TCR transgenic ovalbumin specific CD8 + T-cell mouse; Hogquist et al., 1994. Cell 76:17-27
  • OT-I mouse TCR transgenic ovalbumin specific CD8 + T-cell mouse; Hogquist et al., 1994. Cell 76:17-27
  • the cell medium applied for culturing the T cells should at least contain the minimum: a buffer system, protein, trace elements, vitamins, inorganic salts, and energy sources, as is known to a person skilled in the art. Suitable media are known to a person skilled in the art.
  • RPMI 1640 is used as medium.
  • IL-7 and/or IL-15 are supplemented to the medium.
  • both IL-7 and IL-15 are supplemented. If only one of IL-7 or IL-15 is provided, it is supplemented to the medium in a high concentration, preferably 20-100 ng/mL, most preferably 25-80 ng/mL.
  • a lower concentration of cytokines can be applied, such as a IL-7 concentration of 1- 50 ng/mL and a IL-15 concentration of 1-50 ng/mL, more preferably with a IL-7 concentration of 3-25 ng/mL and a IL-15 concentration of 3-25 ng/mL, most preferably a IL-7 concentration of 5-13 ng/mL and a IL-15 concentration of 5-13 ng/mL.
  • antigen stimulation may be provided by antibodies such as anti-CD3 antibodies and anti-CD28 antibodies.
  • antigen stimulation may be provided by antibodies such as anti-CD3 antibodies and anti-CD28 antibodies.
  • the method of the current invention is in no way limited to any of these antigens, other antigens and corresponding T-cell receptors can be found in literature and can be
  • the antigen preferably is an antigen-specific peptide.
  • Said antigen-specific peptide preferably is selected from the group of globular proteins and fragments thereof, preferably ovalbumin and fragments thereof, most preferably OVA257-264 peptide.
  • the concentration of the antigen for repeated antigen stimulation can be varied.
  • the concentration of the antigen can depend on the chosen antigen, and the particular reactivity of the T-cell receptor against the antigen.
  • the concentration of the antigen preferably is from aboutl ng/mL to about 10 mg/mL, more preferably between 5 ng/mL - 5 mg/mL, most preferably between 10 ng/mL - 1 mg/mL.
  • Repeated antigen stimulation is carried out for at least one day, preferably for multiple days, preferably 2 to 15 days, more preferably 3 to 10 days, most preferably 5 days. It is known to the person skilled in the art, that the cells preferably are checked daily, antigen is refreshed daily. Feeding with fresh media and splitting should be implemented as necessary. Repeated antigen stimulation should be understood in such a way that the chosen antigen specific for the T-cell receptor is added repeatedly, preferably once daily or once every other day, to the medium of the cells.
  • T cells should be understood to be exhausted when three conditions are met:
  • the concentration of IL-2, IFN g and/or TNFa may be measured by any technique known in the art, for example ELISA, antibody arrays, bead-based assays, Western blots. Said technique preferably comprises a fluorescence assay.
  • Said technique preferably comprises a fluorescence assay.
  • an indirect sandwich enzyme-linked immunosorbent assay ELISA
  • antibodies interact with the antigens present in the supernatant, followed by enzyme- or fluorescently labeled antibody detection via a colorimetric or fluorescent assay.
  • Signal is measured via optical density or fluorescence and compared to a standard to determine the concentration of IL-2, IFN g and/or TNFa. Results can be compared between different levels of exhausted T cells.
  • cells are stimulated for 6h with antibody or peptide, in the presence of a Golgi plug and intracellular cytokine accumulation can be detected with fluorescently labeled antibodies against IL-2, IFN g and/or TNFa.
  • Cell may be analyzed on a flow cytometer and the simultaneous production of intracellular cytokines can be measured by measuring fluorescence emission at different wavelengths.
  • cells are harvested, washed and resuspended in a buffer.
  • the cells can then be fixed, for example with chilled ethanol and incubated at -20°C.
  • the cells are again washed and a fluorescently tagged anti-nuclear proliferation antibody, such as for example an anti-Ki67 antobody and/or an anti-proliferating cell nuclear antigen (PCNA) antibody, is added.
  • a fluorescently tagged anti-nuclear proliferation antibody such as for example an anti-Ki67 antobody and/or an anti-proliferating cell nuclear antigen (PCNA) antibody
  • PCNA anti-proliferating cell nuclear antigen
  • cells are transferred into an animal, preferably a rodent.
  • the animal can be vaccinated with a protein or peptides that are recognized by the T cells.
  • animals can be vaccinated or infected with a vector with a DNA or RNA sequence that encodes for the cognate antigen of the T cell, such as a viral vector, can be introduced after a incubation period, for example after 3 hours.
  • donor cells can be counted with any cell counter, automatically or manually, known to a person skilled in the art. Proliferating T cells will result in a higher number of donor cells, when compared to exhausted, non proliferating T cells.
  • One of the embodiments of this invention is a method for testing treatment to prevent, alleviate or accelerate exhaustion of T cells, comprising culturing T cells in a medium comprising at least IL-7 and/or IL-15, and repeated antigen stimulation of said T cells to generate exhausted T cells, wherein said T cells are treated before, during and/or after the culturing and/or the stimulation.
  • a medium comprising at least IL-7 and/or IL-15
  • repeated antigen stimulation of said T cells to generate exhausted T cells, wherein said T cells are treated before, during and/or after the culturing and/or the stimulation.
  • T cells are treated by administration of a chemical compound, preferably a small molecule, a peptide, a protein (including antibodies), a nucleotide and/or a carbohydrate, more preferably a peptide or a protein
  • a chemical compound preferably a small molecule, a peptide, a protein (including antibodies), a nucleotide and/or a carbohydrate, more preferably a peptide or a protein
  • T cells are harvested from an animal, preferably a rodent, and cultured with IL-7 and/or IL-15, an antigen which can induce repeated antigen stimulation, and a chemical compound that is to be tested.
  • the T cells are cultured over multiple days, preferably while daily adding or refreshing the antigen and the tested chemical compound.
  • the cell medium with IL-7 and/or IL-15 can be refreshed when necessary.
  • the level of exhaustion of the T cells can be evaluated, together with cell survivability.
  • Cell survivability can for example be evaluated with a live/death assay. The latter one may also be used to evaluate the toxicity of the tested chemical compound. If a notable difference in exhaustion levels is detected while cell survivability is not altered, a chemical compound could be considered of particular interest for the treatment of cancer and/or chronic infections, especially as an additional treatment next to an anticancer treatment, antiviral or antibacterial treatment.
  • Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme. Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the crRNA. The target specificity of Cas9 stems from the crRNA:DNA complementarity and not on modifications to the protein itself (like TALENs and Zinc-fingers). Hence, engineering Cas9 to target non- self DNA is straightforward. While native Cas9 requires a so-called guide RNA composed of the two disparate RNAs that associate to make the guide - the
  • CRISPR RNA crRNA
  • tracrRNA trans-activating RNA
  • T cells preferably are provided in an arrayed format, for example a multi well plate such as a 96 well plate, 192 well plate of 384 well plate.
  • Each well may be provided with a specific CRISPR-Cas combination that targets a specific gene. Screening each well for the ability of the T cells to generate exhausted T cells will result in the identification of one or more genes that play a role in the process of exhaustion of T cells.
  • Zinc finger nuclease Another gene-editing method is Zinc finger nuclease.
  • DNA sequences are specifically targeted with artificial zinc fingers.
  • the sequences are cleaved at a specific site. This can be performed by fusing a zinc finger DNA- binding domain, for recognition of the DNA sequence, to a DNA-cleavage domain, which will be responsible for the DNA cleavage. After cleavage, foreign DNA could be introduced, for example with a plasmid vector.
  • zinc finger nucleases could be generated that are able to introduce a gene of a molecular target.
  • the generated zinc finger nucleases are added to T cells and the DNA is cleaved in order to allow a new gene to enter the DNA of the T cell.
  • the T cells can be exhausted and the exhaustion levels can be evaluated to determine the impact of the introduced gene.
  • gene expression can be altered by knock in experiments employing, for example, DNA or RNA transfection or viral infection for example adenovirus, retroviruses or lentiviruses.
  • DNA or RNA transfection or viral infection for example adenovirus, retroviruses or lentiviruses.
  • viral infection for example adenovirus, retroviruses or lentiviruses.
  • T cells can be transfected or infected and then subjected to the exhaustion method to identify genes that can prevent, stimulate, or revert exhaustion.
  • T cells subjected to in vitro exhaustion can be tested in vitro or in vivo for the restoration of important functional characteristics and the decrease in inhibitory receptor expression.
  • the T cells are evaluated in vivo, comprising the following steps: in vitro generation of exhausted T cells, transfer of the aforementioned in vitro-generated exhausted T cells into an animal, stimulation of the animal with the antigen, and detection of exhaustion markers.
  • the transfer of the in vitro- generated exhausted T cells into an animal is preferably carried out intravenously.
  • live exhausted T cells can be transferred into an animal, preferably a rodent, most preferably a mouse.
  • a treatment such as a small compound, protein, or genetic modification that is identified as preventing, restoring or accelerating exhaustion of T cells in the in vitro methods of the invention, preferably is tested in vivo.
  • animals are infected, for example acute or chronic infection with a bacterium or virus, or animals carrying a tumor that can be targeted by the exhausted T cells, can be tested for their ability to enhance efficacy of the exhausted cells to control or clear said bacterial or viral infections or said tumor.
  • An embodiment of the current invention is a method for identifying molecular targets to prevent, restore or accelerate T cell exhaustion, comprising culturing T cells in a medium comprising at least IL-7 and/or IL-15; repeated antigen stimulation of said T cells to generate exhausted T cells; treating said T cells with a treatment that is able to prevent, restore or accelerate T cell exhaustion; and comparing the treated T cells with untreated exhausted and non-exhausted T cells, to find relevant changes in said T cells.
  • an inhibitor of a specific molecule and/or receptor is added to the T cells in culture, either before, during and/or after inducing T cell exhaustion. Subsequently, the level of exhaustion of the T cells can be evaluated, together with cell survivability. Cell survivability can for example be evaluated with a live/death assay. The latter one is necessary to evaluate the toxicity of the tested chemical compound. If a notable difference in exhaustion levels is detected while cell survivability stays stabile, the specific molecule and/or receptor could be considered of particular interest.
  • Another embodiment of the current invention is a cell culture comprising viable exhausted T cells. Further provided is a cell culture comprising viable exhausted T cells obtained by any method according to the invention. As shown in the Examples, no T cells obtained with a method of the invention make IL-2 and only a small percentage makes TNFa. Nearly all cells (> 95%) express Tox and PD- 1 ((> 95%) and the vast majority (> 80%) expresses multiple inhibitory receptors. Hence, in a preferred embodiment, in the cell culture:
  • - more than 70% of the cells express two or more inhibitory receptors, preferably two or more inhibitory receptors selected from the group consisting of PD-1, Tim-3, TIGIT, LAG 3, CD 160 and CD244,
  • mice on the C57BL6/J background were generated by backcrossing C57BL/6 Tg (TcraTcrb)1100Mjb/J (OT-I) with B6.SJL-Ptprca
  • Pepcb/BoyJ mice both from the Jackson Laboratory. C57BL/6J mice and OT-I mice were housed in a certified barrier facility at Erasmus University Medical Center. Animal work was performed under Project Proposal
  • mice 8-12 weeks old Female mice were anesthetized with 2.5% isoflurane gas and were infected intranasally with Influenza virus strain A/WSN/33-expressing OVA(257-264) (WSN-OVA, a gift from D. Topham, University of Rochester Medical Center).
  • WSN-OVA Influenza virus strain A/WSN/33-expressing OVA(257-264)
  • spleen was harvested from OT-1 mouse and the tissue was processed to get single cell suspension. Then a CD8 purification kit (EasySep; Stemcell Technologies) was used to isolate CD8+ T cells from these splenocytes. 0.5 *10 ⁇ 6 cells/ml of the Purified CD8+ T cells were cultured in complete media presented with cytokines IL-15 (5ng/ml, Peprotech, Cat 210-15) and IL-7 (5ng/ml, Peprotech, Cat 210-07) with or without lOng/ml OVA257- 264 peptide (Anaspec, Cat AS-60193).
  • cytokines IL-15 5ng/ml, Peprotech, Cat 210-15
  • IL-7 5ng/ml, Peprotech, Cat 210-07
  • peptide stimulation For single peptide stimulation, cells were cultured in the presence of a peptide for 48 hours, before the peptide was removed by washing the cells two times with 10% RPMI medium. For the remaining 3 days, the single peptide stimulated cells were cultured in the media with cytokines. For repeated peptide stimulation, lOng/ml OVA257-264 peptide was added daily for five days. Repeated peptide stimulated cells were washed on day 2 to allow for comparable culture conditions. No peptide stimulation control cells were cultivated in media with cytokines but without adding a peptide. Cells from all these three conditions were checked daily, feeding with fresh media or splitting were implemented as necessary.
  • DAPI Viability dye (Beckman Coulter, Cat B30437) and Acridine Orange (Biotium, Cat 40039) were used to dye the cell to distinguish live and dead cells.
  • AE17 cells were maintained in RPMI 1640 supplemented with 10% FBS (Gibco), 100 units/mL Penicillin/Streptomycin (Life Technologies), 2 mM L-glutamine (Life Technologies), 0.05 mM 2-mercaptoethanol (Sigma), and were cultured at 37°C in 5% CO2.
  • AE17 cells were pulsed with 1 mg/ml OVA(257-264) Peptide (Anaspec Cat AS-60193) for 1 hour and then the cells were washed thoroughly before they were labelled with the CellTraceTM Far Red fluorescent dye (ThermoFisher Scientific Cat C34564/15396613). Un-pulsed cells were not labeled.
  • a 1:1 mix of peptide pulsed and un-pulsed AE17 cells (105 each) were mixed and different amounts of T cells (Effector: Target ratios: 3:1, 1:1, 0.3:1) were added. The cells were harvested after 16 hours, the ratio of labeled and unlabeled tumor cell were detected by flow cytometry.
  • inhibitory receptors and ligand antibodies were: CD8-ef450 (53-6.7, eBioscience); Lag3-APC (C9B7W, BD); PD-l-APC-Cy7 (19F.1A12, Biolegend); CD244-PE (2B4, BD; eBio244F4, eBioscience); Tim3-PE- Cy7 (RMT3-23, Invitrogen); CD160-CF594 (CNX46-3, BD); TIGIT-FITC (GIGD7, eBioscience); PD-L1-BV711 (MIH5, BD).
  • CD8 function differentiation and development related markers were: CD44-BV785 (IM7, BD) ; CD25-APC-Cy7 (PC61, BD); CD127-PerCP-Cy5.5 (A7R34, Biolegend); KLRG1-PE-Dazzle594 (2F1, Biolegend); CD28-FITC (E18, Biolegend). Intracellular expression of transcription factors was determined with Tbet-PE-Cy7 (4B10, Biolegend); TCF1-APC (C63D9, Cell Signaling); EOMES-PE-eF610 (Danllmag, eBioscience); Tox-PE (TXRX10, eBioscience). To exclude dead cells, Annexin V (APC, BD; Cy5.5, BD; PerCP-Cy5.5, BD) stain was included in all the stains and 2.5 mM CaCl 2 was added to all solutions.
  • Annexin V APC, BD; Cy5.5, BD; PerCP-Cy5.5, BD
  • cells were harvested and immediately stained for surface and intracellular antigens.
  • FACS wash 3% FBS in Hanks' balanced salt solution (HBSS) + 2.5 mM CaCl 2 and incubated with 20 mL mix of the pre-determined optimal concentrations of the fluorochrome-conjugated monoclonal antibodies on ice in the dark for 20 minutes.
  • the cells were again washed one time with FACS wash and fixed with 1% PFA + 2.5 mM CaC12.
  • the cells were first stained for surface antigens as described above.
  • anti-EOMES-PE-eF610 (Danllmag, eBioscience); anti-Tox-PE
  • TXRX10 TXRX10, eBioscience
  • Appropriate isotype controls were included for staining of transcription factors.
  • Cytokines were measured with anti- IFN- U -APC (XMG1.2, eBioscience); anti-TNF-a-AF488 (MP6-XT22, eBioscience); anti-IL-2-PE (JES6-5H4, eBioscience); anti-GranzymB-PE-Cy7 (NGZB,
  • in vitro generated cells were adoptively transferred into wild type mice.
  • CD45.1+ OT-I cells from in vitro cultured cells that were first were sorted on a FACSAria III (BD
  • lungs were digested for 2 h at 37°C with 3.0 mg/ml collagenase A and 0.15 mg/ml DNase I (Roche) in RPMI 1640 containing 5% heat-inactivated FBS, 2 mM 1-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin.
  • Digested lung was then run through a 40-pm cell strainer (Falcon ) and washed in the same media as above.
  • PE-conjugated tetramers of H-2b major histocompatibility complex class I loaded with OVA(257-264) was used to identified the antigen-specific CTLs in the lung.
  • Part of the lung tissue was frozen in TRIzol LS reagent (Life Technologies), RNA was isolated (described below) and the viral load was determined by qPCR.
  • RNA sequencing To compare the transcription expression level among those cells, RNA sequencing was performed. On day 5, 1*10 ⁇ 6 live CD8 + cells were sorted from the three different cultured cells and immediately lysed with TRIzol LS reagent (Life Technologies) and stored at -80°C. RNA was extracted according to manufacture’s instructions and a bioanalyzer (Agilent) was used to determine the integrity of the extracted RNA. Barcoded sequencing libraries were generated using a KAPA RNA Hyper+RiboErase HMR (Roche Diagnostics). Library quality was assessed with the bioanalyzer and Kapa qPCR was performed before cluster generation and 100-bp paired-end sequencing. Libraries were sequenced with HTSeq2000 Illumina technology.
  • Count data was preprocessed to remove the very low expression genes, then, an rlog transformation (DESeq2 R package (vl.22.2) was performed with the clean count files. This transformation was exclusively for visualization and comparison purpose which included correlation and clustering analysis of the data (heatmaps and PCA plots). For differential expression analysis DESeq2 package was used in the raw data (not rlog transformed).
  • IPA Ingenuity Pathway analysis
  • Qiagen Qiagen, USA version
  • Pathway enrichment P values Fisher’s exact test
  • activation Z-scores were calculated by IPA and used to rank the significant pathways.
  • GSEA gene set enrichment analysis
  • DNA methylation profiling detection DNA methylation profiling detection. DNA methylation profiling was done as previously described by the MeD-seq method. For MeD-seq sample preparation LpnPI (New England Biolabs) digestions were carried out on DNA samples according to manufacturer’s protocol. Reactions contained 50 ng and digestion took place overnight in the absence of enzyme activators. Digests of genomic DNA with LpnPI resulted in snippets of 32 bp around the fully- methylated recognition site that contains CpG. The DNA concentration was determined by the Quant-iTTM High- Sensitivity assay (Life Technologies) and 50 ng dsDNA was prepared using the ThruPlex DNA-seq 96D kit (Takara).
  • MeD-seq data processing was carried out using specifically created scripts in Python version 2.7.5.
  • Raw fastq files were subjected to Illumina adaptor trimming and reads were filtered based on LpnPI restriction site occurrence between 13-17 bp from either 5’ or 3’ end of the read.
  • Reads that passed the filter were mapped to mmlO using bowtie2.1.0. Multiple and unique mapped reads were used to assign read count scores to each individual LpnPI site in the mmlO genome.
  • BAM files were generated using SAMtools for visualization.
  • MeD-seq data analysis was carried out in Python 2.7.5. DMR detection was performed between two datasets containing the regions of interest (TSS, genebody or CpG islands) using the Chi-Squared test on read counts. Significance was called by either Bonferroni or FDR using the Benjamini-Hochberg procedure. Differently methylated regions were used for unsupervised hierarchical clustering, the Z-score of the read counts was used for normalization and is also shown in the heatmaps.
  • a genome wide sliding window was used to detect sequentially differentially methylated LpnPI sites. Statistical significance was called between LpnPI sites in predetermined groups using the Chi-squared test. Neighbouring significantly called LpnPI sites were binned and reported, DMR threshold was set at a minimum of ten LpnPI sites, a minimum size of 100 bp and either a twofold or fivefold change in read counts. Overlap of genome wide detected DMRs was reported for TSS, CpG island and gene body regions
  • CD8+ T cells purified from OT-I mice were stimulated daily for five days with 10ng/ml OVA(257-264) peptide in the presence of IL-15 and IL-7 (Repeat peptide stimulation, Figure 1A).
  • IL-15 and IL-7 Repeat peptide stimulation
  • cells were either left unstimulated or stimulated only once with OVA(257-264) peptide for 48 hours and then washed and cultured without peptide for an additional 3 days. All cells were cultured with IL-7 and IL-15. On day 5, cells were either harvested for analysis or in some instances cells were cultured an additional 3 days in the presence of IL-7 and IL-15 without any additional peptide stimulation ( Figure 1A).
  • the total amount of viable exhausted cells yielded in the repeated peptide stimulated cultures was comparable for cells cultured with IL-7 or IL-15 on their own and cells cultured with a combination of both cytokines. However, the combination of both IL-7 and IL-15 yielded more viable exhausted cells in the single peptide stimulation cultures
  • Degranulation is an important step for the CD8+ T cell cytotoxicity.
  • CD 107a and Granzyme B (GzmB) expression were analyzed.
  • cells either unstimulated or single peptide stimulated were negative for CD107a and GzmB ( Figure 3A and 3B), while repeat peptide stimulated cells showed a significantly higher level of both markers ( Figure 3A and 3B).
  • OVA(257-264) peptide cells cultivated in the presence of no peptide and single peptide stimulated cells upregulated CD107a ( Figure 3A).
  • inhibitory receptors upregulated after repeated peptide stimulation Having demonstrated that repeated peptide stimulated CD8 + T cells were deficient in cytokine production upon re-stimulation, we sought to determine the inhibitory receptors expressing on these cells. Normally, an increase in inhibitory receptor expression means that the cells are more exhausted and there are more inhibitory pathways that can be researched with this in vitro model. After harvesting the cells on day five, surface staining was used to measure the inhibitory receptors expressed on the differently treated cells. As expected, inhibitory receptors were barely expressed on the cells without peptide stimulation and single peptide stimulated cells (Figure 4A). Unsurprisingly, roughly 20 percent of the cells were PD-1 positive cells in the single peptide stimulated cells.
  • Tcfl T cell factor- 1
  • Tox Thymocyte selection-associated HMG box protein
  • T-box transcription factor 21 T-bet
  • EOMES Eomesodermin
  • T-bet was highly upregulated, while there was no detectable difference of EOMES expression from single peptide stimulated cells ( Figure 5). The increased Tox and T-bet
  • mice that received the multiple times peptide stimulated cells possessed larger amount of endogenous OVA-specific CD8 + T cells in their lungs than the mice that received no peptide treated cells or single time peptide stimulated cells (0.63*10 ⁇ 6 versus 0.13*10 ⁇ 6 and 0.22*10 ⁇ 6), indicating that repeatedly stimulated cells were less fit to compete with the endogenous CD8+ T cell response ( Figure 6c and d).
  • the genes for the markers of the terminally differentiated effector cells were also expressed significantly higher in the repeated peptide stimulated cells.
  • the transcription factors EOMES and TCF7 were significantly downregulated in the repeated peptide stimulated cells, while Tox was found to be three times more expressed in these cells than in single peptide stimulated cells ( Figure 7B).
  • the above changes of single gene expression detected by flow cytometry were in line with the findings of RNA level modification.
  • transcription factors, which are associated with CTL exhaustion such as IRF4, NR4a and Batf were also upregulated in the repeat peptide stimulated cells in comparison to single peptide and no peptide stimulated cells ( Figure 7B).

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Abstract

L'invention concerne des procédés de génération in vitro rapide de des lymphocytes T épuisés viables, consistant à cultiver des lymphocytes T dans un milieu comprenant au moins IL-7 et/ou IL-15, et à réaliser une stimulation d'antigène répétée desdits lymphocytes T pour générer rapidement des lymphocytes T épuisés. L'invention concerne en outre un procédé de traitement desdits lymphocytes T pour prévenir, soulager ou accélérer la génération de lymphocytes T épuisés. En outre, la présente invention concerne une culture cellulaire comprenant des lymphocytes T épuisés viables.
PCT/NL2020/050446 2019-07-08 2020-07-08 Épuisement rapide in vitro de lymphocytes t WO2021006733A1 (fr)

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