WO2022133061A1 - Infusion ready cryopreservation compositions - Google Patents

Infusion ready cryopreservation compositions Download PDF

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Publication number
WO2022133061A1
WO2022133061A1 PCT/US2021/063757 US2021063757W WO2022133061A1 WO 2022133061 A1 WO2022133061 A1 WO 2022133061A1 US 2021063757 W US2021063757 W US 2021063757W WO 2022133061 A1 WO2022133061 A1 WO 2022133061A1
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WIPO (PCT)
Prior art keywords
cells
cell
pharmaceutical composition
dextran
cancer
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PCT/US2021/063757
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French (fr)
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WO2022133061A9 (en
Inventor
Yu Kyeong Hwang
Jung Hyun Her
Yusun KIM
Eunji Kim
Hyojin Kim
Bitna YANG
Bokyung MIN
Sungyoo CHO
Peter Flynn
Jason B. LITTEN
Thomas James FARRELL
John Kin Chuan Lim
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Artiva Biotherapeutics, Inc.
GC Cell Corporation
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Application filed by Artiva Biotherapeutics, Inc., GC Cell Corporation filed Critical Artiva Biotherapeutics, Inc.
Publication of WO2022133061A1 publication Critical patent/WO2022133061A1/en
Publication of WO2022133061A9 publication Critical patent/WO2022133061A9/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • Cell based therapies such as natural killer (NK) cell therapy, e.g., CAR-NK cell therapy, T-cell therapy, e.g., CAR-T cell therapy, and others can be administered to patients intravenously.
  • NK natural killer
  • the cells may be stored frozen.
  • compositions that are suitable both for cryopreservation of cells and administration to patients, e.g., by intravenous infusion.
  • cryopreservation compositions e.g., infusion-ready cryopreservation compositions
  • pharmaceutical compositions comprising cryopreservation compositions, e.g., infusion-ready cryopreservation compositions
  • methods of treating disorders comprising administering cryopreservation compositions, e.g., infusion-ready cryopreservation compositions, and therapeutic cells.
  • cryopreservation compositions comprising albumin, e.g., human albumin, dextran, e.g., Dextran 40, glucose, DMSO, and a buffer, e.g., phosphate buffered saline.
  • albumin e.g., human albumin
  • dextran e.g., Dextran 40
  • glucose e.g., DMSO
  • a buffer e.g., phosphate buffered saline.
  • the cryopreservation composition comprising: (a) about 40 mg/mL human albumin; (b) about 25 mg/mL Dextran 40; (c) about 12.5 mg/mL glucose; (d) about 55 mg/mL DMSO; and (e) about 0.5 mL/mL phosphate buffered saline.
  • the cryopreservation composition further comprises (f) cells.
  • compositions consisting of: (a) about 40 mg/mL human albumin; (b) about 25 mg/mL Dextran 40; (c) about 12.5 mg/mL glucose; (d) about 55 mg/mL DMSO; and (e) about 0.5 mL/mL phosphate buffered saline.
  • pharmaceutical compositions comprising a cry opreservation composition and cells, e.g., therapeutic cells.
  • the pharmaceutical composition comprises: (a) human albumin; (b) dextran; (c) glucose; (d) DMSO; (e) a buffer; and (f) therapeutic cells.
  • the pharmaceutical composition comprises from 30 to 50 mg/mL human albumin.
  • the pharmaceutical composition comprises 50 mg/mL human albumin.
  • the pharmaceutical composition comprises 20 to 30 mg/mL dextran.
  • the pharmaceutical composition comprises 25 mg/mL dextran.
  • the dextran is Dextran 40.
  • the pharmaceutical composition comprises from 12 to 15 mg/mL glucose.
  • the pharmaceutical composition comprises 12.5 mg/mL glucose.
  • the pharmaceutical composition comprises less than 27.5 g/L glucose.
  • the pharmaceutical composition comprises from 50 to 60 ml/mL DMSO.
  • the pharmaceutical composition comprises 55 mg/mL DMSO.
  • the pharmaceutical composition comprises 40 to 60 % v/v buffer.
  • the pharmaceutical composition comprises the buffer is phosphate buffered saline.
  • the therapeutic cells are animal cells.
  • the animal cells are human cells.
  • the therapeutic cells are immune cells.
  • the immune cells are selected from the group consisting of basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.
  • the immune cell is a T cell.
  • the T cell is a CAR-T cell.
  • the immune cell is an NK cell.
  • the NK cell is a CAR-NK cell.
  • the pharmaceutical composition comprises: (a) about 40 mg/mL human albumin; (b) about 25 mg/mL Dextran 40; (c) about 12.5 mg/mL glucose;
  • Also provided herein are methods for treating a disorder comprising: (a) administering a therapeutically effective amount of a pharmaceutical composition described herein to a patient in need thereof.
  • the patient has a cellular proliferative and/or differentiative disorder.
  • the patient has cancer.
  • FIG. 1 shows an exemplary embodiment of a method for NK cell expansion and stimulation.
  • FIG. 2 shows key steps in the manufacture of the AB-101 drug product.
  • cryopreservation compositions e.g., cryopreservation compositions suitable for direct intravenous infusion into a subject, e.g., a human subject, as well as compositions comprising the cryopreservation compositions described herein and cells, e.g., therapeutic cells.
  • the compositions described herein find use as a cryopreservation composition, e.g., to preserve the viability of cells in compositions comprising the cryopreservation compositions described herein and cells.
  • the compositions comprising the cryopreservation compositions described herein and cells can, but need not be, administered directly to a patient, e.g., by intravenous infusion.
  • the cells are selected from the group consisting of animal cell(s), plant cell(s), fungal cell(s), bacterial cell(s), protist cell(s), and combinations thereof.
  • the animal cell(s) are mammalian cell(s).
  • the mammalian cell(s) are selected from the group consisting of human cell(s), monkey cell(s), pig cell(s), horse cell(s), cow cell(s), sheep cell(s), dog cell(s), cat cell(s), mouse cell(s), hamster cell(s), rat cell(s), rabbit cell(s), and combinations thereof.
  • the cell(s) are immune cell(s).
  • the immune cell(s) are selected from basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.
  • the cell(s) are selected from the group consisting of tumor cell(s), blood cell(s), stem cell(s), epithelial cell(s), primary cell(s), and combinations thereof.
  • the cell(s) are selected from the group consisting of peripheral blood cell(s), peripheral blood lymphocytes (PBLs), peripheral blood mononuclear cell(s) (PBMCs), bone marrow cell(s), umbilical cord blood cell(s), isolated NK cell(s), NK cell(s) derived from induced pluripotent stem cell(s), NK cell(s) derived from embryonic stem cell(s), and combinations thereof.
  • PBLs peripheral blood lymphocytes
  • PBMCs peripheral blood mononuclear cell
  • bone marrow cell(s) bone marrow cell(s)
  • umbilical cord blood cell(s) isolated NK cell(s), NK cell(s) derived from induced pluripotent stem cell(s), NK cell(s) derived from embryonic stem cell(s), and combinations thereof.
  • the NK cell(s) are CAR-NK cell(s).
  • the T cell(s) are CAR-T cell(s).
  • the cells are therapeutic cells.
  • the cell(s) are from a cell line, e.g., an immortalized cell line.
  • the cell(s) are feeder cell(s).
  • the feeder cell(s) are selected from the group consisting of peripheral blood mononuclear cell(s) (PBMC), Epstein-Barr virus-transformed B-lymphoblastoid cell(s) (e.g., EBV-LCL), myelogenous leukemia cell(s) (e.g., K562), and CD4(+) T cell(s) (e.g., HuT), and derivatives thereof.
  • the feeder cell(s) are inactivated CD4(+) T cell(s).
  • the inactivated CD4(+) T cell(s) are HuT-78 cells (ATCC® TIB-161TM) or variants or derivatives thereof.
  • the HuT-78 derivative is H9 (ATCC® HTB-176TM).
  • the inactivated CD4(+) T cell(s) are HuT-78 (ATCC® TIB- 161TM) cells or variants or derivatives thereof that express an ortholog of OX40L, or variant thereof.
  • the feeder cells are HuT-78 cells engineered to express at least one gene selected from the group consisting of an 4-1BBL ortholog or variant thereof, a membrane bound IL-21 ortholog or variant thereof, and membrane bound TNFalpha ortholog, or variant thereof.
  • cryopreservation compositions suitable for intravenous administration, e.g., intravenous administration of immune cells, e.g., NK cells, e.g., CAR-NK cells.
  • a pharmaceutical composition comprises the cry opreservation composition and cells, e.g., the NK cells described herein.
  • the cryopreservation composition comprises albumin protein, e.g., human albumin protein (UniProtKB Accession P0278, SEQ ID NO: 1) or variant thereof.
  • the cryopreservation composition comprises an ortholog of an albumin protein, e.g., human albumin protein, or variant thereof.
  • the cryopreservation composition comprises a biologically active portion of an albumin protein, e.g., human albumin, or variant thereof.
  • the albumin e.g., human albumin
  • the cryopreservation composition is or comprises an albumin solution, e.g., a human albumin solution.
  • the albumin solution is a serum-free albumin solution.
  • the albumin solution is suitable for intravenous use.
  • the albumin solution comprises from or from about 40 to or to about 200 g/L albumin.
  • the albumin solution comprises from or from about 40 to or to about 50 g/L albumin, e.g., human albumin.
  • the albumin solution comprises about 200 g/L albumin, e.g., human albumin.
  • the albumin solution comprises 200 g/L albumin, e.g., human albumin.
  • the albumin solution comprises a protein composition, of which 95% or more is albumin protein, e.g., human albumin protein. In some embodiments, 96%, 97%, 98%, or 99% or more of the protein is albumin, e.g., human albumin.
  • the albumin solution further comprises sodium. In some embodiments, the albumin solution comprises from or from about 100 to or to about 200 mmol sodium. In some embodiments, the albumin solution comprises from or from about 130 to or to about 160 mmol sodium.
  • the albumin solution further comprises potassium. In some embodiments, the albumin solution comprises 3 mmol or less potassium. In some embodiments, the albumin solution further comprises 2 mmol or less potassium.
  • the albumin solution further comprises one or more stabilizers.
  • the stabilizers are selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as acetyl tryptophan, N-Acetyl-L-tryptophan and Acetyl-L-tryptophan), 2-acetamido-3-(1H-indol- 3-yl)propanoic acid (also referred to as N-acetyltryptophan, DL-Acetyltroptohan and N-Acetyl- DL-tryptophan).
  • the solution comprises less than 0.1 mmol of each of the one or more stabilizers per gram of protein in the solution. In some embodiments, the solution comprises from or from about 0.05 to or to about 0.1, e.g., from or from about 0.064 to or to about 0.096 mmol of each of the stabilizers per gram of protein in the solution. In some embodiments, the solution comprises less than 0.1 mmol of total stabilizer per gram of protein in the solution. In some embodiments, the solution comprises from or from about 0.05 to or to about 0.1, e.g., from or from about 0.064 to or to about 0.096 mmol of total stabilizer per gram of protein in the solution.
  • the albumin solution consists of a protein composition, of which 95% or more is albumin protein, sodium, potassium, and one or more stabilizers selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3- yl)propanoic acid (also referred to as acetyl tryptophan, N-Acetyl-L-tryptophan and Acetyl-L- tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as N- acetyltryptophan, DL-Acetyltroptohan and N- Acetyl -DL-tryptophan) in water.
  • stabilizers selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3- yl)propanoic acid (also referred
  • the cryopreservation composition comprises from or from about 10% v/v to or to about 50% v/v of an albumin solution, e.g., an albumin solution described herein.
  • the cryopreservation composition comprises from or from about 10% to or to about 50%, from or from about 10% to or to about 45%, from or from about 10% to or to about 40%, from or from about 10% to or to about 35%, from or from about 10% to or to about 30%, from or from about 10% to or to about 25%, from or from about 10% to or to about 20%, from or from about 10% to or to about 15%, from or from about 15% to or to about 50%, from or from about 15% to or to about 45%, from or from about 15% to or to about 40%, from or from about 15% to or to about 35%, from or from about 15% to or to about 30%, from or from about 15% to or to about 25%, from or from about 15% to or to about 20%, from or from about 20% to or to about 50%, from or from about 20% to or to or to or to about 20% to or to or
  • the cryopreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v/v of an albumin solution described herein. In some embodiments, the cry opreservation composition comprises 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v of an albumin solution described herein.
  • the cryopreservation composition comprises from or from about 20 to or to about 100 g/L albumin, e.g., human albumin.
  • the cryopreservation composition comprises from or from about 20 to or to about 100, from or from about 20 to or to about 90, from or from about 20 to or to about 80, from or from about 20 to or to about 70, from or from about 20 to or to about 60, from or from about 20 to or to about 50, from or from about 20 to or to about 40, from or from about 20 to or to about 30, from or from about 30 to or to about 100, from or from about 30 to or to about 90, from or from about 30 to or to about 80, from or from about 30 to or to about 70, from or from about 30 to or to about 60, from or from about 30 to or to about 50, from or from about 30 to or to about 40, from or from about 40 to or to about 100, from or from about 40 to or to about 90, from or from about 40 to or to about 80, from
  • the cryopreservation composition comprises 20 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises 40 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises 70 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises 100 g/L albumin, e.g., human albumin.
  • the cryopreservation composition comprises about 20 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises about 40 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises about 70 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises about 100 g/L albumin, e.g., human albumin.
  • the cryopreservation composition further comprises a stabilizer, e.g., an albumin stabilizer.
  • the stabilizers are selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3- yl)propanoic acid (also referred to as acetyl tryptophan, N-Acetyl-L-tryptophan and Acetyl-L- tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as N- acetyltryptophan, DL-Acetyltroptohan and N-Acetyl-DL-tryptophan).
  • the cryopreservation composition comprises less than .1 mmol of each of the one or more stabilizers per gram of protein, e.g., per gram of albumin protein, in the composition. In some embodiments, the cryopreservation composition comprises from or from about 0.05 to or to about 0.1, e.g., from or from about 0.064 to or to about 0.096 mmol of each of the stabilizers per gram of protein, e.g., per gram of albumin protein in the composition. In some embodiments, the cryopreservation composition comprises less than 0.1 mmol of total stabilizer per gram of protein, e.g., per gram of albumin protein in the cryopreservation composition.
  • the cryopreservation composition comprises from or from about 0.05 to or to about 0.1, e.g., from or from about 0.064 to or to about 0.096 mmol of total stabilizer per gram of protein, e.g., per gram of albumin protein, in the cryopreservation composition.
  • the cryopreservation composition comprises Dextran, or a derivative thereof.
  • Dextran is a polymer of anhydroglucose composed of approximately 95% a-D-(1-6) linkages (designated (C 6 H 10 O 5 ) n ).
  • Dextran fractions are supplied in molecular weights of from about 1,000 Daltons to about 2,000,000 Daltons. They are designated by number (Dextran X), e.g., Dextran 1, Dextran 10, Dextran 40, Dextran 70, and so on, where X corresponds to the mean molecular weight divided by 1,000 Daltons. So, for example, Dextran 40 has an average molecular weight of or about 40,000 Daltons.
  • the average molecular weight of the dextran is from or from about 1,000 Daltons to or to about 2,000,000 Daltons. In some embodiments, the average molecular weight of the dextran is or is about 40,000 Daltons. In some embodiments, the average molecular weight of the dextran is or is about 70,000 Daltons.
  • the dextran is selected from the group consisting of Dextran 40, Dextran 70, and combinations thereof. In some embodiments, the dextran is Dextran 40.
  • the dextran e.g., Dextran 40
  • the composition comprises a dextran solution, e.g., a Dextran 40 solution.
  • the dextran solution is suitable for intravenous use.
  • the dextran solution comprises about 5% to about 50% w/w dextran, e.g., Dextran 40.
  • the dextran solution comprises from or from about 5% to or to about 50%, from or from about 5% to or to about 45%, from or from about 5% to or to about 40%, from or from about 5% to or to about 35%, from or from about 5% to or to about 30%, from or from about 5% to or to about 25%, from or from about 5% to or to about 20%, from or from about 5% to or to about 15%, from or from about 5% to or to about 10%, from or from about 10% to or to about 50%, from or from about 10% to or to about 45%, from or from about 10% to or to about 40%, from or from about 10% to or to about 35%, from or from about 10% to or to about 30%, from or from about 10% to or to about 25%, from or from about 10% to or to about 20%, from or from about 10% to or to about 15%, from or from about or from about 5% to or to about 10%,
  • the dextran solution comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% w/w dextran, e.g., Dextran 40.
  • the dextran solution comprises from or from about 25 g/L to or to about 200 g/L dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises from or from about 35 to or to about 200, from or from about 25 to or to about 175, from or from about 25 to or to about 150, from or from about 25 to or to about 125, from or from about 25 to or to about 100, from or from about 25 to or to about 75, from or from about 25 to or to about 50, from or from about 50 to or to about 200, from or from about 50 to or to about 175, from or from about 50 to or to about 150, from or from about 50 to or to about 125, from or from about 50 to or to about 100, from or from about 50 to or to about 75, from or from about 75 to or to about 200, from or from about 75 to or to about 175, from or from about 75 to or to about 150, from or from about 75 to or to about 125, from or from about 75 to
  • the dextran solution comprises 25, 50, 75, 100, 125, 150, 175, or 200 g/L dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises 100 g/L dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises about 25, about 50, about 75, about 100, about 125, about 150, about 175, or about 200 g/L dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises about 100 g/L dextran, e.g., Dextran 40.
  • the dextran solution further comprises glucose (also referred to as dextrose).
  • the dextran solution comprises from or from about 10 g/L to or to about 100 g/L glucose.
  • the dextran solution comprises from or from about 10 to or to about 100, from or from about 10 to or to about 90, from or from about 10 to or to about 80, from or from about 10 to or to about 70, from or from about 10 to or to about 60, from or from about 10 to or to about 50, from or from about 10 to or to about 40, from or from about 10 to or to about 30, from or from about 10 to or to about 20, from or from about 20 to or to about 100, from or from about 20 to or to about 90, from or from about 20 to or to about 80, from or from about 20 to or to about 70, from or from about 20 to or to about 60, from or from about 20 to or to about 50, from or from about 20 to or to about 40, from or from about 20 to or to about 30, from
  • the dextran solution comprises 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g/L glucose. In some embodiments, the dextran solution comprises 50 g/L glucose. In some embodiments, the dextran solution comprises about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 g/L glucose. In some embodiments, the dextran solution comprises 50 g/L glucose.
  • the dextran solution consists of dextran, e.g., Dextran 40, and glucose in water.
  • the cryopreservation composition comprises from or from about 10% v/v to or to about 50% v/v of a dextran solution described herein. In some embodiments, the cryopreservation composition comprises from or from about 10% to 50%, from or from about 10% to or to about 45%, from or from about 10% to or to about 40%, from or from about 10% to or to about 35%, from or from about 10% to or to about 30%, from or from about 10% to or to about 25%, from or from about 10% to or to about 20%, from or from about 10% to or to about 15%, from or from about 15% to or to about 50%, from or from about 15% to or to about 45%, from or from about 15% to or to about 40%, from or from about 15% to or to about 35%, from or from about 15% to or to about 30%, from or from about 15% to or to about 25%, from or from about 15% to or to about 20%, from or from about 20% to or to about 50%, from or from about 20% to or to about 45%, from or from about 20% to or to or to about 50%, from or
  • the cryopreservation composition comprises 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v of a dextran solution, e.g., a dextran solution described herein.
  • the cry opreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v/v of a dextran solution, e.g., a dextran solution described herein.
  • the cryopreservation composition comprises from or from about 10 to or to about 50 g/L dextran, e.g., Dextran 40.
  • the cryopreservation composition comprises from or from about 10 to or to about 50, from or from about 10 to or to about 45, from or from about 10 to or to about 40, from or from about 10 to or to about 35, from or from about 10 to or to about 30, from or from about 10 to or to about 25, from or from about 10 to or to about 20, from or from about 10 to or to about 15, from or from about 15 to or to about 50, from or from about 15 to or to about 45, from or from about 15 to or to about 40, from or from about 15 to or to about 35, from or from about 15 to or to about 30, from or from about 15 to or to about 25, from or from about 15 to or to about 20, from or from about 20 to or to about 50, from or from about 20 to or to about 45, from or from about 20 to or to about 40, from or from about 20
  • the cryopreservation composition comprises 10, 15, 20, 25, 30, 30, 35, 40, 45, or 50 g/L dextran, e.g., Dextran 40. In some embodiments, the cryopreservation composition comprises about 10, about 15, about 20, about 25, about 30, about 30, about 35, about 40, about 45, or about 50 g/L dextran, e.g., Dextran 40.
  • the cry opreservation composition comprises glucose
  • the cryopreservation composition comprises a Dextran solution comprising glucose.
  • the cryopreservation composition comprises a Dextran solution that does not comprise glucose.
  • glucose is added separately to the cryopreservation composition.
  • the cryopreservation composition comprises from or from about 5 to or to about 25 g/L glucose. In some embodiments, the cryopreservation composition comprises from or from about 5 to or to about 25, from or from about 5 to or to about 20, from or from about 5 to or to about 15, from or from about 5 to or to about 10, from or from about 10 to or to about 25, from or from about 10 to or to about 20, from or from about 10 to or to about 15, from or from about 15 to or to about 25, from or from about 15 to or to about 20, or from or from about 20 to or to about 25 g/L glucose. In some embodiments, the cryopreservation composition comprises 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, or 25 g/L glucose.
  • the cryopreservation composition comprises 12.5 g/L glucose. In some embodiments, the cryopreservation composition comprises about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, about 22.5, or about 25 g/L glucose. In some embodiments, the cryopreservation composition comprises about 12.5 g/L glucose.
  • the cry opreservation composition comprises less than 2.75% w/v glucose. In some embodiments, the cryopreservation composition comprises less than 27.5 g/L glucose. In some embodiments, the cryopreservation composition comprises less than 2% w/v glucose. In some embodiments, the cry opreservation composition comprises less than 1.5% w/v glucose. In some embodiments, the cryopreservation composition comprises about 1.25% w/v or less glucose.
  • the cryopreservation composition comprises dimethyl sulfoxide (DMSO, also referred to as methyl sulfoxide and methylsulfinylmethane).
  • DMSO dimethyl sulfoxide
  • methyl sulfoxide and methylsulfinylmethane dimethyl sulfoxide
  • the DMSO is provided as a solution, also referred to herein as a DMSO solution.
  • the cryopreservation composition comprises a DMSO solution.
  • the DMSO solution is suitable for intravenous use.
  • the DMSO solution comprises 1.1 g/mL DMSO. In some embodiments, the DMSO solution comprises about 1.1 g/mL DMSO.
  • the cry opreservation composition comprises from or from about 1% to or to about 10% v/v of the DMSO solution. In some embodiments, the cry opreservation composition comprises from or from about 1% to or to about 10%, from or from about 1% to or to about 9%, from or from about 1% to or to about 8%, from or from about 1% to or to about 7%, from or from about 1% to or to about 6%, from or from about 1% to or to about 5%, from or from about 1% to or to about 4%, from or from about 1% to or to about 3%, from or from about 1% to or to about 2%, from or from about 2% to or to about 10%, from or from about 2% to or to about 9%, from or from about 8%, from or from about 2% to or to about 7%, from or from about 2% to or to about 6%, from or from about 2% to or to about 5%, from or from about 2% to or to about 4%, from or from or from or from about 1% to
  • the cryopreservation composition comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% v/v of the DMSO solution. In some embodiments, the cryopreservation composition comprises 5% of the DMSO solution. In some embodiments, the cryopreservation composition comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% v/v of the DMSO solution. In some embodiments, the cry opreservation composition comprises about 5% of the DMSO solution.
  • the cryopreservation composition comprises from or from about 11 to or to about 110 g/L DMSO. In some embodiments, from or from about the cryopreservation composition comprises from or from about 11 to or to about 110, from or from about 11 to or to about 99, from or from about 11 to or to about 88, from or from about 11 to or to about 77, from or from about 11 to or to about 66, from or from about 11 to or to about 55, from or from about 11 to or to about 44, from or from about 11 to or to about 33, from or from about 11 to or to about 22, from or from about 22 to or to about 110, from or from about 22 to or to about 99, from or from about 22 to or to about 88, from or from about 22 to or to about 77, from or from about 22 to or to about 77, from or from about 22 to or to about 66, from or from about 22 to or to about 55, from or from about 22 to or to about 44, from or from about 22 to or to about 33
  • the cryopreservation composition comprises 11, 22, 33, 44, 55, 66, 77, 88, 99, or 110 g/L DMSO. In some embodiments, the cryopreservation composition comprises 55 g/L DMSO. In some embodiments, the cryopreservation composition comprises about 11, about 22, about 33, about 44, about 55, about 66, about 77, about 88, about 99, or about 110 g/L DMSO.
  • the cryopreservation composition comprises about 55 g/L DMSO.
  • the cry opreservation composition comprises a buffer solution, e.g., a buffer solution suitable for intravenous administration.
  • Buffer solutions include, but are not limited to, phosphate buffered saline (PBS), Ringer’s Solution, Tyrode’s buffer, Hank’s balanced salt solution, Earle’s Balanced Salt Solution, saline, and Tris.
  • the buffer solution is phosphate buffered saline (PBS).
  • the cryopreservation composition comprises or consists of: 1) albumin, e.g., human albumin, 2) dextran, e.g., Dextran 40, 3) DMSO, and 4) a buffer solution.
  • the cryopreservation composition further comprises glucose.
  • the cryopreservation composition consists of 1) albumin, e.g., human albumin, 2) dextran, e.g., Dextran 40, 3) glucose, 4) DMSO, and 5) a buffer solution.
  • the cryopreservation composition comprises: 1) an albumin solution described herein, 2) a dextran solution described herein, 3) a DMSO solution described herein, and 4) a buffer solution.
  • the cryopreservation composition consists of: 1) an albumin solution described herein, 2) a dextran solution described herein, 3) a DMSO solution described herein, and 4) a buffer solution.
  • the cryopreservation composition does not comprise a cell culture medium.
  • the cryopreservation composition comprises or comprises about 40 mg/mL human albumin, 25 mg/mL Dextran 40, 12.5 mg/mL glucose, and 55 mg/mL DMSO.
  • the cryopreservation composition comprises or comprises about or consists of or consists of about 40 mg/mL human albumin, 25 mg/mL Dextran 40, 12.5 mg/mL glucose, 55 mg/mL DMSO, and 0.5 mL/mL 100% phosphate buffered saline (PBS) in water.
  • PBS phosphate buffered saline
  • the cryopreservation composition comprises or comprises about 32 mg/mL human albumin, 25 mg/mL Dextran 40, 12.5 mg/mL glucose, and 55 mg/mL DMSO.
  • the cryopreservation composition comprises or comprises about or consists of or consists of about of 32 mg/mL human albumin, 25 mg/mL Dextran 40, 12.5 mg/mL glucose, 55 mg/mL DMSO, and 0.54 mL/mL 100% phosphate buffered saline (PBS) in water.
  • PBS phosphate buffered saline
  • cryopreserving the cell(s) comprises: mixing the cell(s) with a cryopreservation composition or components thereof described herein to produce a composition, e.g., a pharmaceutical composition; and freezing the mixture.
  • cryopreserving the cell(s) comprises: mixing a composition comprising the cell(s) with a cryopreservation composition or components thereof described herein to produce a composition, e.g., a pharmaceutical composition; and freezing the mixture.
  • the composition comprising the cell(s) comprises: the cell(s) and a buffer. Suitable buffers are described herein.
  • cryopreserving the cell(s) comprises: mixing a composition comprising the cell(s) and a buffer, e.g., PBS, with a composition comprising albumin, Dextran, and DMSO, e.g., as described herein; and freezing the mixture.
  • a buffer e.g., PBS
  • cryopreserving the cell(s) comprises: mixing a composition comprising the cell(s) and a buffer, e.g., PBS 1 : 1 with a composition comprising 40 mg/mL albumin, e.g., human albumin, 25 mg/mL Dextran, e.g., Dextran 40, 12.5 mg/mL glucose and 55 mg/mL DMSO.
  • a buffer e.g., PBS 1 : 1
  • a composition comprising 40 mg/mL albumin, e.g., human albumin, 25 mg/mL Dextran, e.g., Dextran 40, 12.5 mg/mL glucose and 55 mg/mL DMSO.
  • the composition comprising the cell(s) and the buffer comprises from or from about 2x10 4 to or to about 2x10 10 cells/mL. In some embodiments, the composition comprising the cell(s) and the buffer, e.g., PBS, comprises 2x10 8 cells/mL. In some embodiments, the composition comprising the cell(s) and the buffer, e.g., PBS, comprising about 2x10 8 cells/mL, but the concentration may vary depending on the type of cell(s).
  • cryopreserving the cell(s) comprising mixing: the cell(s), a buffer, e.g., PBS, albumin, e.g., human albumin, Dextran, e.g., Dextran 40, and DMSO; and freezing the mixture.
  • a buffer e.g., PBS
  • albumin e.g., human albumin
  • Dextran e.g., Dextran 40
  • DMSO DMSO
  • the mixture comprises from or from about 1x10 4 to or to about 1x10 10 cells/mL. In some embodiments, the mixture comprises 1x10 8 cells/mL. In some embodiments, the mixture comprises about 1x10 8 cells/mL, but the concentration may vary depending on the type of cell(s).
  • albumin Suitable ranges for albumin, Dextran, and DMSO are set forth above.
  • the composition is frozen at or below -135°C.
  • the composition is frozen at a controlled rate.
  • IV. CELL COMPOSITIONS / PHARMACEUTICAL COMPOSITIONS
  • cell composition comprising cell(s) and a cryopreservation composition as described herein.
  • the cell composition is a pharmaceutical composition.
  • compositions typically include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the pharmaceutical composition comprises: a) cell(s) described herein; and b) a cryopreservation composition described herein.
  • the pharmaceutical composition comprises: a) a cryopreservation composition described herein; and b) therapeutic cell(s).
  • the therapeutic cell(s) are animal cell(s). In some embodiments, the therapeutic cell(s) are human cell(s).
  • the therapeutic cell(s) are immune cell(s).
  • the immune cell(s) are selected from basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.
  • the pharmaceutical composition further comprises: c) a buffer solution.
  • Suitable buffer solutions are described herein, e.g., as for cryopreservation compositions.
  • the pharmaceutical composition comprises from or from about 1x10 4 to or to about 1x10 10 cells/mL. In some embodiments, the mixture comprises 1x10 8 cells/mL. In some embodiments, the pharmaceutical composition comprises about 1x10 8 cells/mL.
  • the pharmaceutical composition further comprises an antibody or antigen binding fragment thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • determining means determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
  • in vivo is used to describe an event that takes place in a subject’s body.
  • ex vivo is used to describe an event that takes place outside of a subject’s body.
  • An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.
  • An example of an ex vivo assay performed on a sample is an “in vitro” assay.
  • in vitro is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained.
  • in vitro assays can encompass cell-based assays in which living or dead cells are employed.
  • In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
  • the term “about” a number refers to that number plus or minus 10% of that number.
  • the term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.
  • buffer solution refers to an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa.
  • cell culture medium refers to a mixture for growth and proliferation of cells in vitro, which contains essential elements for growth and proliferation of cells such as sugars, amino acids, various nutrients, inorganic substances, etc.
  • a buffer solution as used herein, is not a cell culture medium.
  • cry opreservation compositions described herein find use in methods of treating disorders, e.g., cancer or other proliferative disorders.
  • a pharmaceutical composition comprising a cryopreservation composition described herein and cells, e.g., therapeutic cells.
  • Also provided herein are methods for inducing the immune system in a subject in need thereof comprising administering a pharmaceutical composition comprising a cryopreservation composition described herein and cells.
  • the methods described herein include methods for the treatment of disorders associated with abnormal apoptotic or differentiative processes, e.g., cellular proliferative disorders or cellular differentiative disorders, e.g., cancer, including both solid tumors and hematopoietic cancers.
  • the methods include administering a therapeutically effective amount of a treatment as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
  • treatment “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disorder associated with abnormal apoptotic or differentiative processes.
  • a treatment can result in a reduction in tumor size or growth rate.
  • Administration of a therapeutically effective amount of a compound described herein for the treatment of a condition associated with abnormal apoptotic or differentiative processes will result in a reduction in tumor size or decreased growth rate, a reduction in risk or frequency of reoccurrence, a delay in reoccurrence, a reduction in metastasis, increased survival, and/or decreased morbidity and mortality, among other things.
  • treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
  • the terms “inhibition”, as it relates to cancer and/or cancer cell proliferation, refer to the inhibition of the growth, division, maturation or viability of cancer cells, and/or causing the death of cancer cells, individually or in aggregate with other cancer cells, by cytotoxicity, nutrient depletion, or the induction of apoptosis.
  • “delaying” development of a disease or disorder, or one or more symptoms thereof means to defer, hinder, slow, retard, stabilize and/or postpone development of the disease, disorder, or symptom thereof. This delay can be of varying lengths of time, depending on the history of the disease and/or subject being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the subject does not develop the disease, disorder, or symptom thereof.
  • a method that “delays” development of cancer is a method that reduces the probability of disease development in a given time frame and/or reduces extent of the disease in a given time frame, when compared to not using the method. Such comparisons may be based on clinical studies, using a statistically significant number of subjects.
  • prevention refers to a regimen that protects against the onset of the disease or disorder such that the clinical symptoms of the disease do not develop.
  • prevention relates to administration of a therapy (e.g., administration of a therapeutic substance) to a subject before signs of the disease are detectable in the subject and/or before a certain stage of the disease (e.g., administration of a therapeutic substance to a subject with a cancer that has not yet metastasized).
  • the subject may be an individual at risk of developing the disease or disorder, or at risk of disease progression, e.g., cancer metastasis. Such as an individual who has one or more risk factors known to be associated with development or onset of the disease or disorder.
  • an individual may have mutations associated with the development or progression of a cancer. Further, it is understood that prevention may not result in complete protection against onset of the disease or disorder. In some instances, prevention includes reducing the risk of developing the disease or disorder. The reduction of the risk may not result in complete elimination of the risk of developing the disease or disorder.
  • An “increased” or “enhanced” amount refers to an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) an amount or level described herein.
  • It may also include an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% of an amount or level described herein.
  • a “decreased” or “reduced” or “lesser” amount refers to a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6
  • an amount or level described herein may also include a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% of an amount or level described herein.
  • Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias.
  • a metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.
  • cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state.
  • pathologic i.e., characterizing or constituting a disease state
  • non-pathologic i.e., a deviation from normal but not associated with a disease state.
  • the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
  • cancer or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renalcell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • the disease is renal carcinoma or melanoma.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • sarcoma is art recognized and refers to malignant tumors of mesenchymal derivation.
  • hematopoietic neoplastic disorders includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia.
  • myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol ./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • HLL hairy cell leukemia
  • malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.
  • the cancer is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, typical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid, cardiac tumors, medulloblastoma, germ cell tumor, primary CNS lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lympho
  • ALL acute lymphoblastic
  • the cancer is a solid tumor.
  • the cancer is metastatic.
  • the methods of treatment provided herein may be used to treat a subject (e.g., human, monkey, dog, cat, mouse) who has been diagnosed with or is suspected of having a cellular proliferative and/or differentiative disorder, e.g., a cancer.
  • a subject e.g., human, monkey, dog, cat, mouse
  • the subject is a mammal.
  • the subject is a human.
  • a subject refers to a mammal, including, for example, a human.
  • the mammal is selected from the group consisting of an armadillo, an ass, a bat, a bear, a beaver, a cat, a chimpanzee, a cow, a coyote, a deer, a dog, a dolphin, an elephant, a fox, a panda, a gibbon, a giraffe, a goat, a gopher, a hedgehog, a hippopotamus, a horse, a humpback whale, a jaguar, a kangaroo, a koala, a leopard, a lion, a llama, a lynx, a mole, a monkey, a mouse, a narwhal, an orangutan, an orca, an otter, an ox, a pig, a polar bear, a porcupine, a puma,
  • the mammal is a human.
  • the subject e.g., the human subject
  • the subject can be a youth, e.g., from or from about 15 to or to about 24 years in age.
  • the subject can be an adult, e.g., from or from about 25 to or to about 64 years in age.
  • the subject can be a senior, e.g, 65+ years in age.
  • the subject may be a human who exhibits one or more symptoms associated with a cellular proliferative and/or differentiative disorder, e.g., a cancer, e.g., a tumor.
  • a cancer e.g., a tumor.
  • Any of the methods of treatment provided herein may be used to treat cancer at various stages.
  • the cancer stage includes but is not limited to early, advanced, locally advanced, remission, refractory, reoccurred after remission and progressive.
  • the subject is at an early stage of a cancer.
  • the subject is at an advanced stage of cancer.
  • the subject has a stage I, stage n, stage III or stage IV cancer.
  • the methods of treatment described herein can promote reduction or retraction of a tumor, decrease or inhibit tumor growth or cancer cell proliferation, and/or induce, increase or promote tumor cell killing. I n some embodiments, the subject is in cancer remission. The methods of treatment described herein can prevent or delay metastasis or recurrence of cancer.
  • the subject is at risk, or genetically or otherwise predisposed (e.g., risk factor), to developing a cellular proliferative and/or differentiative disorder, e.g., a cancer, that has or has not been diagnosed.
  • a cellular proliferative and/or differentiative disorder e.g., a cancer
  • an “at risk” individual is an individual who is at risk of developing a condition to be treated, e.g., a cellular proliferative and/or differentiative disorder, e.g., a cancer.
  • a condition to be treated e.g., a cellular proliferative and/or differentiative disorder, e.g., a cancer.
  • an “at risk” subject may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein.
  • “At risk” denotes that an individual has one or more so-called risk factors, which are measurable parameters that correlate with development of a disease or condition and are known in the art.
  • an at risk subject may have one or more risk factors, which are measurable parameters that correlate with development of cancer.
  • risk factors may include, for example, age, sex, race, diet, history of previous disease, presence of precursor disease, genetic (e.g., hereditary) considerations, and environmental exposure.
  • the subjects at risk for cancer include, for example, those having relatives who have experienced the disease, and those whose risk is determined by analysis of genetic or biochemical markers.
  • the subject may be undergoing one or more standard therapies, such as chemotherapy, radiotherapy, immunotherapy, surgery, or combination thereof.
  • one or more kinase inhibitors may be administered before, during, or after administration of chemotherapy, radiotherapy, immunotherapy, surgery or combination thereof.
  • the subject may be a human who is (i) substantially refractory to at least one chemotherapy treatment, or (ii) is in relapse after treatment with chemotherapy, or both (i) and (ii). In some of embodiments, the subject is refractory to at least two, at least three, or at least four chemotherapy treatments (including standard or experimental chemotherapies). VII. TREATMENT OF CANCER WITH NK CELLS AND A CD20 TARGETED
  • NHLs are a heterogeneous group of lymphoproliferative malignancies that usually originate in lymphoid tissues and can spread to other organs. Prognosis for NHL patients depends on histologic type, stage, and response to treatment. NHL can be divided into 2 prognostic groups: the indolent lymphomas and the aggressive lymphomas. Indolent NHLs offer a relatively good prognosis with a median survival of up to 20 years and are generally responsive to immunotherapy, radiation therapy, and chemotherapy. However, a continuous rate of relapse is seen in advanced stages of indolent NHLs. In contrast, aggressive NHLs present acutely and are more commonly resistant or refractory to frontline therapy.
  • NHL patients with newly diagnosed NHL are treated with chemotherapy combined with rituximab that confers long-term remissions in most patients.
  • NHL patients who are refractory to front-line treatment or those who relapse soon after completing front-line therapies have poor outcomes.
  • ICE or DHAP second line of chemotherapy
  • mAb therapeutic monoclonal antibody
  • ASCT autologous stem cell transplant
  • CAR-T chimeric antigen receptor T- cell therapy
  • Described herein are methods for treating a patient suffering from a CD20+ cancer the methods include: administering allogenic natural killer cells (NK cells) and an antibody targeted to human CD20, wherein the NK cells are allogenic to the patient, are KIR-B haplotype and express CD16 having the V/V polymorphism at F158.
  • NK cells allogenic natural killer cells
  • an antibody targeted to human CD20 wherein the NK cells are allogenic to the patient, are KIR-B haplotype and express CD16 having the V/V polymorphism at F158.
  • the cancer is non-Hodgkins lymphoma (NHL) (e.g., indolent NHL or aggressive NHL); the patient has relapsed after treatment with an anti-CD20 antibody;patient has the experienced disease progression after treatment with autologous stem cell transplant or chimeric antigen receptor T-cell therapy (CAR-T); the patient is administered 1 x 10 8 to 1 x 10 10 NK cells; the patient is administered 1 x 10 9 to 8 x 10 9 NK cells; the patient is administered 4 x 10 8 , 1 x 10 9 , 4 x 10 9 , or 8 x 10 9 NK cells; 100 to 500 mg/m 2 of the antibody targeted to human CD20; each administration of NK cells is administration of 1 x 10 9 to 5 x 10 9 NK cells; each administration of NK cells is administration of 1 x 10 9 to 5 x 10 9 NK cells; the patient is administered 375 mg/m 2 of the antibody targeted to human CD20; the antibody targeted to human CD20 is
  • the lymphodepleting chemotherapy can include, in various embodiments: treatment with cyclophosphamide and fludarabine, administration of cyclophosphamide at between 100 and 500 mg/m 2 /day; administration of cyclophosphamide at 250 mg/m 2 /day; administration of fludarabine at between 10 and 50 mg/m 2 /day or at 30mg/m 2 /day.
  • the method further comprising administering IL-2 (e.g., a dose of 1 x 10 6 IU/m 2 of IL-2).
  • administration of IL-2 occurs within 1-4 hrs of administration of the NK cells.
  • the administration of the NK cells and the antibody targeted to human CD20 occurs weekly; the NK cells and the antibody targeted to human CD20 are administered weekly for 4 to 8 weeks; the NK cells are not genetically modified; at least 70% of the NK cells are CD56+ and CD16+; at least 85% of the NK cells are CD56+ and CD3-; 1% or less of the NK cells are CD3+, 1% or less of the NK cells are CD19+ and 1% or less of the NK cells are CD14+.
  • the indolent NHL is selected from the group consisting of Follicular lymphoma, Lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, Gastric MALT, Non-gastric MALT, Nodal marginal zone lymphoma, Splenic marginal zone lymphoma, Small-cell lymphocytic lymphoma (SLL), and Chronic lymphocytic lymphoma (CLL); the Small-cell lymphocytic lymphoma (SLL) or Chronic lymphocytic lymphoma (CLL) comprises nodal or splenic involvement; the aggressive NHL is selected from the group consisting of Diffuse large B-cell lymphoma, Mantle cell lymphoma, Transformed follicular lymphoma, Follicular lymphoma (Grade IIIB), Transformed mucosa-associated lymphoid tissue (MALT) lymphoma, Primary mediastinal B-cell lymphoma, Lymphoblastic lymph
  • Suitable NK cells for use in treatment of NHL can be prepared as described in US 2020/0108096 or WO 2020/101361, both of which are incorporated herein by refemce. Briefly, the source cells are cultured on modified HuT-78 (ATCC® TIB-161TM) cells that have been engineered to express 4-1BBL, membrane bound IL-21 and a mutant TNFalpha as described in US 2020/0108096. [0174J As one example, suitable NK cells can be prepared as follows using HuT-78 cells transduced to express 4-1BBL, membrane bound IL-21 and mutant TNFalpha (“eHut-78P cells”) as feeder cells.
  • the feeder cells are suspended in 1% (v/v) CellGro medium at 2.5x10 6 cells/ml and are irradiated with 20,000 cGy in a gamma-ray irradiator.
  • Seed cells e.g., CD3-depleted PBMC or CD3-depleted cord blood cells
  • CellGro medium containing 1% (v/v) human plasma, glutamine, 500 IU of IL-2, 10 ng/ml of OKT-3 at a ratio of 1:2.5 (seed cells: feeder cells) in in static culture at 37° C.
  • the cells are split every 2-4 days.
  • the total culture time can be 19 days.
  • the NK cells are harvested by centrifugation and cryopreserved.
  • Thawed NK are administration to patients in infusion medium consisting of Phosphate Buffered Saline (50% v/v) with albumin (human) 20% (20% v/v), Dextran 40 in Dextrose (25% v/v) and dimethyl sulfoxide (DMSO) (5% v/v).
  • the seed cells are CD3-depleted cord blood cells.
  • the cord blood seed cells are selected to express CD16 having the V/V polymorphism at F158 (Fc gamma RIIIa-158 V/V genotype) (Musolino et al. 2008 J Clin Oncol 26 :1789).
  • the cord blood seed cells are KIR-B haplotype.
  • a cell fraction can be depleted of CD3 cells by immunomagnetic selection, for example, using a CliniMACS T cell depletion set ((LS Depletion set (162-01) Miltenyi Biotec).
  • Rituximab e.g., Rituxan®
  • Rituximab is a preferred IL-20 targeted antibody.
  • Rituximab is preferably administered at 375 mg/m 2 , preferably at least 1 hour prior to each administration of NK cells.
  • IL-2 is preferably administered at 1 x 10 6 IU/m 2 , will be administered subcutaneously, at least 1 hour and no more than 4 hours following the conclusion of each administration.
  • the methods described herein can be used to treat patients suffering from a CD20+ cancer, for example, indolent or aggressive non-Hodgkin’s lymphoma (NHL), particularly relapsed or refractory indolent or aggressive NHL of B-cell origin.
  • a CD20+ cancer for example, indolent or aggressive non-Hodgkin’s lymphoma (NHL), particularly relapsed or refractory indolent or aggressive NHL of B-cell origin.
  • NHL non-Hodgkin’s lymphoma
  • NHL relapsed or refractory indolent or aggressive NHL of B-cell origin.
  • aggressive and indolent subtypes are those in Table 4.
  • the patient Prior to treatment, the patient is preferably lymphodepleted by intravenous administration of cyclophosphamide (250 mg/m 2 /day) and fludarabine (30 mg/m 2 /day) daily for
  • NK cells 3 consecutive days, starting 5 days before the first dose of NK cells (i.e., from Day -5 through Day -3).
  • the NK cells are preferably administered weekly with each administration of 1 x 10 9 or 4 x 10 9 NK cells.
  • the cells are preferably ciyopreserved NK cells suspended in infusion-ready media (50% PBS, 25%Dextran 40, 20% albumin (human), 5% DMSO) in vials containing approximately 1 x 10 9 cells.
  • the cells are thawed in a 37°C water bath prior to administration.
  • the thawed vial(s) of NK cells are aseptically transferred to a single administration bag using a vial adapter and a sterile syringe.
  • the NK cells are administered to the patient from the bag through a Y-type blood/solution set with filter as an IV infusion, by gravity.
  • the NK cells are preferably should be administered as soon as practical, preferably within 30 minutes and no longer than 90 minutes after thawing.
  • IL-2 dosed at 1 x 10 6 IU/m 2 , is administered subcutaneously, at least 1 hour and no more than 4 hours following the conclusion of each dose of NK cells.
  • Rituximab is preferably administered at 375 mg/m 2 , preferably at least 1 hour prior to each administration of NK cells.
  • Administration of the NK cells preferably occurs weekly for 8 weeks.
  • NK cells allogenic natural killer cells
  • an antibody targeted to human CD20 wherein the NK cells are allogenic to the patient, are KIR-B haplotype and express CD16 having the V/V polymorphism at F158.
  • the cancer is non-Hodgkins lymphoma (NHL).
  • NDL non-Hodgkins lymphoma
  • the NHL is indolent NHL.
  • the NHL is aggressive NHL.
  • the patient has relapsed after treatment with an anti-CD20 antibody.
  • the patient has experienced disease progression after treatment with autologous stem cell transplant or chimeric antigen receptor T-cell therapy (CAR-T).
  • CAR-T chimeric antigen receptor T-cell therapy
  • the patient is administered 1 x 10 8 to 1 x 10 10 NK cells.
  • the patient is administered 1 x 10 9 to 8 x 10 9 NK cells.
  • the patient is administered 4 x 10 8 , 1 x 10 9 , 4 x 10 9 , or 8 x 10 9
  • the patient is administered 100 to 500 mg/m 2 of the antibody.
  • the patient is administered 375 mg/m 2 of the antibody.
  • the antibody is rituximab.
  • the patient is subjected to lymphodepl eting chemotherapy prior to treatment.
  • the lymphodepleting chemotherapy is non-myeloablative chemotherapy.
  • the lymphodepleting chemotherapy comprises treatment with at least one of cyclophosphamide and fludarabine.
  • the lymphodepleting chemotherapy comprises treatment with cyclophosphamide and fludarabine.
  • the cyclophosphamide is administered between 100 and 500 mg/m 2 /day.
  • the cyclophosphamide is administered 250 mg/m 2 /day.
  • the fludarabine is administered between 10 and 50 mg/m 2 /day.
  • the fludarabine is administered 30mg/m 2 /day.
  • the method further comprises administering IL-2.
  • the patient is administered 1 x 10 6 IU/m 2 of IL-2.
  • administration of IL-2 occurs within 1-4 hrs of administration of the NK cells.
  • the administration of the NK cells and the antibody targeted to human CD20 occurs weekly.
  • the NK cells and the antibody targeted to human CD20 are administered weekly for 4 to 8 weeks.
  • the NK cells are not genetically modified.
  • At least 70% of the NK cells are CD56+ and CD16+.
  • At least 85% of the NK cells are CD56+ and CD3-.
  • the indolent NHL is selected from the group consisting of Follicular lymphoma, Lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, Gastric MALT, Non-gastric MALT, Nodal marginal zone lymphoma, Splenic marginal zone lymphoma, Small-cell lymphocytic lymphoma (SLL), and Chronic lymphocytic lymphoma (CLL).
  • the Small-cell lymphocytic lymphoma (SLL) or Chronic lymphocytic lymphoma (CLL) comprises nodal or splenic involvement.
  • the aggressive NHL is selected from the group consisting of Diffuse large B-cell lymphoma, Mantle cell lymphoma, Transformed follicular lymphoma, Follicular lymphoma (Grade IIIB), Transformed mucosa-associated lymphoid tissue (MALT) lymphoma, Primary mediastinal B-cell lymphoma, Lymphoblastic lymphoma, High-grade B-cell lymphomas with translocations of MYC and BCL2.
  • the high-grade B-cell lymphomas with translocations of MYC and BLC2 further comprises a translocation of BCL6.
  • each administration of NK cells is administration of 1 x 10 9 to 5 x 10 9 NK cells. In some embodiments, each administration of NK cells is administration of 1 x 10 9 to 5 x 10 9 NK cells.
  • a single unit of FDA-licensed, frozen cord blood that has a high affinity variant of the receptor CD16 (the 158 V/V variant, see, e.g., Koene et al., “Fc ⁇ RIIIa-158V/F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcgammaRIIIa, Independently of the FcgammaRIIIa-48L/R/H Phenotype,” Blood 90: 1109-14 (1997).) and the KIR-B genotype (KIR B allele of the KIR receptor family, see, e.g., Hsu et al., “The Killer Cell Immunoglobulin-Like Receptor (KIR) Genomic Region: Gene-Order, Haplotypes and Allelic Polymorphism,” Immunological Review 190:40-52 (2002); and Pyo et al., “Different Patterns of Evolution in the Centromeric and Telomeric Regions of Group A and B Haplo
  • the cord blood unit was thawed and the freezing medium was removed via centrifugation.
  • the cell preparation was then depleted of T cells using the QuadroMACS Cell Selection System (Miltenyi) and CD3 (T cell) MicroBeads.
  • T cells total nucleated cells
  • CD3 T cell
  • a population of 6 x 10 8 total nucleated cells (TNC) were labelled with the MicroBeads and separated using the QuadroMACS device and buffer.
  • the remaining cells which were predominantly monocytes and NK cells, were washed and collected in antibiotic-free medium (CellgroSCGM).
  • the cell preparation was then evaluated for total nucleated cell count, viability, and % CD3+ cells. As shown in FIG. 1, the cord blood NK cells were CD3 depleted.
  • the CD3- cell preparation was inoculated into a gas permeable cell expansion bag at 0.3 x 10 6 viable cells/mL containing growth medium. As shown in FIG. 1, the cells were cocultured with replication incompetent engineered HuT-78 (eHUT-78) feeder cells to enhance expansion for master cell bank (MCB) production.
  • the CellgroSCGM growth media was initially supplemented with 10 ng/mL of anti-CD3 antibody (OKT3), 1.0% (v/v) human plasma, 4 mM glutamine, and 80 ng/mL IL-2.
  • the NK cells are optionally engineered, e.g., to introduce CARs into the NK cells, e.g., with a lentaviral vector, during one of the co-culturing steps.
  • the cells were incubated as a static culture for 12-16 days at 37°C in a 5% CO2 balanced air environment, with additional exchanges of media occurring every 2 to 4 days. After the culture has expanded more than 100-fold, the cultured cells were harvested and then suspended in freezing medium at 1.0 x 10 8 cells/mL and filled into 5 mL cryobags. In this example, 80 bags or vials at 10 8 cells per bag or vial were produced during the co-culture. The cryobags were frozen using a controlled rate freezer and stored in vapor phase liquid nitrogen (LN 2 ) tanks below -150°C. These cryopreserved NK cells derived from the FDA-licensed cord blood unit served as the master cell bank (MCB).
  • MBC master cell bank
  • a bag of frozen cells from the MCB was thawed and the freezing medium was removed.
  • the thawed cells were inoculated into a disposable culture bag and co-cultured with feeder cells, e.g., eHUT78 feeder cells to produce the drug product.
  • the cells are cultured in a 50 L bioreactor to produce thousands of lots of the drug product per unit of cord blood (e.g., 4,000-8,000 cryovials at 10 9 cells/vial), which are mixed with a cryopreservation composition and frozen in a plurality of storage vessels such as cryovials.
  • the drug product is an off-the-shelf infusion ready product that can be used for direct infusion.
  • Each lot of the drug product can be used to infuse hundreds to thousands of patients (e.g., 100-1,000 patients, e.g. with a target dose of 4 x 10 9 cells).
  • Example 2 AB-101
  • AB-101 is a universal, off-the-shelf, cryopreserved allogeneic cord blood derived NK cell therapy product comprising ex vivo expanded and activated effector cells designed to enhance ADCC anti-tumor responses in patients, e.g., patients treated with monoclonal antibodies or NK cell engagers.
  • AB-101 is comprised of cord blood derived mononuclear cells (CBMCs) enriched for NK cells by depletion of T lymphocytes, and co-cultured with an engineered, replication incompetent T cell feeder line supplemented with IL-2 and anti-CD3 antibody (OKT3).
  • CBMCs cord blood derived mononuclear cells
  • AB-101 is an allogeneic NK-cell product derived from FDA licensed cord blood, specifically designed to treat hematological and solid tumors in combination with therapeutic monoclonal antibodies (mAbs).
  • mAbs therapeutic monoclonal antibodies
  • NK cell profile High surface receptor expression of antibody engaging CD 16 and tumor antigen-engaging/activating receptors such as NKG2D, NKp46, Nkp30 and NKp44.
  • KIR-B-haplotype has been associated with improved clinical outcomes in the haploidentical transplant setting and greater therapeutic potential in the allogeneic setting
  • CD16 F158V polymorphism The higher-affinity CD16 F158V variant binding to mAb Fc-domain is seen to facilitate enhanced antibody dependent cellular cytotoxicity (ADCC).
  • AB-101 is comprised of NK cells (CD16 + , CD56 + ) expressing the natural cytotoxicity receptors NKp30 and NKp46 indicative of mature NK cells.
  • AB-101 contains negligible T cells, B cells and macrophages ( ⁇ 0.2% CD3 + , ⁇ 1.0% CD19 + , ⁇ 1.0% CD14 + ).
  • Residual eHuT-78P feeder cells used in the culturing of AB-101 are ⁇ 0.2% of the drug product. Table 5.
  • Components and Compositions of AB-101 are listed in Table 5.
  • AB-101 cells were prepared by the process shown in FIG. 2. At the end of the culture period the cells were harvested through the use of a Sartorius kSep® 400 Single-Use Automated Centrifugation System at Relative Centrifugal Field (RCF): 800 - 1200 g with a flow rate at 60 to 120 mL/min, and washed two times with Phosphate Buffer Solution (PBS). After washing, the AB-101 cells were formulated with: (1) Albumin (human); (2) Dextran 40; (3) DMSO and (4) PBS to a target concentration of 1 x 10 8 cells/mL (exemplary cryopreservation composition #1, Table 2).
  • RCF Relative Centrifugal Field
  • the formulated suspension was then filled at a target volume of 11 mL into 10 mL AT-Closed vial®. Filled vials were inspected, labeled and cryopreserved in a controlled rate freezer at ⁇ -135°C.
  • the stability storage freezer is a validated vapor phase LN 2 storage freezer which is set to maintain a temperature of ⁇ -135°C.

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Abstract

Provided herein are cryopreservation compositions, e.g., infusion-ready cryopreservation compositions, pharmaceutical compositions comprising cryopreservation compositions, e.g., infusion-ready cryopreservation compositions, and methods of treating disorders comprising administering cryopreservation compositions, e.g., infusion-ready cryopreservation compositions, and therapeutic cells

Description

INFUSION READY CRYOPRESERVATION COMPOSITIONS
CLAIM OF PRIORITY
[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63/127,098, filed on December 17, 2020, and U.S. Provisional Application Serial No. 63/172,435, filed on April 8, 2021. The entire contents of the foregoing are incorporated herein by reference.
BACKGROUND
[0002] Cell based therapies such as natural killer (NK) cell therapy, e.g., CAR-NK cell therapy, T-cell therapy, e.g., CAR-T cell therapy, and others can be administered to patients intravenously. The cells may be stored frozen. Thus, there is a need for compositions that are suitable both for cryopreservation of cells and administration to patients, e.g., by intravenous infusion.
[0003] The present invention addresses these and other deficiencies in the art.
SUMMARY
[0004] Provided herein are cryopreservation compositions, e.g., infusion-ready cryopreservation compositions, pharmaceutical compositions comprising cryopreservation compositions, e.g., infusion-ready cryopreservation compositions, and methods of treating disorders comprising administering cryopreservation compositions, e.g., infusion-ready cryopreservation compositions, and therapeutic cells.
[0005] Provided herein are cryopreservation compositions comprising albumin, e.g., human albumin, dextran, e.g., Dextran 40, glucose, DMSO, and a buffer, e.g., phosphate buffered saline.
[0006] In some embodiments, the cryopreservation composition comprising: (a) about 40 mg/mL human albumin; (b) about 25 mg/mL Dextran 40; (c) about 12.5 mg/mL glucose; (d) about 55 mg/mL DMSO; and (e) about 0.5 mL/mL phosphate buffered saline.
[0007] In some embodiments, the cryopreservation composition further comprises (f) cells.
[0008] Also provided herein is a composition consisting of: (a) about 40 mg/mL human albumin; (b) about 25 mg/mL Dextran 40; (c) about 12.5 mg/mL glucose; (d) about 55 mg/mL DMSO; and (e) about 0.5 mL/mL phosphate buffered saline. [0009] Also provided herein are pharmaceutical compositions comprising a cry opreservation composition and cells, e.g., therapeutic cells.
[0010] In some embodiments, the pharmaceutical composition comprises: (a) human albumin; (b) dextran; (c) glucose; (d) DMSO; (e) a buffer; and (f) therapeutic cells.
[0011] In some embodiments, the pharmaceutical composition comprises from 30 to 50 mg/mL human albumin.
[0012] In some embodiments, the pharmaceutical composition comprises 50 mg/mL human albumin.
[0013] In some embodiments, the pharmaceutical composition comprises 20 to 30 mg/mL dextran.
[0014] In some embodiments, the pharmaceutical composition comprises 25 mg/mL dextran.
[0015] In some embodiments, the dextran is Dextran 40.
[0016] In some embodiments, the pharmaceutical composition comprises from 12 to 15 mg/mL glucose.
[0017] In some embodiments, the pharmaceutical composition comprises 12.5 mg/mL glucose.
[0018] In some embodiments, the pharmaceutical composition comprises less than 27.5 g/L glucose.
[0019] In some embodiments, the pharmaceutical composition comprises from 50 to 60 ml/mL DMSO.
[0020] In some embodiments, the pharmaceutical composition comprises 55 mg/mL DMSO.
[0021] In some embodiments, the pharmaceutical composition comprises 40 to 60 % v/v buffer.
[0022] In some embodiments, the pharmaceutical composition comprises the buffer is phosphate buffered saline.
[0023] In some embodiments, the therapeutic cells are animal cells.
[0024] In some embodiments, the animal cells are human cells.
[0025] In some embodiments, the therapeutic cells are immune cells.
[0026] In some embodiments, the immune cells are selected from the group consisting of basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.
[0027] In some embodiments, the immune cell is a T cell.
[0028] In some embodiments, the T cell is a CAR-T cell.
[0029] In some embodiments, the immune cell is an NK cell. [0030] In some embodiments, the NK cell is a CAR-NK cell.
[0031] In some embodiments, the pharmaceutical composition comprises: (a) about 40 mg/mL human albumin; (b) about 25 mg/mL Dextran 40; (c) about 12.5 mg/mL glucose;
[0032] (d) about 55 mg/mL DMSO; and (e) about 0.5 mL/mL phosphate buffered saline.
[0033] Also provided herein are methods for treating a disorder comprising: (a) administering a therapeutically effective amount of a pharmaceutical composition described herein to a patient in need thereof.
[0034] In some embodiments, the patient has a cellular proliferative and/or differentiative disorder.
[0035] In some embodiments, the patient has cancer.
[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
[0037] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
INCORPORATION BY REFERENCE
[0038] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [0040] FIG. 1 shows an exemplary embodiment of a method for NK cell expansion and stimulation.
[0041] FIG. 2 shows key steps in the manufacture of the AB-101 drug product.
DETAILED DESCRIPTION
[0042] Provided herein are, amongst other things, cryopreservation compositions, e.g., cryopreservation compositions suitable for direct intravenous infusion into a subject, e.g., a human subject, as well as compositions comprising the cryopreservation compositions described herein and cells, e.g., therapeutic cells. The compositions described herein find use as a cryopreservation composition, e.g., to preserve the viability of cells in compositions comprising the cryopreservation compositions described herein and cells. The compositions comprising the cryopreservation compositions described herein and cells can, but need not be, administered directly to a patient, e.g., by intravenous infusion.
I. CELLS
[0043] In some embodiments, the cells are selected from the group consisting of animal cell(s), plant cell(s), fungal cell(s), bacterial cell(s), protist cell(s), and combinations thereof.
[0044] In some embodiments, the animal cell(s) are mammalian cell(s).
[0045] In some embodiments, the mammalian cell(s) are selected from the group consisting of human cell(s), monkey cell(s), pig cell(s), horse cell(s), cow cell(s), sheep cell(s), dog cell(s), cat cell(s), mouse cell(s), hamster cell(s), rat cell(s), rabbit cell(s), and combinations thereof.
[0046] In some embodiments, the cell(s) are immune cell(s). In some embodiments, the immune cell(s) are selected from basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.
[0047] In some embodiments, the cell(s) are selected from the group consisting of tumor cell(s), blood cell(s), stem cell(s), epithelial cell(s), primary cell(s), and combinations thereof.
[0048] In some embodiments, the cell(s) are selected from the group consisting of peripheral blood cell(s), peripheral blood lymphocytes (PBLs), peripheral blood mononuclear cell(s) (PBMCs), bone marrow cell(s), umbilical cord blood cell(s), isolated NK cell(s), NK cell(s) derived from induced pluripotent stem cell(s), NK cell(s) derived from embryonic stem cell(s), and combinations thereof.
[0049] In some embodiments, the NK cell(s) are CAR-NK cell(s). In some embodiments, the T cell(s) are CAR-T cell(s). [0050] In some embodiments, the cells are therapeutic cells.
[0051] In some embodiments, the cell(s) are from a cell line, e.g., an immortalized cell line.
[0052] In some embodiments, the cell(s) are feeder cell(s). In some embodiments, the feeder cell(s) are selected from the group consisting of peripheral blood mononuclear cell(s) (PBMC), Epstein-Barr virus-transformed B-lymphoblastoid cell(s) (e.g., EBV-LCL), myelogenous leukemia cell(s) (e.g., K562), and CD4(+) T cell(s) (e.g., HuT), and derivatives thereof. In some embodiments, the feeder cell(s) are inactivated CD4(+) T cell(s). In some embodiments, the inactivated CD4(+) T cell(s) are HuT-78 cells (ATCC® TIB-161TM) or variants or derivatives thereof. In some embodiments, the HuT-78 derivative is H9 (ATCC® HTB-176™).
[0053] In some embodiments, the inactivated CD4(+) T cell(s) are HuT-78 (ATCC® TIB- 161™) cells or variants or derivatives thereof that express an ortholog of OX40L, or variant thereof. In some embodiments, the feeder cells are HuT-78 cells engineered to express at least one gene selected from the group consisting of an 4-1BBL ortholog or variant thereof, a membrane bound IL-21 ortholog or variant thereof, and membrane bound TNFalpha ortholog, or variant thereof. n. CRYOPRESERVATION COMPOSITIONS
[0054] Provided herein are cryopreservation compositions, e.g., cry opreservation compositions suitable for intravenous administration, e.g., intravenous administration of immune cells, e.g., NK cells, e.g., CAR-NK cells. In some embodiments, a pharmaceutical composition comprises the cry opreservation composition and cells, e.g., the NK cells described herein.
A. Albumin
[0055] In some embodiments, the cryopreservation composition comprises albumin protein, e.g., human albumin protein (UniProtKB Accession P0278, SEQ ID NO: 1) or variant thereof. In some embodiments, the cryopreservation composition comprises an ortholog of an albumin protein, e.g., human albumin protein, or variant thereof. In some embodiments, the cryopreservation composition comprises a biologically active portion of an albumin protein, e.g., human albumin, or variant thereof.
[0056] In some embodiments, the albumin, e.g., human albumin, is provided as a solution, also referred to herein as an albumin solution or a human albumin solution. Thus, in some embodiments, the cryopreservation composition is or comprises an albumin solution, e.g., a human albumin solution. In some embodiments, the albumin solution is a serum-free albumin solution.
[0057] In some embodiments, the albumin solution is suitable for intravenous use. [0058] In some embodiments, the albumin solution comprises from or from about 40 to or to about 200 g/L albumin. In some embodiments, the albumin solution comprises from or from about 40 to or to about 50 g/L albumin, e.g., human albumin. In some embodiments, the albumin solution comprises about 200 g/L albumin, e.g., human albumin. In some embodiments, the albumin solution comprises 200 g/L albumin, e.g., human albumin.
[0059] In some embodiments, the albumin solution comprises a protein composition, of which 95% or more is albumin protein, e.g., human albumin protein. In some embodiments, 96%, 97%, 98%, or 99% or more of the protein is albumin, e.g., human albumin.
[0060] In some embodiments, the albumin solution further comprises sodium. In some embodiments, the albumin solution comprises from or from about 100 to or to about 200 mmol sodium. In some embodiments, the albumin solution comprises from or from about 130 to or to about 160 mmol sodium.
[0061] In some embodiments, the albumin solution further comprises potassium. In some embodiments, the albumin solution comprises 3 mmol or less potassium. In some embodiments, the albumin solution further comprises 2 mmol or less potassium.
[0062] In some embodiments, the albumin solution further comprises one or more stabilizers. In some embodiments, the stabilizers) are selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as acetyl tryptophan, N-Acetyl-L-tryptophan and Acetyl-L-tryptophan), 2-acetamido-3-(1H-indol- 3-yl)propanoic acid (also referred to as N-acetyltryptophan, DL-Acetyltroptohan and N-Acetyl- DL-tryptophan). In some embodiments, the solution comprises less than 0.1 mmol of each of the one or more stabilizers per gram of protein in the solution. In some embodiments, the solution comprises from or from about 0.05 to or to about 0.1, e.g., from or from about 0.064 to or to about 0.096 mmol of each of the stabilizers per gram of protein in the solution. In some embodiments, the solution comprises less than 0.1 mmol of total stabilizer per gram of protein in the solution. In some embodiments, the solution comprises from or from about 0.05 to or to about 0.1, e.g., from or from about 0.064 to or to about 0.096 mmol of total stabilizer per gram of protein in the solution.
[0063] In some embodiments, the albumin solution consists of a protein composition, of which 95% or more is albumin protein, sodium, potassium, and one or more stabilizers selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3- yl)propanoic acid (also referred to as acetyl tryptophan, N-Acetyl-L-tryptophan and Acetyl-L- tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as N- acetyltryptophan, DL-Acetyltroptohan and N- Acetyl -DL-tryptophan) in water. [0064] In some embodiments, the cryopreservation composition comprises from or from about 10% v/v to or to about 50% v/v of an albumin solution, e.g., an albumin solution described herein. In some embodiments, the cryopreservation composition comprises from or from about 10% to or to about 50%, from or from about 10% to or to about 45%, from or from about 10% to or to about 40%, from or from about 10% to or to about 35%, from or from about 10% to or to about 30%, from or from about 10% to or to about 25%, from or from about 10% to or to about 20%, from or from about 10% to or to about 15%, from or from about 15% to or to about 50%, from or from about 15% to or to about 45%, from or from about 15% to or to about 40%, from or from about 15% to or to about 35%, from or from about 15% to or to about 30%, from or from about 15% to or to about 25%, from or from about 15% to or to about 20%, from or from about 20% to or to about 50%, from or from about 20% to or to about 45%, from or from about 20% to or to about 40%, from or from about 20% to or to about 35%, from or from about 20% to or to about 30%, from or from about 20% to or to about 25%, from or from about 25% to or to about 50%, from or from about 25% to or to about 45%, from or from about 25% to or to about 40%, from or from about 25% to or to about 35%, from or from about 25% to or to about 30%, from or from about 30% to or to about 50%, from or from about 30% to or to about 45%, from or from about 30% to or to about 40%, from or from about 30% to or to about 35%, from or from about 35% to or to about 50%, from or from about 35% to or to about 45%, from or from about 35% to or to about 40%, from or from about 40% to or to about 50%, from or from about 40% to or to about 45%, or from or from about 45% to or to about 50% v/v of an albumin solution described herein. In some embodiments, the cryopreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v/v of an albumin solution described herein. In some embodiments, the cry opreservation composition comprises 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v of an albumin solution described herein.
[0065] In some embodiments, the cryopreservation composition comprises from or from about 20 to or to about 100 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises from or from about 20 to or to about 100, from or from about 20 to or to about 90, from or from about 20 to or to about 80, from or from about 20 to or to about 70, from or from about 20 to or to about 60, from or from about 20 to or to about 50, from or from about 20 to or to about 40, from or from about 20 to or to about 30, from or from about 30 to or to about 100, from or from about 30 to or to about 90, from or from about 30 to or to about 80, from or from about 30 to or to about 70, from or from about 30 to or to about 60, from or from about 30 to or to about 50, from or from about 30 to or to about 40, from or from about 40 to or to about 100, from or from about 40 to or to about 90, from or from about 40 to or to about 80, from or from about 40 to or to about 70, from or from about 40 to or to about 60, from or from about 40 to or to about 50, from or from about 50 to or to about 100, from or from about 50 to or to about 90, from or from about 50 to or to about 80, from or from about 50 to or to about 70, from or from about 50 to or to about 60, from or from about 60 to or to about 100, from or from about 60 to or to about 90, from or from about 60 to or to about 80, from or from about 60 to or to about 70, from or from about 70 to or to about 100, from or from about 70 to or to about 90, from or from about 70 to or to about 80, from or from about 80 to or to about 100, from or from about 80 to or to about 90, or from or from about 90 to or to about 100 g/L albumin, e.g., human albumin.
[0066] In some embodiments, the cryopreservation composition comprises 20 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises 40 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises 70 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises 100 g/L albumin, e.g., human albumin.
[0067] In some embodiments, the cryopreservation composition comprises about 20 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises about 40 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises about 70 g/L albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises about 100 g/L albumin, e.g., human albumin.
[0068] In some embodiments, the cryopreservation composition further comprises a stabilizer, e.g., an albumin stabilizer. In some embodiments, the stabilizers) are selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3- yl)propanoic acid (also referred to as acetyl tryptophan, N-Acetyl-L-tryptophan and Acetyl-L- tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as N- acetyltryptophan, DL-Acetyltroptohan and N-Acetyl-DL-tryptophan). In some embodiments, the cryopreservation composition comprises less than .1 mmol of each of the one or more stabilizers per gram of protein, e.g., per gram of albumin protein, in the composition. In some embodiments, the cryopreservation composition comprises from or from about 0.05 to or to about 0.1, e.g., from or from about 0.064 to or to about 0.096 mmol of each of the stabilizers per gram of protein, e.g., per gram of albumin protein in the composition. In some embodiments, the cryopreservation composition comprises less than 0.1 mmol of total stabilizer per gram of protein, e.g., per gram of albumin protein in the cryopreservation composition. In some embodiments, the cryopreservation composition comprises from or from about 0.05 to or to about 0.1, e.g., from or from about 0.064 to or to about 0.096 mmol of total stabilizer per gram of protein, e.g., per gram of albumin protein, in the cryopreservation composition.
B. Dextran
[0069] In some embodiments, the cryopreservation composition comprises Dextran, or a derivative thereof.
[0070] Dextran is a polymer of anhydroglucose composed of approximately 95% a-D-(1-6) linkages (designated (C6H10O5)n). Dextran fractions are supplied in molecular weights of from about 1,000 Daltons to about 2,000,000 Daltons. They are designated by number (Dextran X), e.g., Dextran 1, Dextran 10, Dextran 40, Dextran 70, and so on, where X corresponds to the mean molecular weight divided by 1,000 Daltons. So, for example, Dextran 40 has an average molecular weight of or about 40,000 Daltons.
[0071] In some embodiments, the average molecular weight of the dextran is from or from about 1,000 Daltons to or to about 2,000,000 Daltons. In some embodiments, the average molecular weight of the dextran is or is about 40,000 Daltons. In some embodiments, the average molecular weight of the dextran is or is about 70,000 Daltons.
[0072] In some embodiments, the dextran is selected from the group consisting of Dextran 40, Dextran 70, and combinations thereof. In some embodiments, the dextran is Dextran 40.
[0073] In some embodiments, the dextran, e.g., Dextran 40, is provided as a solution, also referred to herein as a dextran solution or a Dextran 40 solution. Thus, in some embodiments, the composition comprises a dextran solution, e.g., a Dextran 40 solution.
[0074] In some embodiments, the dextran solution is suitable for intravenous use.
[0075] In some embodiments, the dextran solution comprises about 5% to about 50% w/w dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises from or from about 5% to or to about 50%, from or from about 5% to or to about 45%, from or from about 5% to or to about 40%, from or from about 5% to or to about 35%, from or from about 5% to or to about 30%, from or from about 5% to or to about 25%, from or from about 5% to or to about 20%, from or from about 5% to or to about 15%, from or from about 5% to or to about 10%, from or from about 10% to or to about 50%, from or from about 10% to or to about 45%, from or from about 10% to or to about 40%, from or from about 10% to or to about 35%, from or from about 10% to or to about 30%, from or from about 10% to or to about 25%, from or from about 10% to or to about 20%, from or from about 10% to or to about 15%, from or from about 15% to or to about 50%, from or from about 15% to or to about 45%, from or from about 15% to or to about 40%, from or from about 15% to or to about 35%, from or from about 15% to or to about 30%, from or from about 15% to or to about 25%, from or from about 15% to or to about 20%, from or from about 20% to or to about 50%, from or from about 20% to or to about 45%, from or from about 20% to or to about 40%, from or from about 20% to or to about 35%, from or from about 20% to or to about 30%, from or from about 20% to or to about 25%, from or from about 25% to or to about 50%, from or from about 25% to or to about 45%, from or from about 25% to or to about 40%, from or from about 25% to or to about 35%, from or from about 25% to or to about 30%, from or from about 30% to or to about 50%, from or from about 30% to or to about 45%, from or from about 30% to or to about 40%, from or from about 30% to or to about 35%, from or from about 35% to or to about 50%, from or from about 35% to or to about 45%, from or from about 35% to or to about 40%, from or from about 40% to or to about 50%, from or from about 40% to or to about 45%, or from or from about 45% to or to about 50% w/w dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% w/w dextran, e.g., Dextran 40.
[0076] In some embodiments, the dextran solution comprises from or from about 25 g/L to or to about 200 g/L dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises from or from about 35 to or to about 200, from or from about 25 to or to about 175, from or from about 25 to or to about 150, from or from about 25 to or to about 125, from or from about 25 to or to about 100, from or from about 25 to or to about 75, from or from about 25 to or to about 50, from or from about 50 to or to about 200, from or from about 50 to or to about 175, from or from about 50 to or to about 150, from or from about 50 to or to about 125, from or from about 50 to or to about 100, from or from about 50 to or to about 75, from or from about 75 to or to about 200, from or from about 75 to or to about 175, from or from about 75 to or to about 150, from or from about 75 to or to about 125, from or from about 75 to or to about 100, from or from about 100 to or to about 200, from or from about 100 to or to about 175, from or from about 100 to or to about 150, from or from about 100 to or to about 125, from or from about 125 to or to about 200, from or from about 125 to or to about 175, from or from about 125 to or to about 150, from or from about 150 to or to about 200, from or from about 150 to or to about 175, or from or from about 175 to or to about 200 g/L dextran e.g., Dextran 40. In some embodiments, the dextran solution comprises 25, 50, 75, 100, 125, 150, 175, or 200 g/L dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises 100 g/L dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises about 25, about 50, about 75, about 100, about 125, about 150, about 175, or about 200 g/L dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises about 100 g/L dextran, e.g., Dextran 40.
[0077] In some embodiments, the dextran solution further comprises glucose (also referred to as dextrose). In some embodiments, the dextran solution comprises from or from about 10 g/L to or to about 100 g/L glucose. In some embodiments, the dextran solution comprises from or from about 10 to or to about 100, from or from about 10 to or to about 90, from or from about 10 to or to about 80, from or from about 10 to or to about 70, from or from about 10 to or to about 60, from or from about 10 to or to about 50, from or from about 10 to or to about 40, from or from about 10 to or to about 30, from or from about 10 to or to about 20, from or from about 20 to or to about 100, from or from about 20 to or to about 90, from or from about 20 to or to about 80, from or from about 20 to or to about 70, from or from about 20 to or to about 60, from or from about 20 to or to about 50, from or from about 20 to or to about 40, from or from about 20 to or to about 30, from or from about 30 to or to about 100, from or from about 30 to or to about 90, from or from about 30 to or to about 80, from or from about 30 to or to about 70, from or from about 30 to or to about 60, from or from about 30 to or to about 50, from or from about 30 to or to about 40, from or from about 40 to or to about 100, from or from about 40 to or to about 90, from or from about 40 to or to about 80, from or from about 40 to or to about 70, from or from about 40 to or to about 60, from or from about 40 to or to about 50, from or from about 50 to or to about 100, from or from about 50 to or to about 90, from or from about 50 to or to about 80, from or from about 50 to or to about 70, from or from about 50 to or to about 60, from or from about 60 to or to about 100, from or from about 60 to or to about 90, from or from about 60 to or to about 80, from or from about 60 to or to about 70, from or from about 70 to or to about 100, from or from about 70 to or to about 90, from or from about 70 to or to about 80, from or from about 80 to or to about 90, from or from about 80 to or to about 100, from or from about 80 to or to about 90, or from or from about 90 to or to about 100 g/L glucose. In some embodiments, the dextran solution comprises 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g/L glucose. In some embodiments, the dextran solution comprises 50 g/L glucose. In some embodiments, the dextran solution comprises about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 g/L glucose. In some embodiments, the dextran solution comprises 50 g/L glucose.
[0078] In some embodiments, the dextran solution consists of dextran, e.g., Dextran 40, and glucose in water.
[0079] In some embodiments, the cryopreservation composition comprises from or from about 10% v/v to or to about 50% v/v of a dextran solution described herein. In some embodiments, the cryopreservation composition comprises from or from about 10% to 50%, from or from about 10% to or to about 45%, from or from about 10% to or to about 40%, from or from about 10% to or to about 35%, from or from about 10% to or to about 30%, from or from about 10% to or to about 25%, from or from about 10% to or to about 20%, from or from about 10% to or to about 15%, from or from about 15% to or to about 50%, from or from about 15% to or to about 45%, from or from about 15% to or to about 40%, from or from about 15% to or to about 35%, from or from about 15% to or to about 30%, from or from about 15% to or to about 25%, from or from about 15% to or to about 20%, from or from about 20% to or to about 50%, from or from about 20% to or to about 45%, from or from about 20% to or to about 40%, from or from about 20% to or to about 35%, from or from about 20% to or to about 30%, from or from about 20% to or to about 25%, from or from about 25% to or to about 50%, from or from about 25% to or to about 45%, from or from about 25% to or to about 40%, from or from about 25% to or to about 35%, from or from about 25% to or to about 30%, from or from about 30% to or to about 50%, from or from about 30% to or to about 45%, from or from about 30% to or to about 40%, from or from about 30% to or to about 35%, from or from about 35% to or to about 50%, from or from about 35% to or to about 45%, from or from about 35% to or to about 40%, from or from about 40% to or to about 50%, from or from about 40% to or to about 45%, or from or from about 45% to or to about 50% v/v of a dextran solution, e.g., a dextran solution described herein. In some embodiments, the cryopreservation composition comprises 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v of a dextran solution, e.g., a dextran solution described herein. In some embodiments, the cry opreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v/v of a dextran solution, e.g., a dextran solution described herein.
[0080] In some embodiments, the cryopreservation composition comprises from or from about 10 to or to about 50 g/L dextran, e.g., Dextran 40. In some embodiments, the cryopreservation composition comprises from or from about 10 to or to about 50, from or from about 10 to or to about 45, from or from about 10 to or to about 40, from or from about 10 to or to about 35, from or from about 10 to or to about 30, from or from about 10 to or to about 25, from or from about 10 to or to about 20, from or from about 10 to or to about 15, from or from about 15 to or to about 50, from or from about 15 to or to about 45, from or from about 15 to or to about 40, from or from about 15 to or to about 35, from or from about 15 to or to about 30, from or from about 15 to or to about 25, from or from about 15 to or to about 20, from or from about 20 to or to about 50, from or from about 20 to or to about 45, from or from about 20 to or to about 40, from or from about 20 to or to about 30, from or from about 20 to or to about 25, from or from about 25 to or to about 50, from or from about 25 to or to about 45, from or from about 25 to or to about 40, from or from about 25 to or to about 35, from or from about 25 to or to about 30, from or from about 30 to or to about 50, from or from about 30 to or to about 45, from or from about 30 to or to about 40, from or from about 30 to or to about 35, from or from about 35 to or to about 50, from or from about 35 to or to about 45, from or from about 35 to or to about 40, from or from about 40 to or to about 50, from or from about 40 to or to about 45, or from or from about 45 to or to about 50 g/L dextran, e.g., Dextran 40. In some embodiments, the cryopreservation composition comprises 10, 15, 20, 25, 30, 30, 35, 40, 45, or 50 g/L dextran, e.g., Dextran 40. In some embodiments, the cryopreservation composition comprises about 10, about 15, about 20, about 25, about 30, about 30, about 35, about 40, about 45, or about 50 g/L dextran, e.g., Dextran 40.
C. Glucose
[0081] In some embodiments, the cry opreservation composition comprises glucose.
[0082] In some embodiments, as described above, the cryopreservation composition comprises a Dextran solution comprising glucose.
[0083] In some embodiments, the cryopreservation composition comprises a Dextran solution that does not comprise glucose. In some embodiments, e.g., when the Dextran solution does not comprise glucose, glucose is added separately to the cryopreservation composition.
[0084] In some embodiments, the cryopreservation composition comprises from or from about 5 to or to about 25 g/L glucose. In some embodiments, the cryopreservation composition comprises from or from about 5 to or to about 25, from or from about 5 to or to about 20, from or from about 5 to or to about 15, from or from about 5 to or to about 10, from or from about 10 to or to about 25, from or from about 10 to or to about 20, from or from about 10 to or to about 15, from or from about 15 to or to about 25, from or from about 15 to or to about 20, or from or from about 20 to or to about 25 g/L glucose. In some embodiments, the cryopreservation composition comprises 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, or 25 g/L glucose. In some embodiments, the cryopreservation composition comprises 12.5 g/L glucose. In some embodiments, the cryopreservation composition comprises about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, about 22.5, or about 25 g/L glucose. In some embodiments, the cryopreservation composition comprises about 12.5 g/L glucose.
[0085] In some embodiments, the cry opreservation composition comprises less than 2.75% w/v glucose. In some embodiments, the cryopreservation composition comprises less than 27.5 g/L glucose. In some embodiments, the cryopreservation composition comprises less than 2% w/v glucose. In some embodiments, the cry opreservation composition comprises less than 1.5% w/v glucose. In some embodiments, the cryopreservation composition comprises about 1.25% w/v or less glucose.
D. Dimethyl Sulfoxide
[0086] In some embodiments, the cryopreservation composition comprises dimethyl sulfoxide (DMSO, also referred to as methyl sulfoxide and methylsulfinylmethane).
[0087] In some embodiments, the DMSO is provided as a solution, also referred to herein as a DMSO solution. Thus, in some embodiments, the cryopreservation composition comprises a DMSO solution.
[0088] In some embodiments, the DMSO solution is suitable for intravenous use.
[0089] In some embodiments, the DMSO solution comprises 1.1 g/mL DMSO. In some embodiments, the DMSO solution comprises about 1.1 g/mL DMSO.
[0090] In some embodiments, the cry opreservation composition comprises from or from about 1% to or to about 10% v/v of the DMSO solution. In some embodiments, the cry opreservation composition comprises from or from about 1% to or to about 10%, from or from about 1% to or to about 9%, from or from about 1% to or to about 8%, from or from about 1% to or to about 7%, from or from about 1% to or to about 6%, from or from about 1% to or to about 5%, from or from about 1% to or to about 4%, from or from about 1% to or to about 3%, from or from about 1% to or to about 2%, from or from about 2% to or to about 10%, from or from about 2% to or to about 9%, from or from about 8%, from or from about 2% to or to about 7%, from or from about 2% to or to about 6%, from or from about 2% to or to about 5%, from or from about 2% to or to about 4%, from or from about 2% to or to about 3%, from or from about 3% to or to about 10%, from or from about 3% to or to about 9%, from or from about 3% to or to about 8%, from or from about 3% to or to about 7%, from or from about 3% to or to about 6%, from or from about 3% to or to about 5%, from or from about 3% to or to about 4%, from or from about 4% to or to about 10%, from or from about 4% to or to about 9%, from or from about 4% to or to about 8%, from or from about 4% to or to about 7%, from or from about 4% to or to about 6%, from or from about 4% to or to about 5%, from or from about 5% to or to about 10%, from or from about 5% to or to about 9%, from or from about 5% to or to about 8%, from or from about 5% to or to about 7%, from or from about 5% to or to about 6%, from or from about 6% to or to about 10%, from or from about 6% to or to about 9%, from or from about 6% to or to about 8%, from or from about 6% to or to about 7%, from or from about 7% to or to about 10%, from or from about 7% to or to about 9%, from or from about 7% to or to about 8%, from or from about 8% to or to about 10%, from or from about 8% to or to about 9%, or from or from about 9% to or to about 10% v/v of the DMSO solution. In some embodiments, the cryopreservation composition comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% v/v of the DMSO solution. In some embodiments, the cryopreservation composition comprises 5% of the DMSO solution. In some embodiments, the cryopreservation composition comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% v/v of the DMSO solution. In some embodiments, the cry opreservation composition comprises about 5% of the DMSO solution.
[0091] In some embodiments, the cryopreservation composition comprises from or from about 11 to or to about 110 g/L DMSO. In some embodiments, from or from about the cryopreservation composition comprises from or from about 11 to or to about 110, from or from about 11 to or to about 99, from or from about 11 to or to about 88, from or from about 11 to or to about 77, from or from about 11 to or to about 66, from or from about 11 to or to about 55, from or from about 11 to or to about 44, from or from about 11 to or to about 33, from or from about 11 to or to about 22, from or from about 22 to or to about 110, from or from about 22 to or to about 99, from or from about 22 to or to about 88, from or from about 22 to or to about 77, from or from about 22 to or to about 77, from or from about 22 to or to about 66, from or from about 22 to or to about 55, from or from about 22 to or to about 44, from or from about 22 to or to about 33, from or from about 33 to or to about 110, from or from about 33 to or to about 99, from or from about 33 to or to about 88, from or from about 33 to or to about 77, from or from about 33 to or to about 66, from or from about 33 to or to about 55, from or from about 33 to or to about 44, from or from about 44 to or to about 110, from or from about 44 to or to about 99, from or from about 44 to or to about 88, from or from about 44 to or to about 77, from or from about 44 to or to about 66, from or from about 44 to or to about 55, from or from about 55 to or to about 110, from or from about 55 to or to about 99, from or from about 55 to or to about 88, from or from about 55 to or to about 77, from or from about 55 to or to about 66, from or from about 66 to or to about 110, from or from about 66 to or to about 99, from or from about 66 to or to about 88, from or from about 66 to or to about 77, from or from about 77 to or to about 119, from or from about 77 to or to about 88, from or from about 88 to or to about 110, from or from about 88 to or to about 99, or from or from about 99 to or to about 110 g/L DMSO. In some embodiments, the cryopreservation composition comprises 11, 22, 33, 44, 55, 66, 77, 88, 99, or 110 g/L DMSO. In some embodiments, the cryopreservation composition comprises 55 g/L DMSO. In some embodiments, the cryopreservation composition comprises about 11, about 22, about 33, about 44, about 55, about 66, about 77, about 88, about 99, or about 110 g/L DMSO.
In some embodiments, the cryopreservation composition comprises about 55 g/L DMSO.
E. Buffers
[0092] In some embodiments, the cry opreservation composition comprises a buffer solution, e.g., a buffer solution suitable for intravenous administration.
[0093] Buffer solutions include, but are not limited to, phosphate buffered saline (PBS), Ringer’s Solution, Tyrode’s buffer, Hank’s balanced salt solution, Earle’s Balanced Salt Solution, saline, and Tris.
[0094] In some embodiments, the buffer solution is phosphate buffered saline (PBS).
F. Exemplary Cryopreservation Compositions
[0095] In some embodiments, the cryopreservation composition comprises or consists of: 1) albumin, e.g., human albumin, 2) dextran, e.g., Dextran 40, 3) DMSO, and 4) a buffer solution. In some embodiments, the cryopreservation composition further comprises glucose. In some embodiments, the cryopreservation composition consists of 1) albumin, e.g., human albumin, 2) dextran, e.g., Dextran 40, 3) glucose, 4) DMSO, and 5) a buffer solution.
[0096] In some embodiments, the cryopreservation composition comprises: 1) an albumin solution described herein, 2) a dextran solution described herein, 3) a DMSO solution described herein, and 4) a buffer solution.
[0097] In some embodiments, the cryopreservation composition consists of: 1) an albumin solution described herein, 2) a dextran solution described herein, 3) a DMSO solution described herein, and 4) a buffer solution.
[0098] In some embodiments, the cryopreservation composition does not comprise a cell culture medium.
[0099] In one embodiment, the cryopreservation composition comprises or comprises about 40 mg/mL human albumin, 25 mg/mL Dextran 40, 12.5 mg/mL glucose, and 55 mg/mL DMSO.
[0100] In one embodiment, the cryopreservation composition comprises or comprises about or consists of or consists of about 40 mg/mL human albumin, 25 mg/mL Dextran 40, 12.5 mg/mL glucose, 55 mg/mL DMSO, and 0.5 mL/mL 100% phosphate buffered saline (PBS) in water.
[0101] In one embodiment, the cryopreservation composition comprises or comprises about 32 mg/mL human albumin, 25 mg/mL Dextran 40, 12.5 mg/mL glucose, and 55 mg/mL DMSO.
[0102] In one embodiment, the cryopreservation composition comprises or comprises about or consists of or consists of about of 32 mg/mL human albumin, 25 mg/mL Dextran 40, 12.5 mg/mL glucose, 55 mg/mL DMSO, and 0.54 mL/mL 100% phosphate buffered saline (PBS) in water.
[0103] Exemplary Cryopreservation Compositions are shown in Table 1.
Table 1. Exemplary Cryopreservation Compositions
Figure imgf000018_0001
Table 2. Exemplary Cryopreservation Composition #1
Figure imgf000018_0002
Table 3. Exemplary Cry opreservation Composition #2
Figure imgf000018_0003
III. METHODS OF CRYOPRESERVING
[0104] In some embodiments, cryopreserving the cell(s) comprises: mixing the cell(s) with a cryopreservation composition or components thereof described herein to produce a composition, e.g., a pharmaceutical composition; and freezing the mixture.
[0105] In some embodiments, cryopreserving the cell(s) comprises: mixing a composition comprising the cell(s) with a cryopreservation composition or components thereof described herein to produce a composition, e.g., a pharmaceutical composition; and freezing the mixture. In some embodiments, the composition comprising the cell(s) comprises: the cell(s) and a buffer. Suitable buffers are described herein.
[0106] In some embodiments, cryopreserving the cell(s) comprises: mixing a composition comprising the cell(s) and a buffer, e.g., PBS, with a composition comprising albumin, Dextran, and DMSO, e.g., as described herein; and freezing the mixture.
[0107] In some embodiments, cryopreserving the cell(s) comprises: mixing a composition comprising the cell(s) and a buffer, e.g., PBS 1 : 1 with a composition comprising 40 mg/mL albumin, e.g., human albumin, 25 mg/mL Dextran, e.g., Dextran 40, 12.5 mg/mL glucose and 55 mg/mL DMSO.
[0108] In some embodiments, the composition comprising the cell(s) and the buffer, e.g., PBS, comprises from or from about 2x104 to or to about 2x1010 cells/mL. In some embodiments, the composition comprising the cell(s) and the buffer, e.g., PBS, comprises 2x108 cells/mL. In some embodiments, the composition comprising the cell(s) and the buffer, e.g., PBS, comprising about 2x108 cells/mL, but the concentration may vary depending on the type of cell(s).
[0109] In some embodiments, cryopreserving the cell(s) comprising mixing: the cell(s), a buffer, e.g., PBS, albumin, e.g., human albumin, Dextran, e.g., Dextran 40, and DMSO; and freezing the mixture.
[0110] In some embodiments, the mixture comprises from or from about 1x104 to or to about 1x1010cells/mL. In some embodiments, the mixture comprises 1x108 cells/mL. In some embodiments, the mixture comprises about 1x108 cells/mL, but the concentration may vary depending on the type of cell(s).
[0111] Suitable ranges for albumin, Dextran, and DMSO are set forth above.
[0112] In some embodiments, the composition is frozen at or below -135°C.
[0113] In some embodiments, the composition is frozen at a controlled rate. IV. CELL COMPOSITIONS / PHARMACEUTICAL COMPOSITIONS
[0114] Provided herein are cell composition comprising cell(s) and a cryopreservation composition as described herein. In some embodiments, the cell composition is a pharmaceutical composition.
[0115] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
[0116] In some embodiments, the pharmaceutical composition comprises: a) cell(s) described herein; and b) a cryopreservation composition described herein.
[0117] In some embodiments, the pharmaceutical composition comprises: a) a cryopreservation composition described herein; and b) therapeutic cell(s).
[0118] In some embodiments, the therapeutic cell(s) are animal cell(s). In some embodiments, the therapeutic cell(s) are human cell(s).
[0119] In some embodiments, the therapeutic cell(s) are immune cell(s). In some embodiments, the immune cell(s) are selected from basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.
[0120] In some embodiments, the pharmaceutical composition further comprises: c) a buffer solution. Suitable buffer solutions are described herein, e.g., as for cryopreservation compositions.
[0121] In some embodiments, the pharmaceutical composition comprises from or from about 1x104 to or to about 1x1010 cells/mL. In some embodiments, the mixture comprises 1x108 cells/mL. In some embodiments, the pharmaceutical composition comprises about 1x108 cells/mL.
[0122] In some embodiments, the pharmaceutical composition further comprises an antibody or antigen binding fragment thereof.
V. DEFINITIONS
[0123] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0124] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
[0125] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.
[0126] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
[0127] The terms “subject,” “individual,” or “patient” are often used interchangeably herein.
[0128] The term “in vivo" is used to describe an event that takes place in a subject’s body.
[0129] The term “ex vivo” is used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “in vitro" assay.
[0130] The term “in vitro" is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. [0131] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.
[0132] As used herein, the term "buffer solution" refers to an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa.
[0133] As used herein, the term "cell culture medium" refers to a mixture for growth and proliferation of cells in vitro, which contains essential elements for growth and proliferation of cells such as sugars, amino acids, various nutrients, inorganic substances, etc.
[0134] A buffer solution, as used herein, is not a cell culture medium.
[0135] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
VI. METHODS OF TREATMENT
[0136] The cry opreservation compositions described herein find use in methods of treating disorders, e.g., cancer or other proliferative disorders.
[0137] Thus, also provided herein are methods of treating a patient suffering from a disorder, comprising administering a pharmaceutical composition comprising a cryopreservation composition described herein and cells, e.g., therapeutic cells.
[0138] Also provided herein are methods of preventing, reducing and/or inhibiting the recurrence, growth, proliferation, migration and/or metastasis of a cancer cell or population of cancer cells in a subject in need thereof, comprising administering a pharmaceutical composition comprising a cryopreservation composition described herein and cells, e.g., therapeutic cells.
[0139] Also provided herein are methods of enhancing, improving, and/or increasing the response to therapy, e.g., a cancer therapy, to a subject in need thereof, comprising administering the NK cells, e.g., the NK cells described herein, e.g., the CAR-NK cells described herein.
[0140] Also provided herein are methods for inducing the immune system in a subject in need thereof comprising administering a pharmaceutical composition comprising a cryopreservation composition described herein and cells.
[0141] The methods described herein include methods for the treatment of disorders associated with abnormal apoptotic or differentiative processes, e.g., cellular proliferative disorders or cellular differentiative disorders, e.g., cancer, including both solid tumors and hematopoietic cancers. Generally, the methods include administering a therapeutically effective amount of a treatment as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. [0142] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disorder associated with abnormal apoptotic or differentiative processes. For example, a treatment can result in a reduction in tumor size or growth rate. Administration of a therapeutically effective amount of a compound described herein for the treatment of a condition associated with abnormal apoptotic or differentiative processes will result in a reduction in tumor size or decreased growth rate, a reduction in risk or frequency of reoccurrence, a delay in reoccurrence, a reduction in metastasis, increased survival, and/or decreased morbidity and mortality, among other things. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
[0143] As used herein, the terms "inhibition”, as it relates to cancer and/or cancer cell proliferation, refer to the inhibition of the growth, division, maturation or viability of cancer cells, and/or causing the death of cancer cells, individually or in aggregate with other cancer cells, by cytotoxicity, nutrient depletion, or the induction of apoptosis.
[0144] As used herein, “delaying” development of a disease or disorder, or one or more symptoms thereof, means to defer, hinder, slow, retard, stabilize and/or postpone development of the disease, disorder, or symptom thereof. This delay can be of varying lengths of time, depending on the history of the disease and/or subject being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the subject does not develop the disease, disorder, or symptom thereof. For example, a method that “delays” development of cancer is a method that reduces the probability of disease development in a given time frame and/or reduces extent of the disease in a given time frame, when compared to not using the method. Such comparisons may be based on clinical studies, using a statistically significant number of subjects.
[0145] As used herein, “prevention” or “preventing” refers to a regimen that protects against the onset of the disease or disorder such that the clinical symptoms of the disease do not develop. Thus, “prevention” relates to administration of a therapy (e.g., administration of a therapeutic substance) to a subject before signs of the disease are detectable in the subject and/or before a certain stage of the disease (e.g., administration of a therapeutic substance to a subject with a cancer that has not yet metastasized). The subject may be an individual at risk of developing the disease or disorder, or at risk of disease progression, e.g., cancer metastasis. Such as an individual who has one or more risk factors known to be associated with development or onset of the disease or disorder. For example, an individual may have mutations associated with the development or progression of a cancer. Further, it is understood that prevention may not result in complete protection against onset of the disease or disorder. In some instances, prevention includes reducing the risk of developing the disease or disorder. The reduction of the risk may not result in complete elimination of the risk of developing the disease or disorder.
[0146] An “increased” or “enhanced” amount (e.g., with respect to antitumor response, cancer cell metastasis) refers to an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) an amount or level described herein. It may also include an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% of an amount or level described herein.
[0147] A “decreased” or “reduced” or “lesser” amount (e.g., with respect to tumor size, cancer cell proliferation or growth) refers to a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6
1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6,
1.7. 1.8, etc.) an amount or level described herein. It may also include a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% of an amount or level described herein.
A. Disorders
[0148] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.
[0149] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
[0150] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renalcell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
[0151] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the disease is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
[0152] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
[0153] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol ./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.
[0154] In some embodiments, the cancer is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, typical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid, cardiac tumors, medulloblastoma, germ cell tumor, primary CNS lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer (e.g., intraocular melanoma or retinoblastoma), fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumor, hepatocellular cancer, histiocytosis, Hodgkin lymphomas, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney (renal cell) carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, pleuropulmonary blastoma, and tracheobronchial tumor), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, plasma cell neoplasm, multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., childhood rhabdomyosarcoma, childhood vascular tumors, Ewing sarcoma, Kaposi sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomach cancer, T-cell lymphomas, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thryomoma and thymic carcinomas, thyroid cancer, tracheobronchial tumors, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, and Wilms tumor.
[0155] In some embodiments, the cancer is a solid tumor.
[0156] In some embodiments, the cancer is metastatic.
B. Patients
[0157] In some embodiments, the methods of treatment provided herein may be used to treat a subject (e.g., human, monkey, dog, cat, mouse) who has been diagnosed with or is suspected of having a cellular proliferative and/or differentiative disorder, e.g., a cancer. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.
[0158] As used herein, a subject refers to a mammal, including, for example, a human.
[0159] In some embodiments, the mammal is selected from the group consisting of an armadillo, an ass, a bat, a bear, a beaver, a cat, a chimpanzee, a cow, a coyote, a deer, a dog, a dolphin, an elephant, a fox, a panda, a gibbon, a giraffe, a goat, a gopher, a hedgehog, a hippopotamus, a horse, a humpback whale, a jaguar, a kangaroo, a koala, a leopard, a lion, a llama, a lynx, a mole, a monkey, a mouse, a narwhal, an orangutan, an orca, an otter, an ox, a pig, a polar bear, a porcupine, a puma, a rabbit, a raccoon, a rat, a rhinoceros, a sheep, a squirrel, a tiger, a walrus, a weasel, a wolf, a zebra, a goat, a horse, and combinations thereof.
[0160] In some embodiments, the mammal is a human.
[0161] The subject, e.g., the human subject, can be a child, e.g., from or from about 0 to or to about 14 years in age. The subject can be a youth, e.g., from or from about 15 to or to about 24 years in age. The subject can be an adult, e.g., from or from about 25 to or to about 64 years in age. The subject can be a senior, e.g, 65+ years in age.
[0162] In some embodiments, the subject may be a human who exhibits one or more symptoms associated with a cellular proliferative and/or differentiative disorder, e.g., a cancer, e.g., a tumor. Any of the methods of treatment provided herein may be used to treat cancer at various stages. By way of example, the cancer stage includes but is not limited to early, advanced, locally advanced, remission, refractory, reoccurred after remission and progressive. In some embodiments, the subject is at an early stage of a cancer. In other embodiments, the subject is at an advanced stage of cancer. In various embodiments, the subject has a stage I, stage n, stage III or stage IV cancer. The methods of treatment described herein can promote reduction or retraction of a tumor, decrease or inhibit tumor growth or cancer cell proliferation, and/or induce, increase or promote tumor cell killing. I n some embodiments, the subject is in cancer remission. The methods of treatment described herein can prevent or delay metastasis or recurrence of cancer.
[0163] In some embodiments, the subject is at risk, or genetically or otherwise predisposed (e.g., risk factor), to developing a cellular proliferative and/or differentiative disorder, e.g., a cancer, that has or has not been diagnosed.
[0164] As used herein, an “at risk” individual is an individual who is at risk of developing a condition to be treated, e.g., a cellular proliferative and/or differentiative disorder, e.g., a cancer. Generally, an “at risk” subject may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein. “At risk” denotes that an individual has one or more so-called risk factors, which are measurable parameters that correlate with development of a disease or condition and are known in the art. For example, an at risk subject may have one or more risk factors, which are measurable parameters that correlate with development of cancer. A subject having one or more of these risk factors has a higher probability of developing cancer than an individual without these risk factor(s). In general, risk factors may include, for example, age, sex, race, diet, history of previous disease, presence of precursor disease, genetic (e.g., hereditary) considerations, and environmental exposure. In some embodiments, the subjects at risk for cancer include, for example, those having relatives who have experienced the disease, and those whose risk is determined by analysis of genetic or biochemical markers.
[0165] In addition, the subject may be undergoing one or more standard therapies, such as chemotherapy, radiotherapy, immunotherapy, surgery, or combination thereof. Accordingly, one or more kinase inhibitors may be administered before, during, or after administration of chemotherapy, radiotherapy, immunotherapy, surgery or combination thereof.
In certain embodiments, the subject may be a human who is (i) substantially refractory to at least one chemotherapy treatment, or (ii) is in relapse after treatment with chemotherapy, or both (i) and (ii). In some of embodiments, the subject is refractory to at least two, at least three, or at least four chemotherapy treatments (including standard or experimental chemotherapies). VII. TREATMENT OF CANCER WITH NK CELLS AND A CD20 TARGETED
ANTIBODY
[0166] NHLs are a heterogeneous group of lymphoproliferative malignancies that usually originate in lymphoid tissues and can spread to other organs. Prognosis for NHL patients depends on histologic type, stage, and response to treatment. NHL can be divided into 2 prognostic groups: the indolent lymphomas and the aggressive lymphomas. Indolent NHLs offer a relatively good prognosis with a median survival of up to 20 years and are generally responsive to immunotherapy, radiation therapy, and chemotherapy. However, a continuous rate of relapse is seen in advanced stages of indolent NHLs. In contrast, aggressive NHLs present acutely and are more commonly resistant or refractory to frontline therapy.
[0167] In general, patients with newly diagnosed NHL are treated with chemotherapy combined with rituximab that confers long-term remissions in most patients. NHL patients who are refractory to front-line treatment or those who relapse soon after completing front-line therapies, have poor outcomes. These patients are typically treated with a second line of chemotherapy (ICE or DHAP), often combined with an approved therapeutic monoclonal antibody (mAb). Depending on their response to this therapy and the patient’s physical condition, autologous stem cell transplant (ASCT) or an approved chimeric antigen receptor T- cell therapy (CAR-T) may be offered. For patients who are ineligible for ASCT, treatment options are limited, and median overall survival is 3.3 months. For patients who have experienced disease progression after ASCT or CAR-T, treatment options and survival are poor (Van Den Neste 2016 Bone Marrow Transplantation 51:51-57). Relapsed and refractory NHL of B-cell origin is, therefore, an area of unmet medical need.
[0168] Described herein are methods for treating a patient suffering from a CD20+ cancer, the methods include: administering allogenic natural killer cells (NK cells) and an antibody targeted to human CD20, wherein the NK cells are allogenic to the patient, are KIR-B haplotype and express CD16 having the V/V polymorphism at F158.
[0169] In various embodiments: the cancer is non-Hodgkins lymphoma (NHL) (e.g., indolent NHL or aggressive NHL); the patient has relapsed after treatment with an anti-CD20 antibody;patient has the experienced disease progression after treatment with autologous stem cell transplant or chimeric antigen receptor T-cell therapy (CAR-T); the patient is administered 1 x 108 to 1 x 1010 NK cells; the patient is administered 1 x 109 to 8 x 109 NK cells; the patient is administered 4 x 108, 1 x 109, 4 x 109, or 8 x 109 NK cells; 100 to 500 mg/m2 of the antibody targeted to human CD20; each administration of NK cells is administration of 1 x 109 to 5 x 109 NK cells; each administration of NK cells is administration of 1 x 109 to 5 x 109 NK cells; the patient is administered 375 mg/m2 of the antibody targeted to human CD20; the antibody targeted to human CD20 is rituximab; the patient is subjected to lymphodepleting chemotherapy (e.g., non-myeloablative chemotherapy by administering at least one of or both of cyclophosphamide and fludarabine) prior to treatment with the NK cells. The lymphodepleting chemotherapy can include, in various embodiments: treatment with cyclophosphamide and fludarabine, administration of cyclophosphamide at between 100 and 500 mg/m2/day; administration of cyclophosphamide at 250 mg/m2/day; administration of fludarabine at between 10 and 50 mg/m2/day or at 30mg/m2/day.
[0170] In various embodiments: the method further comprising administering IL-2 (e.g., a dose of 1 x 106 IU/m2 of IL-2). In some embodiments, administration of IL-2 occurs within 1-4 hrs of administration of the NK cells.
[0171] In various embodiments: the administration of the NK cells and the antibody targeted to human CD20 occurs weekly; the NK cells and the antibody targeted to human CD20 are administered weekly for 4 to 8 weeks; the NK cells are not genetically modified; at least 70% of the NK cells are CD56+ and CD16+; at least 85% of the NK cells are CD56+ and CD3-; 1% or less of the NK cells are CD3+, 1% or less of the NK cells are CD19+ and 1% or less of the NK cells are CD14+.
[0172] In various embodiments: the indolent NHL is selected from the group consisting of Follicular lymphoma, Lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, Gastric MALT, Non-gastric MALT, Nodal marginal zone lymphoma, Splenic marginal zone lymphoma, Small-cell lymphocytic lymphoma (SLL), and Chronic lymphocytic lymphoma (CLL); the Small-cell lymphocytic lymphoma (SLL) or Chronic lymphocytic lymphoma (CLL) comprises nodal or splenic involvement; the aggressive NHL is selected from the group consisting of Diffuse large B-cell lymphoma, Mantle cell lymphoma, Transformed follicular lymphoma, Follicular lymphoma (Grade IIIB), Transformed mucosa-associated lymphoid tissue (MALT) lymphoma, Primary mediastinal B-cell lymphoma, Lymphoblastic lymphoma, High-grade B-cell lymphomas with translocations of MYC and BCL2; the high-grade B-cell lymphomas with translocations of MYC and BLC2 further comprises a translocation of BCL6.
[0173] Suitable NK cells for use in treatment of NHL can be prepared as described in US 2020/0108096 or WO 2020/101361, both of which are incorporated herein by refemce. Briefly, the source cells are cultured on modified HuT-78 (ATCC® TIB-161™) cells that have been engineered to express 4-1BBL, membrane bound IL-21 and a mutant TNFalpha as described in US 2020/0108096. [0174J As one example, suitable NK cells can be prepared as follows using HuT-78 cells transduced to express 4-1BBL, membrane bound IL-21 and mutant TNFalpha (“eHut-78P cells”) as feeder cells. The feeder cells are suspended in 1% (v/v) CellGro medium at 2.5x106 cells/ml and are irradiated with 20,000 cGy in a gamma-ray irradiator. Seed cells (e.g., CD3-depleted PBMC or CD3-depleted cord blood cells) are grown on the feeder cells in CellGro medium containing 1% (v/v) human plasma, glutamine, 500 IU of IL-2, 10 ng/ml of OKT-3 at a ratio of 1:2.5 (seed cells: feeder cells) in in static culture at 37° C. The cells are split every 2-4 days. The total culture time can be 19 days. The NK cells are harvested by centrifugation and cryopreserved. Thawed NK are administration to patients in infusion medium consisting of Phosphate Buffered Saline (50% v/v) with albumin (human) 20% (20% v/v), Dextran 40 in Dextrose (25% v/v) and dimethyl sulfoxide (DMSO) (5% v/v).
[0175] In some case, the seed cells are CD3-depleted cord blood cells. Preferably, the cord blood seed cells are selected to express CD16 having the V/V polymorphism at F158 (Fc gamma RIIIa-158 V/V genotype) (Musolino et al. 2008 J Clin Oncol 26 :1789). Preferably, the cord blood seed cells are KIR-B haplotype. A cell fraction can be depleted of CD3 cells by immunomagnetic selection, for example, using a CliniMACS T cell depletion set ((LS Depletion set (162-01) Miltenyi Biotec).
[0176] Rituximab (e.g., Rituxan®) is a preferred IL-20 targeted antibody. Rituximab is preferably administered at 375 mg/m2, preferably at least 1 hour prior to each administration of NK cells.
[0177] IL-2 is preferably administered at 1 x 106 IU/m2, will be administered subcutaneously, at least 1 hour and no more than 4 hours following the conclusion of each administration.
The methods described herein can be used to treat patients suffering from a CD20+ cancer, for example, indolent or aggressive non-Hodgkin’s lymphoma (NHL), particularly relapsed or refractory indolent or aggressive NHL of B-cell origin. Among the aggressive and indolent subtypes are those in Table 4.
Table 4. Exemplary Aggressive and Indolent NHL
Figure imgf000031_0001
Figure imgf000032_0001
[0178] Prior to treatment, the patient is preferably lymphodepleted by intravenous administration of cyclophosphamide (250 mg/m2/day) and fludarabine (30 mg/m2/day) daily for
3 consecutive days, starting 5 days before the first dose of NK cells (i.e., from Day -5 through Day -3).
[0179] The NK cells (for example AB-101, Artiva Biotherapeutics, Inc.) are preferably administered weekly with each administration of 1 x 109 or 4 x 109 NK cells. The cells are preferably ciyopreserved NK cells suspended in infusion-ready media (50% PBS, 25%Dextran 40, 20% albumin (human), 5% DMSO) in vials containing approximately 1 x 109 cells. The cells are thawed in a 37°C water bath prior to administration. The thawed vial(s) of NK cells are aseptically transferred to a single administration bag using a vial adapter and a sterile syringe. The NK cells are administered to the patient from the bag through a Y-type blood/solution set with filter as an IV infusion, by gravity. The NK cells are preferably should be administered as soon as practical, preferably within 30 minutes and no longer than 90 minutes after thawing.
[0180] IL-2, dosed at 1 x 106 IU/m2, is administered subcutaneously, at least 1 hour and no more than 4 hours following the conclusion of each dose of NK cells. Rituximab is preferably administered at 375 mg/m2, preferably at least 1 hour prior to each administration of NK cells.
[0181] Administration of the NK cells preferably occurs weekly for 8 weeks.
[0182] Thus, described herein are methods for treating a patient suffering from a CD20+ cancer, the method comprising administering allogenic natural killer cells (NK cells) and an antibody targeted to human CD20, wherein the NK cells are allogenic to the patient, are KIR-B haplotype and express CD16 having the V/V polymorphism at F158.
[0183] In some embodiments, the cancer is non-Hodgkins lymphoma (NHL).
[0184] In some embodiments, the NHL is indolent NHL.
[0185] In some embodiments, the NHL is aggressive NHL.
[0186] In some embodiments, the patient has relapsed after treatment with an anti-CD20 antibody.
[0187] In some embodiments, the patient has experienced disease progression after treatment with autologous stem cell transplant or chimeric antigen receptor T-cell therapy (CAR-T). [0188] In some embodiments, the patient is administered 1 x 108 to 1 x 1010 NK cells.
[0189] In some embodiments, the patient is administered 1 x 109 to 8 x 109 NK cells.
[0190] In some embodiments, the patient is administered 4 x 108, 1 x 109, 4 x 109, or 8 x 109
NK cells.
[0191] In some embodiments, the patient is administered 100 to 500 mg/m2 of the antibody.
[0192] In some embodiments, the patient is administered 375 mg/m2 of the antibody.
[0193] In some embodiments, the antibody is rituximab.
[0194] In some embodiments, the patient is subjected to lymphodepl eting chemotherapy prior to treatment.
[0195] In some embodiments, the lymphodepleting chemotherapy is non-myeloablative chemotherapy.
[0196] In some embodiments, the lymphodepleting chemotherapy comprises treatment with at least one of cyclophosphamide and fludarabine.
[0197] In some embodiments, the lymphodepleting chemotherapy comprises treatment with cyclophosphamide and fludarabine.
[0198] In some embodiments, the cyclophosphamide is administered between 100 and 500 mg/m2/day.
[0199] In some embodiments, the cyclophosphamide is administered 250 mg/m2/day.
[0200] In some embodiments, the fludarabine is administered between 10 and 50 mg/m2/day.
[0201] In some embodiments, the fludarabine is administered 30mg/m2/day.
[0202] In some embodiments, the method further comprises administering IL-2.
[0203] In some embodiments, the patient is administered 1 x 106 IU/m2 of IL-2.
[0204] In some embodiments, administration of IL-2 occurs within 1-4 hrs of administration of the NK cells.
[0205] In some embodiments, the administration of the NK cells and the antibody targeted to human CD20 occurs weekly.
[0206] In some embodiments, the NK cells and the antibody targeted to human CD20 are administered weekly for 4 to 8 weeks.
[0207] In some embodiments, the NK cells are not genetically modified.
[0208] In some embodiments, at least 70% of the NK cells are CD56+ and CD16+.
[0209] In some embodiments, at least 85% of the NK cells are CD56+ and CD3-.
[0210] In some embodiments, 1% or less of the NK cells are CD3+, 1% or less of the NK cells are CD19+ and 1% or less of the NK cells are CD14+. [0211] In some embodiments, the indolent NHL is selected from the group consisting of Follicular lymphoma, Lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, Gastric MALT, Non-gastric MALT, Nodal marginal zone lymphoma, Splenic marginal zone lymphoma, Small-cell lymphocytic lymphoma (SLL), and Chronic lymphocytic lymphoma (CLL).
[0212] In some embodiments, the Small-cell lymphocytic lymphoma (SLL) or Chronic lymphocytic lymphoma (CLL) comprises nodal or splenic involvement.
[0213] In some embodiments, the aggressive NHL is selected from the group consisting of Diffuse large B-cell lymphoma, Mantle cell lymphoma, Transformed follicular lymphoma, Follicular lymphoma (Grade IIIB), Transformed mucosa-associated lymphoid tissue (MALT) lymphoma, Primary mediastinal B-cell lymphoma, Lymphoblastic lymphoma, High-grade B-cell lymphomas with translocations of MYC and BCL2.
[0214] In some embodiments, the high-grade B-cell lymphomas with translocations of MYC and BLC2 further comprises a translocation of BCL6.
[0215] In some embodiments, each administration of NK cells is administration of 1 x 109 to 5 x 109 NK cells. In some embodiments, each administration of NK cells is administration of 1 x 109 to 5 x 109 NK cells.
VIIL EXAMPLES
[0216] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Off-the-Shdf NK Cell Therapy Platform
[0217] One example of a method by which NK cells were expanded and stimulated is shown
FIG. 1.
[0218] A single unit of FDA-licensed, frozen cord blood that has a high affinity variant of the receptor CD16 (the 158 V/V variant, see, e.g., Koene et al., “FcγRIIIa-158V/F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcgammaRIIIa, Independently of the FcgammaRIIIa-48L/R/H Phenotype,” Blood 90: 1109-14 (1997).) and the KIR-B genotype (KIR B allele of the KIR receptor family, see, e.g., Hsu et al., “The Killer Cell Immunoglobulin-Like Receptor (KIR) Genomic Region: Gene-Order, Haplotypes and Allelic Polymorphism,” Immunological Review 190:40-52 (2002); and Pyo et al., “Different Patterns of Evolution in the Centromeric and Telomeric Regions of Group A and B Haplotypes of the Human Killer Cell Ig-like Receptor Locus,” PLoS One 5:el5115 (2010)) was selected as the source of NK cells. [0219] The cord blood unit was thawed and the freezing medium was removed via centrifugation. The cell preparation was then depleted of T cells using the QuadroMACS Cell Selection System (Miltenyi) and CD3 (T cell) MicroBeads. A population of 6 x 108 total nucleated cells (TNC) were labelled with the MicroBeads and separated using the QuadroMACS device and buffer. Following depletion of T cells, the remaining cells, which were predominantly monocytes and NK cells, were washed and collected in antibiotic-free medium (CellgroSCGM). The cell preparation was then evaluated for total nucleated cell count, viability, and % CD3+ cells. As shown in FIG. 1, the cord blood NK cells were CD3 depleted.
[0220] The CD3- cell preparation was inoculated into a gas permeable cell expansion bag at 0.3 x 106 viable cells/mL containing growth medium. As shown in FIG. 1, the cells were cocultured with replication incompetent engineered HuT-78 (eHUT-78) feeder cells to enhance expansion for master cell bank (MCB) production. The CellgroSCGM growth media was initially supplemented with 10 ng/mL of anti-CD3 antibody (OKT3), 1.0% (v/v) human plasma, 4 mM glutamine, and 80 ng/mL IL-2.
[0221] As shown in FIG. 1, the NK cells are optionally engineered, e.g., to introduce CARs into the NK cells, e.g., with a lentaviral vector, during one of the co-culturing steps.
[0222] The cells were incubated as a static culture for 12-16 days at 37°C in a 5% CO2 balanced air environment, with additional exchanges of media occurring every 2 to 4 days. After the culture has expanded more than 100-fold, the cultured cells were harvested and then suspended in freezing medium at 1.0 x 108 cells/mL and filled into 5 mL cryobags. In this example, 80 bags or vials at 108 cells per bag or vial were produced during the co-culture. The cryobags were frozen using a controlled rate freezer and stored in vapor phase liquid nitrogen (LN2) tanks below -150°C. These cryopreserved NK cells derived from the FDA-licensed cord blood unit served as the master cell bank (MCB).
[0223] To produce the drug product, a bag of frozen cells from the MCB was thawed and the freezing medium was removed. The thawed cells were inoculated into a disposable culture bag and co-cultured with feeder cells, e.g., eHUT78 feeder cells to produce the drug product. In this example, the cells are cultured in a 50 L bioreactor to produce thousands of lots of the drug product per unit of cord blood (e.g., 4,000-8,000 cryovials at 109 cells/vial), which are mixed with a cryopreservation composition and frozen in a plurality of storage vessels such as cryovials. The drug product is an off-the-shelf infusion ready product that can be used for direct infusion. Each lot of the drug product can be used to infuse hundreds to thousands of patients (e.g., 100-1,000 patients, e.g. with a target dose of 4 x 109 cells). Example 2: AB-101
[0224] AB-101 is a universal, off-the-shelf, cryopreserved allogeneic cord blood derived NK cell therapy product comprising ex vivo expanded and activated effector cells designed to enhance ADCC anti-tumor responses in patients, e.g., patients treated with monoclonal antibodies or NK cell engagers. AB-101 is comprised of cord blood derived mononuclear cells (CBMCs) enriched for NK cells by depletion of T lymphocytes, and co-cultured with an engineered, replication incompetent T cell feeder line supplemented with IL-2 and anti-CD3 antibody (OKT3).
[0225] AB-101 is an allogeneic NK-cell product derived from FDA licensed cord blood, specifically designed to treat hematological and solid tumors in combination with therapeutic monoclonal antibodies (mAbs). The AB- 101 manufacturing process leads to an NK cell product with the following attributes:
• Consistent NK cell profile. High surface receptor expression of antibody engaging CD 16 and tumor antigen-engaging/activating receptors such as NKG2D, NKp46, Nkp30 and NKp44.
• KIR-B-haplotype. KIR-B haplotype has been associated with improved clinical outcomes in the haploidentical transplant setting and greater therapeutic potential in the allogeneic setting
• CD16 F158V polymorphism. The higher-affinity CD16 F158V variant binding to mAb Fc-domain is seen to facilitate enhanced antibody dependent cellular cytotoxicity (ADCC).
• Unmodified NK cells. No genetic enhancement or gene editing is required for, or is a part of, the AB-101 drug product.
[0226] The components and composition of AB-101 are listed in Table 5. AB-101 is comprised of NK cells (CD16+, CD56+) expressing the natural cytotoxicity receptors NKp30 and NKp46 indicative of mature NK cells. AB-101 contains negligible T cells, B cells and macrophages (≤ 0.2% CD3+, ≤ 1.0% CD19+, ≤ 1.0% CD14+). Residual eHuT-78P feeder cells used in the culturing of AB-101 are ≤ 0.2% of the drug product. Table 5. Components and Compositions of AB-101
Figure imgf000037_0001
[0227] Initial stability studies indicate that AB-101 is stable for up to six months in the vapor phase of liquid nitrogen. Long-term stability studies to assess product stability beyond six months are ongoing, and the most current stability information will be captured on the certificate of analysis.
Example 3: Crvopreservation of NK Cells
[0228] AB-101 cells were prepared by the process shown in FIG. 2. At the end of the culture period the cells were harvested through the use of a Sartorius kSep® 400 Single-Use Automated Centrifugation System at Relative Centrifugal Field (RCF): 800 - 1200 g with a flow rate at 60 to 120 mL/min, and washed two times with Phosphate Buffer Solution (PBS). After washing, the AB-101 cells were formulated with: (1) Albumin (human); (2) Dextran 40; (3) DMSO and (4) PBS to a target concentration of 1 x 108 cells/mL (exemplary cryopreservation composition #1, Table 2). The formulated suspension was then filled at a target volume of 11 mL into 10 mL AT-Closed vial®. Filled vials were inspected, labeled and cryopreserved in a controlled rate freezer at ≤ -135°C.
[0229] Stability studies were carried out with time=0 as the initial release testing data. The stability storage freezer is a validated vapor phase LN2 storage freezer which is set to maintain a temperature of ≤ -135°C. For sterility timepoints, 10% of the batch size or 4 vials, whichever is greater, was tested. Test articles were thawed at 37°C to mimic clinical thawing conditions.
[0230] As shown in Table 6, viability and activity of cryopreserved AB-101 was shown to be preserved through at least nine months.
Table 6. Long Term Viability and Activity of Cryopreserved AB-101
Figure imgf000038_0001
[0231] To understand the stability characteristics of AB-101 during handling just prior to administration, a “bedside” short-term stability study was performed. Samples were thawed, transferred to 10 mL syringes, filtered, and the contents stored in Falcon tubes, and kept at that temperature for defined time periods as shown. The collected product was then tested. Short-
Term Stability Data for two lots of AB-101 is shown in Table 7.
Table 7. Short Term Stability Data for AB-101
Figure imgf000038_0002
Figure imgf000039_0001
SEQUENCES
Figure imgf000040_0001
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A cryopreservation composition comprising:
(a) about 40 mg/mL human albumin;
(b) about 25 mg/mL Dextran 40;
(c) about 12.5 mg/mL glucose;
(d) about 55 mg/mL DMSO; and
(e) about 0.5 mL/mL phosphate buffered saline.
2. The cryopreservation composition of claim 1, further comprising: (f) cells.
3. A composition consisting of:
(a) about 40 mg/mL human albumin;
(b) about 25 mg/mL Dextran 40;
(c) about 12.5 mg/mL glucose;
(d) about 55 mg/mL DMSO; and
(e) about 0.5 mL/mL phosphate buffered saline.
4. A pharmaceutical composition comprising:
(a) human albumin;
(b) dextran;
(c) glucose;
(d) DMSO;
(e) a buffer; and
(f) therapeutic cells.
5. The pharmaceutical composition of claim 4 comprising from 30 to 50 mg/mL human albumin.
6. The pharmaceutical composition of claim 4 comprising 50 mg/mL human albumin.
7. The pharmaceutical composition of any one of claims 4-6 comprising 20 to 30 mg/mL dextran.
8. The pharmaceutical composition of any one of claims 4-6 comprising 25 mg/mL dextran.
9. The pharmaceutical composition of any one of claims 4-8, wherein the dextran is Dextran 40.
10. The pharmaceutical composition of any one of claims 4-9 comprising from 12 to 15 mg/mL glucose.
11. The pharmaceutical composition of any one of claims 4-9 comprising 12.5 mg/mL glucose.
12. The pharmaceutical composition of any one of claims 4-9 comprising less than 27.5 g/L glucose.
13. The pharmaceutical composition of any one of claims 4-12 comprising from 50 to 60 ml/mL DMSO.
14. The pharmaceutical composition of any one of claims 4-12 comprising 55 mg/mL DMSO.
15. The pharmaceutical composition of any one of claims 4—14 comprising 40 to 60 % v/v buffer.
16. The pharmaceutical composition of any one of claims 4—15, wherein the buffer is phosphate buffered saline.
17. The pharmaceutical composition of any one of claims 1-16 wherein the therapeutic cells are animal cells.
18. The pharmaceutical composition of claim 17, wherein the animal cells are human cells.
19. The pharmaceutical composition of claim 17 or claim 18, wherein the therapeutic cells are immune cells.
20. The pharmaceutical composition of claim 19, wherein the immune cells are selected from the group consisting of basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.
21. The pharmaceutical composition of claim 20, wherein the immune cell is a T cell.
22. The pharmaceutical composition of claim 21, wherein the T cell is a CAR-T cell.
23. The pharmaceutical composition of claim 20, wherein the immune cell is an NK cell.
24. The pharmaceutical composition of claim 23, wherein the NK cell is a CAR-NK cell.
25. The pharmaceutical composition of any one of claims 4-24, wherein the composition comprises:
(a) about 40 mg/mL human albumin;
(b) about 25 mg/mL Dextran 40;
(c) about 12.5 mg/mL glucose;
(d) about 55 mg/mL DMSO; and
(e) about 0.5 mL phosphate buffered saline per mL of the pharmaceutical composition.
26. The pharmaceutical composition of any one of claims 4-24, further comprising 0.5 mL water per mL of the pharmaceutical composition.
27. A method for treating a disorder, the method comprising:
(a) administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 4-25 to a patient in need thereof.
28. The method of claim 26, wherein the patient has a cellular proliferative and/or differentiative disorder.
29. The method of claim 27, wherein the patient has cancer.
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