WO2019014684A1 - Expansion de cellules immunitaires avec des composés inhibant la kinase des lymphocytes t inductibles par l'interleukine 2 - Google Patents

Expansion de cellules immunitaires avec des composés inhibant la kinase des lymphocytes t inductibles par l'interleukine 2 Download PDF

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WO2019014684A1
WO2019014684A1 PCT/US2018/042349 US2018042349W WO2019014684A1 WO 2019014684 A1 WO2019014684 A1 WO 2019014684A1 US 2018042349 W US2018042349 W US 2018042349W WO 2019014684 A1 WO2019014684 A1 WO 2019014684A1
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cells
cell
ibrutinib
subject
itk
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PCT/US2018/042349
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John Byrd
Meixiao LONG
Natarajan Muthusamy
Michael Caligiuri
Bethany MUNDY-BOSSE
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Ohio State Innovation Foundation
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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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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]
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • CLL Chronic lymphocytic leukemia
  • BlOpro regulatory B (Breg) cell types that produce IL-10, a major immunosuppressive cytokine.
  • CLL cells can also increase the number of regulatory T (Treg) cells that diminish cellular immune responses.
  • CLL cells can induce IDO M CD14 + HLA-DR l0 myeloid-derived suppressor cells (MDSC), which inhibit T-cell responses both directly and indirectly through promoting Treg induction and expansion.
  • MDSC HLA-DR l0 myeloid-derived suppressor cells
  • CLL T cell subsets are skewed toward a terminally differentiated phenotype, with significant reduction in naive T cells and expansion of effector memory/effector T cells.
  • CLL patient T-cells also demonstrate features of pseudo-exhaustion, with significant up- regulation of checkpoint molecules and exhaustion markers such as PD-1, CTLA-4, CD 160, CD244 and CD57. These phenotypic changes are closely associated with profound functional defects including reduced cytotoxic capacity of CD8 T cells and defective immunologic synapse formation.
  • a shift in the balance between Thl, Th2 and Thl7 immune subsets in CLL patients can also be an important factor in driving disease progression, and the T cell response in CLL patients is skewed toward the Th2 polarization.
  • Studies in CLL patients have also shown that a decrease in IL-17-producing T cells is associated with Treg expansion and disease progression, while increased Thl7 cell numbers correlate with improved overall survival.
  • success in therapeutically enhancing cell-mediated immunity in CLL has been limited.
  • Lenalidomide has favorable immune modulating properties on T-cells in CLL patients via down-regulation of IKZFl through a cereblon-dependent mechanism, and can promote durable, sustained complete remissions in 50% or more of patients receiving this treatment.
  • lenalidomide in this setting also can produce significant morbidity and sometimes fatal outcome from early-onset tumor flare.
  • New to the field of B-cell cancer therapy are agents that irreversibly target Bruton' s tyrosine kinase (BTK) such as ibrutinib, which yields high response rates and durable remissions in patients with CLL.
  • BTK Bruton' s tyrosine kinase
  • Ibrutinib irreversibly inhibits interleukin-2 inducible T cell kinase (ITK), leading to enhanced Thl response in-vitro and in-vivo.
  • Ibrutinib treatment led to a more than two-fold increase in expansion of Thl pathogen-specific T cells and corresponding suppression of Th2 immunity.
  • Ibrutinib treatment also leads to a shift in macrophages toward a Thl -supportive phenotype and increases CD8 T cell tumor infiltration in a mouse model of pancreatic cancer.
  • BTK Bruton' s tyrosine kinase
  • the expanded T cells are effector memory (CD45RA-CCR7-) T cells or CD45RA+CCR7- T cells.
  • Also disclosed herein are methods of inhibiting activation induced cell death of T cells and/or NK cells in a subject with chronic lymphocytic leukemia comprising administering to the subject an inhibitor of interleukin-2 inducible T cell kinase (ITK).
  • ITK interleukin-2 inducible T cell kinase
  • NK cells comprising contacting NK cells with a stimulatory molecule and ibrutinib; wherein the stimulatory molecule is selected from the group consisting of IL-15, IL-21, IL-2, 41BBL, IL-12, IL-18, MICA, 2B4, LFA-1, and BCM1/SLAMF2.
  • Also disclosed herein are methods of treating cancer in a subject comprising administering to a subject NK cell therapy, wherein the NK cell therapy comprises a) expanding NK cells by stimulating NK cells with a stimulatory molecule ibrutinib; wherein the stimulatory molecule is selected from the group consisting of IL-15, IL-21, IL-2, 41BBL, IL-12, IL-18, MICA, 2B4, LFA-1, and BCM1/SLAMF2; and b)administering to the subject the expanded NK cell population.
  • the stimulatory molecule is selected from the group consisting of IL-15, IL-21, IL-2, 41BBL, IL-12, IL-18, MICA, 2B4, LFA-1, and BCM1/SLAMF2
  • an immune cell such as, for example, a chimeric antigen receptor (CAR) T cell, tumor infiltrating lymphocyte (TIL), or marrow-infiltrating lymphocyte (MIL), natural killer (NK) cell, NK-T cell, a cytokine-induced memory NK cell, or a cytokine-induced killer (CIK) cell
  • an immune cell comprising contacting the isolated immune cell with an effective amount of interleukin-2 inducible T cell kinase (ITK) inhibitor (such as, for example, ibrutinib) to expand the immune cell in an amount effective for immunotherapy; and culturing the isolated immune cells in the presence of the ITK inhibitor (for example, culturing the cells in the presence of the ITK inhibitor.
  • ITK interleukin-2 inducible T cell kinase
  • a tumor cell such as, for example an autologous tumor
  • methods of increasing the cytotoxicity and survival of T cells to a tumor cell comprising contacting CD4 and/or CD8 T cells with an ITK inhibitor (such as, for example ibrutinib).
  • an ITK inhibitor such as, for example ibrutinib
  • contacting a T cell population with an ITK inhibitor comprising contacting a T cell population with an ITK inhibitor.
  • checkpoint inhibition such as, for example, checkpoint inhibition by immunosuppressive ligands such as PD-1/PD-L1, CTLA-4 and/or CD200
  • ITK inhibitor such as, for example, ibrutnib
  • Figures 1 A and IB show that ibrutinib but not acalabrutinib treatment of CLL patients increases total T cell numbers:
  • Cycle 3 indicates samples obtained after two cycles (8 weeks into treatment), and "cycle 6” indicates samples obtained after 5 cycles (20 weeks into treatment).
  • T cells were differentiated into subsets based on expression of CCR7 and CD45RA: naive T cells (CCR7+CD45RA+), central memory T cells (CCR7+CD45RA-), effector memory T cells (CCR7-CD45RA-), and more differentiated effector memory T cells (T- EMRA; CCR7-CD45RA+).
  • Figures 2 A and 2B show the effect of ibrutinib or acalabrutinib treatment on the frequency of different subsets of peripheral T cells.
  • T cells are differentiated into subsets based on their expression of CCR7 and CD45RA: naive T cells (CCR7+CD45RA+), central memory T cells
  • CCR7+CD45RA- effector memory T cells
  • CCR7-CD45RA- effector memory T cells
  • T-EMRA most differentiated effector memory T cells
  • Figures 3A, 3B, 3C, 3D, and 3E show that ibrutinib treatment of human T cells or
  • NK cells protects against activation induced cell death in a dose dependent manor.
  • Figures 3 A, 3B, and 3C show that T cells were isolated from healthy human donors blood samples, stimulated in vitro with CD3/CD28 for 3 days, rested in culture medium containing 50 R7 IL-2 for 1 1 days, then were restimulated with plate bound CD3 for 6 hours (as in 3 A. and 3B.) or 3 hours (as in 3C.) in the presence of IL2 to induce activation induced cell death in the presence of absence of ibrutinib. Each indicated condition.
  • Figure 3 A shows a representative FACS dot plots of Annexin V & PI staining.
  • Figure 3B shows a Bar graphs that show the percentage of nonviable was done in triplicate. Data shown are representative of three independent experiments (apoptotic + necrotic, as defined by Annexin V positive and PI positive cells) cells after induction of AICD.
  • Figure 3C shows a FAS-L mRNA upregulation in activated T cells upon induction of AICD was impaired by ibrutinib treatment. mRNA was isolated from the T cells after induction of AICD, cDNA was synthesized and qPCR for FAS-L and GAPDH was done.
  • Figure A, B and C represent 3 independent experiments.
  • Figures 4A, 4B, and 4C show that ibrutinib treatment does not affect total numbers of stem memory T cells:
  • Figure 4A shows the gating strategy for stem memory T cells.
  • Naive CD4 T cells (CCR7+CD45RA+) expressing CD62L were selected for further analysis. Of these, CD95+CD122hi cells were defined as stem memory T cells.
  • Figure 4B left shows representative plots showing stem memory T cells in samples from a healthy donor and a CLL patient.
  • Figure 4B right shows representative plots showing Tbet and Eomesodermin expression in naive CD8 and CD4 T cells from a healthy donor and a CLL patient.
  • FIGS. 6A and 6B show that treatment with ibrutinib, as well as with acalabrutinib, leads to a significant reduction in the frequency of PD-1 positive cells in CD4 T cell
  • FIG. 7A and 7B show that treatment with BTK inhibitors significantly reduces the frequency of T cells expressing intracellular CTLA4: Percentages of cells positive for intracellular CTLA4 expression among total CD4 T cells, CD45RA- CD4 T cells, and
  • Figures 8A and 8B show that treatment with ibrutinib, as well as with acalabrutinib, leads to a significant reduction in the frequency of intracellular CTLA4 positive cells in CD8 T cell populations.
  • FIGS 10A, 10B, and I OC show that Ibrutinib treatment of CLL patients leads to a reduced frequency, but not reduced absolute number, of CD4+CD25+ Foxp3+ Treg cells:
  • Fgiure 10A shows representative plots showing CD25 and intranuclear Foxp3 staining in CD4+CD3+ (upper panel) and CD8+CD3+ (lower panel) T cells.
  • MFI mean fluorescence intensity
  • Figures 12A, 12B, and 12C show the ability of CLL cells to produce IL-10 is impaired with BTK inhibitor treatment: PBMC from CLL patients were collected before and during treatment with ibrutinib (12A, 12B, 12C). Cells were stimulated in-vitro with CpG and PMA/ionomycin for 5 hours (B IO conditions) or with CpG/CD40L for 48 hours, with
  • FIG. 12A shows representative flow cytometry plots of IL-10 expression in CLL cells under B IO conditions (top) and B lOPro conditions (bottom).
  • Figure 12C shows that PBMC were collected from CLL patients at baseline and at the beginning of cycles 3 and 6 (8 and 20 weeks, respectively) after starting acalabrutinib treatment.
  • FIG. 29 Figure 13 shows that ibrutinib treatment increases the number of activated leukemia specific T cells.
  • Mice were engrafted with AML cell line (C1498) expressing OVA (a model antigen).
  • OVA a model antigen
  • Figure 15 shows that ibrutinib treatment in CLL patients improves their T cells' capability to mediated cytotoxicity against autologous CLL cells in the presence of
  • CLL patent's T cells collected pre-ibrutinib vs. post-ibrutinib treatment
  • autologous CLL cells pre-ibrutinib
  • Percentage of live CLL cells at the end of the assay were shown in the left lower corner of each plot. Data shown are representative of three independent experiments.
  • FIG. 32 Figure 16 shows that ibrutinib rescues CLL patient's T cells from AICD triggered by blinatumomab.
  • CLL patent's T cells collected pre-ibrutinib vs. post-ibrutinib treatment
  • autologous CLL cells pre-ibrutinib
  • Upper panel shows the number of viable CLL cells at the end of the assay.
  • Lower panel shows the number of the viable T cells at the end of the assay.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • the term "subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician. 39.
  • the term "therapeutically effective" refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • An "increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant. 44.
  • various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
  • Immune cell therapy (such as NK cell therapy or T cell therapies including chimeric antigen receptor (CAR) T cell therapy and transfer of tumor infiltrating lymphocytes (TIL), or marrow-infiltrating lymphocyte (MIL)) has significant potential as a cancer therapy because immune cells can expand in large numbers to eradicate high volume disease, can traffic throughout disparate areas of the body to eradicate residual tumor sites, and can endow patients with long-lived tumor immunity.
  • major disadvantages that limit the utility of adoptive immune cell therapy include the MHC restriction of antigen presentation to T cell receptors (TCR), MHC downregulation as a mechanism of immune escape, and the lengthy production time required to create a sufficient number of immune cells (including NK cells and tumor- specific T cells).
  • T-cells isolated from CLL patients prior to starting ibrutinib expand ex vivo chimeric antigen receptor (CAR) T-cells poorly, whereas those derived during treatment expand significantly better.
  • CAR chimeric antigen receptor
  • addition of ibrutinib to anti-CD 19 CAR T Cells improves responses against mantle cell lymphoma.
  • ibrutinib enhances T cell-dependent antitumor immune responses and further potentiates the efficacy of immune checkpoint blockade.
  • Immune modulatory effects have been preliminarily reported with the BTK inhibitor acalabrutinib (ACP-196), which also demonstrates promising clinical activity in CLL.
  • acalabrutinib lacks inhibitory activity against the BTK-related kinase ITK. Clinically, this raises the question of whether more selective BTK inhibition will promote effective immune modulation and avoid the off-target effects observed with ibrutinib.
  • ibrutinib in one aspect, disclosed are methods and compositions related to expanding T cell and NK cell populations by contacting said cells with an interleukin-2 inducible T cell inhibitor.
  • the effects of ibrutinib on T-cells were comprehensively studied in-vivo and the ability of this agent to modulate the immune suppressive capacity of CLL cells, and compare the results to those achieved with acalabrutinib.
  • the results indicate that while both agents diminish tumor- mediated immune suppressive molecules, ibrutinib has unique immune modulating capability in promoting expansion of chronically activated T-cells by diminishing activation-induced cell death.
  • this expansion was not extended to Treg (CD25+Foxp3+) cells.
  • CD8 and CD4 T cells such as, for example, effector memory (CD45RA-CCR7-) T cells or CD45RA+CCR7- T cells
  • CD25+Foxp3+ T cells in a subject comprising administering to a subject an agent that inhibits interleukin-2 inducible T cell kinase (ITK) (such as, for example ibrutinib).
  • ITK interleukin-2 inducible T cell kinase
  • the immune cells can be expanded in the presence of the ITK inhibitor for any amount of time sufficient to generate a therapeutically effective amount of immune cells for the adoptive immune cell therapy.
  • methods of expanding immune cells such as, for example CD4 T cells, CD8 T cells, and/or NK cells wherein the immune cells are incubated with the ITK inhibitor for at least 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 32, 34, 36, 48 hours, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, or 14 days.
  • the expansion of immune cells can occur ex vivo, in situ, or in vitro.
  • the effect of the ITK inhibitor on immune cells can also occur in vivo.
  • disclosed herein are methods of expanding immune cells (such as, for example CD4 T cells, CD8 T cells, and/or NK cells) wherein the ITK inhibitor (such as, for example, ibrutinib) is administered to the subject.
  • This method can not only expand endogenous CD4, CD8, and/or NK cells, but will also work on endogenous TILs, MILs, or adoptively transferred TILs, MILs, and/or CAR T cells.
  • IL-2 inducible T cell kinase INK
  • ITK inhibitors including, but not limited to broadly reactive BTK inhibitors that are also ITK inhibitors
  • the ITK inhibitor can be used to maximize cells being used for immune cell therapy for a cancer.
  • an immune cell such as, for example, a chimeric antigen receptor (CAR) T cell, tumor infiltrating lymphocyte (TIL), or marrow-infiltrating lymphocyte (MIL), natural killer (NK) cell, NK-T cell, a cytokine- induced memory NK cell, or a cytokine-induced killer (CIK) cell
  • an immune cell comprising contacting the isolated immune cell with an effective amount of interleukin-2 inducible T cell kinase (ITK) inhibitor (such as, for example, ibrutinib) to expand the immune cell in an amount effective for immunotherapy; and culturing the isolated immune cells in the presence of the ITK inhibitor (for example, culturing the cells in the presence of the ITK inhibitor.
  • ITK interleukin-2 inducible T cell kinase
  • ibrutinib is an ITK inhibitor and an inhibitor of Bruton's tyrosine kinase (BTK).
  • BTK Bruton's tyrosine kinase
  • ibrutinib inhibits both BTK and ITK, mere inhibition of BTK alone is not sufficient to perform the methods disclosed herein.
  • acalabrutinib (a BTK inhibitor) does not expand T cells.
  • T-EM effector memory T cells
  • T-EMRA CD45RA+ effector memory T cell
  • ITK inhibitors can also have an effect on the cytotoxicity and survival of T cells.
  • a tumor cell such as, for example an autologous tumor
  • an ITK inhibitor such as, for example ibrutinib
  • the contact of the ITK inhibitor with the target T cell can occur in vitro or ex vivo (such as to effect T cells that would be adoptively transferred to subject for a cancer treatment) and/or in vivo (to increase the cytotoxicity and/or survival of endogenous T cells or TILs, MILs, or CAR T cells that have previously been transferred to a subject or are being transferred concurrent with or following administration of the ITK inhibitor).
  • Such methods can further comprise the administration of blinatumomab.
  • an ITK inhibitor can have on the cytotoxicity and survival of T cells
  • the ITK inhibitor (such as, for example, ibrutinib) can also polarize the T cell response to a Thl7 response. Studies have shown that increased Thl7 cell numbers correlate with improved overall survival. Accordingly, in one aspect, disclosed herein are method of increasing the percentage of Thl7 T cells comprising contacting a T cell population with an ITK inhibitor.
  • IL-2 inducible T cell kinase IGF
  • NK cells are typically expanded with cytokine activation, but the expanded NK cells are susceptible to AICD during expansion and after transfer to a subject to be treated.
  • Administration of an ITK can reduce AICD of the expanded NK cells making more available for treatment.
  • NK cells comprising contacting NK cells with a stimulatory molecule and ibrutinib; wherein the stimulatory molecule is a cytokine (such as, for example IL-15, IL-21, IL-2, 41BBL, IL-12, IL-18, MICA, 2B4, LFA-1, and BCM1/SLAMF2).
  • a stimulatory molecule such as, for example IL-15, IL-21, IL-2, 41BBL, IL-12, IL-18, MICA, 2B4, LFA-1, and BCM1/SLAMF2
  • NK cell therapy comprises expanding NK cells (including, but not limited to natural killer (NK) cell, NK-T cell, a cytokine-induced memory NK cell, or a cytokine-induced killer (CIK) cell) by stimulating NK cells with a stimulatory molecule ibrutinib; wherein the stimulatory molecule is a cytokine (such as, for example IL-15, IL-21, IL-2, 41BBL, IL-12, IL-18, MICA, 2B4, LFA-1, and BCM1/SLAMF2); and administering to the subject the expanded NK cell population.
  • a cytokine such as, for example IL-15, IL-21, IL-2, 41BBL, IL-12, IL-18, MICA, 2B4, LFA-1, and BCM1/SLAMF2
  • PD-1 Programmed death-1
  • T cells T cells, B cells and natural killer cells.
  • PD-L1/PD-L2 PD-1 functions by inhibiting an activated T cell response.
  • Tumor cells up-regulate PD-L1 in response to inflammation thereby suppressing an anti-tumor immune response. Similar effects occur via CTL-4 and CD200.
  • immunosuppressive ligands such as PD-1, CD200, and CTLA-4 serve as checkpoint inhibitors to reduce the immune response.
  • the immune response to the cancer can be thwarted.
  • ibrutinib treatment can reduce the percentage of PD-1 positive CD4 and CD8 T cells as well as CTLA-4 CD4 and CD 8 T cells.
  • an ITK inhibitor such as, for example, ibrutnib
  • the disclosed methods of reducing checkpoint inhibition can be used to augment any cancer immune cell therapy, including, but not limited to direct application to the subject for in vivo applications or used in conjunction with MIL, TIL, or CAR T cell therapy and thus, can also be applied to T cells ex vivo or in vitro.
  • the disclosed methods of reducing checkpoint blockade can further comprise the administration of any known immune checkpoint inhibitor, such as for example, a PD-1 inhibitor, a PD-L1 inhibitor, or CTLA-4 inhibitor (such as, for example, nivolumab, pembrolizumab, pidilizumab, BMS-936559, Atezolizumab, Durvalumab, or Avelumab).
  • a PD-1 inhibitor such as for example, a PD-1 inhibitor, a PD-L1 inhibitor, or CTLA-4 inhibitor
  • CTLA-4 inhibitor such as, for example, nivolumab, pembrolizumab, pidilizumab, BMS-936559, Atezolizumab, Durvalumab, or Avelumab.
  • NK cell therapy and/or T cell therapy can also occur in vivo after transfer of expanded cells to the subject with a cancer
  • ITK such as ibrutinib
  • methods of treating cancer of any preceding aspect further comprising administering to the subject ibrutinib prior to, concurrent with, or after administration of the expanded NK cells or T cells to the subject.
  • cancers that the disclosed compositions can be used to treat is the following: leukema (including, but not limited to, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), lymphoma; B cell lymphoma,; T cell lymphoma, mantle cell lymphoma, mycosis fungoides; Hodgkin's Disease; leukemias, including but not limited to myeloid leukemia;
  • leukema including, but not limited to, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), lymphoma
  • B cell lymphoma including, but not limited to, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), lymph
  • cervical cancer cervical carcinoma
  • breast cancer epithelial cancer
  • renal cancer genitourinary cancer
  • pulmonary cancer esophageal carcinoma
  • head and neck carcinoma large bowel cancer
  • hematopoietic cancers testicular cancer
  • colon and rectal cancers prostatic cancer
  • AIDS- related lymphomas or sarcomas metastatic cancers, or cancers in general
  • pancreatic cancer pancreatic cancer
  • NK and/or T cells can be used as one aspect of treatment of a cancer (such as, for example, CLL) and can be used in conjunction with additional anti-cancer agents.
  • Anti-cancer agents that can be used in the disclosed methods can comprise any anti-cancer agent known in the art, the including, but not limited to Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor
  • Daunorubicin Hydrochloride and Cytarabine Liposome Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt
  • Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2
  • Ondansetron Hydrochloride Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab,
  • Panobinostat Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride , Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride,
  • Trifluridine and Tipiracil Hydrochloride Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VelP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta
  • Example 1 Ibrutinib Treatment Improves T-cell Number and Function of CLL in patients
  • Ibrutinib treatment increases numbers of both CD4 and CD8 T cells via a BTK independent mechanism.
  • Both CD4 and CD8 T cells were further categorized into naive (CD45RA+CCR7+), central memory (T-CM; CD45RA-CCR7+), effector memory (T-EM; CD45RA-CCR7-) and CD45RA+ effector memory T cells (T-EMRA; CD45RA+CCR7-) ( Figure 1 A).
  • the T-EMRA subset is considered to consist of more terminally differentiated effector memory/effector T cells.
  • the increase in total cell number was most prominent in the T-EMRA and T-EM compartments, whereas in the naive and T-CM subsets, the increase was more modest and not significant at several time points examined.
  • CD4 T-EMRA cell numbers increased by more than three-fold (from 0.055 to 0.18 ⁇ 10 3 / ⁇ 1) 8 weeks into treatment (beginning of cycle 3), while naive and central memory CD4 T cell numbers increased by about two fold at the same time point (from 0.065 to 0.147 ⁇ 10 3 / ⁇ 1 for naive CD4 T cells).
  • This same pattern coincides with the frequencies of different T-cell subsets, where the proportion of both CD4 and CD8 T- EM cells were increased modestly by cycle 6 of ibrutinib treatment (Figure 2A).
  • the mean proportion of CD4 T-EMRA cells increased from 5.1% to 7.3% (P ⁇ 0.05).
  • T cells numbers after ibrutinib treatment may merely reflect the release of T-cells from the secondary lymphoid organs as opposed to true T-cell expansion.
  • CLL engrafted mice were treated with ibrutinib and monitored peripheral blood T cell numbers before starting ibrutinib, 2 days and 4 days post starting ibrutinib. These time points correspond to the period when CLL cells numbers were transiently increased in peripheral blood post ibrutinib treatment. If ibrutinib causes translocation of T cells from secondary lymphoid organ to peripheral circulation like it does to CLL cells, an increase in T cell numbers was noticed in peripheral blood.
  • ibrutinib can enhance the expansion of activated antigen specific T cells using a mouse leukemia model. As shown in Figure 13, ibrutinib increased the number of tumor-antigen specific T cells in the secondary lymphoid organ (spleen) by approximately two fold.
  • NK cells can also undergo apoptosis following activation via cytokine receptors.
  • Ibrutinib targets such as ITK and BTK also play an important role in NK cell function and signaling processes. Therefore, it was investigated if ibrutinib can also ameliorate "AICD" of NK cells utilizing an established assay system in which NK cells are co- stimulated with IL-2/IL- 12 or IL-15/IL-12.
  • ibrutinib a similar protection by ibrutinib, but not acalabrutinib was found (Figure 3).
  • Ibrutinib treatment does not compromise stem memory T cells localized in the naive T cell compartment.
  • TSCM Next stem memory T cells
  • Eomesodermin are established Thl differentiation markers. As expected, few naive T cells from healthy donors express these markers ( Figure 4B). However, a significant percentage of naive T cells (more than 10% naive CD4 cells) from CLL patients express Tbet or Eomesodermin.
  • naive T cells from CLL patients are not bona fide naive T cells, but rather TSCM cells.
  • CD27+ T-EM and T-EMRA cells showed a statistically significant reduction in PD-1 after ibrutinib treatment (p ⁇ 0.001), while their CD27- counterparts did not ( Figure 6).
  • Ibrutinib enhances Thl polarization in-vitro and in-vivo in murine models.
  • PBMCs were re-stimulated from ibrutinib-treated patients with PMA/ionomycin and assessed their cytokine production profile. As shown in
  • Ibrutinib decreases the T-reg:CD4 ratio but not absolute number of CD25+Foxp3+ Treg cells.
  • Ibrutinib down-regulates immunosuppressive molecules CD200 and BTLA on CLL cells.
  • CLL cells have been reported to express a variety of immunosuppressive ligands; the expression levels of these immunosuppressive molecules was evaluated in CLL cells before and after ibrutinib treatment.
  • the surface expression of PD-L1, HLA-G and CD276 was low in general, and a significant change of their expression in CLL cells was not detected after ibrutinib treatment.
  • BlOPro conditioning is impaired after ibrutinib treatment. 73. It has been reported that CLL cells share phenotypic and functional features with regulatory-B cells and can produce IL-10 after in-vitro stimulation under "B 10" (5 hour stimulation) or "B lOPro" (48 hour stimulation) conditions. Activation through the BCR, TLRs and CD40 is required for production of IL-10 by B cells or CLL cells. As BTK is involved in signal transduction of all these receptors, BTK inhibition can affect IL-10 production in CLL cells.
  • CLL patient T cells were collected pre-ibrutinib treatment and post-ibrutinib treatment and mixed with autologous CLL cells (pre-ibrutinib treatment). Cells were then either cultured with or without the addition of blinatumab and with or without Treg depletion. Cells were then stained for annexin V and propidium iodide ( Figure 15). After treatment with ibrutinib, CLL patients' T cells demonstrated superior survival after being stimulated with blinatumomab plus autologous CLL cells. The underlying mechanism is likely rescuing activated T cells from activation induced cell death(AICD) by inhibition of ITK ( Figure 16).
  • AICD activation induced cell death
  • ibrutinib also modulates the expression of several immune suppressive molecules on/in CLL cells including CD200, BTLA4 and IL-10.
  • Acalabrutinib is a second generation, selective BTK inhibitor. As shown table 1, while ibrutinib has comparable IC50 for BTK and ITK, acalabrutinib has virtually no affinity for ITK. The pharmacologic studies in patients clearly differentiate ibrutinib from the more selective BTK inhibitor acalabrutinib in its ability to inhibit AICD via ITK inhibition. Collectively, ibrutinib represents a novel T-cell immune modulating agent, and the data clearly differentiates it from other immunotherapeutics used in cancer.
  • Table 1 IC50 values for inhibition of enzymatic activity by ibrutinib versus acalabrutinib
  • ITK-deficient T cells have been found to have impaired proliferation whereas in-vivo, activated ITK _/" T cells survived to a much greater degree than normal T cells, leading to a greater accumulation.
  • Targeting ITK with kinase inhibitors showed a similar pattern.
  • ITK inhibitors inhibit IL-2 secretion and T- cell proliferation, whereas in-vivo ITK inhibitor was found to reduce AICD, leading to a 2-3 fold increase of activated T cell numbers.
  • CLL cells promote chronic stimulation of T cells and lead to an "exhaustion" phenpotype by inducing a CLL-specific immune response, or by modifying T cell response to chronic infections including cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • CLL cells cause chronic activation of T cells, and it is the activated T cells, but not resting T cells that are susceptible to AICD and can be rescued by ibrutinib. Therefore, this effect on T cell numbers by ibrutinib is likely only to be seen in patients who still have significant tumor burden, since those patients who have achieved remission will no longer have CLL-induced aberrant activation of T cells.
  • earlier time points (8 and 20 weeks into treatment) were selected and studied patients with persistent lymphocytosis (absolute lymphocyte counts at cycle 1 and cycle 6 are comparable; Table 2) to address this confounding factor.
  • ibrutinib strongly increased the number of activated T cells during listeria infection, while it has no significant impact on the resting T cell populations from healthy non-infected mice. Furthermore, in ibrutinib-treated patients, the increase in T cell numbers was most prominent in the effector and effector memory T cells compared to the resting naive T cells and central memory T cells. As a side note, it is possible that some of the changes in T cell populations are due to redistribution rather than expansion, as has been observed in HIV patients after institution of antiretroviral therapy. However, the durable effects make it unlikely that all the effects are due to redistribution.
  • Ibrutinib treatment leads to preferential expansion of more differentiated T cells subsets (e.g. T-EM and EMRA), but it does not have a deleterious effect on the absolute number of naive and central memory T cells. This is another feature that is desired for cancer immunotherapy.
  • IL-2 is able to increase effector cell proliferation it also compromises the persistence of the less differentiated memory T cells, and therefore has deleterious effect on the long term persistence of antitumor immunity.
  • ibrutinib treatment does not compromise the total numbers of the stem memory T cells (TSCM), which represent the earliest and long-lasting memory T cells. The self-renewal capacity and long-term survival of these cells make them ideal vehicle for the cancer immunotherapy.
  • TSCM stem memory T cells
  • T cells from CLL patients demonstrate features of exhaustion similar to those exposed to chronic stimulation by viral infections and ibrutinib preferentially increases the number of these exhausted T cells from AICD, enrichment of such cells was not observed. Instead, diminished PD-1 surface and intracellular CTLA-4 expression was detected.
  • Thl7 cells T cells capable of producing IL-17
  • Thl7 cells Thl7 cells
  • IFNy intracellular Thl
  • IL4 Th2
  • Ibrutinib has also been recently found to enhance IL-17 response indirectly by modulating the function of antigen presenting cells such as dendritic cells.
  • Thl7 cells have been found to undergo FAS-mediated AICD, a process that can also be blocked by ITK inhibition. The findings indicate that the net effect of ibrutinib treatment in CLL patients is the increased percentage of Thl7 cells.
  • Thl7 response can play a role in CLL pathogenesis.
  • Decreased frequency of Thl7 cells has been found to be associated with regulatory T cell expansion and disease progression in CLL patients.
  • elevated Thl7 cells in CLL patients is associated with improved survival.
  • CD200 and BTLA are significantly down-regulated on the surface of CLL cells as early as cycle 3 of ibrutinib treatment. While the function of BTLA on CLL cells is uncertain, CD200 regulates both innate and adaptive immunity and plays a key role in both tumor-specific and global immune suppression in CLL patients. Moreover, CD200 expression on tumor cells has been found to promote the expansion of Tregs, and CD200 blockade significantly decreases Treg cell numbers.
  • Ibrutinib treatment of CLL patients dramatically reduced the frequency of malignant B lOpro cells, which can express IL-10 after prolonged in-vitro stimulation, and similar findings with acalabrutinib indicate this is a BTK-dependent effect.
  • BTK is involved in signaling transduction of all these receptors.
  • chemokine CXCL12 enhances IL-10 production in CLL cells via the CXCR4-STAT3 pathway, and BTK inhibition was reported to impair CXCR4 surface expression and signaling in CLL cells.
  • mice with BTK deficiency showed a more severe reduction in the numbers of B la cells, which are also CD5+ B cells and are enriched with "B 10" (-30%) and "B 10pro"(30-40%) cells. Therefore, BTK inhibition can reduce the frequency of B lOPro-like CLL cells in two mutually non-exclusive mechanisms: by directly inhibiting the IL-10 production in CLL cells, and/or by selective depleting B lOPro-like CLL cells.
  • IL-10 is a major immunosuppressive cytokine that can be produced by multiple cell types. Surprisingly, it has been found that B cells are actually a dominant source of IL-10 in-vivo in both naive and immune system-activated mice. Secretion of IL-10 by CLL cells can be triggered by Infections or host inflammatory responses in CLL.
  • the disclosure includes comparative data from CLL patient samples obtained at matched time points during treatments with either an irreversible ITK/BTK inhibitor or more selective BTK inhibitor.
  • these studies identify effects such as expansion of effector T-cells, increased proportion of Thl7 producing cells, and distinct changes in CTLA- 4 intracellular expression between CD4 and CD8 subsets that are likely attributable to alternative, non-BTK targets such as ITK that are inhibited by ibrutinib but not acalabrutinib.
  • non-BTK targets such as ITK that are inhibited by ibrutinib but not acalabrutinib.
  • ITK intracellular expression between CD4 and CD8 subsets
  • ITK that are inhibited by ibrutinib but not acalabrutinib.
  • in-vitro evidence that ibrutinib but not acalabrutinib prevents AICD of activated T-cells and NK cells.
  • Ibrutinib induces significant increases in T cell numbers that are not achieved by a more selective BTK inhibitor.
  • the underlying mechanism is likely to be ITK inhibition that leads to the rescue of chronically stimulated T cells from AICD.
  • the data therefore provide support for ibrutinib therapy as an ideal cellular immune modulating agent for CLL and potentially other types of hematologic and solid cancers.
  • ibrutinib can be incorporated as part of cellular immune therapy. In-vivo persistence and expansion of antigen-specific T cells is the most critical determining factor for the success of adoptive immunotherapy with TIL cells and CAR T cells.
  • IL-2 Expanding such cells with systemic administration of IL-2 is toxic and can have deleterious effect on the long term persistence of antitumor immunity.
  • IL-2 also leads to preferentially expansion of Treg cells.
  • Pre-conditioning with lymphocyte depletion enhances homeostatic proliferation and depletes host Tregs.
  • it also carry along significant toxicities, and Tregs can out-proliferate conventional T cells in the lymphopenic environment.
  • Low persistence of infused T cells can also be a result of T cell exhaustion, and ongoing clinical trials are investigating immune checkpoint blockade to boost the persistence of tumor-specific T cells.
  • checkpoint blockade of CTLA-4 has been found to expand functional Treg cells.
  • ibrutinib enhances persistence/expansion of activated T cells and shows the following desirable qualities: a) it has no deleterious effects on the central memory or naive T cells; b) it does not cause collateral expansion of the Treg cells; and c) it partially reverses the exhausted T cell phenotype by reducing the expression of PD-1 and CTLA-4.
  • PBMC peripheral blood mononuclear cells
  • PMA 25ng/ml, Sigma-Aldrich
  • Ionomycin 500ng/ml, Sigma-Aldrich
  • monensin 2mM; eBioscience
  • Cells were then harvested and were stained with surface markers and then LIVE/DEAD® Fixable Near-IR stain (Thermo Fisher Scientific) as described above, with the exception that monensin was added to all the staining buffers.
  • PBMC cells were resuspended (2 x 10 cells/mL) in in Iscove's Modified Dulbecco's Media (FMDM) containing 10% fetal bovine serum (FBS), 200 ⁇ g/mL penicillin, 200 U/mL
  • FMDM Iscove's Modified Dulbecco's Media
  • CD3, CD4, and CD8 PECF-594 labeled CD14, CDl lb, CD16, CD56 and CD123 were added as a "dump channel" to gate out corresponding cell types. After surface staining, cells were labeled with LIVE/DEAD® Fixable Dead Cell Stains from ThermoFisher before being fixed with 1.5% Formaldehyde. Fixed cells were then permeablized with FACS buffer containing 0.25% Saponin and were stained with IL-10 antibody.
  • T cells were isolated from healthy human donors using EasySepTM Human T Cell Isolation Kit. Isolated T cells were stimulated in vitro with plate bound CD3/CD28 for 3 days. Cells were then rested in complete medium containing 50IU/ml IL-2 for additional 7-1 1 days before they were treated with vehicle, Ibrutinib or acalabrutinib for 30 minutes. Cells were then plated on to 48 well plates coated with CD3; incubate for 6 hours (for flow cytometry based apoptosis assay) or 3 hours (to isolate mRNA for qPCR to quantify FAS-L expression.) in the presence of IL2 to induce AICD.
  • FAS Ligand mRNA quantification mRNA were extracted from T cells after 3 hours of re-stimulation using QIAGEN "RNeasy Mini”RNA Isolation Kit. mRNA was then reverse transcribed to cDNA using the M-MLV Reverse Transcriptase from Thermo Fisher. Quantitative PCR for FAS-L were performed using the Taqman probe/primer mix (FAM labeled) from Thermo Fisher using GAPDH as internal control.
  • NK cells were then sorted to greater than 99% purity with a
  • NK cells were plated at 5x10 /CD3 /14 120 NK cells were isolated from peripheral blood leuko-Paks from normal donors (American Red Cross) by incubation with an NK cell RosetteSep negative enrichment cocktail (Stem Cell Technology), followed by Ficoll-Hypaque density gradient centrifugation (cells/well in a 96- well round bottom plate and cultured for three days at 37°C.
  • Medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum (FBS), and 1% antibiotic/antimycotic (Life
  • cytokines IL-2 Pieris X (Peprotech) and IL-15 (National Cancer Institute) were supplemented as indicated for a final concentration of lOng/mL.
  • IL-12 (Miltenyi Biotec) was added where indicated at a concentration of lOng/mL to induce activation induced cell death.
  • NK cells were harvested and stained with annexin V per manufacturer's instructions (BD Biosciences).
  • TO-PRO-3 was added immediately prior to acquisition, and all samples were analyzed with a LSRII cytometer (BD Biosciences) within one hour of annexin V staining. Analysis of dual staining of annexin V and TO-PRO-3 was analyzed using FlowJo (TreeStar).
  • PBMC peripheral blood cells
  • TCL-1 transgenic mice on C57BL/6 background were used for these experiments. Breeding pairs were provided by Dr. Carlo M. Croce (the Ohio State University, Columbus, OH). OT-1 TCR transgenic mice were purchased from The Jackson Laboratory. C1498-OVA is a murine myeloid leukemia cell line (H-2b, C57BL/6 background) expressing the experimental surrogate antigen ovalbumin. It is kindly provided by Dr. Bruce R. Blazar (University of Minnesota, Minneapolis, MN). (7) Statistical analysis:
  • Results are shown for the updated data with a total of 19 ibrutinib patients and 13 acalabrutinib patients. Not all experiments were performed on each patient's serial sample, therefore the actual "N" for each experiment was less than 19 and 13 for ibrutinib and acalabrutinib treated patients, respectively. All clinical-sample analyses were performed using SAS/STAT software, Version 9.4 of the SAS System for Windows (SAS Institute Inc., Cary, NC). For the in-vitro/animal experiments described Figures 3 and 13, two tailed Student' s t test were used. A p-value less than 0.05 was considered significant for all the experiments.
  • Attia MA Nosair NA, Gawally A, Elnagar G, and Elshafey EM.
  • HLA-G Expression as a Prognostic Indicator in B-Cell Chronic Lymphocytic Leukemia. Acta Haematol.
  • Ibrutinib is an irreversible molecular inhibitor of ITK driving a Thl-selective pressure in T lymphocytes. Blood. 2013; 122(15):2539-49.
  • CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion. Blood. 2008; 1 12(4): 1 175-83.
  • T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production. Blood. 2013; 121(9): 1612-21.
  • Ibrutinib enhances the antitumor immune response induced by intratumoral injection of a TLR9 ligand in mouse lymphoma. Blood. 2015; 125(13):2079-86.

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Abstract

L'invention concerne des procédés d'expansion sélective de populations de lymphocytes T et de cellules NK.
PCT/US2018/042349 2017-07-14 2018-07-16 Expansion de cellules immunitaires avec des composés inhibant la kinase des lymphocytes t inductibles par l'interleukine 2 WO2019014684A1 (fr)

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US11066644B2 (en) 2018-02-01 2021-07-20 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer
WO2022228539A1 (fr) * 2021-04-30 2022-11-03 四川大学华西医院 Procédé de préparation de cellules car-cik ayant une proportion élevée de cellules nkt, et son application
CN115989038A (zh) * 2020-04-10 2023-04-18 北卡罗莱纳州立大学 使用材料支架的增强的哺乳动物细胞病毒转导

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WO2019140137A1 (fr) * 2018-01-10 2019-07-18 The Board Of Trustees Of The Leland Stanford Junior University Compositions et procédés d'expansion de populations de lymphocytes t
US11066644B2 (en) 2018-02-01 2021-07-20 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer
WO2020198031A1 (fr) * 2019-03-22 2020-10-01 Windmil Therapeutics Inc. Lymphocytes infiltrant la moelle spécifiques au cancer du poumon et utilisations associées
CN115989038A (zh) * 2020-04-10 2023-04-18 北卡罗莱纳州立大学 使用材料支架的增强的哺乳动物细胞病毒转导
WO2022228539A1 (fr) * 2021-04-30 2022-11-03 四川大学华西医院 Procédé de préparation de cellules car-cik ayant une proportion élevée de cellules nkt, et son application

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