Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • 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
• • 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
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4225—Growth factors
  • A61K40/4229—Transforming growth factor [TGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0646—Natural killers cells [NK], NKT cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/515—Animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/515—Animal cells
  • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/47—Brain; Nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/55—Lung
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • 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
  • C12N2501/10—Growth factors
  • C12N2501/15—Transforming growth factor beta (TGF-β)
• • 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
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/30—Coculture with; Conditioned medium produced by tumour cells
• • 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
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Definitions

• NK cells natural killer cells
• NK cells represent only a small fraction of the cells in blood and isolation from a typical blood draw does not yield many cells.
• NK. cells must be purified away from contaminating PBMCs such as T and B cells by CD3 and CD 19 depletion, respectively.
• PBMCs such as T and B cells
• NK cells expand poorly in vitro compared to others kinds of cells due mainly to early senescence. Using even the most effective methods, NK cells are susceptible to telomere shortening and senescence after only a few passages. Denman et al., PLoS ONE 7(1): e30264 (2012). The most effective method for increasing NK cell viability and proliferation in vitro is co-culturing with feeder cells.
• NK cell expansion Commonly used feeder cells for NK cell expansion include irradiated peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL), gene-modified K562 cells constitutively expressing IL-15 or 21, and other irradiated tumor cell lines.
• PBMCs peripheral blood mononuclear cells
• EBV-LCL Epstein-Barr virus-transformed lymphoblastoid cell lines
• K562 cells constitutively expressing IL-15 or 21, and other irradiated tumor cell lines.
• OS including chemotherapy-resistdnt OS
• NK cells are readily killed in vitro by both autologous and allogeneic activated NK cells.
• the tumor microenvironment in OS, and other solid tumors has elevated levels of the highly immunosuppressive cytokine, transforming growth factor-beta (TGF-b).
• TGF-b transforming growth factor-beta
• TGF ⁇ suppression of NK cell function is multi-faceted.
• TGF-b modulates the development of NK cells and subsequently negatively affects their function upon reaching maturation.
• TGF-b ptomotes an immature NK cell lineage, by preventing the progression of NK cells into CD16+ NK cells.
• TGF-b can also induce formerly CD 16+ NK cells to become CD16-.
• Keskin et al Proc Natl Acad Sci U S A, 104: 3378-3383 (2007).
• mice expressing a dominant negative T ⁇ RbK on NK cells have increased numbers of mature NK cells compared to wild-type mice.
• Dahl et al Science signaling 9: ral9 (2016).
• TGF-b inhibits anti-tumor activity through multiple mechanisms.
• TGF-b decreases IL-2 and IL-15 induced NK cell proliferation (Wilson et al, PloS one 6: e22842 (2011)), and IL-15 induced mTOR activation.
• TGF-b also inhibits IFNy secretion, which is important for stimulating the adaptive immune system and can sensitive tumors to NK cell lysis.
• TGF-b inhibits IFNy both directly and indirectly.
• SMAD3 directly binds to the IFNy promoter and can also inhibit IFNy indirectly by decreasing expression of the IFNy-promoting transcription factors, T-bet and E4BP4. Tang et al, Nat Commun, 8: 14677 (2017). However, TGF-b mediated inhibition of IFNy secretion can be partially alleviated by pre-incubation of NK cells with IL-12, IL-15, or IL-18. Yu et al, Immunity, 24: 575-590 (2006). Furthermore, TGF-b also inhibits ⁇ NFa and GM-CSF secretion and modulates chemokine receptor expression to promote the retention of NK cells within the bone marrow. Castriconi et al, J Immunol, 190: 5321-5328 (2013).
• TGF-b mediates its’ inhibition of NK cell cytotoxicity by decreasing Granzyme and Perforin secretion, and expression of the following activating receptors: NKG2D, NKp30, KIRs, DNAM-l, NKp44, TRAIL, and CD 16. This inhibits NK cell recognition of malignant cells expressing their cognate ligands.
• TGF-b has been several approaches towards generating NK and T-cells resistant to TGF-b. These include dominant negative TGFpRII expressing cells and combination therapies using TGF-b small molecule inhibitors with immune-based therapies. Importantly, all of these methods have demonstrated increased in vitro and in vivo efficacy of NK and T- cell therapies. Wallace et ah, Clin Cancer Res., 14(12):3966-74 (2008); Bollard, C., Blood, 99: 3179-3187 (2002). However, broad spectrum inhibition of TGF-b has potential for adverse side effects; since TGF-b signaling is context dependent and can have both tumor- promoting and tumor-suppressive effects. For example, the inhibition of TGF-b in murine models increased the number of circulating tumor cells (Wrzesinski et ai, Clin Cancer Res., 13: 5262-5270 (2007)), and broad spectrum inhibition of TGF-b causes profound
• TGF-b inprinted NK (TORb ⁇ NK) cells were developed by adding TGF-b during stimulation of NK cells with IL- 12/15/18.
• TORb ⁇ NK maintained their cytotoxicity following stimulation better than normal NK cells.
• TGFpi N cells cultured with TGF-b have increased secretion ofIFN-g, TNF-a, and GM-CSF, which can increase tumor killing and broadly stimulate the adaptive immune response. Increased cytokine secretion persists for more than one month.
• the resistance to TGF-b persists for at least 1 week in vitro, and is mediated by SMAD3 downregulation.
• Figure 1 provides graphs showing that NK cell activation with parental (unmodified) K562 in the presence of TGFp induces TGFpi NK cells with cytokine hypersecretion in response to tumor targets.
• NK cells were stimulated weekly with K562 and cultured in the media containing IL-2 (control) or IL-2 and 10 ng/mL TGFP (TGFpi) for 14 days. Following culture, IFNy and TNFa secretion was assessed in supernatant after co- culture with MG63 tumor targets. Lines and bars represent Mean ⁇ SD.
• FIG. 2 provides graphs showing that NK cell activation with pro-inflammatory cytokines in the presence of TGFP induces TGFpi NK cells with cytokine hypersecretion in response to tumor targets.
• NK cells were activated overnight with IL-12, -15, and -18 (10 ng/mL, 50 ng/mL, and 50 ng/mL respectively) with or without IL-2 and TGFP, followed by culture in IL-15 (1 ng/mL) with or without IL-2 and TGFp. After 7-14 days of culture, antitumor IFNy and TNFa production in response to MG63 was measured by intracellular flow cytometry (n-4). Percent IFNy+ and TNFa+ NK cells normalized to no target.
• FIG. 3 provides graphs showing that NK cell expansion with K562 mbIL-l5 (clone 4) feeder cells in the presence of TGFp induces TGFpi NK cells with cytokine hypersecretion in response to tumor targets.
• TGFpi feeder cells with
• control TGFP
• NK cells were rested overnight in 50 IU/mL IL-2 with or without 10 ng/mL TGFp. NK cells were then co-cultured with tumor targets in the same media and supernatants were collected to measure cytokine secretion. Control in black,
• FIG. 4 provides graphs showing that NK cell expansion with K562 mbIL-21 feeder cells in the presence of TGFp induces TGFpi NK cells with cytokine hypersecretion in response to tumor targets.
• TGFpi feeder cells with
• control control
• NK cells were rested overnight in 50 IU/mL IL-2 with or without 10 ng/mL TGFp.
• NK cells were then co-cultured with tumor targets in the same media and supernatants were collected to measure cytokine secretion. Control in black, TGFpi in red.
• Statistical differences were determined by two-way repeated measures ANOVA with Holm-Sidak’s multiple comparisons test for all others.
• FIG. 5 provides a graph showing that the addition of TGFp to generate TGFpi NK cells does not reduce proliferative potential in expansion cultures with K562 mbIL-2l (CSTX002) feeder cells.
• NK cells were expanded for 14 days on feeder cells with (TGFpi) or without (control) TGFp in paired cultures from 5 donors starting from the same number of cells at Day 0. Total number of viable cells after 14 days is shown, normalized to control NK cells for each paired expansion. Non-significant by Student’s paired t test.
• FIG. 6 provides graphs showing that TGFpi NK cells maintain cytokine hypersecretion against tumor cells for weeks.
• Figure 9 provides a graph showing that TGFpi NK cells hypersecrete GM-CSF, TNFa, and IFNy. Control and TGFpi NK cells were stimulated with 10 pg/mL of PHA at 2 x l0e6 NK cells/mL for 4 hours and cytokine secretion was measured. Lines and bars represent Mean ⁇ SD. Statistical differences were determined by paired t-test.
• FIG. 10 provides graphs showing that TGFpi NK. cells expanded with parental K562 have similar cytotoxicity to control NK cells. Control and TGFpi NK cell cytotoxicity was measured using a 4-hour calcein-release cytotoxicity assay, following overnight treatment in IL-2 alone or IL-2 and TGFp. Lines and bars represent Mean ⁇ SD. Statistical differences were determined by two-way repeated measures ANOVA with Holm-Sidak’s multiple comparisons test. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001.
• Figure 11 provides a graph showing that expanded TGFpi NK cells control tumor growth better than control expanded NK cells in a mouse model of osteosarcoma.
• FIG. 12 provides graphs showing that TGFpi NK cells have a gene expression profile that distinguishes them from standard expanded NK cells.
• Standard and TGFpi NK cells were expanded in pairs on CSTX002 from 4 donors. mRNA was isolated and subjected to RNA sequencing. The top 100 differentially-expressed genes were identified.
• A) Principle components analysis was performed in which original values are ln(x + l)-transformed. Unit variance scaling is applied to rows; SVD with imputation is used to calculate principal components. X and Y axis show principal component 1 and principal component 2 that explain 95.4% and 1.6% of the total variance, respectively.
• TGFpi NK cells are identified in the red box.
• the present invention provides TGF-b Imprinted Natural Killer (TGFpi NK) cells, which are highly cytotoxic, produce high levels of cytokine, and are resistant to the TGF-b Superfamily of immunosuppressive cytokines. These cells can be prepared by chronic in vitro activation of natural killer cells in the presence of a TGF-b Superfamily cytokine.
• the invention also provides a method of treating cancer or infection in a subject in need thereof by administering a therapeutically effective number of TGFpi NK cells to the subject.
• the term“subject” can refer to any warm-blooded organism including, but not limited to, human beings, rats, mice, dogs, goats, sheep, horses, monkeys, apes, pigs, rabbits, cattle, etc.
• the term when the term is used in the context of a subject needing or requiring compositions of the present application, the term may be referred to as“a subject in need thereof’ and include subjects that have been clinically diagnosed (e.g., by a medical professional, e.g., a physician) as being in need of compositions of the present application, subjects that are suspected of being in need of compositions of the present application, subjects at risk for a disease or condition and who may benefit from compositions of the present application, and subjects that are already suffering from a disease or condition and who may benefit from compositions of the present application.
• a subject in need thereof include subjects that have been clinically diagnosed (e.g., by a medical professional, e.g., a physician) as being in need of compositions of the present application, subjects that are suspected of being in need of compositions of the present application, subjects at risk for a disease or condition and who may benefit from compositions of the present application, and subjects that are already suffering from a disease or condition and who may benefit from compositions of the present
• pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• therapeutically effective is intended to qualify the number or amount of an agent which will achieve the goal of decreasing disease severity while avoiding adverse side effects such as those typically associated with alternative therapies.
• a therapeutically effective amount may be administered in one or more doses.
• Treatments that are therapeutically effective include treatments that improve a subject's quality of life even if they do not improve the disease outcome per se
• An“Effective amount” generally means an amount which provides the desired local or systemic effect, e.g., effective to stimulate cytokine formation, including achieving the specific desired effects described in this application.
• an effective amount is an amount sufficient to effectuate a beneficial or desired clinical result.
• Treatment are used broadly in relation to the invention and each such term encompasses, among others, preventing, ameliorating, inhibiting, or curing a deficiency, dysfunction, disease, or other deleterious process, including those that interfere with and/or result from a therapy.
• the symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%* at least 30%, at least 40%, or at least 50%.
• administer refers to the placement of a composition (e.g., a cell composition) into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced.
• a resistant natural killer cell or composition described herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.
• cytokine refers to a small protein ( ⁇ 5 ⁇ 20 kDa) that is important in cell signaling, and in particular immunomodulation.
• cytokines include chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors.
• the present invention provides a method of treating cancer or infection in a subject in need thereof.
• the method includes administering a therapeutically effective number of TGF-b Imprinted Natural Killer (TORb ⁇ NK) cells to the subject.
• TORb ⁇ NK TGF-b Imprinted Natural Killer
• T ⁇ Rb ⁇ NK cells can be more effective and persist longer in vivo compared with typical expanded natural killer cells because their resistance to cytokines of the TGF-b superfamily protects them from suppression by the immune system.
• NK cells Natural Killer Cells
• MHC Major Histocompatibility Complex
• NK cells are large granular lymphocytes (LGL) and are known to differentiate and mature in the bone marrow from where they then enter into the circulation.
• the invention includes NK cells that are resistant to cytokines of the transforming growth factor-beta (TGF-b) superfamily, and methods of making and using such resistant NK cells.
• TGF-b superfamily is a large group of structurally related cell regulatory proteins.
• TGF-b is a multifunctional peptide that controls proliferation, differentiation and other functions in many cell types.
• TGF-b-! is a peptide of 112 amino acid residues derived by proteolytic cleavage from the C-terminal of a precursor protein. These proteins interact with a conserved family of cell surface serine/threonine-specific protein kinase receptors, and generate intracellular signals using a conserved family of proteins called SMADs.
• the major subfamilies of the TGF-b superfamily include the TGF-b subfamily (including the TGF-b 1 to 4 isoforms), the decapentaplegic Vg-related (DVR) related proteins (e.g., bone morphogenic protein), growth differentiation factors (e.g., GDF-l through GDF-l 5), and the activin and inhibin subfamily.
• the T ⁇ Rb ⁇ NK cells are resistant to TGF-b.
• the TORb ⁇ N cells can be used to treat cancer or infection in a subject.
• the TORb ⁇ NK cells are typically administered by adoptive transfer of the cells.
• the subject has been diagnosed as having cancer.
• Cancer as defined herein, is a disease based on the development of cells that contain genetic damage resulting in the relatively unrestrained growth of the cells. The genetic damage present in a cancer cell is maintained as a heritable trait in subsequent generations of the cancer cell line.
• the caiicer treated by the method of the invention may be any of the forms of cancer known to those skilled in the art or described herein. Cancer that manifests as both solid tumors and cancer that instead forms non-solid tumors as typically seen in leukemia can be treated.
• the present invention provides methods for treating a subject that is afflicted with various different types of cancers, including carcinoma, sarcom , and lymphoma.
• the cancer being treated is a leukemia (e.g., acute lymphoblastic leukemia; acute myeloid leukemia; chronic myelogenous leukemia, chronic lymphocytic leukemia), a myelodysplastic syndrome, a lymphoma (e.g., B cell non-Hodgkin lymphoma, Hddgkin lymphoma, T-cell lymphoblastic lymphoma, anaplastic large cell lymphoma), a solid tumor (e.g., a breast cancer, prostate cancer, gastric cancer, colon cancer, hepatocellular carcinoma, nasopharyngeal carcinoma, neuroblastoma, high grade glioma), a sarcoma (e.g., Ewing sarcoma, rhabdomyosarcoma, non-rhabdomyosarcoma soft-tissue sarcoma, osteosarcoma).
• the cancer is selected from
• the effectiveness of cancer treatment may be measured by evaluating a reduction in tumor load or decrease in tumor growth in a subject in response to the administration of the TGFpi NK cells.
• the reduction in tumor load may be represent a direct decrease in mass, or it may be measured in terms of tumor growth delay, which is calculated by subtracting the average time for control tumors to grow over to a certain volume from the time required for treated tumors to grow to the same volume.
• the subject being treated has an infectious disease.
• the TGFpi NK cells have broad-band systemic effects and can be used to treat infection by a variety of different microorganisms.
• infectious diseases is means to include all diseases which are caused by infection with viruses, pathogenic bacteria, or fungi, and can be infected through respiratory organ, blood or skin contact.
• infectious diseases include, but are not limited to, hepatitis B, hepatitis C, human papilloma virus (HPV) infection, human immunodeficiency disease (HIV), cytomegalovirus infection, viral respiratory disease, influenza and so on.
• TGFpi NK cells a natural killer (NK) cell or NK cell line cultured in the presence of a TGF-b superfamily cytokine, referred to herein as TGFpi NK cells.
• TGFpi NK cells a natural killer cell or NK cell line cultured in the presence of a TGF-b superfamily cytokine, referred to herein as TGFpi NK cells.
• TGFpi NK cells TGF-b superfamily cytokine
• the TGFpi NK cells can be allogenic or autologous cells.
• the NK cell is a mammalian NK cell.
• mammals include primates (e.g., human), canines, felines, rodents, porcine, ruminants, and the like. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats and mice.
• the mammalian NK cell is a human NK cell.
• the TGFpi NK cells exhibit a number of characteristics that distinguish them from naturally occurring NK cells.
• the NK cell or cell lines exhibit increased resistance to TGF-b.
• the NK cells produce and increased amount of interferon-g (IFN-g), and/or tutaor necrosis factor-a (TNF-a), and/or Granulocyte- macrophage colony-stimulating factor (GM-CSF).
• the NK cells show decreased levels of SMAD family member 3 (SMAD3) protein and/or Transforming growth factor beta receptor III (TGFBR3) protein.
• SMAD proteins received their name as a contraction of of the names of the C. elegans Sma and Drosophila Mad (Derynck et ai, Cell, 95(6), p737 ⁇ 740, 1998) and are transcriptional activators of TGF-b responses.
• the TGFpi NK cells exhibit a number of characteristics that distinguish them from naturally occurring NK cells.
• the NK cells have a gene expression profile substantially similar to that shown in Figure 12.
• a gene expression profile that is substantially similar is one in which the gene expression is within 10% of that shown.
• the TGFpi NK cells produce increased amounts of one or more of IFN-g, TNF-a and GM-CSF protein.
• the NK cell or cell lines exhibit increased expression of SCUBE1 , MY07A, KLF3, WIPF3, and EPHA1.
• the TGFpi NK cells exhibit a number of characteristics that distinguish them from naturally occurring NK cells.
• the TGFpi NK cells show decreased levels of SMAD3 protein and/or TGFBR3 protein.
• the NK cell or cell lines exhibit decreased expression of CD300A, SGSM1 , SMAD3, TBX21, and GZMK, TGFBR3, and GZMA.
• TGF-P-Superfamilv Imprinted Natural Killer (TGFBi NK) cell line Another aspect of the invention provides a method of making a TGF-b- Sujperfamily Imprinted Natural Killer (T ⁇ Rb ⁇ NK) cell line, comprising the in vitro activation of natural killer cells in the presence of a TGF ⁇ -superfamily cytokine.
• the methods can further comprise isolating or separating the one or more TORb ⁇ NK cells produced by the methods provided herein.
• the methods can further comprise culturing the one or more T ⁇ Rb ⁇ NK cells.
• a TORb ⁇ NK cell line is produced.
• the T ⁇ Rb ⁇ NK cell line is expanded in the presence of TGF-b.
• a cell line is a plurality of cells that can be maintained ih cell culture.
• Expansion refers to the ex vivo proliferation of NK cells so that the population of NK cells is increased.
• NK cells can be expanded, for example, from peripheral blood mononuclear cells.
• NK cells can also be expanded from other types of cells, such as hematopoietic stem cells or progenitor cells.
• the initial blood or stem cells can be isolated from a variety of different sources, such placenta, umbilical cord blood, placental blood, peripheral blood, spleen or liver. Expansion occurs in a cell culture medium.
• Suitable cell culture mediums are known to those skilled in the art, and include Basal Medium Eagle (BME), Dulbecco's Modified Eagle's Medium (DMEM), Glasgow Minimum Essential Medium (GMEM), Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/F-12), Minimum Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), Nutrient Mixture F-10 Ham (Ham's F-10), Nutrient Mixture F-12 Ham (Ham's F-12), RPMI-1640 Medium, Williams' Medium E, STEMSPAN ® (Cat. No.
• Glycostem Basal Growth Medium GBGM ®
• AIM-V ® medium Invitrogen
• X-VIVOTM 10 Lihza
• X-VIVO.TM. 15 Lihza
• OPTMIZER Invitrogen
• STEMSPAN ® H3000 STEMCELL Technologies
• CELLGRO COMPLETETM Mediatech
• feeder cells refers to cells which do not have the ability to divide and proliferate, but have metabolic activity, and thus produce various metabolic products assisting in the proliferation of target NK cells.
• feeder cells include, but are not limited to, animal cell lines introduced with genes, peripheral blood leukocytes (PBL) treated with various cytokines or compounds, autologous or allogeneic peripheral blood leukocytes (PBL), T-cells, B -cells, monocytes and the like.
• the feeder cells are K562 feeder cells.
• the K562 feeder cells are selected from clone 4 cells, clone 9 cells, and CSTX002 cells.
• the ih vitro activation of natural killer cells is carried out in the presence of an NK-stimulating exosome or NIC-stimulating nanoparticle.
• Exosomes are small extracellular vesicles derived from endosomes, with a diameter between 30-100 nm. Tumor-derived exosomes carry many molecules and factors from tumor cells, and can be used to stimulate natural killer cells. See Li et ai, Exp Cell Res., 363(2): 141 -150 (2016). Nanoparticles can also be used to stimulate natural killer cells. Nanoparticles are particles between 1 and 2500 nm in size with a surrounding interfacial layer.
• Nanoparticles having a size from 1 to 100 nm, and fine nanoparticles having a size from 100 to 2500 nm.
• Nanoparticles can be prepared using a polymer, or minerals such as graphene oxide.
• the nanoparticles are functionalized to include additional groups such as antibodies that help to stimulate natural killer cells. See, for example, Loftus et ai, Nano Lett., 18(5):3282-3289 (2016).
• the TGFpi NK cells should be administered and dosed in accordance with good medical practice, taking into account the site and method of administration, scheduling of administration, patient age, sex, body weight, the nature and severity of the disorder to be treated or prevented, and other factors known to medical practitioners.
• the cells may be administered in a single dose or in divided doses.
• the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement, including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
• said dose is about 10 x IQ 6 cells/kg of subject weight or lower, is about 9 x 10 6 cells/kg or lower, is about 8 x 10 6 cells/kg or lower, is about 7 x 10 6 cells/kg or lower, is about 6 x 10 6 cells/kg or lower, is about 5 x 10 6 cells/kg or lower.
• said dose may be between about 0.25 x 10 6 cells/kg to about 5 x 10 6 cells/kg; or more preferably about 1 x 10 6 cells/kg to about 5 x 10 6 cells/kg.
• the dose may be about 0.25 x IQ 6 cells/kg, 0.5 x 10 6 cells/kg, 0.6 x 10 6 cells/kg, 0.7 x 10 6 cells/kg; 0.8 x 10 6 cells/kg; 0.9 x 10 6 cells/kg; 1.1 x 10 6 cells/kg; 1.2 x 10 6 cells/kg; 1.3 x 10 6 cells/kg; 1.4 x 10 6 cells/kg; 1.5 x 10 6 cells/kg; 1.6 x 10 6 cells/kg; 1.7 x 10 6 cells/kg; 1.8 x 10 6 cells/kg; 1.9 x 10 6 cells/kg or 2 x 10 6 cells/kg.
• the dose may, in other embodiments, be between 0.1 and 1 million cells/kg; or between 1 and 2 million cells/kg; or between 2 and 3 million cells/kg; or between 3 and 4 million cells/kg; or between 4 and 5 million cells/kg; or between 5 and 6 million cells/kg; or between 6 and 7 million cells/kg; or between 7 and 8 million cells/kg; or between 8 and 9 million cells/kg; or between 9 dnd 10 million cells/kg.
• Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion.
• injection includes, without limitation, intfavenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular,
• compositions are intfaorbital, intracardiac, intradermal, int aperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.
• the compositions are intfaorbital, intracardiac, intradermal, int aperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.
• the compositions are intfaorbital, intracardiac, intradermal, int aperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.
• the compositions are intfaorbital, intracardiac, intradermal,
• TGFpi NK cells can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration.
• the composition can be sterile.
• the formulation should suit the mode of administration.
• Cell Therapy Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.
• Choice of the cellular excipient and any accompanying elements of the composition comprising a population of TGFpi NK cells will be adapted in accordance with the route and device used for administration.
• the TGFpi NK cells are administered together with a pharmaceutically acceptable carrier.
• suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose of starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
• the pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the active compounds.
• auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the active compounds.
• Example 1 Imprinting of NK cells by TGF-B to be TGF-B resistant
• TGFpi NK human NK cells expanded with TGF-b culture
• TGFpi NK TGF-b culture
• the inventors demonstrate: a) TGFpi NK cells have rerharkably increased IFN-g, TNF-a, and GM-CSF secretion against tumor targets and that b) TGFpi NK cells retain cytokine hypersecretion for at least 1 month post-activation.
• NK cells were cultured in RPMI 1640 media supplemented with Glutamax, 10% FBS, and antibiotics. All cells were routinely tested for Mycoplasma contamination using Lonza MycoAlert (Lonza, LT027-58) and found to be negative at all time points.
• K562 feeder cells were purchased from ATCC (CCL-243) and irradiated at 100
• NK cell expansion Purified primary human NK Cells were stimulated at Day 0 1 :2 with irradiated K562 and 1 : 1 at Day 7.
• the K562 cell lines used are indicated in the figure legends, as untnodified (parental), expressing 4-1BBL and membrane-bound IL-15 (Clone 4), or expressing 4-1BBL and metnbrane-bound IL-21 (Clone 9 or CSTX002).
• Standard expanded NK cells were supplemented with 50 IU/mL recombinant human IL-2, and Resistant (TGFpi NK) expanded NK cells received 50 IU/mL IL-2 and 10 ng/mL TGF-b (Biolegend, 580706). Fresh media and cytokines were added every 2-3 days. NK Cell Expansion was calculated based on the percentage of CD37CD56 + cells.
• Cytofix/Cytoperm Fixation/Permeabilization Kit with GolgiStop (BD Biosciences, 554715).
• Antibodies for the following proteins were used to assess NK phenotype and function: CD3 PeCy7/APC-H7,CD56 FITC/BV421, NKG2D Pe-CF594/BV5lO, TRAIL PE/APC/BV421, FasL PE, NKp30 PE/Alexa Fluor 647/PE-Vio6l5, Granzyme A APC, Granzyme B BV510, Perforin BV421, DNAM-l BV711, CD 107a BV510, IFN-g APC, TNF-ct BV42I, CD16 PE, and Tonbo Ghost Dye 510/780.
• Cell events were acquired on a LSR Fortessa. Flow cytometry gating was determined using cells stained with viability dye only and single color controls were analyzed using FlowJo 7.6.5/10. SMAD
• Cytotoxicity assay NK cells were prepared for cytotoxicity assays by resting overnight in either human IL-2 alone or IL-2 (50 IU/mL) with 10 ng/mL soluble TGF-b (Biolegend). Cytotoxicity assays with calcein-AM based method were conducted in at least duplicate using 3 mg calcein AM/mL/ 1 ,000,000 target cells in complete media. Calcein assays were conducted in the same cytokines as the NK cells were rested in overnight. Sofnachi et ai, Journal of visualized experiments: JoVE 48, 2540 (2011).
• Intracellular function flow cytometry To determine degranulation by CD 107a expression and intracellular cytokine production in response to tumors, 300,000 NK cells were co-cultured in a 96-well round-bottom plate with 60,000 tumor cells (5:1 E:T ratio) or no target for a control in 200 m ⁇ media as described for cytotoxicity assays. One m ⁇ of monensin was added to each sample along with CD 107a at the beginning of the assay. Plates were spun down at 100 g x 2 minutes to promote cell-cell contact, and placed in a 37°C incubator for 3 hours. After 3 hours, media was removed and staining began for cell surface and intracellular proteins as detailed.
• Cytometric bead array TO determine the NK cell release of IFNy and TNPa, NK cells were cultured as described for intracellular functional flow cytometry with the exception of the monensin and CD 107a antibody. After 3 h co-culture with tumof targets or 4 h stimulation with 10 pg/mL PHA, supernatants were collected and frozen at -75 °C until use.
• the supernatants were thawed and 50 pL of undiluted supernatant was used according to the manufacturer’s instructions for the BD CBA Soluble Protein Master Kit (BD Biosciences, Cat#: 558265) and IFNy and TNFa Flex Set (BD Biosciences, Cat: 558269, 560112) or MACSPlex Cytokine 12 Kit ( iltenyi, Cat: 130-099- 169).
• the analytes were acquired on a BD LSR II or a MACSQuant. The geometric mean for each analyte was determined in Flow Jo v.
• NK cell activation with cytokines For NK cell stimulation with IL-12, IL-15, and IL-18, primary NK cells were stimulated overnight With 10 ng/mL IL-12 (Biolegend, 573002), 50 ng/mL IL-15 (Biolegend, 570302) and 50 ng/mL IL-18 (Biolegend, 592102) as described and rested in 1 ng/mL IL-15 for 7 14 days following overnight stimulation with IL-12, IL-15, and IL-18.
• the NK cells were treated as described but with the addition of IL-2 and/or TGF-b as ihdicated in the overnight stimulation with IL-12, IL-15, and IL-18, and along with 1 ng/mL IL-15 for 7- 14 days.
• the NK cells were rested in 1 lig/mL IL-15 only overnight and throughout the assay and co-cultured with MG63 at a 5: 1 ratio or equal numbers of NK cells only ds a no target control and intracellular flow staining was conducted as described below.”
• RNA from fresh, never frozen, Day 14 expanded human NK cells was isolated using RNAeasy Kit, QiaShredder Columns, and RNAase-Free DNase Set (all Qiagen, 74104, 79654, 79254), and cDNA was synthesized High Capacity cDNA Reverse Transcription Kit (Thermo Fish r, 4368814).
• PCR for the TGF-b pathway was done usirig Taqman Fast PCR Mastermix and kuman Fast 96-well TGF-b Pathway Array
• IFNy and TNFa are two pro-inflammatory cytokines important in the anti-tumor response and their production has been reported to be inhibited by TGF-b.
• NK cells were cultured for 2 weeks with parental (unmodified K562) plus or minus TGFp.
• parental (unmodified K562) plus or minus TGFp At the end of 2 weeks, control and TGFpi NK cells were incubated with tumor targets as described and supernatants were collected to measure IFN-g and TNF-a secretion with Cytometric Bead Array.
• a significant increase in IFN-g and TNFa secretion in both the presence and absence of TGF-b compared to control expanded NK cells was observed ( Figure 1).
• K562 expressing mbIL-l5 ( Figure 3) or mbIL-21 ( Figure 4) were co-cultured with NK cells for 2 weeks plus or minus TGFp. Both feeder cells induced TGFpi NK cells with increased cytokine production.
• TGFpi NK and donor-matched Standard NK cells were rested in low-dose IL-2 alone (e.g. TGFpi NK were removed from TGF-b) after completion of 2 weeks of activation.
• TGFpi NK cells were assessed for secretion of IFN-g and TNF-a at the end of expansion and 7-33 days days post-activation. After activation, TGFpi NK cells maintained their increase in IFN-g and TNF-a secretion at both baseline and with TGF-b treatment (Figure 6).
• TGFpi NK cells maintained cytokine hyperproduction in response to various stimuli.
• TGFpi NK celsl produced increased IFNy and TNFa in response to medulloblastoma and neuroblastoma cell lines and that this heightened production of cytokines could be induced with PHA stimulation suggesting an innate ability to produce increased anti-tutnor cytokines ( Figure 7 & 9).
• SMAD3 as a suppressor of NK cell anti-tumor function has beeh clear.
• SMAD3 binds directly to the IFNy promoter to inhibit IFNy expression ahd SMAD3 7 mice have enhanced NK cell function and decreased tumor growth.
• the inventors determined if SMAD3 was decreased at the protein level by western blot, and found TGFpi NK to have significiantly decreased SMAD3 protein (Figure 8).
• TGFpi NK human NK ceils expanded with TGF-P culture
• TGFpi NK have reduced sensitivity to TGF-P, most likely through loss of one of the key TGF-P signaling proteins- SMAD3.
• TGFpi NK cells have remarkably increased IFN-g, TNF-a, and GM-CSF secretion.
• TGF-P did not inhibit the overall 2 week proliferation of TGFpi NK cells.
• TGFpi NK cells The ability of TGFpi NK cells to produce IFN-g and TNF-a was assessed because these cytokines can both inhibit TGF-P and conversely, TGF-P can inhibit the production ofIFN-g and TNF-ct. Surprisingly, remarkably increased anti-tumor IFN-g and TNF-a secretion in TGFpi NK cells compared to Standard NK cells both with and without TGF-p treatment was found. Previous studies have demonstrated that SMAD3 deletion increases baseline IFN-g production, therefore, it is likely that a similar mechanism is occurring in TGFpi NK cells which do not express SMAD3. Tang et al, Nat Commun 8: 14677 (2017).
• TGF-b is reported to inhibit TNFa production in primary NK cells (Bellone et al., J Immunol 155: 1066-1073 (1995)). Unexpectedly, TGFpi NK cells had significantly increased secretion of TNF-a with or without TGF-b in the assay media compared to Standard NK cells. Regulation of TNF-a production is less well-understood, but is also known to be inhibited by T ⁇ Rb, so it was unexpected to find increased TNF-a secretion in TORb ⁇ NK cells.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Epidemiology (AREA)
• Medicinal Chemistry (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Pharmacology & Pharmacy (AREA)
• Immunology (AREA)
• Biomedical Technology (AREA)
• Zoology (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Genetics & Genomics (AREA)
• Cell Biology (AREA)
• Hematology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Mycology (AREA)
• Wood Science & Technology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Biochemistry (AREA)
• Virology (AREA)
• Oncology (AREA)
• General Engineering & Computer Science (AREA)
• Developmental Biology & Embryology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Communicable Diseases (AREA)
• Gastroenterology & Hepatology (AREA)
• Toxicology (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Priority Applications (15)

Applications Claiming Priority (2)

Publications (1)

Family

ID=67478491

Family Applications (1)

Country Status (12)

Cited By (2)

Families Citing this family (1)

Citations (1)

Family Cites Families (4)

• 2019
  • 2019-01-29 IL IL276374A patent/IL276374B2/en unknown
  • 2019-01-29 US US16/966,367 patent/US20200368281A1/en active Pending
  • 2019-01-29 KR KR1020257019712A patent/KR20250093419A/ko active Pending
  • 2019-01-29 BR BR112020015490-3A patent/BR112020015490A2/pt unknown
  • 2019-01-29 CA CA3090096A patent/CA3090096A1/en active Pending
  • 2019-01-29 AU AU2019213678A patent/AU2019213678B2/en active Active
  • 2019-01-29 CN CN201980016864.3A patent/CN111818941A/zh active Pending
  • 2019-01-29 JP JP2020562093A patent/JP7591406B2/ja active Active
  • 2019-01-29 KR KR1020207024676A patent/KR102822300B1/ko active Active
  • 2019-01-29 WO PCT/US2019/015617 patent/WO2019152387A1/en not_active Ceased
  • 2019-01-29 IL IL309656A patent/IL309656A/en unknown
  • 2019-01-29 SG SG11202007288VA patent/SG11202007288VA/en unknown
  • 2019-01-29 EP EP19747119.6A patent/EP3746118A4/en active Pending
  • 2019-01-29 MX MX2020008044A patent/MX2020008044A/es unknown
• 2024
  • 2024-11-18 JP JP2024200825A patent/JP2025026937A/ja active Pending

Patent Citations (1)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events