WO2024040201A1 - Méthode et système améliorés de thérapie cellulaire - Google Patents

Méthode et système améliorés de thérapie cellulaire Download PDF

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WO2024040201A1
WO2024040201A1 PCT/US2023/072432 US2023072432W WO2024040201A1 WO 2024040201 A1 WO2024040201 A1 WO 2024040201A1 US 2023072432 W US2023072432 W US 2023072432W WO 2024040201 A1 WO2024040201 A1 WO 2024040201A1
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cells
optionally
cell
disease
subject
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PCT/US2023/072432
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English (en)
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Jake REDER
Joana M. MURAD
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Celdara Medical Llc
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Publication of WO2024040201A1 publication Critical patent/WO2024040201A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/20—Supervised data analysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • CAR chimeric antigen receptor
  • T cells have shown promise in the field of oncology, especially hematopoietic cancer.
  • Autoimmune diseases are another area in which CARbased therapy may show success in the near future.
  • Stem cell therapy has great potential to treat many of the diseases for which effective therapy does not exist, such as neurodegenerative diseases and genetic diseases.
  • the current manufacturing and administration of such therapeutic cells involve complex and prolonged procedures and large variabilities. There exists a need for improved methods and systems for cell therapy, particularly those that make cell therapy less time consuming, more automated, more controllable, more personalized, and/or more feasible.
  • the present disclosure provides methods for cell therapy.
  • the method may comprise: (A) collecting a biological sample containing cells from a subject; (B) selecting cells of at least one cell type of interest, such as enriching cells of at least one cell type of interest and/or depleting cells of at least one cell type not of interest, from the biological sample from step (A); (C) treating such as modifying, e.g., genetically modifying, transdifferentiating, dedifferentiating, the cells selected in step (B); and (D) transferring one or more of the cells treated in step (C) to the subject.
  • the steps (A)-(D) may occur in a closed system.
  • the biological sample may comprise: a sample of blood, lymphatic fluid, ascitic fluid, cerebrospinal fluid, adipose tissue, or bone marrow.
  • the blood may be peripheral blood.
  • the biological sample may comprise a sample of a disease site.
  • the disease site may be blood or a solid tumor or an infection site or inflammation site.
  • the subject in step (A), may have and/or the method may be for treating a disease, such as but not limited to cancer, an autoimmune disease, a neurode generative disease, an infectious disease, an inflammatory disease, or another disease.
  • a disease such as but not limited to cancer, an autoimmune disease, a neurode generative disease, an infectious disease, an inflammatory disease, or another disease.
  • the cancer may be a solid cancer, optionally chosen from: one or more of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, renal cancer, liver cancer, brain cancer, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, pharynx cancer, head and neck cancer, rectal cancer, esophagus cancer, or bladder cancer, or a metastasis thereof.
  • mesothelioma malignant pleural mesothelioma
  • non-small cell lung cancer small cell lung
  • the cancer may be a liquid cancer, optionally chosen from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, M
  • the autoimmune or inflammatory disease may be psoriasis, rheumatoid arthritis, autoimmune arthritis, type I diabetes, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, scleroderma, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, pemphigus vulgaris, Sjorgen syndrome, Addison disease, Bahcet’s disease, Schmidt syndrome, celiac disease. dermatomyositis, autoimmune vitiligo, Graves disease, Hashimoto thyroiditis, Kawasaki disease, pernicious anemia, autoimmune vasculitis.
  • the neurodegenerative disease may be Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, Creutzfeldt- Jakob disease.
  • the infectious disease may be a viral, bacterial, fungal, yeast, protozoan, prion or parasitic disease, optionally wherein (1) the viral disease is human immunodeficiency virus, hepatitis C virus, hepatitis B virus, human cytomegalovirus, Pox virus, Influenza virus, or coronavirus infection, (2) the bacterial disease is Mycobacterium tuberculosis, and/or (3) the fungal infection is aspergillosis.
  • the viral disease is human immunodeficiency virus, hepatitis C virus, hepatitis B virus, human cytomegalovirus, Pox virus, Influenza virus, or coronavirus infection
  • the bacterial disease is Mycobacterium tuberculosis
  • the fungal infection is aspergillosis.
  • the other disease may be age-related macular degeneration (AMD), neuromyelitis optica spectrum disorder, atopic dermatitis, asthma, rhinosinusitis, nasal polypsosis, or hemophilia.
  • AMD age-related macular degeneration
  • neuromyelitis optica spectrum disorder atopic dermatitis, asthma, rhinosinusitis, nasal polypsosis, or hemophilia.
  • the at least one cell type in step (B) may comprise at least one immune cell, optionally one or more of the following: leukocyte or erythrocyte, optionally cell of the lymphoid lineage or cell of the myeloid lineage, optionally T cell, B cell, plasma cell, NK cell, NK T cell, macrophage, dendritic cell, monocyte, eosinophil, neutrophil, basophil, or mast cell; progenitor of any of the aforementioned cell types, optionally lymphoid progenitor, myeloid progenitor, lymphoblast, or myeloblast; CD8+ T cell, CD4+ T cell, or any combination thereof, optionally Thl cell, Th2 cell, Th9 cell, Th 17 cell, Th22 cell, regulatory T (Treg) cell, or follicular helper (Tfh) cell; or any combination of the aforementioned cell types.
  • leukocyte or erythrocyte optionally cell of the lymphoid lineage or cell of the myeloid
  • the at least one cell type in step (B) may comprise at least one stem cell, optionally: hematopoietic stem cell, neural stem cell, mesenchymal stem cell, epithelial stem cell, or skin stem cell, optionally lymphoid stem cell or myeloid stem cell.
  • stem cell optionally: hematopoietic stem cell, neural stem cell, mesenchymal stem cell, epithelial stem cell, or skin stem cell, optionally lymphoid stem cell or myeloid stem cell.
  • step (B) may comprise centrifugation-based selection, antibody -based selection, bead-based selection, magnetic selection, filtration-based selection, or any combination thereof.
  • step (B) may comprise one or more of the following: (B-l) separating leukocytes from non-leukocyte elements of the biological sample from step (A), optionally by lysing erythrocytes and/or centrifuging all or part of the biological sample from (A); and (B-2) separating the at least one cell type of interest from other elements of the biological sample from step (A) or from other elements of the leukocytes from (B-l); and (B-3) optionally removing at least one undesirable cell type from the cells obtained in (B-l) and/or (B-2).
  • the method may further comprise returning all or part of the elements of the biological sample not selected in step (B) to the subject.
  • said all or part of the elements of the biological sample may be returned to the subject together with or separately from the one or more of the cells transferred in step (D).
  • one or more sub-elements of said all or part of the elements of the biological sample may be removed before the returning.
  • the one or more sub-elements may comprise: (a) an immunosuppressive cell, optionally a Treg cell and/or myeloid-derived suppressor cell (MDSC); (b) an immunosuppressive molecule, optionally an immunosuppressive cytokine, further optionally IL-10 or TGF-P; or (c) any combination thereof.
  • an immunosuppressive cell optionally a Treg cell and/or myeloid-derived suppressor cell (MDSC);
  • an immunosuppressive molecule optionally an immunosuppressive cytokine, further optionally IL-10 or TGF-P; or (c) any combination thereof.
  • step (C) may comprise (C-l) genetically modifying the cells.
  • step (C-l) may comprise transfecting or transducing the cells with at least one nucleic acid.
  • the at least one nucleic acid comprises a DNA or RNA.
  • the at least one nucleic acid is contained in a vector, optionally a plasmid, a viral vector (optionally adenoviral, lentiviral, or retroviral), a lipid-based vector, a self-replicating RNA vector, a virus-like particle, a polymer-based vector, and/or a nanoparticle, optionally a lipid-based nanoparticle.
  • the transfecting or transducing is via electroporation.
  • step (C-l) may comprise editing the genome in the cells.
  • the editing may comprise clustered regularly interspaced short palindromic repeats (CRISPR)-(CRISPR associated protein) Cas gene editing, editing via a Zinc finger nuclease, and/or editing via a transcription activator-like effector nuclease (TALEN).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • TALEN transcription activator-like effector nuclease
  • step (C-l) may comprise transdiffcrcntiating or dedifferentiating the cells.
  • step (C) may optionally comprise (C-2) separating successfully treated cells from unsuccessfully treated cells
  • step (C) may optionally comprise (C-3) culturing the cells selected in step (B), the cells modified in (C-l), and/or the cells optionally separated in (C-2).
  • the cells may be cultured in the presence of at least one stimulus, optionally one or more cytokines, an antigen, or a growth factor.
  • step (C) may further comprise (C-4) (I) selecting one or more desirable cells and/or (II) removing (i) one or more undesirable cells and/or (ii) one or more undesirable factors from the sample obtained from (C-l), (C-2), or (C-3).
  • (C-5) resuspending the cells obtained from (C-l), (C-2), (C-3), or (C- 4) in a solution of a desired cell concentration rage to provide a formulation suited for administration to the subject.
  • the cell concentration rage in the formulation may be about 100-500 cells/mL, about 500-1000 cells/mL, about 1000-5000 cells/mL, about 5000-10,000 cells/mL, about 10,000-50,000 cells/mL, about 50,000-100,000 cells/mL, about 100,000-500,000 cells/mL, about 500,000-1,000,000 cells/mL, about 1,000,000-5,000,000 cells/mL, about 5,000,000-10,000,000 cells/mL, or about 10,000,000-50,000,000 cells/mL.
  • step (C-l) comprises transfecting or transducing the cells with at least one nucleic acid
  • the at least one nucleic acid may encode at least one membrane protein
  • the membrane protein when expressed may render the one or more cells specific to a target of interest, optionally wherein the target is a target molecule, a target cell, and/or a target site in the subject.
  • the membrane protein may be a receptor, optionally a chimeric antigen receptor (CAR), an T cell receptor, a B cell receptor, a pathogen receptor, e.g., a viral, fungal, yeast, bacterial or parasite receptor, a receptor comprising an antibody or antigen-binding antibody fragment, or a cytokine receptor or chemokine receptor, or any combination thereof, optionally wherein the chemokine receptor is CCR1, CCR2, CCR5, CCR6, CXCR3, CXCR4, CXCR6, CX3CR1, or S1PR.
  • CAR chimeric antigen receptor
  • the membrane protein may be a membrane -bound ligand, optionally a membrane-bound cytokine, membrane-bound chemokine, adhesion molecule, optionally wherein the adhesion molecule is selectin ligand, integrin, LFA-1, or VAL-4.
  • step (C-l) comprises transfecting or transducing the cells with at least one nucleic acid
  • the at least one nucleic acid may encode at least one soluble protein.
  • the soluble protein may comprise a cytokine or chemokine.
  • the at least one nucleic acid may further encode a marker.
  • step (C-l) comprises transfecting or transducing the cells with at least one nucleic acid
  • the at least one nucleic acid may comprise or encode an mRNA, siRNA, miRNA, and/or a shRNA.
  • step (C-l) comprises editing the genome in the cells
  • the editing causes: (i) elimination of a gene or a potion thereof associated with a disease or condition; and/or (ii) correction of the sequence of a gene associated with a disease or condition, which optionally comprises elimination and/or addition of one or more nucleic acids in the gene.
  • the cells in step (C-3), may be cultured in the presence of at least one stimulus, which may comprise a cytokine or chemokine.
  • the cytokine or chemokine may be IL-2, IL-7, IL-15, IL-21, IL-ip, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IL-23, IL- 27, IFN-y, TNF-a, TGF-0, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, LLC13, CCL19, CCL21, CCL22, CXCL12, TSLP, or any combination thereof, optionally a combination of IL-2 and one or both of IL- 15 and IL-21.
  • the cells in step (C-3), may be cultured in the presence of at least one stimulus, which may comprise a molecule which binds to a surface molecule of one or more of the cells.
  • the surface molecule may be one or more of CD3, CD27, CD28, ICOS, 4-1BBL, CTLA-4, PD-1, NKp30, CD28H, CD80, CD86, PD-L1, PD-L2, ICOSLG, B7-H3, B7-H4, VISTA, B7-H6, or B7-H7, and optionally wherein the molecule is an antibody or a binding partner of the surface molecule, further optionally wherein the molecule is soluble, fixed optionally on a bead or a resin, or expressed by another cell.
  • the cells in step (C-3), may be cultured in the presence of at least one stimulus, which may comprise a growth factor and/or a differentiation factor.
  • the cells in step (C-3), may be cultured in the presence of at least one stimulus, which may comprise a (the) target of interest, optionally an antigen of interest, optionally a cognate antigen of or an agent which stimulates a CAR, T cell receptor, B cell receptor, and/or CD28, optionally T cell TransActTM.
  • a stimulus which may comprise a (the) target of interest, optionally an antigen of interest, optionally a cognate antigen of or an agent which stimulates a CAR, T cell receptor, B cell receptor, and/or CD28, optionally T cell TransActTM.
  • the cells may not be cultured in the presence of at least one stimulus, which does not target of interest, optionally an antigen of interest, optionally a cognate antigen of or an agent which stimulates a CAR, T cell receptor, B cell receptor, and/or CD28.
  • the culturing is for: about 24 hours or less, about 18 hours or less, about 12 hours or less, about 6 horns or less, about 4 horns or less, about 3 hours or less, about 2 hours or less, about 1 hours or less, about 30 minute or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, about 3 minutes or less, about 2 minutes or less, or about 1 minute or less; or about 1 minute to about 24 hours, about 1 minute to about 18 hours, about 2 minutes to about 12 horns, about 3 minutes to about 6 hours, about 5 minutes to about 4 hours, about 10 minutes to about 3 hours, about 1 minutes to about 2 horns, or about 30 minutes to about 1 horn.
  • the one or more desirable cells may comprise one or more of memory T cells, central memory T (Tcm) cells, CD62L+CCR7+ T cells, stem cell memory T (Tscm) cells, CD45RA+CD62L+CCR7+ T cells, CD45RA+CD62L+CCR7+CD27+CD28+ T cells, CD95+ T cells, CD 122+ T cells, effector memory T (Tern) cells, CD62L-CCR7- T cells.
  • memory T cells central memory T (Tcm) cells
  • CD62L+CCR7+ T cells CD62L+CCR7+ T cells
  • stem cell memory T (Tscm) cells CD45RA+CD62L+CCR7+ T cells
  • CD45RA+CD62L+CCR7+CD27+CD28+ T cells CD95+ T cells
  • CD 122+ T cells effector memory T (Tern) cells
  • CD62L-CCR7- T cells CD62L-CCR7- T cells.
  • the one or more undesirable factors may comprise a cytokine or chcmokinc, optionally IL-10, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL- 21, IL-23, IL-27, IFN-y, TNF-a, TGF-0, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, LLC13, CCL19, CCL21, CCL22, CXCL12, TSLP, or any combination thereof.
  • a cytokine or chcmokinc optionally IL-10, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL- 21, IL-23, IL-27, IFN-y, TNF-a, TGF-0, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, LLC13, CCL19
  • the one or more undesirable factors may comprise a growth factor and/or a differentiation factor.
  • the one or more undesirable factors may comprise a cytokine or chemokine and a growth factor and/or a differentiation factor.
  • the one or more undesirable cells comprise one or more of exhausted T cells, overactivated or hyperactivated T cells, Treg cells, and/or T cells expressing one or more of PD-1, TIM-3, CTLA-4, LAG-3, T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), B and T lymphocyte attenuator (BTLA), 2B4, CD38, CD43, CD101, and/or CD160 optionally at mid or high levels, optionally PD-1+ LAG-3+ T cells.
  • the selecting and/or removing comprises centrifugationbased selection and/or removal, antibody -based selection and/or removal, bead -based selection and/or removal, magnetic selection and/or removal, filtration-based selection and/or removal, or any combination thereof.
  • step (D) the one or more of the cells treated in step (C) are transferred to: the blood, lymphatic fluid, ascitic fluid, cerebrospinal fluid, or bone marrow of the subject.
  • the blood may be peripheral blood.
  • step (D) the one or more of the cells treated in step (C) are transferred to a disease site of the subject.
  • the disease site is blood or a solid tumor, an infection site or inflammation site.
  • the transferring may be performed at a speed of about 1000- 5000 cells per minute, about 5,000-10,000 cells per minute, about 10,000-50,000 cells per minute, about 50,000-100,000 cells per minute, about 100,000-500,000 cells per minute, about 500,000- 1,000,000 cells per minute, about 1,000,000-5,000,000 cells per minute, about 5,000,000-10,000,000 cells per minute, about 1,000,000-5,000,000 cells per minute, about 5,000,000-10,000,000 cells per minute, about 10,000,000-50,000,000 cells per minute, about 50,000,000-100,000,000 cells per minute, about 100,000,000-500,000,000 cells per minute, about 500,000,000-1,000,000,000 cells per minute.
  • a cell solution in (D), may be transferred to the subject at about 25-150 mL/minute, about 50-120 mL/minute, about 60-100 mL/minute, or about 70-80 mL/minute.
  • the one or more of the cells treated in step (C) may be transferred at a concentration of about 100-500 cells/mL, about 500-1000 cells/mL, about 1000-5000 cells/mL, about 5000-10,000 cells/mL, about 10,000-50,000 cells/mL, about 50,000-100,000 cells/mL, about 100,000-500,000 cells/mL, about 500,000-1,000,000 cells/mL, about 1,000,000- 5,000,000 cclls/mL, about 5,000,000-10,000,000 cclls/mL, or about 10,000,000-50,000,000 cclls/mL [0056] In some embodiments, the transferring may take about 1-60 minutes, about 5-45 minutes, about 10-30 minutes, or about 15-20 minutes.
  • the method may further comprise (E) conditioning the subject.
  • step (E) may be performed before step (A), during step (A), after step (A), during one or more of steps (A)-(D), during one or more of steps (B)-(D), during step (D), and/or after step (D).
  • the conditioning may comprise depleting or inactivating an undesired cell type in the subject.
  • the undesired cell type may comprise one or more of: lymphocyte, T cell, B cell, NK cell, Treg cell, MDSC, and/or cancer cell.
  • the depleting or inactivating may comprise administering one or more small molecules to the subject, optionally the one or more small molecules comprise fludarabine, cyclophosphamide, bendamustine, a checkpoint inhibitor, or any combination thereof.
  • the depleting or inactivating may comprise administering one or more antibodies specific to the undesired cell type to the subject, optionally wherein the one or more antibodies comprise a checkpoint inhibitor antibody, an antibody specific to Treg cells, an antibody specific to MDSCs, an antibody specific to cancer cells, an anti-CD19 antibody, an anti-CD52 antibody, or any combmation thereof.
  • the depleting or inactivating may comprise administering one or more cytokines which inhibit one or more activities of the undesired cell type.
  • the conditioning may comprise increasing or activating a desired cell type in the subject.
  • the desired cell type may comprises the at least one cell type of interest of step (B), i.e.,: at least one immune cell, optionally: leukocyte or er throcyte, optionally cell of the lymphoid lineage or cell of the myeloid lineage, optionally T cell, B cell, plasma cell, NK cell, NK T cell, macrophage, dendritic cell, monocyte, eosinophil, neutrophil, basophil, or mast cell; progenitor of any of the aforementioned cell types, optionally lymphoid progenitor, myeloid progenitor, lymphoblast, or myeloblast; CD8+ T cell, CD4+ T cell, or any combination thereof, optionally Thl cell, Th2 cell, Th9 cell, Thl7 cell, Th22 cell, regulatory T (Treg) cell, or follicular helper (Tfh) cell; or any combination of the aforementioned cell types; or at least one stem cell, optionally: hematoma, hematom,
  • the increasing or activating comprises one or more of: (i) administering one or more agonistic antibodies specific to the desired cell type to the subject; and/or (ii) administering one or more cytokines which activate one or more activities of the undesired cell type.
  • the conditioning may comprise eliminating or reducing an undesired factor in the subject.
  • the undesired factor comprises one or more of: an immunosuppressive cytokine, optionally IL-10 or TGF-[>, or an inflammatory cytokine, optionally IL- 2, IL-6, IL-7, IL-15, and/or IFNy.
  • the depleting or inactivating comprises administering an antibody against the undesired factor, optionally a blocking or neutralizing antibody.
  • the conditioning may comprise reducing or preventing a toxic and/or side effect in the subject.
  • the toxic and/or side effect may comprise cytokine release syndrome (CRS), macrophage activation syndrome (MAS), hemophagocytic lymphohistiocytosis (HLH), neurological side effects (e.g., confusion, delirium, word-finding aphasia, coma, seizures, irritability, and/or delirium), tumor lysis syndrome (TLS), on-target/off-tumor toxicities (e.g., hypogammaglobulinemia).
  • CRS cytokine release syndrome
  • MAS macrophage activation syndrome
  • HH hemophagocytic lymphohistiocytosis
  • neurological side effects e.g., confusion, delirium, word-finding aphasia, coma, seizures, irritability, and/or delirium
  • TLS tumor lysis syndrome
  • on-target/off-tumor toxicities e.g., hypogammaglobulinemia
  • the reducing or preventing may comprise administering to the subject one or more of: (i) an anti-cytokine or anti-cytokine receptor antibody, optionally anti-IL-6 or anti-IL-6 receptor antibody for treating or preventing CRS; (ii) a corticosteroid for treating or preventing MAS or HLH; and/or (iii) prophylactic allopurinol for treating or preventing TLS; and/or (iv) intravenous immuno globulin (IVIG) replacement therapy for treating or preventing hypogammaglobulinemia
  • the method may further comprise (F) monitoring the subject.
  • step (F) may be performed before step (A), during step (A), after step (A), during one or more of steps (A)-(D), during one or more of steps (B)-(D), during step (D), and/or after step (D).
  • step (F) may comprise (F-l) collecting a monitoring sample from the subject.
  • the monitoring sample comprises a sample of blood, lymphatic fluid, ascitic fluid, cerebrospinal fluid, adipose tissue, or bone marrow, optionally wherein the blood is peripheral blood.
  • the monitoring sample comprises a sample of a disease site, optionally blood or a solid tumor or an infection site or inflammation site.
  • step (F) may comprise (F-2) analyzing: (i) all or part of the biological sample collected in step (A); (ii) all or part of the cells selected in step (B); (iii) all or part of the elements of the biological sample not selected in step (B), optionally plasma; (iv) all or part of the cells treated in step (C); (v) all or part of the cells being transferred in step (D) or a formulation containing such cells; (vi) all or part of the monitoring sample collected in (F-l); and/or (vii) body temperature, breathing rate, blood pressure, heart rate, oxygen levels, or any combination thereof of the subject,
  • the analyzing may be by measuring the level of a cytokine of interest, optionally IL-10, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN-y, TNF-a, MCP-1, GM-CSF, TGF-f>. or any combination thereof, or a receptor thereof.
  • a cytokine of interest optionally IL-10, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN-y, TNF-a, MCP-1, GM-CSF, TGF-f>. or any combination thereof, or a receptor thereof.
  • the analyzing may be by measuring the level of a chemokine of interest, optionally CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL12, CCL13, CCL19, CCL21, CCL22, CXCL8, CXCL9, CXCL10, CXCL12, TSLP, MIP-1, or any combination thereof, or a receptor thereof.
  • a chemokine of interest optionally CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL12, CCL13, CCL19, CCL21, CCL22, CXCL8, CXCL9, CXCL10, CXCL12, TSLP, MIP-1, or any combination thereof, or a receptor thereof.
  • the analyzing may be by measuring the level of cells of at least one undesired cell type, optionally exhausted T cells, overactivated or hyperactivated T cells, Treg cells, T cells expressing one or more of PD-1, TIM-3, CTLA-4, LAG-3, T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), B and T lymphocyte attenuator (BTLA), 2B4, CD38, CD43, CD101, and/or CD160 optionally at mid or high levels, PD-1+ LAG-3+ T cells, Treg cells, MDSCs, or any combination thereof.
  • step (F) may comprise (F-3) optionally adjusting the method based on the analysis result obtained in (F-2).
  • the adjusting may be by changing the treating condition in step (C), optionally increasing or decreasing the time length of the culturing and/or adding, reducing, or removing a stimulus in the culture in (C-3).
  • the adjusting may be by increasing or decreasing the speed of transferring and/or the amount of cells transferred in step (D).
  • the adjusting may be by administering a drug or therapy to the subject.
  • a drug which reduces or blocks a cytokine, a chemokine, or a receptor thereof further optionally a drug which reduces or blocks IL-ip, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL- 13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN-y, TNF-a, MCP-1, GM-CSF, TGF-P, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL12, CCL13, CCL19, CCL21, CCL22, CXCL8, CXCL9, CXCL10, CXCL12, TSLP, MIP-1, or a receptor thereof, further optionally an anti-IL-6 or anti-IL-6 receptor antibody may be administered.
  • a corticosteroid may be administered.
  • IV fluid, oxygen or ventilator support
  • the present disclosure provides computer-readable medium.
  • One or more computer-readable media may have encoded thereon computer-readable instructions for causing one or more processing devices to control operations of various portions of a closed system apparatus for cell therapy.
  • the processing devices may optionally transmit data in real-time to a controller or operator on-site or off-site relative to where the processes are conducted.
  • Such an apparatus may comprise: (a) a first connector configured to be operably and/or directly connectable to a subject to allow for collection of a biological sample from the subject through the first connector; (b) a selector, such as a separator, configured to separate cells of at least one cell ty pe of interest from elements not of interest in the biological sample; (c) a treatment chamber configured to treat cells; and (d) a second connector configured to be operably and/or directly connectable to the subject to allow for transfer of the treated cells to the subject through the second connector.
  • a first connector configured to be operably and/or directly connectable to a subject to allow for collection of a biological sample from the subject through the first connector
  • a selector such as a separator, configured to separate cells of at least one cell ty pe of interest from elements not of interest in the biological sample
  • a treatment chamber configured to treat cells
  • a second connector configured to be operably and/or directly connectable to the subject to allow for transfer of the treated cells to the subject through the second connector
  • Such operations may comprise: (A) optionally collecting a biological sample containing cells from a subject and optionally transferring the biological sample through the first connector (i) to the treatment chamber or (ii) to the selector; (B) selecting cells of at least one cell type of interest, such as enriching cells of at least one cell type of interest and/or depleting cells of at least one cell type not of interest, from a/the biological sample (i) in the treatment chamber and then in the selector or (ii) in the selector and transferring the selected cells to the treatment chamber; and (C) treating such as modifying, e.g., genetically modifying, transdifferentiating, the selected cells (i) in the treatment chamber or (ii) in the treatment chamber and then in the selector and optionally then in the treatment chamber; and (D) optionally transferring one or more of the cells treated in (C) to the subject.
  • A optionally collecting a biological sample containing cells from a subject and optionally transferring the biological sample through the first connector (i) to the treatment chamber
  • such operations may further comprise: (I) presenting, on a display, a user interface configured to accept one or more user-inputs pertaining to one or more of the operations, optionally wherein the one or more user-inputs identify (i) a condition of the selecting in the operation(s) of (B), optionally the at least one cell type of interest and/or a least one cell type not to be selected and/or (ii) a condition of the treating in the operation(s) of (C), optionally the duration of the treating, a temperature used during the treating, and/or a volume of a solution containing the cells during the treating; and (II) optionally adjusting at least one of the operations in accordance with the one or more user-inputs.
  • the biological sample in tire operation(s) of (A), may comprise a sample of blood, lymphatic fluid, ascitic fluid, cerebrospinal fluid, adipose tissue, or bone marrow, optionally wherein the blood is peripheral blood.
  • the biological sample may comprise a sample of a disease site, optionally blood or a solid tumor, or an infection site or inflammation site.
  • the operation(s) of (A) may comprise (i) said collecting and said transferring or (ii) said transferring.
  • the transferring may comprise electronically controlling at least one valve and/or a pump configured to control fluid flow between the first connector and the treatment chamber and/or the selector and/or between the treatment chamber and the selector.
  • the subject in the operation(s) of (A), may have a disease, optionally cancer, an autoimmune disease, an inflammatory disease, an infectious disease, a neurodegenerative disease, or another disease.
  • a disease optionally cancer, an autoimmune disease, an inflammatory disease, an infectious disease, a neurodegenerative disease, or another disease.
  • the cancer may be a solid cancer, optionally chosen from: one or more of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, renal cancer, liver cancer, brain cancer, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, pharynx cancer, head and neck cancer, rectal cancer, esophagus cancer, or bladder cancer, or a metastasis thereof
  • the cancer may be a liquid cancer, optionally chosen from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, M
  • the autoimmune or inflammatory disease may be psoriasis, rheumatoid arthritis, autoimmune arthritis, type I diabetes, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, scleroderma, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, pemphigus vulgaris, Sjorgen syndrome, Addison disease, Bahcet’s disease, Schmidt syndrome, celiac disease, dermatomyositis, autoimmune vitiligo, Graves disease, Hashimoto thyroiditis, Kawasaki disease, pernicious anemia, autoimmune vasculitis, or fibrosis.
  • the neurode generative disease may be Alzheimer’s disease. Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, or Creutzfeldt-Iakob disease.
  • the infectious disease may be a viral, bacterial, fungal, yeast, protozoan, prion or parasitic disease, optionally wherein (1) the viral disease is human immunodeficiency virus, hepatitis C virus, hepatitis B virus, human cytomegalovirus, Pox virus, Influenza virus, or coronavirus infection, (2) the bacterial disease is Mycobacterium tuberculosis, and/or (3) the fungal infection is aspergillosis.
  • the viral disease is human immunodeficiency virus, hepatitis C virus, hepatitis B virus, human cytomegalovirus, Pox virus, Influenza virus, or coronavirus infection
  • the bacterial disease is Mycobacterium tuberculosis
  • the fungal infection is aspergillosis.
  • the other disease may be age-related macular degeneration (AMD), neuromyelitis optica spectrum disorder, atopic dermatitis, asthma, rhinosinusitis, nasal polypsosis, or hemophilia.
  • AMD age-related macular degeneration
  • neuromyelitis optica spectrum disorder atopic dermatitis, asthma, rhinosinusitis, nasal polypsosis, or hemophilia.
  • the at least one cell type may comprise at least one immune cell, optionally: leukocyte or er throcyte, optionally cell of the lymphoid lineage or cell of the myeloid lineage, optionally T cell, B cell, plasma cell, NK cell, NK T cell, macrophage, dendritic cell, monocyte, eosinophil, neutrophil, basophil, or mast cell; progenitor of any of lhe aforementioned cell types, optionally lymphoid progenitor, myeloid progenitor, lymphoblast, or myeloblast; CD8+ T cell, CD4+ T cell, or any combination thereof, optionally Thl cell, Th2 cell, Th9 cell, Thl 7 cell, Th22 cell, regulatory T (Treg) cell, or follicular helper (Tfh) cell; or any combination of the aforementioned cell types.
  • leukocyte or er throcyte optionally cell of the lymphoid lineage or cell of the myeloid lineage, optionally T
  • the at least one cell type may comprise at least one stem cell, optionally: hematopoietic stem cell, neural stem cell, mesenchymal stem cell, epithelial stem cell, or skin stem cell, optionally lymphoid stem cell or myeloid stem cell.
  • the selecting may comprise centrifugationbased selection, antibody-based selection, bead-based selection, magnetic selection, magnetic activated cell sorting (MACS)-based selection, filtration-based selection, or any combination thereof.
  • the selecting may comprise: (i-1) priming a MACS column contained in the selector using a buffer solution, optionally wherein the priming is controlled by controlling at least one valve and/or a pump configured to control fluid flow between a buffer reservoir and the MACS column; (i-2) applying all or part of the biological sample to the MACS column, optionally comprising controlling a rate at which the biological sample is applied to the MACS column; (i-3) flowing a buffer solution through the MACS column to obtain one or more flow through fractions, optionally wherein the rinsing is controlled by controlling at least one valve and/or a pump configured to control fluid flow between a/the buffer reservoir and the MACS column; and (i-4) harvesting the cells of at least one cell type of interest from the MACS column by eluting the column or from the one or more flow through fractions, optionally wherein the eluting is controlled by controlling at least one valve and/or a pump configured to control fluid flow between a/the buffer reservoir and the MACS column;
  • the selecting may comprise one or more of the following: (B-l) separating leukocytes from non-leukocyte elements of the biological sample from step (A), optionally by lysing erythrocytes and/or centrifuging all or part of the biological sample from (A), optionally in the treatment chamber and/or the selector; and (B-2) separating the at least one cell type of interest from other elements of the biological sample from step (A) or from other elements of the leukocytes from (B-l), optionally in the treatment chamber and/or the selector; and (B-3) optionally removing at least one undesirable cell type from the cells obtained in (B-l) and/or (B-2), optionally in the treatment chamber and/or the selector [0092]
  • the method may further comprise returning all or part of the elements of the biological sample not selected to the subject, c.g., not selected in step (B).
  • said all or part of the elements of the biological sample is returned to tire subject together with or separately from the one or more of the cells transferred in (D).
  • one or more sub-elements of said all or part of the elements of the biological sample is removed before the returning.
  • the one or more subelements may comprise an immunosuppressive cell, optionally a Treg cell and/or myeloid-derived suppressor cell (MDSC).
  • the one or more sub-elements may comprise an immunosuppressive molecule, optionally an immunosuppressive cytokine, further optionally IL-10 or TGF-0.
  • the one or more sub-elements may comprise one or more immunosuppressive cells and one or more immunosuppressive molecules.
  • the transferring may comprise electronically controlling at least one valve and/or a pump configured to control fluid flow between the selector, optionally a receptacle configured to hold the selected cells within the selector, and the treatment chamber.
  • the operation(s) of (C) may comprise (C-l) genetically modifying the cells.
  • the genetically modifying the cells may comprise transfecting or transducing the cells with at least one nucleic acid.
  • the at least one nucleic acid may comprise a DNA or RNA.
  • the at least one nucleic acid may be contained in a vector, optionally a plasmid, a viral vector (optionally adenoviral, lentiviral, or retroviral), a lipid-based vector, a self-replicating RNA vector, a virus-like particle, a polymer-based vector, and/or a nanoparticle, optionally a lipid-based nanoparticle.
  • the genetically modifying the cells may comprise editing the genome in the cells.
  • the editing comprises clustered regularly interspaced short palindromic repeats (CRISPR)-(CRISPR associated protein) Cas gene editing, editing via a Zinc finger nuclease, and/or editing via a transcription activator-like effector nuclease (TALEN).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • TALEN transcription activator-like effector nuclease
  • the genetically modifying the cells may comprise transdifferentiating or dedifferentiating the cells.
  • the operation(s) of (C) may comprise optionally separating successfully treated cells from unsuccessfully treated cells.
  • the operation(s) of (C) may comprise culturing the cells selected in step (B), the cells modified in (C-l), and/or the cells optionally separated in (C-2).
  • the culturing may be performed in the presence of at least one stimulus and/or feeder cells.
  • the operation(s) of (C) may further comprise (C-4) (I) selecting one or more desirable cells and/or (II) removing (i) one or more undesirable cells and/or (ii) one or more undesirable factors from the sample obtained from (C-l), (C-2), or (C-3).
  • the operation(s) of (C) may further comprise resuspending the cells obtained from (C-l), (C-2), (C-3), or (C-4) in a solution of a desired cell concentration rage to provide a formulation suited for administration to the subject.
  • the cell concentration rage may be about 100-500 cells/mL, about 500-1000 cells/mL, about 1000-5000 cells/mL, about 5000-10,000 cells/mL, about 10,000-50,000 cells/mL, about 50,000-100,000 cells/mL, about 100,000- 500,000 cells/mL, about 500,000-1,000,000 cells/mL, about 1,000,000-5,000,000 cells/mL, about 5,000,000-10,000,000 cells/mL, or about 10,000,000-50,000,000 cells/mL.
  • the operation(s) of (C-l) comprises introducing a medium into the treatment chamber subsequent to the transferring of the operation(s) of (B).
  • the operation(s) of (C-l) and/or (C-3) comprise executing a motion of the treatment chamber to facilitate contact between the cells and the medium.
  • the introducing and/or said executing is/are repeated at least once.
  • the at least one nucleic acid may encode at least one membrane protein.
  • the membrane protein when expressed may render the one or more cells specific to a target of interest, optionally wherein the target is a target molecule, a target cell, and/or a target site in the subject.
  • the membrane protein may be a receptor, optionally a chimeric antigen receptor (CAR), an T cell receptor, a B cell receptor, a pathogen receptor, e g., a viral, fungal, yeast, bacterial or parasite receptor, a receptor comprising anantibody or antigen-binding antibody fragment, or a cytokine receptor or chemokine receptor, or any combination thereof, optionally wherein the chemokine receptor is CCR1, CCR2, CCR5, CCR6, CXCR3, CXCR4, CXCR6, CX3CR1, or S1PR.
  • CAR chimeric antigen receptor
  • the membrane protein may be a membrane-bound ligand, optionally a membrane-bound cytokine, membrane-bound chemokine, adhesion molecule, optionally wherein the adhesion molecule is selectin ligand, integrin, LFA-1, or VAL-4.
  • the genetically modifying the cells comprises transfecting or transducing the cells with at least one nucleic acid
  • the at least one nucleic acid may encode at least one soluble protein, optionally cytokine or chemokine.
  • the at least one nucleic acid may encode a marker, e.g., a marker which indicates successful transduction/transfection, such as truncated CD 19 or trCD19.
  • the at least one nucleic acid may comprise or encode an mRNA, siRNA, miRNA, and/or a shRNA.
  • the editing may cause elimination of a gene or a potion thereof associated with a disease or condition.
  • the editing may cause correction of the sequence of a gene associated with a disease or condition.
  • such correction may comprise elimination and/or addition of one or more nucleic acids in the gene.
  • the separating may comprise: (i) receiving user-input indicative of a supernatant volume; (ii) executing a centrifugal motion of the treatment chamber to pellet the genetically modified cells; and (iii) removing the supernatant material in accordance with the supernatant volume.
  • the at least one stimulus may comprise a cytokine or chemokine, optionally IL-2. IL-7, IL-15. IL-21, IL-10, IL-4, IL-6, IL-10, IL-12, IL-13, IL- 18, IL-23, IL-27, IFN-y, TNF-a, TGF-0, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, LLC13, CCL19, CCL21, CCL22, CXCL12, TSLP, or any combination thereof.
  • the at least one stimulus may comprise a molecule which binds to a surface molecule of one or more of the cells.
  • the surface molecule may be one or more of CD3, CD27, CD28, ICOS, 4-1BBL, CTLA-4, PD-1, NKp30, CD28H, CD80, CD86, PD-L1, PD-L2, ICOSLG, B7-H3, B7-H4, VISTA, B7-H6, or B7-H7.
  • the molecule may be an antibody or a binding partner of the surface molecule.
  • the molecule may be soluble, fixed optionally on a bead or a resin, or expressed by another cell.
  • the at least one stimulus may comprise a growth factor and/or a differentiation factor.
  • the at least one stimulus may comprise a (the) target of interest, optionally an antigen of interest, optionally a cognate antigen of or an agent which stimulates a CAR, T cell receptor, B cell receptor, and/or CD28, optionally T cell TransActTM.
  • the at least one stimulus may comprise a (the) target of interest, optionally an antigen of interest, optionally a cognate antigen of or an agent which stimulates a CAR, T cell receptor, B cell receptor, and/or CD28.
  • the at least one stimulus may comprise any combination of the stimuli described above.
  • the culturing in the operation(s) of (C-3), may be for: about 24 hours or less, about 18 hours or less, about 12 hours or less, about 6 horns or less, about 4 hours or less, about 3 horns or less, about 2 hours or less, about 1 hours or less, about 30 minute or less, about 1 minutes or less, about 10 minutes or less, about 5 minutes or less, about 3 minutes or less, about 2 minutes or less, or about 1 minute or less; or about 1 minute to about 24 horns, about 1 minute to about 18 hours, about 2 minutes to about 12 hours, about 3 minutes to about 6 hours, about 5 minutes to about 4 hours, about 10 minutes to about 3 hours, about 15 minutes to about 2 hours, or about 30 minutes to about 1 hour.
  • the culturing may comprise: (i) controlling the temperature of the treatment chamber for a predetermined amount of time; and/or (ii) controlling flow of at least one gas into the treatment chamber
  • the one or more desirable cells may comprise one or more of memory T cells, central memory T (Tcm) cells, CD62L+CCR7+ T cells, stem cell memory T (Tscm) cells, CD45RO-/CCR7+ T cells, CD45RA+CD62L+CCR7+ T cells, CD45RA+CD62L+CCR7+CD27+CD28+ T cells, CD95+ T cells, CD 122+ T cells, effector memory T (Tem) cells, CD62L-CCR7- T cells.
  • Tcm central memory T
  • Tcm central memory T
  • Tscm stem cell memory T
  • CD45RO-/CCR7+ T cells CD45RA+CD62L+CCR7+ T cells
  • CD45RA+CD62L+CCR7+CD27+CD28+ T cells CD95+ T cells
  • CD 122+ T cells effector memory T (Tem) cells, CD62L-CCR7- T cells.
  • the one or more undesirable factors may comprise one or more cytokines and/or chemokines, optionally IL-10, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-y, TNF-a, TGF- , CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, LLC13, CCL19, CCL21, CCL22, CXCL12, TSLP, or any combination thereof.
  • cytokines and/or chemokines optionally IL-10, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-y, TNF-a, TGF- , CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, LLC13, CCL19, CCL
  • the one or more undesirable factors may comprise one or more growth factors and/or differentiation factors.
  • the one or more undesirable factors may comprise one or more cytokines and/or chemokines and/or one or more growth factors and/or differentiation factors.
  • the one or more undesirable cells may comprise one or more one or more of exhausted T cells, overactivated or hyperactivated T cells, Treg cells, and/or T cells expressing one or more of PD-1, TIM-3, CTLA-4, LAG-3, T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), B and T lymphocyte attenuator (BTLA), 2B4, CD38, CD43, CD101, and/or CD160 optionally at mid or high levels, optionally PD- 1+ LAG-3+ T cells.
  • the selecting and/or removing may comprise centrifugation-based selection and/or removal, antibody -based selection and/or removal, bead-based selection and/or removal, magnetic selection and/or removal, MACS-based selection and/or removal, filtration-based selection and/or removal, or any combination thereof.
  • the resuspending may comprise: (i) controlling the treatment chamber to pellet the cells and to remove supernatant; (ii) controlling a flow of a carrier medium into the treatment chamber; and (iii) controlling a flow of the carrier medium out of the treatment chamber in accordance with a target volume of the formulation to generate the formulation.
  • the operations comprise (D) transferring one or more of the cells treated in (C) to the subject.
  • the one or more of the cells treated in step (C) may be transferred to the blood, lymphatic fluid, ascitic fluid, cerebrospinal fluid, adipose tissue, or bone marrow of the subject.
  • the blood may be peripheral blood.
  • the one or more of the cells treated in step (C) may be transferred to a disease site of the subject.
  • the disease site may be blood or a solid tumor, an infection site or inflammation site.
  • the transferring in the operation(s) of (D), may be performed at a speed of about 1000-5000 cells per minute, about 5,000-10,000 cells per minute, about 10,000-50,000 cells per minute, about 50,000-100,000 cells per minute, about 100,000-500,000 cells per minute, about 500,000-1,000,000 cells per minute, about 1,000,000-5,000,000 cells per minute, about 5,000,000-10,000,000 cells per minute, about 1,000,000-5,000,000 cells per minute, about 5,000,000- 10,000,000 cells per minute, about 10,000,000-50,000,000 cells per minute, about 50,000,000- 100,000,000 cells per minute, about 100,000,000-500,000,000 cells per minute, about 500,000,000- 1,000,000,000 cells per minute.
  • a cell solution is transferred to the subject at about 25-150 mL/minute, about 50-120 mL/minute, about 60-100 mL/minute, or about 70-80 mL/minute.
  • the one or more of the cells treated in step (C) transferred may be at a concentration of about 100-500 cells/mL, about 500-1000 cells/mL, about 1000-5000 cells/mL, about 5000-10,000 cells/mL, about 10,000-50,000 cells/mL, about 50,000- 100,000 cells/mL, about 100,000-500,000 cells/mL, about 500,000-1,000,000 cells/mL, about 1,000,000-5,000,000 cells/mL, about 5,000,000-10,000,000 cells/mL, or about 10,000,000-50,000,000 cells/mL.
  • the transferring in the operation(s) of (D), may take about 1-60 minutes, about 5-45 minutes, about 10-30 minutes, or about 15-20 minutes.
  • the transferring in the operation(s) of (D), may comprise electronically controlling at least one valve and/or a pump configured to control fluid flow between the treatment chamber and the second connector.
  • the operations may further comprise (E) conditioning the subject.
  • the operation(s) of (E) may be performed before (A), during (A), after (A), during one or more of (A)-(D), during one or more of (B)-(D), during (D), and/or after (D).
  • the conditioning may comprise depleting or inactivating an undesired cell type in the subject.
  • the undesired cell type comprises one or more of: lymphocyte, T cell, B cell, NK cell, Treg cell, MDSC, cancer cell.
  • the depleting or inactivating comprises administering one or more small molecules to the subject, optionally the one or more small molecules comprise fludarabinc, cyclophosphamide, bendamustine, a checkpoint inhibitor, or any combination thereof.
  • the depleting or inactivating comprises administering one or more antibodies specific to the undesired cell type to die subject, optionally wherein the one or more antibodies comprise a checkpoint inhibitor antibody, an antibody specific to Treg cells, an antibody specific to MDSCs, an antibody specific to cancer cells, an anti-CD19 antibody, an anti-CD52 antibody, or any combination thereof.
  • the depleting or inactivating comprises administering one or more cytokines which inhibit one or more activities of the undesired cell type.
  • the conditioning may comprise increasing or activating a desired cell type in the subject.
  • the desired cell type comprises the at least one cell type of interest of (B), i.e., (i) at least one immune cell, optionally: leukocyte or erythrocyte, optionally cell of the lymphoid lineage or cell of the myeloid lineage, optionally T cell, B cell, plasma cell, NK cell, NK T cell, macrophage, dendritic cell, monocyte, eosinophil, neutrophil, basophil, or mast cell; progenitor of any of the aforementioned cell types, optionally lymphoid progenitor, myeloid progenitor, lymphoblast, or myeloblast; CD8+ T cell, CD4+ T cell, or any combination thereof, optionally Thl cell, Th2 cell, Th9 cell, Th 17 cell, Th22 cell, regulatory T (Treg) cell, or follicular helper (Till) cell; or any combination of the aforementioned cell types; and/or ii) at least one stem cell, optionally: leukocyte or eryth
  • the increasing or activating a desired cell type may comprise administering one or more agonistic antibodies specific to the desired cell type to the subject.
  • the increasing or activating a desired cell type may comprise administering one or more cytokines which activate one or more activities of the undesired cell type.
  • the conditioning may comprise eliminating or reducing an undesired factor.
  • the undesired factor comprises one or more of: an immunosuppressive cytokine, optionally IL-10 or TGF-[>. or an inflammatory cytokine, optionally IL- 2, IL-6, IL-7, IL-15, and/or IFNy.
  • the depleting or inactivating comprises administering an antibody against the undesired factor, optionally a blocking or neutralizing antibody.
  • the conditioning may comprise reducing or preventing a toxic and/or side effect in the subject.
  • the toxic and/or side effect comprises cytokine release syndrome (CRS), macrophage activation syndrome (MAS), hemophagocytic lymphohistiocytosis (HLH), neurological side effects (e.g., confusion, delirium, word-finding aphasia, coma, seizures, irritability, and/or delirium), tumor lysis syndrome (TLS), on-target/off-tumor toxicities (e.g., hypogammaglobulinemia) .
  • CRS cytokine release syndrome
  • MAS macrophage activation syndrome
  • HH hemophagocytic lymphohistiocytosis
  • neurological side effects e.g., confusion, delirium, word-finding aphasia, coma, seizures, irritability, and/or delirium
  • TLS tumor lysis syndrome
  • on-target/off-tumor toxicities e.g., hypogammaglobulinemia
  • the reducing or preventing may comprise administering to the subject an anti-cytokine or anti-cytokine receptor antibody, optionally anti-IL-6 or anti-IL-6 receptor antibody for treating or preventing CRS.
  • the reducing or preventing may comprise administering to the subject a corticosteroid for treating or preventing MAS or HLH.
  • the reducing or preventing may comprise administering to the subject prophylactic allopurinol for treating or preventing TLS.
  • the reducing or preventing may comprise administering to the subject intravenous immunoglobulin (IVIG) replacement therapy for treating or preventing hypogammaglobulinemia.
  • IVIG intravenous immunoglobulin
  • the operations may further comprise (F) monitoring the subject or cell therapy progress.
  • the operation(s) of (F) may be performed before (A), during (A), after (A), during one or more of (A)-(D), during one or more of (B)-(D), during (D), and/or after (D).
  • the operation(s) of (F) may comprises (F- 1) collecting a monitoring sample from the subject.
  • the monitoring sample comprises a sample of blood, lymphatic fluid, ascitic fluid, cerebrospinal fluid, adipose tissue, or bone marrow.
  • the blood may be peripheral blood.
  • the monitoring sample comprises a sample of a disease site.
  • the disease site may be blood or a solid tumor or an infection site or inflammation site.
  • the operation(s) of (F) may comprises (F-2) analyzing: (i) all or part of the biological sample collected in step (A); (ii) all or part of the cells selected in step (B); (iii) all or part of the elements of the biological sample not selected in step (B), optionally plasma; (iv) all or part of the cells treated in step (C); (v) all or part of the cells being transferred in step (D) or a formulation containing such cells; (vi) all or part of the monitoring sample collected in (F- 1 ); and/or (vii) body temperature, breathing rate, blood pressure, heart rate, oxygen levels, or any combination thereof of the subject.
  • the analyzing may be by measuring the level of a cytokine of interest, optionally IL-10, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN-y, TNF-a, MCP-1, GM-CSF, TGF-0. or any combination thereof, or a receptor thereof.
  • a cytokine of interest optionally IL-10, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN-y, TNF-a, MCP-1, GM-CSF, TGF-0. or any combination thereof, or a receptor thereof.
  • the analyzing may be by measuring the level of a chemokine of interest, optionally CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL12, CCL13, CCL19, CCL21, CCL22, CXCL8, CXCL9, CXCL10, CXCL12, TSLP, MIP-1, or any combination thereof, or a receptor thereof.
  • a chemokine of interest optionally CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL12, CCL13, CCL19, CCL21, CCL22, CXCL8, CXCL9, CXCL10, CXCL12, TSLP, MIP-1, or any combination thereof, or a receptor thereof.
  • the analyzing may be by measuring the level of cells of at least one undesired cell type.
  • the cells of at least one undesired cell type may comprise exhausted T cells, overactivated or hyperactivated T cells, Treg cells, T cells expressing one or more of PD-1, TIM-3, CTLA-4, LAG-3, T cell immunorcccptor with immunoglobulin and ITIM domains (TIGIT), B and T lymphocyte attenuator (BTLA), 2B4, CD38, CD43, CD101, and/or CD160 optionally at mid or high levels, PD-1+ LAG-3+ T cells, Treg cells, MDSCs, or any combination thereof.
  • the operation(s) of (F) may comprises (F-3) optionally adjusting the operations based on the analysis result obtained in (F-2).
  • the adjusting may be by changing the treating condition in the operation(s) of (C).
  • the changing may comprise increasing or decreasing the time length of the culturing and/or adding, reducing, or removing a stimulus in the culture in (C-3).
  • the adjusting may be by increasing or decreasing the speed of transferring and/or the amount of cells transferred in the operation) s) of (D) .
  • the adjusting may be by administering a drug or therapy to the subject.
  • a drug which reduces or blocks a cytokine, a chemokine, or a receptor thereof further optionally a drug which reduces or blocks IL-10, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN-y, TNF-a, MCP-1, GM-CSF, TGF-0, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL12, CCL13, CCL19, CCL21, CCL22, CXCL8, CXCL9, CXCL10, CXCL12, TSLP, MIP- 1, or a receptor thereof, further optionally an anti-IL-6 or anti-IL-6 receptor antibody may be administered.
  • a corticosteroid may be administered.
  • the present disclosure provides apparatuses for cell therapy.
  • the apparatus may be for manufacturing and administering cell therapy.
  • the apparatus may comprise: (a) a first connector configured to be operably and/or directly connectable to a subject to allow for collection of a biological sample from the subject through the first connector; (b) a selector, such as a separator, configured to separate cells of at least one cell type of interest from elements not of interest in the biological sample; (c) a treatment chamber configured to treat cells; and (d) a second connector configured to be operably and/or directly connectable to the subject to allow for transfer of the treated cells to the subject through the second connector.
  • any one or more of (a)-(d) may be entirely or partially disposable.
  • the apparatus is a closed system apparatus.
  • the operation(s) of the apparatus may be controlled by computer- readable instructions encoded on one or more computer-readable media.
  • the apparatus comprises one or more computer-readable media having encoded thereon computer-readable instructions for controlling the operation(s) of the apparatus.
  • the one or more computer-readable media may be any of computer-readable media described herein.
  • the apparatus may comprise a computer which is capable of reading such computer-readable instructions.
  • the apparatus may comprise one or more of: (iii-1) fluid circuitry configured to allow fluid flow between the first connector and the selector; (iii-2) fluid circuitry configured to allow fluid flow between the first connector and the treatment chamber; (iii-3) fluid circuitry configured to allow fluid flow between the selector and the treatment chamber; (iii-4) fluid circuitry configured to allow fluid flow between the treatment chamber and the second connector; and/or (iii-5) fluid circuitry configured to allow fluid flow between the selector and the second connector.
  • one or more of (iii- l)-(iii-5) may be entirely or partially disposable.
  • the apparatus may comprise one or more of: (iv-1) a valve and/or pump configured to control fluid flow between the first connector and the selector; (iv-2) a valve and/or pump configured to control fluid flow between the first connector and the treatment chamber; (iv-3) a valve and/or pump configured to control fluid flow between the selector and the treatment chamber; (iv-4) a valve and/or pump configured to control fluid flow between the treatment chamber and the second connector; and/or (iv-5) a valve and/or pump configured to control fluid flow between the selector and the second connector.
  • one or more of (iv-l)-(iv-5) may be entirely or partially disposable.
  • the first connector comprises a first needle or aspirator or is operably connectable to a first needle or aspirator.
  • the selector comprises (b-1) a separation column, optionally a MACS column and (b-2) optionally a centrifugation chamber.
  • the treatment chamber comprises (c-1) an incubation chamber configured to centrifuge cells contained in the incubation chamber or (c-2) an incubation chamber and a centrifugation chamber.
  • the second connector comprise a needle or is operably connectable to a second needle.
  • the apparatus may further comprise a conditioning element configured to be operably and/or directly connectable to the subject.
  • the operable and/or direct connection may be via the second connector or a third connector, to allow for administration of a conditioning agent to the subject.
  • the apparatus may further comprise a monitoring element.
  • the monitoring element may be configure to analyze the level of one or more molecules and/or cells of interest in the biological sample or any sample materials contained in the apparatus. In certain embodiments, the monitoring element may be configure to analyze one or more conditions in the apparatus, optionally temperature, pressure, pH, or any combination thereof. In certain embodiments, the monitoring element may be configure to analyze body temperature, breathing rate, blood pressure, heart rate, oxygen levels, or any combination thereof of the subject. In further embodiments, the monitoring element may be further configured to trigger an instruction to adjust operations of the apparatus based on the results obtained by the monitoring element.
  • the monitoring element may comprise a fourth connector configured to be operably and/or directly connectable to the subject to allow for collection of a monitoring sample from the subject through the fourth connector.
  • the monitoring element may be contained in one or more of the first connector, the selector, the treatment chamber, and the second connector.
  • the apparatus may further comprise a display.
  • the display may provide information on the progress of cell therapy.
  • the display may provide a user interface configured to accept one or more user-inputs pertaining to one or more of the operations of the apparatus.
  • the one or more user-inputs may identity a condition of the cell separation in the selector, optionally the at least one cell type of interest and/or a least one cell type not to be selected.
  • the one or more user-inputs may identify a condition of the cell treatment in the treatment chamber, optionally the duration of the treatment, a temperature used during the treatment. and/or a volume of a solution containing the cells during the treatment.
  • the apparatus is configured to allow for adjustment of at least one of the operations in accordance with the one or more user-inputs.
  • the apparatus may further comprise a non-target cell container configured to receive elements not to be used for manufacturing cells for cell therapy.
  • such elements may include but are not limited to plasma, platelet, erythrocytes, non-T cell leukocytes, and/or non-stem cells.
  • the non-target cell container may be operably and/or directly connectable to a subject to allow for transferring or administering the all or part of the elements.
  • the apparatus may further comprise a waste container configured to receive one or materials not to be transferred to the patients.
  • the waste container may be operably connected to the selector and/or the treatment chamber.
  • Any of the one or more computer-readable media described herein may encode computer- readable instructions for causing one or more processing devices to control operations of various portions of any of the apparatuses for cell therapy described herein.
  • Any of the methods described herein may be effected by any of the apparatuses for cell therapy described herein and/or any of the computer-readable media described herein.
  • FIG 1 provides a schematic of an exemplary method for cell therapy according to the present disclosure.
  • a method may comprise (A) a step of collecting a biological sample containing cells from a subject, (B) a step or selecting cells of at least one cell type of interest from the biological sample, (C) a step of treating the selected cells, and (D) a step of transferring one or more of the treated cells to the subject, in which (A) -(D) are performed in a closed system and may be performed using an apparatus according to the present disclosure. Open arrows indicate steps to be performed in the method.
  • FIGS 2A-2D provide schematics of an exemplary computer-readable medium according to the present disclosure.
  • a computer-readable medium may store instructions that, when executed by a computer, cause it to perform a method of cell therapy, which may be a method according to the present disclosure.
  • the instructions may cause one or more processing devices to control operations of a closed system apparatus for cell therapy, which may be an apparatus according to the present disclosure, comprising (a) a first connector configured to be operably and/or directly connectable to a subject to allow for collection of a biological sample from the subject through the first connector, (b) a selector configured to separate cells of at least one cell type of interest from elements not of interest in the biological sample, (c) a treatment chamber configured to treat cells, and (d) a second connector configured to be operably and/or directly connectable to the subject to allow for transfer of the treated cells to the subject through the second connector.
  • a closed system apparatus for cell therapy which may be an apparatus according to the present disclosure, comprising (a) a first connector configured to be operably and/or directly connectable to a subject to allow for collection of a biological sample from the subject through the first connector, (b) a selector configured to separate cells of at least one cell type of interest from elements not of interest in the biological sample, (c) a treatment chamber configured to treat
  • Open arrows indicate steps to be performed in a method of cell therapy according to the present disclosure, and two headed arrows indicate instructions causing operations of different elements of an apparatus which may be encoded on a CRM (stripe indicates instructions encoded on a CRM; dotted indicates instructions optionally encoded on a CRM or may instead be caused manually). Doited line indicates a closed system apparatus.
  • the operations may comprise: (A) optionally collecting a biological sample containing cells from a subject and optionally transferring the biological sample through the first connector (i) to the treatment chamber and then to the selector or (ii) to the selector; (B) selecting cells of at least one cell type of interest from a/the biological sample, e.g., (i) in the selector (FIGS 2A and 2C) and or (ii) in the treatment chamber and then in the selector (FIGS 2B and 2C), and transferring the selected cells to the treatment chamber; (C) treating the selected cells, e.g., (i) in the treatment chamber (FIG 2A and 2B), (ii) in the treatment chamber and then in the selector (FIG 2C and 2D), or (iii) in the treatment chamber, then in the selector, and then the selector (FIG 2C and 2D) and (D) optionally transferring one or more of the cells treated in (C) to the subject. Collecting (and transferring)
  • FIGS 3A-3E provide schematics of an exemplary flow of samples/cells in an exemplary apparatus for cell therapy according to the present disclosure comprising (a) a first connector configured to be operably and/or directly connectable to a subject to allow for collection of a biological sample from the subject through the first connector; (b) a selector configured to separate cells of at least one cell type of interest from elements not of interest in the biological sample; (c) a treatment chamber configured to treat the cells of interest; and (d) a second connector configmed to be operably and/or directly connectable to the subject to allow for transfer of the treated cells to the subject through the second connector.
  • Black arrows illustrate an exemplary flow of samplcs/cclls.
  • an apparatus comprises necessary mechanisms to allow for such a flow of samples/cells, such as one or more fluid circuits betw een elements of the apparatus and/or one or more valves and/or pumps for turning on and off the circuit as needed.
  • Various mechanisms and structures for allowing a flow of samples/cells are known in the art, including but not limited to those depicted in FIG 4C.
  • a biological sample e.g., blood
  • cells of at least one cell type of interest e.g., T cells
  • the selected cells are transferred to the treating chamber
  • the selected cells are treated (e.g., transduced with a nucleic acid encoding a CAR) in treatment chamber (e.g., comprising an incubator, a centrifuge, a spinoculator, any combination thereof); and (D) one or more of the treated cells are transferred to the subject.
  • a biological sample e.g., blood
  • certain elements e.g., leukocytes
  • tire treatment chamber e.g., comprising a centrifuge (e.g., for apheresis or leukapheresis)
  • selector cells of at least one cell ty pe of interest (e.g., T cells) are selected in the selector (e.g., comprising a MACS column), and the selected cells are transferred to the treating chamber
  • the selected cells are treated (e.g., transduced with a nucleic acid encoding a CAR) in treatment chamber (e.g., comprising an incubator, a centrifuge, a spinoculator, any combination thereof); and (D) one or more of the treated cells are transferred to the subject.
  • a biological sample e.g., blood
  • cells of at least one cell type of interest e.g., T cells
  • the selected cells are transferred to the treating chamber
  • the selected cells are treated (e.g., transduced with a nucleic acid encoding a CAR) in treatment chamber (e.g., comprising an incubator, a centrifuge, a spinoculator, any combination thereof) and transferred to the selector, and successfully treated cells or cells having a phenoty pe of interest (e.g., memory T cells) are selected in the selector; and (D) one or more of the treated cells are transferred to the subject.
  • a biological sample e.g., blood
  • cells of at least one cell type of interest e.g., T cells
  • the selected cells are treated (e.g., transduced with a nucleic acid encoding a CAR) in treatment chamber (e.g., comprising an incubator, a centrifuge, a spinoculator, any combination thereof
  • a biological sample e.g., blood
  • certain elements e.g., leukocytes
  • the biological sample in the treatment chamber e.g., comprising a centrifuge (e.g., for apheresis or leukapheresis)
  • cells of at least one cell ty pe of interest e.g., T cells
  • the selector e.g., comprising a MACS column
  • the selected cells are transferred to the treating chamber
  • the selected cells are treated (e.g., transduced with a nucleic acid encoding a CAR) and optionally expanded in treatment chamber (e.g., comprising an incubator, a centrifuge, a spinoculator, any combination thereof) and transferred to the selector, and successfully treated cells or cells having a phenotype of interest (e.g., memory
  • a biological sample e.g., blood
  • certain elements e.g., leukocytes
  • the biological sample in the treatment chamber e.g., comprising a centrifuge (e.g., for apheresis or leukapheresis)
  • cells of at least one cell ty pe of interest e.g., T cells
  • the selector e.g., comprising a MACS column
  • the selected cells are transferred to the treating chamber
  • the selected cells are treated (e.g., transduced with a nucleic acid encoding a CAR) in treatment chamber (e.g., comprising an incubator, a centrifuge, a spinoculator, any combination thereof) and transferred to the selector, successfully treated cells are selected in the selector and transferred to the treatment chamber, and the cells are expanded in the treating chamber;
  • FIGS 4A-4C provide various schematics relating to an apparatus for cell therapy.
  • FIG 4A provides a schematic of some of many variations of connections between an apparatus according to the present disclosure and a subject/patient.
  • An apparatus according to the present disclosure comprises (a) a first connector configured to be operably and/or directly connectable to a subject to allow for collection of a biological sample from die subject through the first connector; (b) a selector configured to separate cells of at least one cell type of interest from elements not of interest in the biological sample; (c) a treatment chamber configured to treat the cells of interest; and (d) a second connector configured to be operably and/or directly connectable to the subject to allow for transfer of the treated cells to the subject through the second connector.
  • the apparatus has (i) a first connector configured to be operably and/or directly connectable to a subject and (ii) a second connector configured to be operably and/or directly connectable to the subject, i.e., the connectors of an apparatus are operably and/or directly connectable to a subject.
  • the dotted line indicates that elements inside are part of the apparatus for cell therapy.
  • a (first and/or second) connector of an apparatus has a mechanism to which an adaptor is attachable and the adaptor may be indirectly (e.g., via a tube) connected to a mechanism (e.g., needle) which allows for biological sample collection or therapeutic cell administration.
  • a mechanism e.g., needle
  • a connector of an apparatus comprises an adaptor indirectly (e.g., via a tube) connectable to a mechanism (e.g., needle) which allows for biological sample collection or therapeutic cell administration.
  • the adaptor is considered to be part of a (first and/or second) connector.
  • a connector of an apparatus comprises a mechanism (e.g., a tube or an adaptor with a tube) directly connectable to a mechanism (e.g., needle) which allows for biological sample collection or therapeutic cell administration.
  • a mechanism e.g., needle
  • the directly connecting mechanism e.g., an adaptor with a tube or at least a tube
  • the directly connecting mechanism is considered to be part of a (first and/or second) connector.
  • a connector of an apparatus comprises a mechanism (e.g., needle) which allows biological sample collection or therapeutic cell administration.
  • the mechanism e.g., needle
  • the mechanism which allows for biological sample collection or therapeutic cell administration is considered to be part of a (first and/or second) connector.
  • FIG 4B provides a schematic of an exemplary apparatus for manufacturing and administering cells for cell therapy, in which the first connector is operably connected to a subject via a mechanism exemplified in the second panel of FIG 4A to allow for collection of a biological sample from the subject through the first connector and in which the second connector is operably connected to a subject via a mechanism exemplified in the second panel of FIG 4A to allow for transfer of the treated cells to the subject through the second connector.
  • Dotted line indicates a closed system apparatus.
  • Black arrows illustrate an exemplary flow of samples/cells.
  • an apparatus comprises necessary mechanisms to allow for such a flow of samples/cells, such as fluid circuitry between elements of the apparatus and/or one or more valves and/or pumps for turning on and off the circuit as needed.
  • FIG 4C provides a schematic of an exemplary apparatus for manufacturing cells for cell therapy known in the field (known as CliniMACS Prodigy®) (taken from page 9 of https://www.miltenyibiotec.com/ Resources/Persistent/19dll2a2f0d907d93b8dbe21e2ad4b31d3dl69 53/CliniMACS Prodigy System flyer.pdf).
  • an “Application bag” containing a biological sample already isolated from a subject may be loaded to the apparatus, cells of at least one cell type of interest (e.g., T cells) from the biological sample are selected in the “Separation Column”, and the selected cells are treated (e.g., transduced) and expanded in the “CentriCult” chamber, and the expanded cells are collected in the “Target Cell Bag”.
  • the “Target Cell Bag” is detached from the apparatus and then the cells therein will be administered to a/the subject.
  • FIG 4D provides a schematic of an exemplary apparatus for manufacturing and administering cells for cell therapy, which is designed based on the cell manufacturing device depicted in FIG 4C.
  • the apparatus comprises a mechanism (e.g., needle) which directly connects to the subject to allow for collection of a biological sample from the subject or (ii) is operably, optionally directly, connectable to a mechanism (e g., needle) which directly connects to the subject to allow biological sample collection.
  • a mechanism e.g., needle
  • the apparatus comprises a mechanism (e.g., needle) which directly connects to the subject to allow for therapeutic cell administration or (ii) is operably, optionally directly, connectable to a mechanism (e.g., needle) which directly connects to the subject to allow for therapeutic cell administration.
  • a mechanism e.g., needle
  • essentially the modification(s) may be added to any appropriate therapeutic cell manufacturing apparatuses.
  • the invention disclosed herein generally relates to improved cell therapy. Some aspects of the invention may relate to cell therapy performed in a closed system, in which a patient in part of the closed system. Some embodiments may relate to cell therapy performed in a closed loop, in which a patient in part of the closed loop.
  • Various embodiments of the present invention may provide various technical advantages. Some embodiments may reduce the time needed for therapeutic cell manufacturing and/or administration processes (e.g., reduction from the current clinical standard of weeks to less than a day or several hours such as about 1-4 horns). Some embodiments may reduce the complexity of such processes due to the essentially fully automated/automatable processes. Some embodiments may eliminate the step of freezing cells and the step of thawing cells. Some embodiments increase yield and/or reduce loss of samples/cells. Some embodiments may reduce the risk of an error to occur. Some embodiments may reduce the probability of contamination. Some embodiments may allow less sophisticated medical centers to provide cellular therapies.
  • Some embodiments may provide therapeutic cells that are more effective, longer lasting, less exhausted, more active, less toxic, and/or more controlled. Some embodiments may allow for an autologous therapy with one or more advantages of allogeneic therapies. Some embodiments may make the regulatory application and approval processes simpler. Some embodiments may provide an improved patient experience. Some embodiments may reduce the time needed for treatment or time to treatment. Some embodiments may allow patients to receive cell therapy with a reduced number of office visits (e.g., a single trip to the hospital or cell therapy center for the process starting from host cell collection to therapeutic cell administration) or reduced duration of office stay. Some embodiments may allow monitoring of the cell manufacturing and/or administration processes and/or of the patient as cells for therapy are manufactured and administered.
  • Some embodiments may allow for adjustment of the manufacturing processes (e.g., cell culture condition such as the amount of stimulus, culture duration depending on the cell condition, cell type ratio, etc) and/or the administration (e.g., dose, speed, duration of administration, cell type ratio, inclusion/removal of a particular cell type or a particular molecule such as cytokine, administration of another drug to increase efficacy and/or reduce toxicity or a side effect).
  • the manufacturing processes e.g., cell culture condition such as the amount of stimulus, culture duration depending on the cell condition, cell type ratio, etc
  • the administration e.g., dose, speed, duration of administration, cell type ratio, inclusion/removal of a particular cell type or a particular molecule such as cytokine, administration of another drug to increase efficacy and/or reduce toxicity or a side effect.
  • Some embodiments may provide any combination of the above-described and/or any other advantages.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a “nucleic acid,” “nucleic acid molecule,” “polynucleotide,” or “polynucleotide molecule” comprise a nucleotide/nucleoside derivative or analog. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions, e.g., conservative substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions e.g., conservative substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • nucleotide sequence encoding an ammo acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).
  • a "vector” is a compound or a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Numerous vectors are known in the art including, but not limited to, viruses, virus-like particles (VLPs), linear polynucleotides, plasmids, autonomously replicating plasmids, self-replicating RNAs, polynucleotides associated with ionic or amphiphilic compounds, and the like.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, poly lysine compounds, liposomes, and the like.
  • examples of viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus vectors, and the like.
  • an effective amount or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result of interest.
  • expression refers to the transcription and/or translation of a particular nucleotide sequence. In some embodiments, expression comprises translation of an mRNA introduced into a cell.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • cosmids e.g., naked or contained in liposomes
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • promoter refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • tissue-specific promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of tire tissue type corresponding to tire promoter.
  • lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
  • Other examples of lentivirus vectors that may be used in the clinic include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENT1MAXTM vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • transfected or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • the term “differentiate” herein refers to the conversion of less specialized cells into a more specialized or mature cell type.
  • dedifferentiate refers to the reversal of differentiation and/or the loss of one or more specialized characteristics of a cell.
  • transdifferentiate refers to the conversion of a particular cell into another cell type, e.g., the conversion or differentiation of a stem cell into a more specialized cell type.
  • Gene editing systems are known in the art, and are described more fully below.
  • the methods, apparatuses, and systems of the present invention may involve polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 85%, 90%, or 95% identical or higher to the sequence specified.
  • the term “substantially identical” is used herein to refer to a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity, for example, amino acid sequences that contain a common structural domain having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.
  • nucleotide sequence In the context of a nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity, for example, nucleotide sequences having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.
  • homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or tw o RNA molecules, or between two polypeptide molecules.
  • two nucleic acid molecules such as, two DNA molecules or tw o RNA molecules
  • polypeptide molecules between two polypeptide molecules.
  • a subunit position in both of the two molecules is occupied by the same monomeric submit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten submits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • conservative sequence modifications refer to sequence modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site- directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine.
  • valine isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • variant refers to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence or is encoded by a substantially identical nucleotide sequence.
  • the variant is a functional variant.
  • functional variant refers to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence or is encoded by a substantially identical nucleotide sequence, and is capable of having one or more activities of the reference ammo acid sequence.
  • cytokine e.g., IL-2, IL-7, IL-15, or IL-21
  • chemokine e.g., CCL5 or CCL19
  • cytokine e.g., IL-2, IL-7, IL-15, or IL-21
  • chemokine e.g., CCL5 or CCL19
  • a functional variant including fragments and functional variants thereof having at least 10%, 30%, 50%, or 80% of the activity, e.g., the immunomodulatory activity, of the naturally- occurring cytokine.
  • the cytokine or chemokine has an amino acid sequence that is substantially identical (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to its naturally -occurring counterpart, or is encoded by a nucleotide sequence that is substantially identical (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a naturally-occurring nucleotide sequence encoding a naturally- occurring cytokine or chemokine.
  • the cytokine or chemokine further comprises a receptor domain, e.g., a cytokine receptor domain (e g., an IL-15/IL- 15R) or a chemokine receptor domain (e.g., CCL5/CCR5).
  • a cytokine receptor domain e.g., an IL-15/IL- 15R
  • a chemokine receptor domain e.g., CCL5/CCR5
  • CAR Chimeric Antigen Receptor
  • a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain” or “an ICS domain”) comprising a functional signaling domain derived from a stimulatory molecule as defined herein.
  • the domains in the CAR polypeptide construct are in the same polypeptide chain, e.g., comprise a chimeric fusion protein.
  • the cytoplasmic signaling domain may comprise a primary signaling domain (e.g., a primary signaling domain of CD3C).
  • the cytoplasmic signaling domain may further comprise one or more functional signaling domains derived from at least one costimulatory molecule as defined herein.
  • the costimulatory molecule is chosen from 41BB (i.e., CD137), DAP10, CD27, ICOS, and/or CD28.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising (i) a functional signaling domain derived from a costimulatory molecule and (ii) a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising (i) two functional signaling domains derived from one or more co-stimulatory molecule(s) and (ii) a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising (i) at least two functional signaling domains derived from one or more co-stimulatory molecule(s) and (ii) a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., an scFv) during cellular processing and localization of the CAR to the cellular membrane.
  • a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., an scFv) during cellular processing and localization of the CAR to the cellular membrane.
  • a CAR that comprises an antigen-binding domain e.g., an scFv, a single domain antibody, or TCR (e.g., a TCR alpha binding domain or TCR beta binding domain)
  • a specific marker X e.g., X can be a disease marker such as a cancer or tumor marker
  • XCAR a CAR that comprises an antigen binding domain that targets BCMA
  • BCMA CAR a CAR that comprises an antigen binding domain that targets BCMA.
  • the CAR can be expressed in any cell, e.g., an immune effector cell as described herein (e.g., a T cell, an NK cell, or macrophages).
  • signaling domain refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
  • antibody refers to a protein, or polypeptide sequence derived from an immuno globulin molecule, which specifically binds with an antigen.
  • Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.
  • antibody fragment refers to at least one portion of an intact antibody, or a recombinant variant thereof, and refers to a fragment comprising an antigen-binding domain, e.g., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen.
  • antibody fragments include, but arc not limited to, Fab, Fab’, F(ab’) 2 , Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multi-specific molecules formed from antibody fragments such as a bivalent or multivalent fragment comprising two or more, e.g., two, Fab fragments linked by a disulfide bridge at the hinge region, or two or more, e.g., two isolated CDR or other epitope binding fragments of an antibody linked.
  • An antibody fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).
  • Antibody fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).
  • Fn3 fibronectin type III
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker- VH or may comprise VH-linker-VL.
  • CDR complementarity determining region
  • HCDR1, HCDR2, and HCDR3 three CDRs in each heavy chain variable region
  • LCDR1, LCDR2, and LCDR3 three CDRs in each light chain variable region
  • EU EU numbering scheme
  • the CDRs correspond to the amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.
  • the portion of the CAR composition of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms, for example, where the antigen-binding domain is expressed as part of a polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), or e.g., a human or humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci.
  • sdAb single domain antibody fragment
  • scFv single chain antibody
  • the antigen binding domain of a CAR composition of the invention comprises an antibody fragment.
  • the CAR comprises an antibody fragment that comprises an scFv.
  • binding domain refers to a protein, e.g., an immuno globulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence.
  • binding domain or “antibody molecule” encompasses antibodies and antibody fragments.
  • an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.
  • a multispecific antibody molecule is a bispecific antibody molecule.
  • a bispecific antibody has specificity for no more than two antigens.
  • a bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (K) and lambda (/-) light chains refer to the two major antibody light chain isotypes.
  • recombinant antibody refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
  • antigen refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” al all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule other than a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.
  • the term “specifically binds,” refers to an antibody, or a ligand, which recognizes and binds with a cognate binding partner (e.g., a stimulatory and/or costimulatory molecule present on a T cell) protein present in a sample, but which antibody or ligand does not substantially recognize or bind other molecules in the sample.
  • a cognate binding partner e.g., a stimulatory and/or costimulatory molecule present on a T cell
  • “Derived from” as that term is used herein, indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connotate or include a process or source limitation on a first molecule that is derived from a second molecule.
  • the intracellular signaling domain retains sufficient CD3c structure such that is has the required function, namely, the ability to generate a signal under the appropriate conditions. It does not connotate or include a limitation to a particular process of producing the intracellular signaling domain, e.g., it does not mean that, to provide the intracellular signaling domain, one must start with a CD3zeta sequence and delete unwanted sequence, or impose mutations, to arrive at the intracellular signaling domain.
  • stimulation in the context of stimulation by a stimulatory and/or costimulatory molecule refers to a response, e.g., a primary or secondary response, induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) and/or a costimulatoiy molecule (e.g., CD28 or 4- 1BB) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
  • a stimulatory molecule e.g., a TCR/CD3 complex
  • a costimulatoiy molecule e.g., CD28 or 4- 1BB
  • Stimulation can mediate altered expression of certain molecules and/or reorganization of cytoskeletal structures, and the like.
  • the term “stimulatory molecule,” refers to a molecule expressed by a T cell that provides the cytoplasmic signaling sequence(s) that regulate activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway.
  • an ITAM -containing domain within the CAR expressed by a cell recapitulates TCR signaling or part of such signaling independently of endogenous TCR complexes of the cell.
  • the TCR signaling recapitulated may be the signaling initiated by, for instance, binding of a TCR/CD3 complex with a peptide presented on an MHC molecule, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signal domain transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • the intracellular signaling domain of a CAR which may be used according to the present invention generates a signal that promotes an immune effector function of the CAR-expressing cell, e.g., a CART cell.
  • immune effector function e.g., in a CART cell
  • examples of immune effector function, e.g., in a CART cell include cytolytic activity and helper activity, including the secretion of cytokines.
  • the cytoplasmic signaling domain of a CAR that acts in a stimulatory manner may contain a signaling domain comprising a signaling motif known as immunoreceptor tyrosine-based activation motif or ITAM.
  • an ITAM-containing signaling domain that is of particular use in the invention includes, but is not limited to, those derived from CD3C or TCRL FcRy, FcR[3, CD3y, CD35, CD3E, CD79a, CD79b, FCERI and CD66d, and DAP12.
  • the cytoplasmic signaling domain of a CAR that acts in a stimulatory manner may contain a signaling domain comprising an ITAM motif derived from CD3L
  • CD3 ⁇ or “CD3 zeta/’ or alternatively, “zeta,” “zeta chain,” “CD3-zeta,” “CD3z,” “TCR-zeta,” or “CD247,” is a protein encoded by the CD247 gene on chromosome 1, with gene location lq24.2, in humans.
  • CD3 zeta together with T cell receptor (TCR) and CD3 (a protein complex composed of a CD3 gamma, a CD3 delta, and two CD3 epsilon), forms the TCR complex.
  • Human CD3 zeta may have an amino acid sequence provided as NP 000725 or NP 932170, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • the term "CD3 zeta intracellular signaling domain,” or alternatively “CD3 zeta ICS domain” or a “CD3zICS,” is defined as the amino acid residues from the cytoplasmic domain of the CD3 zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.
  • Costimulatory molecules include, but are not limited to an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDlla/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, I
  • the intracellular signaling domain of a CAR can further comprise a signaling domain derived from a costimulatory molecule, which domain may also be referred to as a "costimulatorv signaling domain”.
  • the CAR may comprise the entire intracellular portion, or the entire native intracellular signaling domain, of a costimulatory molecule, or a functional fragment thereof.
  • signaling motifs include but are not limited to: the IT AM, which may be derived from FcRy, FcR[3, CD79a, CD79b, FCERI and CD66d, and DAP12; the YXXM motif, which may be derived from e.g., CD28, CD278 (or “ICOS”), or DAP10; the PXXP or PXXXP motif, which may be derived from, e.g., CD2 or CD28; the immunoreceptor tyrosine-based switch motif (ITSM), which may be derived from, e.g., CD150; the TRAF-binding motif, which may be derived from, e.g., CD27, 0X40, CD40, 4-1BB, or CD30; and/or the JAKs-binding motif, which may be derived from, e.g., the receptor of IL-2, 4, 7, 15, or 21 (Guo et al., J Immunother Cancer.
  • ITAM which may be
  • the cytoplasmic signaling domain of a CAR may contain a signaling domain derived from CD28, DAP10, 4-1BB, or CD27, or the TRAF-binding motif derived from CD27, the YXXM motif or the PXXP or PXXXP motif derived from CD28, the YXXM motif derived from DAP10, or the TRAF-binding motif derived from 4-1BB.
  • CD28 refers to the protein Cluster of Differentiation 28, one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival.
  • Human CD28 protein may have at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI Reference No: NP 006130 or a fragment thereof that has stimulatory activity.
  • CD28 transmembrane domain also referred to as “CD28 TM domain” or “CD28TM” refers to the amino acid residues derived from the transmembrane domain of CD28.
  • CD28 hinge refers to amino acid residues that may be used to join two domains or two portions within a domain in CARs of some of the embodiments.
  • CD28 costimulatory domain also referred to as “CD28 CS domain” or “CD28CS,” refers to the amino acid residues derived from the cytoplasmic domain of CD28.
  • 4-1BB refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA53133.1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • the "4-1BB costimulatory domain,” also referred to as “4-1BB CS domain” or “41BBCS,” may be derived from the cytoplasmic domain of 4-1BB.
  • DAP 10 refers to a protein, which in humans is encoded by the HSCT gene. It may also be referred to as HSCT, KAP10, PIK3AP, or hematopoietic cell signal transducer. In some embodiments, DAP10 may have the sequence provided in Genbank Accession No.: Q9UBK5.1.
  • DAP10 costimulatory domain also referred to as “DAP10 CS domain” or “DAP10CS,” refers to the amino acid residues derived from the cytoplasmic domain of DAP10.
  • fresh cells refers to cells obtained from patients that have not been frozen. Without wishing to be bound It is a unique aspect of some embodiments according to the present invention that fresh cells from a patient are processed by an apparatus without involving a step of freezing such cells to prepare cells ready for administration and that such fresh cells are transferred back to a/the patient.
  • the term “antigen presenting cell” or “APC” refers to an immune cell such as an accessory cell (e.g., a B-cell, a dendritic cell, a macrophage, and the like) that displays an antigen or a portion thereof complexed with a major histocompatibility' complex (MHC) on its surface. T-cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.
  • Immuno effector cell refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response.
  • immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes such as macrophages and dendritic cells.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • immune effector function or immune effector response refers to function or response of an immune effector cell. In some embodiments, an immune effector function or response may enhance or promote an immune attack of a target cell.
  • an immune effector function or response of a T cell or NK cell refers a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell.
  • antigen-mediated stimulation and co-stimulation are examples of immune effector function or response.
  • a “naive T cell” refers to a T cell that is antigen-inexperienced.
  • naive T cells may be differentiated, but have not yet encountered their cognate antigens in the peripheral.
  • naive T cells are precursors of memory cells.
  • naive T cells express CD45RA and CCR7, but not CD45RO.
  • naive T cells may be characterized by expression of CD62L, CD27, CCR7, CD45RA, CD28, and CD127, and the absence of CD95, or CD45RO isoform.
  • naive T cells express CD62L, IL-7 rcccptor-a, and CD132, but not CD25, CD44, CD69, or CD45RO.
  • central memory T cells refers to a subset of T cells that in humans are CD45RO positive and constitutively express CCR7 and CD62L. In some embodiments, central memory T cells express CD95. In some embodiments, central memory T cells express IL-2R, IL-7R and/or IL-15R.
  • the subject is a human.
  • a subject may have or have a risk of developing a disease.
  • a subject may have or have a risk of developing cancer, an autoimmune disease, a neurodegenerative disease, or another disease.
  • anti-tumor effect and “anti-cancer effect” are used interchangeably and refer to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume or cancer volume, a decrease in the number of tumor cells or cancer cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in tumor cell proliferation or cancer cell proliferation, a decrease in tumor cell survival or cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
  • an “antitumor effect” and “anti-cancer effect” may also be manifested by increased immune cells that help eliminate cancer cells, such as an increase in the number or percentage of cancer-specific lymphocytes, e.g., T cells, or by reduced immune cells that inhibit anti-cancer immune activities, such as a reduction in the number or percentage of regulatory T cells or myeloid derived suppressor cells.
  • An “anti-tumor effect” or “anti-cancer effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor or cancer in the first place.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • an apheresis sample refers to a sample obtained using apheresis.
  • cancer refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and/or lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, skin cancer, pancreatic cancer, colorectal cancer, stomach cancer, renal cancer, liver cancer, lung cancer, testicular cancer, brain cancer, mesothelioma, tongue cancer, lymphoma, myeloma, leukemia, and the like.
  • a “cancer” may be a “solid cancer”, which refers to a malignancy that forms a discrete tumor mass, e.g., breast cancer and pancreatic cancer.
  • a “cancer” may be a “liquid cancer”, which refers to a malignancy present in body fluids such as the blood and bone marrow, e.g., leukemias, lymphomas, and myelomas. While leukemias generally does not form a tumor, lymphomas and myelomas can form solid tumors, e.g., in the lymph nodes and bone marrow, respectively, and therefore lymphomas and myelomas can also be classified as a solid cancer.
  • tumor refers to an abnormal mass of tissue that forms when cells grow and divide more than they should or do not die when they should.
  • Tumors may be benign (in which case the tumor is not cancerous) or malignant (in which case the tumor is cancerous).
  • Benign tumors may grow large but generally do not spread into, or invade, nearby tissues or other parts of the body.
  • Malignant tumors can spread into, or invade, nearby tissues and can also spread to other parts of the body through the blood and/or lymph systems.
  • treating a sample or cells may include placing the sample or cells in a rotating environment such as a centrifuge, culturing/mcubating the sample or cells in the presence of a particular material such as a medium, a buffer, a vector, beads such as MACS beads, and/or cytokines, applying a magnetic field such as a MACS column to the sample or cells, and/or washing cells.
  • treating cells encompasses modifying cells, such as genetically modifying, transdifferentiating, or dedifferentiating cells.
  • the term “treat,” “treatment,” or “treating” in the context of clinical procedures generally refers to the procedure for reducing or ameliorating the progression, severity, and/or duration of a disease or of a condition, or for ameliorating one or more conditions or symptoms (preferably, one or more discernible ones) of a disease.
  • the effect of the “treatment” may be evaluated by the amelioration of at least one measurable physical parameter of a disease, resulting from the administration of one or more therapies.
  • the parameter may be, for example, gene expression profiles, the number of disease- affected cells, the percentage or frequency of disease-affected cells among the cells of the same lineage, disease-associated marker levels, and/or the presence or absence or levels of certain cytokines or chemokines or other disease-associated molecules and may not necessarily discernible by the patient.
  • “treat”, “treatment,” or “treating” may result in and/or be evaluated based on the inhibition of the progression of a disease, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of cancerous tissue or cells.
  • inventive methods can provide any amount of any level of treatment or prevention effects of a disease in a mammal.
  • treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • depletion or “depleting” (such as in “lymphodepletion” or “lymphodepleting”), as used interchangeably herein, refers to the decrease or reduction of the level or amount of a cell, a protein, or macromolecule in a sample after a process, e.g., a selection step, e.g., a negative selection, is performed.
  • the depletion can be a complete or partial depletion of the cell, protein, or macromolecule.
  • the depletion is at least a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% decrease or reduction of the level or amount of a cell, a protein, or macromolecule, as compared to the level or amount of the cell, protein or macromolecule in the sample before the process was performed.
  • the term “bead” refers to a discrete particle with a solid surface, ranging in size from approximately 0.1 pm to several millimeters in diameter. Beads may be spherical (e.g., microspheres) or have an irregular shape. Beads may comprise a variety of materials including, but not limited to, paramagnetic materials, ceramic, plastic, glass, polystyrene, methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose, nylon and the like. In one embodiment, the beads are relatively uniform, about 4.5 pm in diameter, spherical, superparamagnetic polystyrene beads, e.g..
  • the beads are Dynabeads®.
  • both anti-CD3 and anti- CD28 antibodies are coupled to the same bead, mimicking stimulation of T cells by antigen presenting cells.
  • the property of Dynabeads® and the use of Dynabeads® for cell isolation and expansion are well known in the art, e.g., see, Neurauter et al., Cell isolation and expansion using Dynabeads, Adv Biochem Eng Biotechnol. 2007; 106:41-73, herein incorporated by reference in its entirety.
  • the term “closed system” as used herein refers to a system for cell therapy (e.g., wherein one or more cell types in at least one cell sample from a patient are modified, e.g., genetically modified and/or the differentiation status is modified, and/or one or more cell types comprised in at least one cell sample from a subject arc depleted or removed such as tumorigcnic cells or pathogenic cells) which is effected using an apparatus such as one according to the present disclosure, in which the patient receiving cell therapy is physically connected to the apparatus for cell therapy at at least one point/site of the patient’s body at least during the step of collecting a biological sample from a patient and the step of transferring therapeutic cells to the patient; and in some exemplary embodiments wherein the patient receiving cell therapy is physically connected to the apparatus for effecting cell therapy from the step of collecting at least one biological sample from the patient until the step wherein the treated cells are transferred back to the patient.
  • a system for cell therapy e.g., wherein one or more cell types in at least
  • the patient is physically connected to the apparatus at a site for biological sample collection (e.g., vein or bone marrow) via the first connector of the apparatus; and during cell transfer/administration, the patient is physically connected to the apparatus at a site for cell transfer/administration (e.g., vein or a disease site such as a tumor) via the second connector of the apparatus.
  • a site for biological sample collection e.g., vein or bone marrow
  • the patient is physically connected to the apparatus at a site for biological sample collection and/or at a site for cell transfer/administration throughout the duration from biological sample collection to cell administration/transfer.
  • steps of collecting a biological sample a patient, selecting cells of interest from the biological sample, treating the selected cells, and transferring the treated cells to the patient take place in a closed system. It is a unique aspect of some embodiments according to the present invention that steps of collecting a biological sample a patient, selecting cells of interest from the biological sample, treating the selected cells, and transferring the treated cells to the patient take place in a single closed system.
  • closed system apparatus refers to an apparatus which allows for cell therapy manufacturing and administration in a closed system.
  • closed loop refers to a system for cell therapy effected using an apparatus such as one according to the present disclosure, in which the patient receiving cell therapy is physically comiected to the apparatus at at least two points/sites of the patient’s body.
  • the patient is physically connected to the apparatus at a site for biological sample collection (e.g., vein or bone marrow) via the first connector of the apparatus; and the patient is physically connected to the apparatus also at a site for cell transfer/administration (e.g., vein or a disease site such as a tumor) via at least one second connector of the apparatus.
  • a site for biological sample collection e.g., vein or bone marrow
  • a site for cell transfer/administration e.g., vein or a disease site such as a tumor
  • the patient is physically connected to the apparatus at a site for biological sample collection and also at a site for cell transfer/administration throughout the duration from biological sample collection to cell administration/transfer. It is a unique aspect of some embodiments of a method according to the present invention that steps of collecting a biological sample a patient, selecting cells of interest from the biological sample, treating the selected cells, and transferring the treated cells to the patient take place in a closed loop.
  • the phrase “operably connected to” or “operable connection to” relating to the connection between a first connector of an apparatus and a patient is used herein to indicate that the first connector is physically connected to the patient in a manner that at least one biological sample from the patient is transferrable to parts of the apparatus via at least one first comrector.
  • the at least one first connector itself may be directly attached to the patient, and for example the first connector may comprise a needle that is directly applied to the vein of the patient to collect blood.
  • a first connector may be indirectly but physically attached to the patient, and for example the first connector may comprise an adaptor to which such a needle may be directly attached or indirectly but physically (e.g., via a tube or another adaptor) attached.
  • the phrase “operably connected to” or “operable connection to” relating to the connection between a second connector of an apparatus and a patient is used herein to indicate that the second connector is physically connected to the patient in a manner that treated cells prepared by the apparatus is transferrable to the patient via at least one second connector.
  • the at least one second connector itself may be directly attached to the patient, and for example the second connector may comprise a needle that may be directly applied to the vein of the patient to transfer or administer the cells prepared by the apparatus.
  • the second connector may be indirectly but physically attached to the patient, and for example the second connector may comprise an adaptor to which such a needle may be directly attached or indirectly but physically (e.g., via a tube or another adaptor) attached.
  • die first connector since die first connector must be physically connected to the patient or the second connector must be physically connected to the patient, the phrase “operably connected to” or “operable connection to” does not encompass a connection between the first connector and a patient or between a patient and the second connector that is not continuous. For example, if a biological sample is collected in a container (such as a vial or a pouch) not attached to the apparatus and the container is detached from the patient before the container is attached to the apparatus, the patient and the first connector are not operably connected.
  • a container such as a vial or a pouch
  • the second connector and the patient are not operably connected.
  • the phrase “operably connectable to” refers to a mechanism which allows for such an operable connection (e g., the first connector has an adaptor to which a needle can be attached, and/or the second connector has an adaptor to which a needle can be attached). See FIG. 4 for exemplary variations of an apparatus (dotted line) operably connectable to a patient.
  • the phrase “directly connected to” or “direct connection to” relating to the connection between the first connector of an apparatus and a patient is used herein to indicate that the first connector comprises a mechanism, which mechanism attaches to the patient with nothing in between (i.e., there is nothing between the patient and the mechanism of the first connector), in a manner that a biological sample from the patient is transferrable to parts of the apparatus via the first connector.
  • the first connector may comprise a needle that is directly applied to the vein of the patient to collect blood.
  • the phrase “directly connected to” or “direct connection to” relating to the connection between the second connector of an apparatus and a patient is used herein to indicate that the second connector comprises a mechanism which attaches to the patient without having a material between the patient and the mechanism of the second connector, in a manner that cells prepared by the apparatus is transferrable to the patient via the second connector.
  • the second connector may comprise a needle that is directly applied to the vein of the patient to transfer the treated cells to the patient.
  • the phrase “directly connectable to” refers to a mechanism which allows for such a direct connection (e.g., the first connector has a needle, and/or the second connector has a needle). Therefore, “direct connection” is one form of “operable connection”, and “directly connectable” is one form of “operably connectable”.
  • the term “physically connected to” relating to the connection between the connector of an apparatus and a patient is used herein to indicate that the connector is linked to the patient via physical mechanism or a series of physical mechanisms (e.g., tube, valve, adaptor, etc) and there is no physical disconnection or gap between the connector and the patient.
  • physical mechanism e.g., tube, valve, adaptor, etc
  • the connector is directly connected to the mechanism and the mechanism is directly connected to the patient.
  • first and second mechanisms mediate the linkage in tandem
  • the connector is directly connected to a first mechanism
  • die first mechanism is directly connected to a second mechanism
  • the second mechanism is directly connected to the patient.
  • three or more phy sical mechanisms first, second, ...
  • the connector is directly connected to a first mechanism
  • the last mechanism is directly connected to the patient
  • each of the rest of the mechanism(s) is/are directly connected to the previous mechanism and the following mechanism (i.e., N ,h mechanism is directly connected to (N-l) th mechanism and also the (N+l) th mechanism).
  • One aspect of the invention provides a method of cell therapy.
  • the method is performed using or by an apparatus according to the present invention.
  • one or more of the steps of the method is performed using or by an apparatus, operations of which are controlled by computer-readable instructions encoded on one or more computer-readable media according to the present invention. Therefore, any of the variations/parameters described below in the COMPUTER-READABLE MEDIA section and the variations/parameters described below in the APPARATUS section below may apply to any of the CRM described herein. Any other variations/parameters described herein may also apply.
  • a method may comprise: (A) collecting a biological sample containing cells from a subject; (B) selecting cells of at least one cell type of interest from the biological sample from step (A); (C) treating the cells selected in step (B); and (D) transferring one or more of the cells treated in step (C) to a/thc subject, wherein the steps (A)-(D) arc performed in a closed system. Therefore, in such embodiments: once the biological sample leaves the patient, the patient does not become disconnected from the system (such as an apparatus for cell therapy e.g., one according to the present disclosure) until the biological sample reaches the system; and similarly, once treated cells leave the system, the system does not become disconnected from the patient until the treated cells reach the patient. Any apparatus for cell therapy which allows for such method may be used.
  • the steps (A)-(D) are performed in a single closed system.
  • collection is performed in the same system as selection, treatment, and transferring, and therefore, the cells from the subject do not leave the system once the cells leave the patient until the cells are processed (e.g., selected and treated) and ready to be transferred to the subject, with some exception) s), e.g., cells from the biological sample not selected (e.g., cells not suited to be treated to generate therapeutic cells to be transferred, such as erythrocytes or non-leukocytes in case of some CAR cell therapy, or non-T cells in case of some CAR T cell therapy) or cells not successfully treated (e.g., cells that were not successfully transduced or cells that are exhausted, in case of some CAR cell therapy), may be removed from the system (e.g., in a waste bag) or be returned to the subject as appropriate, without being further treated. Therefore, for example, cells that are selected but not yet treated may not leave the system
  • a cycle of the steps (A)-(D) are performed in a closed loop throughout the duration from biological sample collection in step (A) to cell administration/transfer in step (D).
  • a biological sample may be collected continuously as steps (B)-(D) of a cycle is performed. In such embodiments, at least part of the sample collected may be analyzed and used for monitoring described herein.
  • a cycle of the steps (A)-(D) may be performed more than once, optionally in tandem or in a manner that one cycle and another cycle may overlap in time (i.e. a cycle starts while the previous cycle is still being performed).
  • the cycle may be repeated until a certain criterion is met (e.g., total cell number transferred, the number of a particular cell type (e.g., memory T cell) transferred, number or % of transferred cells in the circulation of the subject, or the like).
  • the steps (A)-(D) are performed in a sterile condition.
  • the cells transferred in step (D) are fresh cells, i.e., have never been frozen.
  • freezing and thawing cells may increase the in vitro expansion potential during manufacturing and/or in vivo expansion potential of therapeutic cells once administered to a subject and/or may reduce loss of samples during manufacturing steps.
  • the method of cell therapy is for treating or preventing a disease, which may be for example cancer, an autoimmune disease, a neurode generative disease, or another disease described herein.
  • a disease which may be for example cancer, an autoimmune disease, a neurode generative disease, or another disease described herein.
  • step (A) or the one who provides a biological sample
  • the subject in step (B) or the one who receives the treated cells
  • the subject in step (A) are the same subject (i.e., autologous cell therapy)
  • some embodiments in which the subject in step (A) differs from the subject in step (B) are possible and envisioned according to the present invention.
  • the biological sample collected may be any sample containing cells (e g., blood, another kind of body fluid, or bone marrow) that is appropriate for cell therapy of interest.
  • the type of a biological sample may depend on, e.g., the purpose of therapy (e.g., what disease or condition to be treated) and/or the cell type of interest (e.g., T cell in case of CAR T therapy).
  • blood may be collected for selecting T cells and engineering for CAR expression in later steps
  • stem cell therapy for treating a genetic disease such as hemophilia or sickle cell anemia, a bone marrow sample, or alternatively blood upon stem cell mobilization, may be collected for selecting stem cells and performing gene editing in later steps.
  • the biological sample may be collected using any appropriate technique or mechanism, which may be part of an apparatus for cell therapy or which is physically connected to such an apparatus.
  • a biological sample is blood or the like, any technique or mechanism that allows for blood or fluid draw may be used, such as a needle.
  • a biological sample is bone marrow or another tissue, any technique or mechanism that allows for collection may be used, such as a needle or an aspirator.
  • blood may be collected in the presence of an anti-coagulant (e.g. through a needle and/or a tube containing an anti-coagulant and/or into a container (e g., vial., pouch, or bag).
  • any cell type of interest contained in the biological sample may be selected using any appropriate technique or mechanism, which may be part of an apparatus for cell therapy.
  • a cell type of interest may comprise immune cells such as but not limited to leukocyte or erythrocyte, optionally cell of the lymphoid lineage or cell of the myeloid lineage, optionally T cell, B cell, plasma cell, NK cell, NK T cell, macrophage, dendritic cell, monocyte, eosinophil, neutrophil, basophil, or mast cell; progenitor of any of the aforementioned cell types, optionally lymphoid progenitor, myeloid progenitor, lymphoblast, or myeloblast; CD8+ T cell, CD4+ T cell, or any combination thereof, optionally Thl cell, Th2 cell, Th9 cell, Th 17 cell, Th22 cell, regulatory T (Treg) cell, or follicular helper (Tfh) cell; or any combination of the aforementioned cell types.
  • immune cells such as but not limited to leukocyte or erythrocyte, optionally cell of the lymphoid lineage or cell of the myeloid lineage,
  • a cell type of interest may comprise T cells, optionally CD8+ T cells.
  • a cell type of interest may comprise macrophages.
  • a cell type of interest may comprise Treg cells.
  • a cell type of interest may comprise at least one stem cell, optionally: hematopoietic stem cell, neural stem cell, mesenchymal stem cell, epithelial stem cell, or skin stem cell, optionally lymphoid stem cell or myeloid stem cell.
  • a cell type of interest may comprise hematopoietic stem cells.
  • Selection may be performed using any appropriate mode, technique, or method of selection.
  • Exemplary modes, teclmiques, or methods of selection may include but are not limited to centrifugation-based selection (e.g., apheresis or leukapheresis), antibody-based selection, bead-based selection, magnetic selection, filtration-based selection, or any combination thereof.
  • selection may comprise multiple types of modes selections, two or more of which optionally may be performed in tandem or simultaneously.
  • the biological sample comprises blood and the cell type of interest comprises an immune cell, apheresis or leukapheresis.
  • the sample when the biological sample comprises blood and the cell ty pe of interest comprises an immune cell, the sample may first be centrifuged to be separated into plasma, platelet, leukocytes, and erythrocytes, and then the cell type(s) of interest may be further selected from the leukocytes, e.g., via antibody -based selection, bead-based selection, and/or magnetic selection. In certain embodiments, the selection may be positive selection and/or negative selection.
  • the sample when the biological sample comprises blood and the cell ty pe of interest comprises an immune cell, the sample may first be subject to red blood cell lysis, e.g., using a lysis buffer, followed by centrifugation to obtain leukocytes, and the cell type(s) of interest may be further selected from the leukocytes, e.g., via antibody-based selection, bead-based selection, and/or magnetic selection. In certain embodiments, the selection may be positive selection and/or negative selection.
  • one or more of the unselected elements of the biological sample may be returned to the subject.
  • Such element(s) may be returned as the selection step is performed, shortly after the selection is complete, or while one or more of steps (B)-(D) are performed.
  • such element(s) may be returned to the subject in step (D) together with the treated cells, i.e., therapeutic cells.
  • particular cells e.g., Treg cells
  • factors i.e., immunosuppressive cytokines
  • the step of selection may be performed entirely in a selector comprised in an apparatus for cell therapy, e.g., one described herein.
  • the selector may comprise a centrifuge mechanism and selection mechanism
  • leukocytes may be first selected via the centrifuge mechanism and immune cells of interest may be selected via the selection mechanism.
  • the step of selection may be performed in a treatment chamber and in a selector comprised in an apparatus for cell therapy, e.g., one described herein.
  • the biological sample comprises blood and the cell type of interest comprises an immune cell
  • the treatment chamber comprises a centrifuge mechanism and the selector comprises a selection mechanism
  • leukocytes may be first selected via the centrifuge mechanism of the treatment chamber and immune cells of interest may be selected via the selection mechanism of the selector.
  • FIGS 2B, 2D, and 3B-3E E.g., FIGS 2B, 2D, and 3B-3E.
  • such an apparatus may comprise a selector and a treatment chamber essentially the same as the selector (may be referred to as a separation column) and the treatment chamber (may also be referred to as CentriCult chamber) and the mechanism of operation thereof comprised in an apparatus described in US10705091, US11339407, https://atmpmanufacture.org/wp-content/uploads/2017/10/Jason Jones Miltenyi.pdf.
  • Manufacturing of adoptive cell therapeutic product requires processing the desired cells, e.g., immune effector cells, away from a complex mixture of blood cells and blood elements present in peripheral blood apheresis starting materials.
  • Peripheral blood-derived lymphocyte samples have been successfully isolated using density gradient centrifugation through Ficoll solution.
  • Ficoll is not a preferred reagent for isolating cells for therapeutic use, as Ficoll is not qualified for clinical use.
  • Ficoll contains glycol, which has toxic potential to the cells.
  • Ficoll density gradient centrifugation of thawed apheresis products after cry opreservation yields a suboptimal T cell product, e.g., as described in the Examples herein. For example, a loss of T cells in the final product, with a relative gain of non-T cells, especially undesirable B cells, blast cells and monocytes was observed in cell preparations isolated by density gradient centrifugation through Ficoll solution.
  • a medium with a density greater than Ficoll is believed to provide improved isolation of desired immune effector cells in comparison to Ficoll or other mediums with the same density as Ficoll, e.g., 1.077 g/mL.
  • the density gradient centrifugation method described herein includes the use of a density gradient medium comprising iodixanol.
  • the density gradient medium comprises about 60% iodixanol in water.
  • the density gradient centrifugation method described herein includes the use of a density gradient medium having a density greater than Ficoll. In one embodiment, the density gradient centrifugation method described herein includes the use of a density gradient medium having a density greater than 1.077 g/mL, e.g., greater than 1.077 g/mL, greater than 1.1 g/mL, greater than 1.15 g/mL, greater than 1.2 g/mL, greater than 1.25 g/mL, greater than 1.3 g/mL, greater than 1.31 g/mL. In one embodiment, the density gradient medium has a density of about 1.32 g/mL.
  • the selecting may comprise elutriation that removes unwanted cells, e.g., monocytes and blasts, thereby resulting in an improved enrichment of desired immune effector cells suitable for CAR expression.
  • the elutriation method described herein is optimized for the enrichment of desired immune effector cells suitable for CAR expression from a freshly collected biological sample.
  • the elutriation method described herein provides a preparation of cells with improved purity as compared to a preparation of cells collected from the elutriation protocols known in the art.
  • the elutriation method described herein includes using an optimized viscosity of the starting sample, e.g., cell sample, e.g., thawed cell sample, by dilution with certain isotonic solutions (e.g., PBS), and using an optimized combination of flow rates and collection volume for each fraction collected by an elutriation device.
  • an optimized viscosity of the starting sample e.g., cell sample, e.g., thawed cell sample
  • certain isotonic solutions e.g., PBS
  • the selection comprises a positive selection, e.g., selection for the desired immune effector cells, by enrichment.
  • the selection comprises a negative selection, e.g., selection for unwanted cells, e.g., removal of unwanted cells, by depletion.
  • both positive and negative selections are performed, optionally in tandem.
  • the positive or negative selection methods described herein are performed under flow conditions, e.g., by using a flow-through device, e.g., a flow-through device described herein comprised in an apparatus for cell therapy. Exemplary positive and negative selections are described on pages 53-57 of WO 2017/117112, herein incorporated by reference in its entirety.
  • Selection methods can be performed under flow conditions, e.g., by using a flow-through device, also referred to as a cell processing system, to further enrich a preparation of cells for desired immune effector cells, e.g., T cells, suitable for CAR expression.
  • a flow-through device also referred to as a cell processing system
  • Exemplary flow-through devices are described on pages 57-70 of WO 2017/117112, herein incorporated by reference in its entirety.
  • Exemplary cell separation and de-beading methods are described on pages 70-78 of WO 2017/117112, herein incorporated by reference in its entirety.
  • Selection procedures are not limited to ones described on pages 57-70 of WO 2017/117112. Negative T cell selection via removal of unwanted cells with CD19, CD 14 and CD26 Miltenyi beads in combination with column technology (CliniMACS® Plus or CliniMACS® Prodigy) or positive T cell selection with a combination of CD4 and CD8 Miltenyi beads and column technology (CliniMACS® Plus or CliniMACS® Prodigy) can be used. Alternatively, column-free technology with releasable CD3 beads (GE Healthcare) can be used.
  • bead-free technologies such as ThermoGenesis X-series devices can be utilized as well.
  • Exemplary cells selected in the selecting step for cell therapy manufacturing may be enriched e.g. for CD14 (monocytes), CD56 (natural killer cells), CD335 (NKp46, natural killer cells), CD4 (T helper cells), CD8 (cytotoxic T cells), CDlc (BDCA-1, blood dendritic cell subset), CD303 (BDCA- 2), CD304 (BDCA-4, blood dendritic cell subset), NKp80 (natural killer cells, gamma/delta T cells, cffcctor/mcinorv T cells), “6B11” (Va24/Vbll; invariant natural killer T cells), CD 137 (activated T cells), CD25 (regulatory T cells) or depleted for CD138 (plasma cells), CD4, CD8, CD19, CD25, CD45RA, CD45RO.
  • CD14 monocytes
  • CD56 natural killer cells
  • CD335 NKp46, natural killer cells
  • CD4 T helper cells
  • CD8 cytotoxic T cells
  • NK cells, NK T cells, T cells and their subsets can be utilized as effector cells in donor lymphocyte infusion approaches to eliminate virus infected cells, tumor cells or bacteria.
  • dendritic cells either generated from monocytes in cell culture or directly isolated, can be used to “vaccinate” patients to promote antigen specific and natural immunity against virus infected cells, tumor cells, bacteria, and/or fungi.
  • step (C) the cells selected in step (B) may be treated in any appropriate manner. Genetic modification
  • the cells may be genetically engineered, e.g., transduced/transfected with a nucleic acid and/or the gene may be edited, cultured/expanded in the presence of a factor of interest (e.g., cytokine, growth factor, differentiation factor, another cell type, etc).
  • a factor of interest e.g., cytokine, growth factor, differentiation factor, another cell type, etc.
  • the selected cells when the method is for CAR-based therapy, may be transduced/transfected with a nucleic acid encoding a CAR.
  • the selected cells when the method is for CAR T cell therapy, may be T cells (or one or more subsets thereof) and the T cells may be expanded first (e.g., culturing peripheral blood mononuclear cells (PBMCs) with an anti-CD3 and/or anti-CD28 agent(s) such as MACS beads) before the cells are transduced/transfected.
  • PBMCs peripheral blood mononuclear cells
  • an anti-CD3 and/or anti-CD28 agent(s) such as MACS beads
  • T cells may be simply selected and may not need to be expanded or only need to be expanded for a short period of time or in a milder condition.
  • freshly collected cells as opposed to cells that were frozen and thawed, may be healthier and may not require extensive expansion.
  • the selected cells may be contacted with a viral vector (e.g., lentiviral vector) encoding a CAR.
  • a viral vector e.g., lentiviral vector
  • the selected cells when the method is for CAR-based therapy, may be subject to gene editing, via CRISPR-Cas gene editing, Zinc finger nuclease, and/or TALEN.
  • the cells may be exposed to a Cas protein complexed with a guide RNA or one or more nucleic acids encoding a Cas protein and a guide RNA.
  • the step of treating may further comprise separating successfully treated cells or particular cell types from the rest of the cells.
  • the treating step comprise transfecting/transducing the cells with a nucleic acid (e.g., which may encode a CAR)
  • the nucleic acid may encode a marker (e.g., truncated CD19 or any marker that allows isolation of successfully transfected/transduced cells), and such separation may be based on the marker expression.
  • the separation may separate/isolate e.g., memory T cells or T cells having a memory -like phenotype and may eliminate exhausted T cells.
  • the memory T cells or T cells having a memor -like phenotype may be selected from central memory T (Tcm) cells, CD62L+CCR7+ T cells, stem cell memory T (Tscm) cells, CD45RA+CD62L+CCR7+ T cells, CD45RA+CD62L+CCR7+CD27+CD28+ T cells, CD95+ T cells, CD 122+ T cells, effector memory T (Tem) cells, and CD62L-CCR7- T cells.
  • Tcm central memory T
  • CD62L+CCR7+ T cells CD62L+CCR7+ T cells
  • stem cell memory T (Tscm) cells CD45RA+CD62L+CCR7+ T cells
  • CD45RA+CD62L+CCR7+CD27+CD28+ T cells CD95+ T cells
  • CD 122+ T cells effector memory T (Tem) cells
  • CD62L-CCR7- T cells CD62L-CCR7- T cells
  • the step of treating may further comprise culturing the treated (or successfully treated) cells, optionally for allowing some expansion of the cells.
  • the step of treating may further comprise resuspending the treated cells in a solution having a desired cell concentration to provide a formulation for administration.
  • the step of treating may be performed in a treatment chamber in an apparatus for cell therapy, e.g., one described herein.
  • the selected cells selected in a selector may be transferred to a treatment chamber and the entire treating step may be performed in the treatment chamber (e.g., FIGS 2A-2B and 3A-3B).
  • the selected cells selected in a selector may be transferred to a treatment chamber and the treating step may be performed in the treatment chamber (e.g., transduction, or transduction and expansion) and then in the selector (e.g., separation of successfully transduced cells) (e.g., FIGS 2C-2D and 3C-3D).
  • the selected cells selected in a selector may be transferred to a treatment chamber and the treating step may be performed in the treatment chamber (e.g., transduction) and then in the selector (e.g., separation of successfully transduced cells) and then in the treatment chamber (e.g., expansion of successfully treated cells) (e.g., FIGS 2D and 3E).
  • the treating step may be performed in the treatment chamber (e.g., transduction) and then in the selector (e.g., separation of successfully transduced cells) and then in the treatment chamber (e.g., expansion of successfully treated cells) (e.g., FIGS 2D and 3E).
  • such a treatment chamber may be essentially the same as the treatment chamber (which may also be referred to as CcntriCult chamber) and the mechanism of operation thereof comprised in an apparatus described in US10705091, US11339407, https://atmpmanufacture.org/wp- content/uploads/2017/10/Jason Jones Miltenyi.pdf, https://www.miltenvibiotec.com/ Resources/Persistent/19dl l2a2f0d907d93b8dbe21e2ad4b31d3dl69 53/CliniMACS Prodigy System flver.pdf, and/or comprised in a CliniMACS® device (Miltenyi) such as CliniMACS Prodigy® or CliniMACS® Plus.
  • a CliniMACS® device Miltenyi
  • T cells were first expanded from PBMCs, then transduced, and then expanded, which took about several days to weeks.
  • the recent progress in the manufacturing method cut the manufacturing time to less than 24 horns by eliminating the initial T cell activation/expansion before transduction (https://www.pennmedicine.org/news/news- releases/2022/march/penn-researchers-shorten-manufacturing-time-for-car-t-cell-therapy; https://www.novartis.com/news/media-releases/novartis-announces-t-chargetm-next-generation-car-t- platform-first-human-data-ash-2021).
  • Such shortened protocols may be found for example in US20210171909A1, US20220168389A1, W02020210678A1, and Ghassemi et al., Nat Biomed Eng. 2022 Feb;6(2): 118-128.
  • any of such shortened protocols for generating CAR T cells or an even more shortened protocol e.g., the same culture condition except that the culture duration is shorter may be used.
  • Transduction may be performed using any appropriate methods or techniques and/or transduction reagents.
  • viral or non-viral transduction/transfection may be used.
  • coculturing of cells with viral particles spinoculation, electroporation (e.g., using the mechanism comprised in Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.), or the Gene Pulser II (BioRad, Denver, Colo.)), multiporation (e.g., using the mechanism comprised in Multiporator (Eppendort, Hamburg Germany)), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al.
  • the cells selected in step (B) or the cells treated (e.g., transduced) in step (C) may further be cultured and/or expanded. In some cases, such culturing may be for expanding cells.
  • such cells may be cultured and/or expanded in the presence of at least one stimulus, optionally a cytokine, a chemokine, a molecule which binds to a surface molecule of the cells, a growth factor, a differentiation factor, or a target of interest (e.g., antigen corresponding to a CAR with which cells arc transduced) or in the presence of a particular cell.
  • a target of interest e.g., antigen corresponding to a CAR with which cells arc transduced
  • cells may be cultured in the absence of a target of interest (e.g., antigen corresponding to a CAR with which cells are transduced).
  • such cells may be cultured for about 24 hours or less, about 18 hours or less, about 12 hours or less, about 6 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 1 hours or less, about 30 minute or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, about 3 minutes or less, about 2 minutes or less, or about 1 minute or less; or about 1 minute to about 24 hours, about 1 minute to about 18 horns, about 2 minutes to about 12 hours, about 3 minutes to about 6 hours, about 5 minutes to about 4 hours, about 10 minutes to about 3 hours, about 15 minutes to about 2 hours, or about 30 minutes to about 1 hour.
  • transduced cells were expanded for a longer period of time (e.g., 4 days).
  • prolonged expansion may be associated with exhaustion of T cells and associated reduced in vivo persistence once administered.
  • transduced/transfected T cells may be expanded for about 24 horns or less, about 18 hours or less, about 12 hours or less, about 6 horns or less, about 4 horns or less, about 3 hours or less, about 2 hours or less, about 1 horns or less, about 30 minute or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, about 3 minutes or less, about 2 minutes or less, or about 1 minute or less; or about 1 minute to about 24 hours, about 1 minute to about 18 hours, about 2 minutes to about 12 hours, about 3 minutes to about 6 hours, about 5 minutes to about 4 hours, about 10 minutes to about 3 hours, about 15 minutes to about 2 hours, or about 30 minutes to about 1 hour.
  • T cells having a memory cell phenotype may be selected, optionally after expansion.
  • memory cells may be central memory T (Tcm) cells, CD45RO+CCR7+ T cells, CD62L+CCR7+ T cells, stem cell memory T (Tscm) cells, CD45RO- /CCR7+ T cells, CD45RA+CD62L+CCR7+ T cells, CD45RA+CD62L+CCR7+CD27+CD28+ T cells, CD95+ T cells, CD122+ T cells, effector memory T (Tern) cells, CD62L-CCR7- T cells.
  • Tcm central memory T
  • CD45RO+CCR7+ T cells CD62L+CCR7+ T cells
  • stem cell memory T (Tscm) cells CD45RO- /CCR7+ T cells
  • CD45RA+CD62L+CCR7+ T cells CD45RA+CD62L+CCR7+CD27+CD28+ T cells
  • CD95+ T cells CD122+
  • Tscm cells are associated with better in vivo persistence upon administration. Therefore, in particular embodiments, Tscm cells, CD45RO-/CCR7+ T cells, or CD45RA+CD62L+CCR7+ T cells may be selected, optionally after expansion.
  • any appropriate T cell transduction (and optionally expansion) conditions and/or methods may be used.
  • any conditions and/or methods described in US20210171909A1, US20220168389A1, or W02020210678A1 or used in T- ChargeTM (Novartis) may be used.
  • the treated cells may be transferred back to any appropriate site of the patient.
  • the treated cells may be transferred using any appropriate technique or mechanism, which may be part of an apparatus for cell therapy or which is physically connected to such an apparatus. For example, when the cells are transferred to the patient via the vein, any technique or mechanism that allows for infusion may be used, such as a needle.
  • an administration may take about 0.5-120 minutes, about 1-60 minutes, about 5-45 minutes, about 10-30 minutes, or about 15-20 minutes.
  • an administration dose may be described based on the number of cells/time (such as cells/min) and the administration duration (such as minutes).
  • cells may be administered at about 1000-5000 cells per minute, about 5,000-10,000 cells per minute, about 10,000-50,000 cells per minute, about 50,000-100,000 cells per minute, about 100,000-500,000 cells per minute, about 500,000-1,000,000 cells per minute, about 1,000,000- 5,000,000 cells per minute, about 5,000,000-10,000,000 cells per minute, about 1,000,000-5,000,000 cells per minute, about 5,000,000-10,000,000 cells per minute, about 10,000,000-50,000,000 cells per minute, about 50,000,000-100,000,000 cells per minute, about 100,000,000-500,000,000 cells per minute, about 500,000,000-1,000,000,000 cells per minute.
  • an administration dose when cells are administered at a constant number of cells/time, an administration dose may be described based on administration duration (such as minutes or hours). [0344] In some embodiments, an administration dose may be described based on the number of cells/volume (such as cells/mL), the administration volume/time (such as mL/min), and the administration duration (such as minutes).
  • cells may be administered at a concentration of about 100-500 cells/mL, about 500-1000 cells/mL, about 1000-5000 cells/mL, about 5000-10,000 cells/mL, about 10,000-50,000 cells/mL, about 50,000-100,000 cells/mL, about 100,000- 500,000 cells/mL, about 500,000-1,000,000 cells/mL, about 1,000,000-5,000,000 cells/mL, about 5,000,000-10,000,000 cells/mL, or about 10,000,000-50,000,000 cells/mL.
  • cells may be administered at about 25-150 mL/minute, about 50-120 mL/minute, about 60-100 mL/minute, or about 70-80 mL/minute.
  • an administration dose may be described based on the number of cells/volume (such as cells/mL) and the total administration volume (such as mL). In some embodiments, the total administration volume may be about 5-5000 mL, about 50-2000 mL, about 200-1500 mL, or about 500-1000 mL.
  • an administration dose may be described based on the total number of cells transferred to a patient.
  • the total number of cells transferred may be about IxlO 4 - IxlO 14 cells, about IxlO 5 - IxlO 13 cells, about 1x10 s - IxlO 12 cells, about IxlO 7 - IxlO 11 cells, about IxlO 8 - IxlO 10 cells, about 5xl0 8 - 5xl0 9 cells, about 7.5xl0 8 - 2.5xl0 9 cells, or about IxlO 9 cells.
  • protocols currently used in the clinic often include a step of conditioning a patient. Conditioning may be performed before, during, and/or after the step of administering therapeutic cells. Conditioning performed before administration of cells may also be referred to as preconditioning.
  • conditioning may vary widely. In some cases, conditioning may be performed to increase the efficacy provided by therapeutic cells. In some cases, conditioning may be performed to increase the persistence of therapeutic cells. In some cases, conditioning may be performed to prevent, reduce, or eliminate a side effect or an adverse event that may occur or is occurring.
  • preconditioning may comprise attenuating the host immune system to reduce host responses to the administered cells thereby increasing persistence of the administered cells.
  • conditioning may involve altering the disease site environment such as cancer microenvironment (e.g., reducing Treg cells and/or MDSCs, altering expression of certain molecule such as an immuno stimulatory costimulatory molecule or immunosuppressive indoleamine 2,3- dioxygenrase, or increasing permeability of tumor vasculature, eliminating cytokine sinks (e.g. sinks for IL-2, IL-7, and IL-15)).
  • cytokine sinks e.g. sinks for IL-2, IL-7, and IL-15
  • Such conditioning may work by making tire diseased site or cells such as cancer cells more susceptible/responsive to therapy and/or making a disease site such as a cancer site or a tumor more accessible to therapeutic cells and/or host immune cells.
  • lymphodepleting conditioning is believed to enhance efficacy of therapy at least by making space for therapeutic cells and promoting expansion, function, and persistence of the administered cells.
  • conditioning may further work by eliminating diseased cells (e.g., leukemic lymphocytes) and/or altering disease cells (e.g., reducing production of certain metabolites, modifying the expression of costimulatory molecules).
  • diseased cells e.g., leukemic lymphocytes
  • altering disease cells e.g., reducing production of certain metabolites, modifying the expression of costimulatory molecules.
  • lymphodepleting conditioning is also believed to work by eliminating sinks for homeostatic cytokines such as IL-2, IL-7, and IL-15 (thereby increasing the availability of such cytokines) and/or eradicating or inhibiting immunosuppressive elements such as Treg cells (Neelapu. Blood. 2019 Apr 25;133(17):1799-1800.).
  • Various lymphodepleting conditioning protocols are known in the field. Some of the most commonly used protocols may involve administration of one or more of anti-proliferative/anti- neoplastic/anti-cancer agents (e.g., cyclophosphamide, fhidarabine, bendamustine, -fluorouracil, leucovorin, oxaliplatin and/or anti-CD20 antibody such as rituximab, and/or anti-CD52 antibody such as alemtuzumab) and/or irradiation (e.g., total body irradiation), which may be administered before, typically several days (e.g., 2-14 days) before, administering cell therapy.
  • anti-proliferative/anti- neoplastic/anti-cancer agents e.g., cyclophosphamide, fhidarabine, bendamustine, -fluorouracil, leucovorin, oxaliplatin and/or anti-CD
  • the combination of fludarabine (e.g., at 25-50 mg/m 2 per day for 1-10 days) and cyclophosphamide (e.g., at 100-1000 mg/m 2 per day for 1-10 da s) or lhe combination of folinic acid, fluorouracil, and oxaliplatin (also known as FOLFOX) may be administered.
  • fludarabine e.g., at 25-50 mg/m 2 per day for 1-10 days
  • cyclophosphamide e.g., at 100-1000 mg/m 2 per day for 1-10 da s
  • lhe combination of folinic acid, fluorouracil, and oxaliplatin also known as FOLFOX
  • lymphodepletion practice it is noted that while many benefits of lymphodepletion in cell therapy for cancer have been largely confirmed in the context of treatment of liquid cancer, there is no established lymphodepletion practice (see, e.g., Nissani et al., J Immunother Cancer. 2021 May;9(5):e001743.; Abstract of Owens and Bozic. Bull Math Biol. 2021 Mar 19;83(5):42 ), and this is especially so for treating solid cancer, for which no cell therapy is FDA approved to date.
  • No lymphodepletion Regardless of the disease to be treated and/or whether the cancer to be treated is liquid or solid, the traditional lymphodepletion regimes eliminate at least some of the healthy lymphocytes such as healthy lymphocytes that are useful for engineering and administration and/or that may remain in the host and be successfully activated by therapeutic cells. Therefore, in some embodiments according to the present invention, no lymphodepletion may be performed, regardless of the type of the disease to be treated (and in case of cancer, liquid or solid).
  • lymphodepletion in treating cancer of lymphocytes, which is elimination of cancerous lymphocytes (e.g., elimination of leukemic cells), is not applicable to treatment of solid cancer, in some embodiments particularly relating to treatment of solid cancer, no lymphodepletion may be performed.
  • CAR T cell therapy successfully provided efficacy without lymphodepletion in clinical trials (see, e.g., ClinicalTrials.gov Identifiers: NCT0220382) and in some studies no significant differences in CAR T cell engraftment were observed, for example between “no preconditioning” vs “FOLFOX” (see, e.g., Alcantar-Orozco et al..
  • lymphodepletion may be performed regardless of the disease to be treated and/or whether the cancer to be treated is liquid or solid.
  • T cells surviving fludarabine/cyclophosphamide treatment have a more mature phenotype and fludarabine-treated T cells are more proliferative and more prone to Thl cytokine secretion, i.e., those cells that remain may be ideally suited for engineering and subsequent administration (Gassner et al., Cancer Immunol Irmnunother. 2011 Jan;60(l):75-85. Epub 2010 Sep 21).
  • lymphodepletion can be beneficial for cell therapy even for solid cancer by providing a more suited lymphocyte population.
  • some preclinical studies reported advantages observed with lymphodepletion in cell therapy for solid cancer.
  • preconditioning using cyclophosphamide reversed the immunosuppressive tumor microenvironment, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells as well as endogenous and adoptively transferred T cells, resulting in long-term anti-tumor immunity, which effects allowed for efficacy of CAR T therapy (Murad et al., Mol Ther. 2021 Jul 7;29(7):2335-2349.
  • TILs tumor-infiltrating T lymphocytes
  • Many methods to determine an appropriate conditioning protocol including doses, timing, and durations and/or frequencies, are known in the field.
  • a mathematical model may be applied to determine an appropriate conditioning protocol (e.g., as proposed in Owens and Bozic. Bull Math Biol. 2021 Mar 19;83(5):42 ).
  • a low level of or partial lymphodepletion may be performed, e.g., by using a dose that is lower than most commonly used lymphodepleting doses and/or by administering a lymphodepleting agent for a shorter period of time or shortly before or shortly after (e.g., less than 2 days or less than 1 day, such as less than 18 hours, less than 12 hours less than 8 hours, less than 6 hours, less than 4 horns, less than 3 hours, less than 2 hours, less than 1 hour, or less than 30 minutes before or after) collection and/or processing of cells.
  • a low level of or partial lymphodepletion may minimize depletion of lymphocytes that are suited for cell therapy, while still providing some advantages (e.g., toxicity to cancerous lymphocytes, prevention of T cell exhaustion).
  • cells being processed may be monitored in real time and the conditions used in cell processing may be adjusted based on the monitoring results. Therefore, in some embodiments, the amount or phenoty pe of cells such as particular type(s) of T cells (e g., Treg cells, exhausted T cells, etc) may be monitored and the cell processing condition(s) may be adjusted so that the processed cells ready for administration/transfer have a cell profile of interest (e.g. CD4:CD8 ratio, %exhausted T cells, %memory T cells, % Treg cells, etc).
  • a cell profile of interest e.g. CD4:CD8 ratio, %exhausted T cells, %memory T cells, % Treg cells, etc.
  • non-lymphodepleting or non-pan-lymphodepleting i.e., not depleting all kinds of lymphocytes and rather selective depletion
  • conditioning which aims to increase or reduce one or more particular types of lymphocytes or immune cells may be performed.
  • conditioning for reducing or inhibiting immunosuppressive cells may be performed, e.g., by administering an antibody which targets one or more surface marker of such cells (e.g., a checkpoint inhibitor, such as an anti-PD-1, anti-PD-Ll, anti- CTLA4 antibody) or a cytokine or another molecule which inhibits or reverses the function of such cells.
  • an antibody which targets one or more surface marker of such cells e.g., a checkpoint inhibitor, such as an anti-PD-1, anti-PD-Ll, anti- CTLA4 antibody
  • a cytokine e.g., cytokine or another molecule which inhibits or reverses the function of such cells.
  • such conditioning may for example support the effector function and/or persistence of the therapeutic cells or endogenous cells activated by the therapeutic cells.
  • conditioning for increasing or activating immunosuppressive cells may be performed, e.g., by administering a cytokine or another molecule which supports or enhances the function of such cells. In some cases, such conditioning may for example mitigate, reduce, or prevent overactivation and/or exhaustion of the therapeutic cells and/or overt inflammation such as cytokine release syndrome (CRS).
  • CRS cytokine release syndrome
  • conditioning for reducing or inhibiting immunosuppressive cytokine(s) e.g., IL-10 and/or TGF-P
  • chemokine(s) may be performed, e.g., by administering a blocking or neutralizing antibody.
  • conditioning may for example support the effector function and/or persistence of the therapeutic cells or endogenous cells activated by the therapeutic cells.
  • conditioning for increasing or activating immunosuppressive cytokine(s) e.g., IL-10 and/or TGF-P
  • chemokine(s) may be performed, e.g., by administering or administering a factor which increases such cytokine(s) and/or chemokines.
  • such conditioning may for example mitigate, reduce, or prevent overactivation and/or exhaustion of the therapeutic cells and/or overt inflammation such as cytokine release syndrome (CRS).
  • CRS cytokine release syndrome
  • conditioning for reducing or inhibiting cytokine(s) and/or chemokine(s) related to CRS may be performed, e.g., by administering a blocking or neutralizing antibody (e.g., anti-IL-6 or IL-6 receptor antibody).
  • a blocking or neutralizing antibody e.g., anti-IL-6 or IL-6 receptor antibody.
  • such conditioning may for example mitigate, reduce, or prevent overactivation and/or exhaustion of the therapeutic cells and/or overt inflammation such as CRS.
  • conditioning for increasing or activating cytokine(s) and/or chemokine(s) which support effector function(s) may be performed, e.g., by administering such cytokine(s) and/or chemokine(s).
  • CAR cell therapy side effects or toxic effects may occur.
  • exemplary side effects and/or toxic effects in CAR cell therapy include but are not limited to CRS, macrophage activation syndrome (MAS), hemophagocytic lymphohistiocytosis (HLH), neurological side effects (e.g., confusion, delirium, word-finding aphasia, coma, seizures, irritability, and/or delirium), tumor lysis syndrome (TLS), on-target/off-tumor toxicities (e.g., hypogammaglobulinemia). Therefore, in some embodiments, conditioning which treats, mitigates, or prevents such or other conditions may be performed.
  • MAS macrophage activation syndrome
  • HHLH hemophagocytic lymphohistiocytosis
  • neurological side effects e.g., confusion, delirium, word-finding aphasia, coma, seizures, irritability, and/or delirium
  • TLS tumor lysis syndrome
  • conditioning may be performed at any appropriate timing, including but not limited to before, during, and/or after collecting cells from a patient, during any of the cell processing steps (e.g., while the cells are selected and/or treated (such as genetically modified) or while the treated cells are being administered), or even after completion of administration.
  • some protocols include a step of stem-cell mobilization as a conditioning step.
  • an agent which induces or enhances the movement of stem cells from the bone marrow into the blood may be administered to a patient, and once stem cells are mobilized blood may be collected and stem cells may be isolated and/or treated (e.g., expanded, differentiated, and/or the gene is edited) and transferred the/another patient.
  • Exemplary stem-cell mobilization agents include but are not limited to G-CSF, GM-CSF, Plerixafor, and SCF.
  • stem -cell mobilization agents include but are not limited to CXCL12/CXCR4 antagonists or blockers, SIP agonists, VCAM/VLA-4 inhibitors, parathyroid hormone, proteosome inhibitors, Gro[>. and HIF stabilizers.
  • the amount or phenotype of cells such as particular type(s) of T cells (e.g., Treg cells, exhausted T cells, type of stem cells etc) may be monitored and the cell processing condition(s) may be adjusted so that the processed cells ready for administration/transfer have a cell profile of interest (e.g. CD4:CD8 ratio, %exhausted T cells, %memory T cells, % Treg cells, expression level of differentiation marker etc).
  • a cell profile of interest e.g. CD4:CD8 ratio, %exhausted T cells, %memory T cells, % Treg cells, expression level of differentiation marker etc.
  • a subject receiving cell therapy or a sample collected therefrom may optionally be monitored. In some embodiments, based on the monitoring results, the method may be adjusted.
  • all or part of the biological sample collected in step (A), all or part of cells selected in step (B), all or part of the elements not selected in step (B), all or part of cells treated in step (C), and/or all or part of the cells being transferred in step (D) or a formulation containing such cells may be analyzed for monitoring
  • a monitoring sample is collected separately from the biological sample collected in step (A) and may be analyzed for monitoring.
  • the monitoring step may be also performed in the closed system in which steps (A)-(D) are performed.
  • monitoring may be performed in an automated manner and the method parameters (e.g., selection criteria, culturing condition (stimulus to be used, duration), expansion condition (stimulus to be used, duration), administration dose and speed, administration of an agent other than tire therapeutic cells) may be adjusted based on the monitoring results in an automated manner.
  • the method parameters e.g., selection criteria, culturing condition (stimulus to be used, duration), expansion condition (stimulus to be used, duration), administration dose and speed, administration of an agent other than tire therapeutic cells
  • CRM computer-readable media
  • the CRM is used with the closed system apparatus to perform one or more steps of any of the methods for cell therapy according to the present invention.
  • the closed system apparatus may be any apparatus for cell therapy according to the present invention. Therefore, any of the variations/parameters described above in the METHOD OF CELL THERAPY section above and the variations/parameters described above in the APPARATUS section below may apply to any of the CRM described herein. Any other variations/parameters described herein may also apply.
  • One or more CRM may have encoded thereon computer-readable instructions for causing one or more processing devices to control operations of various portions of a closed system apparatus for cell therapy.
  • the closed system apparatus may comprise: (a) a first connector configured to be operably and/or directly connectable to a subject to allow for collection of a biological sample from the subject through the first connector; (b) a selector configured to separate cells of at least one cell type of interest from elements not of interest in the biological sample; (c) a treatment chamber configured to treat cells; and (d) a second connector configured to be operably and/or directly connectable to the subject to allow for transfer of the treated cells to the subject through the second connector.
  • the operations may comprise: (A) optionally collecting a biological sample containing cells from a subject and optionally transferring the biological sample through the first connector (i) to the treatment chamber or (ii) to the selector; (B) selecting cells of at least one cell type of interest from a/the biological sample (i) in the treatment chamber and then in the selector or (ii) in the selector and transferring the selected cells to the treatment chamber; and (C) treating the selected cells (i) in the treatment chamber or (ii) in the treatment chamber and then in the selector and optionally then in the treatment chamber; and (D) optionally transferring one or more of the cells treated in (C) to the subject.
  • the operations may further comprise: (E) conditioning the subject and/or (F) monitoring the subject.
  • the operations may further comprise: (I) presenting, on a display, a user interface configured to accept one or more user-inputs pertaining to one or more of the operations; and (II) optionally adjusting at least one of the operations in accordance with the one or more user-inputs.
  • the one or more user-inputs may identify (i) a condition of the selecting in the operation(s) of (B), optionally the at least one cell type of interest and/or a least one cell ty pe not to be selected and/or (ii) a condition of the treating in the operation(s) of (C), optionally the duration of the treating, a temperature used during the treating, and/or a volume of a solution containing the cells during the treating.
  • the operations may not comprise (A) collecting a biological sample containing cells from a subject and transferring the biological sample through the first connector (i) to the treatment chamber or (ii) to the selector; and/or (D) transferring one or more of the cells treated in (C) to the subject.
  • a user of the apparatus may manually perform or cause steps (A) and/or (D).
  • the operations may comprise (A) collecting a biological sample containing cells from a subject and transferring the biological sample through the first connector (i) to tire treatment chamber or (ii) to the selector.
  • the operations may comprise (D) transferring one or more of the cells treated in (C) to the subject.
  • the operations comprise (A) collecting a biological sample containing cells from a subject and transferring the biological sample through the first connector (i) to the treatment chamber or (ii) to the selector; and (D) transferring one or more of the cells treated in (C) to the subject.
  • a user of the apparatus dose not manually perform steps (A) and (D), i.e., the operations of the apparatus are more automated.
  • the collecting and transferring may comprise electronically controlling at least one valve and/or a pump configured to control fluid flow between the subject and a component of the apparatus (e.g.. the first connector, the selector, or the treatment chamber).
  • a component of the apparatus e.g. the first connector, the selector, or the treatment chamber.
  • the transferring may comprise electronically controlling at least one valve and/or a pump configured to control fluid flow between the subject and the apparatus (e g., the second connector, the treatment chamber, or the selector).
  • the selecting cells of at least one cell type of interest from a/the biological sample in step (B) may take place in the treatment chamber and then in the selector.
  • the treatment chamber may comprise an input port and an output port coupled to a rotatable container having at least one sample chamber, and the biological sample transferred to the treatment chamber in step (A) may be placed in die sample chamber; and the selector may comprise an input port and an output port coupled to a MACS column (also containing one or more container each of which receives flow-through material(s) and/or elution material(s)).
  • the operations for step (B) may comprise (1) controlling the rotatable container to rotate to centrifuge the biological sample, (2) removing the supernatant material(s) (e.g., plasma), the second layer material(s) (e.g., platelets), and/or the bottom material(s) (e.g., erythrocytes) from the sample chamber to leave cell of interest (e.g., leukocytes) in the sample chamber, (3) adding MACS beads specific to a surface molecule of a cell type of interest (e.g., CD3) or of cell types not of interest to the sample chamber, (4) incubating the cells in the sample chamber in buffer, (5) controlling the rotatable container to rotate to centrifuge the cells in buffer, (6) removing die supernatant from the sample chamber, (7) adding buffer to the sample chamber, (8) transferring die materials in the sample chamber to the selector, (9) priming the MACS column with column priming buffer and apply a magnetic field (if not already applied), (
  • the selecting cells of at least one cell type of interest from a/the biological sample in step (B) may take place in then in the selector.
  • the selector may comprise (i) an input port, (ii) a rotatable container having at least one sample chamber, configured to receive material(s) through the input port, (iii) a MACS column (also containing one or more container each of which receives flow- through material(s) and/or elution material(s)) configured to receive material(s) from the sample chamber, and (iv) an output port configures to receive flow-through material(s) and/or eluted material(s) from the MACS column.
  • the operations for step (B) may comprise (1) controlling the rotatable container to rotate to centrifuge the biological sample, (2) removing the supernatant material(s) (e.g., plasma), the second layer material(s) (e.g., platelets), and/or the bottom material(s) (e g., erythrocytes) from the sample chamber to leave cell of interest (e.g., leukocytes) in the sample chamber, (3) adding MACS beads specific to a surface molecule of a cell ty pe of interest (e.g., CD3) or of cell types not of interest to the sample chamber, (4) incubating the cells in the sample chamber in buffer, (5) controlling the rotatable container to rotate to centrifuge the cells in buffer, (6) removing the supernatant from the sample chamber, (7) adding buffer to the sample chamber, (8) priming the MACS column with column priming buffer and apply a magnetic field (if not already applied), (9) applying the materials from the sample chamber to the MACS
  • the transferring the selected cells to the treatment chamber comprises electronically controlling at least one valve and/or a pump configured to control fluid flow between the selector, optionally a receptacle configured to hold the selected cells within the selector, and the treatment chamber.
  • the treating the selected cells in step (C) may take place solely in the treatment chamber.
  • the treatment chamber may comprise an input port and an output port coupled to a rotatable container having at least one sample chamber, and the cells selected in step
  • step (B) may be placed in the sample chamber.
  • the operations for step (B) may be placed in the sample chamber.
  • (C) may comprise the following steps (1) adding a medium, a nucleic acid (e.g., viral vector optionally encoding a CAR), and optionally a transfeclion/transduction reagent (2) optionally controlling the rotatable container to rotate to centrifuge the cells in the sample chamber (e.g., for spinoculation/spinfection), (3) incubating the cells in the sample chamber, (4) rotating the container to centrifuge the cells in the sample chamber, (5) removing the supernatant material(s) (e.g., medium containing nucleic acids, e.g., viral vectors which have not been taken up by cells (“unused vectors”) leaving cells, e.g., transduced and non-transduced cells in the sample chamber, (6) optionally adding buffer or additional medium to the sample chamber, (7) optionally adding one or more stimuli to the sample chamber and optionally culturing the cells in the sample chamber (e.g., to allow for expansion), (8) optionally rotating the container in order to
  • the cells selected in step (B) may be washed before step (1).
  • washing may be performed once the cells selected in step (B) are placed in the sample chamber of the treatment chamber.
  • such washing procedure may comprise: (i) adding buffer (e.g., washing buffer) to the sample chamber, (ii) rotating the container to centrifuge the cells in the sample chamber, and (iii) removing the supernatant material(s) (e.g., buffer and any element(s) carried over from step (B), such as MACS® buffer) to leave cells in the sample chamber.
  • buffer e.g., washing buffer
  • MACS® buffer e.g., MACS® buffer
  • a buffer may be added (e.g., wash buffer).
  • the addition of buffer to the medium may increase the liquid volume during step (4) and the supernatant volume which is removed in (5), allowing for more efficient and/or more complete removal of some supernatant materials such as unused vectors contained in the medium used in step (3).
  • the cells may be washed.
  • washing may comprise: (i) adding buffer (e.g., washing buffer) to the sample chamber, (ii) rotating the container to centrifuge the cells in the sample chamber, and (iii) removing the supernatant material(s) (e.g., buffer and any element(s) carried over from step (3), such as medium, unused vectors, and/or cytokines released by the cells) to leave cells in the sample chamber.
  • buffer e.g., washing buffer
  • the supernatant material(s) e.g., buffer and any element(s) carried over from step (3), such as medium, unused vectors, and/or cytokines released by the cells
  • (i)-(iii) may be repeated twice, three times, or more.
  • additional buffer may be added (e.g., wash buffer).
  • additional buffer e.g., wash buffer
  • the addition of buffer to the medium may increase the liquid volume during (8) and the supernatant volume to be removed in step (9), allowing for more efficient and/or complete removal of some element(s) such as cell culture debris and/or cytokines contained in the medium used during the culturing in (7).
  • the cells may be washed.
  • washing may comprise: (i) adding buffer (e.g., washing buffer) to the sample chamber, (ii) rotating the container to centrifuge the cells in the sample chamber, and (iii) removing the supernatant material(s) (e.g., buffer and any element(s) carried over from (7), such as cytokines released by the cells) to leave cells in the sample chamber.
  • buffer e.g., washing buffer
  • the supernatant material(s) e.g., buffer and any element(s) carried over from (7), such as cytokines released by the cells
  • (i)-(iii) may be repeated twice, three times, or more.
  • the treating of the selected cells in step (C) may take place in the treatment chamber and then in the selector.
  • the treatment chamber may comprise an input port and an output port coupled to a rotatable container having at least one sample chamber, and the cells selected in step (B) may be placed in the sample chamber; and the selector may comprise an input port and an output port coupled to a MACS® column (may also be containing one or more container each of which receives flow-through material(s) and/or elution material(s)).
  • the selector may be configured to comprise a rotatable container which is capable of providing centrifugation to pre- or post-MACS® separation samples.
  • the operations for step (C) may comprise (1) adding a medium, a nucleic acid (e.g., viral vector optionally encoding a CAR), and optionally a transfection/transduction reagent (2) optionally controlling the rotatable container to rotate to centrifuge the cells in the sample chamber (e.g., for spinoculation/spinfection), (3) incubating the cells in the sample chamber, (4) controlling the rotatable container to rotate to centrifuge the cells in the sample chamber, (5) removing the supernatant material(s) (e.g., medium containing unused vectors) to leave cells in the sample chamber, (6) optionally adding media and optionally one or more stimuli to the sample chamber and optionally culturing the cells in the sample chamber (to allow for expansion), (7) optionally rotating the container to centrifuge the cells in the sample chamber, (8) optionally removing the supernatant material(s) (e.g., medium) to leave cells in the sample chamber, and (9)
  • the cells selected in step (B) may be washed before step (1).
  • such washing may be performed once the cells selected in step (B) are placed in the sample chamber of the treatment chamber.
  • the operation for such a washing may comprise: (i) adding buffer (e.g., washing buffer) to the sample chamber, (ii) rotating the container to centrifuge the cells in the sample chamber, and (iii) removing the supernatant material(s) (e.g., buffer and any element(s) carried over from step (B), such as MACS® buffer) to leave cells in the sample chamber.
  • buffer e.g., washing buffer
  • the supernatant material(s) e.g., buffer and any element(s) carried over from step (B), such as MACS® buffer
  • (i)-(iii) may be repeated tw ice, three times, or more.
  • step (4) additional buffer (e.g., wash buffer) may be added.
  • additional buffer e.g., wash buffer
  • the addition of buffer to the medium may increase the liquid volume during (4) and thus the supernatant volume to be removed in (5), allowing for more efficient removal of some element(s) such as unused vectors contained in the medium used in (3).
  • the cells may be washed.
  • the such washing may comprise: (i) adding buffer (e.g., washing buffer) to the sample chamber, (ii) rotating the container to centrifuge the cells in the sample chamber, and (iii) removing the supernatant material(s) (e g., buffer and any element(s) carried over from step (3), such as medium, unused vectors, and/or cytokines released by the cells) to leave cells in the sample chamber.
  • buffer e.g., washing buffer
  • the supernatant material(s) e.g., buffer and any element(s) carried over from step (3), such as medium, unused vectors, and/or cytokines released by the cells
  • (i)-(iii) may be repeated twice, three times, or more.
  • step (7) additional buffer (e.g., wash buffer) may be added.
  • additional buffer e.g., wash buffer
  • the addition of buffer to the medium increases the liquid volume during step (7) and the supernatant volume to be removed in (8), providing for more efficient and/or complete removal of some element(s) such as cell culture debris and/or cytokines contained in the medium used during the culturing in (6).
  • the cells may be washed.
  • washing may comprise: (i) adding buffer (e.g., washing buffer) to the sample chamber, (ii) rotating the container to centrifuge the cells in the sample chamber, and (iii) removing the supernatant material(s) (e.g., buffer and any element(s) carried over from step (6), such as cytokines released by the cells) to leave cells in the sample chamber.
  • buffer e.g., washing buffer
  • the supernatant material(s) e.g., buffer and any element(s) carried over from step (6), such as cytokines released by the cells
  • (i)-(iii) may be repeated twice, three times, or more.
  • step (12) additional buffer (e.g., wash buffer or MACS® buffer) may be added.
  • additional buffer e.g., wash buffer or MACS® buffer
  • the addition of buffer to the medium increases the liquid volume during step (12) and the supernatant volume to be removed in step (13), providing for more efficient and/or complete removal of some element(s) such as unused MACS® beads contained in the medium used in step (11).
  • the cells in the flow-through material(s) (negative fraction(s)) from step (17) or in the eluted material(s) (positive fraction(s)) from step (18), whichever contains the cells of interest (e.g., depends on whether the MACS® separation was based on positive selection or negative selection), may be washed.
  • the operation for such a washing may comprise: (i) placing the flow-through material(s) (negative fraction(s)) from step (17) or in the eluted material (s) (positive fraction(s)), whichever contains the cells of interest (e.g., depends on whether the MACS® separation was based on positive selection or negative selection), in the rotatable container of the selector (if not already in the rotatable container), (ii) optionally adding buffer (e.g., wash buffer) to the rotatable container, (iii) controlling the rotatable container to rotate to centrifuge the cells in the sample chamber, and (iv) removing the supernatant material(s) (e.g., buffer and any element(s) carried over from steps ( 17)-(l 8), such as MACS® buffer) to leave cells.
  • buffer e.g., wash buffer
  • controlling the rotatable container to rotate to centrifuge the cells in the sample chamber removing the supernatant material(s) (e.g
  • the treating the selected cells in step (C) may take place in the treatment chamber and then in the selector and then in the treatment chamber.
  • the treatment chamber may comprise an input port and an output port coupled to a rotatable container having at least one sample chamber, and the cells selected in step (B) may be placed in the sample chamber; and the selector may comprise an input port and an output port coupled to a MACS® column (may also be containing one or more container each of which receives flow-through material(s) and/or elution material(s)).
  • the operations for step (C) may comprise steps (l)-(l 8) described above (with the understanding that optional steps and elements are still optional) and further optionally including one or more of the additional washing steps described above and may further comprise one or more of the following additional steps (19) transferring the flow-through material(s) (negative fraction(s)) from step (17) and/or in the eluted material(s) (positive fraction(s)), whichever contains the cells of interest (e g., depends on whether the MACS® separation was based on positive selection or negative selection), to the sample chamber of the treating chamber, (20) optionally adding buffer (e.g., wash buffer) to the rotatable container, (21) rotating the container to centrifuge the cells in the sample chamber, and (22) removing the supernatant material(s) (e.g., buffer and any element(s) carried over from steps ( 17)-(l 8), such as MACS® buffer) to leave cells.
  • steps (20)-(l 8) described above (with the understanding that optional steps and elements are
  • the operations for step (C) may comprise the following steps: (1) adding a medium, a nucleic acid (e.g., viral vector optionally encoding a CAR), and optionally a transfection/transduction reagent (2) optionally controlling the rotatable container to rotate to centrifuge the cells in the sample chamber (e.g., for spinoculation/spinfection), (3) incubating the cells in the sample chamber, (4) rotating the container to centrifuge the cells in the sample chamber, (5) removing the supernatant material(s) (e.g., medium containing unused vectors) to leave cells in the sample chamber, (6) adding MACS® beads specific to a surface molecule of a cell type of interest (e.g., marker indicating successful transduction, such as truncated CD19) or of cell types not of interest to the sample chamber, (7) incubating the cells in the sample chamber in buffer, (8) rotating the container to centrifuge the cells in buffer, (9)
  • the transferring the cells from the treatment chamber to the selector comprises electronically controlling at least one valve and/or a pump configured to control fluid flow between the treatment chamber and the selector.
  • the transferring the cells from the selector to the treatment chamber comprises electronically controlling at least one valve and/or a pump configmed to control fluid flow between the selector and the treatment chamber.
  • the cells selected in step (B) may be washed. For example, such washing may be performed once the cells selected in step (B) are placed in the sample chamber of the treatment chamber.
  • the operation for such a washing may comprise: (i) adding buffer (e.g., washing buffer) to the sample chamber, (ii) controlling the rotatable container to rotate to centrifuge the cells in the sample chamber, and (iii) removing the supernatant matcrial(s) (e.g., buffer and any element(s) carried over from step (B), such as MACS® buffer) to leave cells in the sample chamber.
  • buffer e.g., washing buffer
  • step (B)-(iii) may be repeated twice, three times, or more.
  • step (4) additional buffer may be added (e.g., wash buffer).
  • additional buffer e.g., wash buffer.
  • the addition of buffer to the medium increases the liquid volume during (4) and the supernatant volume to be removed in (5), allowing for more efficient and/or complete removal of some element(s) such as unused vectors contained in the medium used in (3).
  • the cells may be washed.
  • washing may comprise: (i) adding buffer (e.g., washing buffer) to the sample chamber, (ii) controlling the rotatable container to rotate to centrifuge the cells in the sample chamber, and (iii) removing the supernatant material(s) (e.g., buffer and any element(s) carried over from step (3), such as medium, unused vectors, and/or cytokines released by the cells) to leave cells in the sample chamber.
  • buffer e.g., washing buffer
  • step (3) such as medium, unused vectors, and/or cytokines released by the cells
  • step (8) additional buffer may be added (e.g., wash buffer).
  • additional buffer e.g., wash buffer.
  • the addition of buffer to the medium increases the liquid volume during step (8) and the supernatant volume to be removed in (9), allowing for more efficient and/or complete removal of some element(s) such as beads that are unbound after incubation step (3).
  • the cells may be washed.
  • washing may comprise: (i) adding additional buffer (e.g., washing buffer) to the sample chamber, (ii) rotating the container to centrifuge the cells in the sample chamber, and (iii) removing the supernatant material(s) (e.g., buffer and any element(s) carried over from step (7), such as unbound MACS® beads) to leave cells in the sample chamber.
  • additional buffer e.g., washing buffer
  • rotating the container to centrifuge the cells in the sample chamber e.g., removing the supernatant material(s) (e.g., buffer and any element(s) carried over from step (7), such as unbound MACS® beads) to leave cells in the sample chamber.
  • supernatant material(s) e.g., buffer and any element(s) carried over from step (7), such as unbound MACS® beads
  • (i)-(iii) may be repeated twice, three times, or more.
  • the cells in the flow-through material(s) (negative fraction(s)) or in the eluted material(s) (positive fraction(s)), whichever contains the cells of interest, transferred to the sample chamber of the treatment chamber in step (15) may be washed before step (16).
  • the operation for such a washing may comprise: (i) adding buffer (e.g., washing buffer) to the sample chamber, (ii) controlling the rotatable container to rotate to centrifuge the cells in the sample chamber, and (iii) removing the supernatant material(s) (e.g., buffer and any element(s) carried over from steps (13)-(14) such as MACS® buffer) to leave cells in the sample chamber.
  • buffer e.g., washing buffer
  • the supernatant material(s) e.g., buffer and any element(s) carried over from steps (13)-(14) such as MACS® buffer
  • (i)-(iii) may be repeated, optionally twice, three times, or more.
  • step (17) additional buffer may be added, (e.g., wash buffer).
  • additional buffer e.g., wash buffer
  • the addition of buffer to the medium increases the liquid volume during step (17) and the supernatant volume to be removed in step (18), allowing for more efficient and/or complete removal of some element(s) such as cytokines produced during the optional culturing in (16).
  • the cells may be washed.
  • the operation for such washing may comprise: (i) adding buffer (e.g., washing buffer) to the sample chamber, (ii) rotating the container to centrifuge the cells in the sample chamber, and (iii) removing the supernatant material(s) (e.g., to remove element(s) carried over from step (16), such as part of the medium and/or cytokines released by tire cells) to leave cells in the sample chamber.
  • buffer e.g., washing buffer
  • removing the supernatant material(s) e.g., to remove element(s) carried over from step (16), such as part of the medium and/or cytokines released by tire cells
  • (i)-(iii) may be repeated, optionally twice, three times, or more.
  • the operations for step (C) may further comprise adding a solution to the selector or the sample chamber of the treatment chamber to resuspend the treated cells to achieve a desired cell concentration range.
  • the optional step (E) of conditioning the subject may also be performed by an apparatus for cell therapy.
  • the apparatus may comprise or may be operably connectable to a conditioning agent container (e.g., vial or pouch) which is operably connectable to the subject to allow for transfer/administration of a conditioning agent to the subject.
  • a conditioning agent container e.g., vial or pouch
  • the operations for step (E) may comprise transferring a conditioning agent from the conditioning agent container to the subject.
  • the transferring a conditioning agent from the conditioning agent container to the subject may comprise electronically controlling at least one valve and/or a pump configured to control fluid flow between the conditioning agent container and the subject.
  • the optional step (F) of monitoring the subject may also be performed by an apparatus for cell therapy .
  • the apparatus may comprise or may be operably connectable to a monitoring element (e.g., a needle (for collecting a monitoring sample), a sensor (for measuring temperature, heart rate, blood pressure, breathing rate, oxygen levels), an analytic device (for analyzing levels of cytokine, chemokine, and/or cells of interest) which is operably connectable to the subject to allow for collection of a monitoring sample from the subject or from materials in the apparatus.
  • a monitoring element e.g., a needle (for collecting a monitoring sample), a sensor (for measuring temperature, heart rate, blood pressure, breathing rate, oxygen levels), an analytic device (for analyzing levels of cytokine, chemokine, and/or cells of interest) which is operably connectable to the subject to allow for collection of a monitoring sample from the subject or from materials in the apparatus.
  • the operations for step (F) may comprise (1) collecting a monitoring sample from the subject or from materials in the apparatus, (2) analyzing a parameter of interest in the monitoring sample, and (3) optionally adjusting
  • Another aspect of the invention provides an apparatus for cell therapy.
  • an apparatus for cell therapy according to the present invention may be for performing and/or may be configured to perform all or part of any of the methods for cell therapy according to the present invention.
  • the operations of various portions of an apparatus for cell therapy according to the present invention may be controlled by computer-readable instructions encoded on any of the CRM according to the present invention. Therefore, any of the variations/parameters described above in the METHOD FOR CELL THERAPY section above and the variations/parameters described above in the COMPUTER-READABLE MEDIA section above may apply to any of the apparatuses described herein. Any other variations/parameters described herein may also apply.
  • an apparatus may comprise such one or more computer-readable media or comprise a computer which is capable of reading such one or more computer-readable media.
  • such an apparatus may be configured to perform steps of such a method in a sterile environment.
  • An apparatus for cell therapy may comprise: (a) a first connector configured to be operably and/or directly connectable to a subject to allow for collection of a biological sample from the subject through tire first connector; (b) a selector configured to separate cells of at least one cell type of interest from elements not of interest in the biological sample; (c) a treatment chamber configured to treat cells; and (d) a second connector configured to be operably and/or directly connectable to the subject to allow for transfer of the treated cells to the subject through the second connector.
  • An apparatus may be a closed system apparatus.
  • the selection chamber and/or the treatment chamber comprises one or more disposable and/or single-use elements.
  • the first connector may comprise a first mechanism (e.g., needle or aspirator) which allows for biological sample collection or is operably connectable to a first mechanism (e.g., needle or aspirator) which allows for biological sample collection (see, e.g., FIG 4A).
  • a first mechanism e.g., needle or aspirator
  • a first mechanism e.g., needle or aspirator
  • the selector may comprise a magnetic column configured to isolated materials bound to beads from materials not bound to beads in the presence of a magnetic field, such as a MACS® column.
  • the selector may comprise an input port and an output port coupled to the MACS® column.
  • the selector may comprise a centrifuge configure to centrifuge cellcontaining samples.
  • the selector may comprise a MACS® column and a centrifuge.
  • a magnetic column that may be comprised in the selector may comprise a nonmagnetic housing that defines a separation chamber, and a fluid permeable matrix of e.g. metallic spheres within the chamber. The spheres form a closely stacked lattice, which creates substantially uniform channels for homogeneous flow during separations.
  • a selector may comprise or use the separation column in conjunction with a prefilter.
  • the column may be used in an instrument having a permanent or electromagnet for use during separations, with an optional retractable arm for moving the magnet, pumping means for washing and separating cells of interest (e.g., T cells), and a microprocessor for controlling the separation fluid flow.
  • the selector may comprise one or more fraction containers each of which receives flow-through material(s) (e.g., comprising negative fraction(s)) and/or elution material(s) (e.g., comprising positive fraction(s)).
  • the selector may be configured to comprise a rotatable container (e.g., comprising a fraction container) providing centrifugation to pre- or post-MACS® separation samples, such as flow-through material(s) (e.g., comprising negative fraction(s)) and/or elution material(s) (e.g., comprising positive fraction(s)).
  • the apparatus may comprise a suction mechanism (e.g., a suction pump and optionally a suction tube or nozzle which may be inserted into the fraction container(s)) which may be configured to apply suction to centrifuged samples or materials, allowing for removal of supernatant and/or collection of a cell compartment such as one comprising pelleted cells.
  • a suction mechanism e.g., a suction pump and optionally a suction tube or nozzle which may be inserted into the fraction container(s)
  • a suction mechanism e.g., a suction pump and optionally a suction tube or nozzle which may be inserted into the fraction container(s)
  • a suction mechanism e.g., a suction pump and optionally a suction tube or nozzle which may be inserted into the fraction container(s)
  • the apparatus may comprise a suction mechanism (e.g., a suction pump and optionally a suction tube or nozzle which may be inserted into the fraction container
  • the apparatus may further comprise a resuspension mechanism, e.g., one or more tubes or nozzles connected or connectable to a reservoir (e.g., containing a medium or buffer (such as wash buffer or MACS® buffer), which may be configured to add medium or buffer to a sample (e.g., pelleted cells obtained by removing supernatant from centrifuged flow-through material(s) and/or elution material(s) (e.g., comprising positive fraction(s))) in the fraction container(s) and optionally resuspending the sample.
  • a resuspension mechanism e.g., one or more tubes or nozzles connected or connectable to a reservoir (e.g., containing a medium or buffer (such as wash buffer or MACS® buffer), which may be configured to add medium or buffer to a sample (e.g., pelleted cells obtained by removing supernatant from centrifuged flow-through material(s) and/or el
  • Exemplary columns that may be comprised in the selector and the mechanism of operations thereof include but are not limited to those described in US10705091, US11339407, or WO2013072288 Al, (entire content of the patent application publications are incorporated herein) or one contained in CliniMACS® apparatuses such as ClimMACS Prodigy® and ClimMACS® Plus (Miltenyi).
  • the treatment chamber may comprise (c-1) an incubation chamber configured to centrifuge cells contained in the incubation chamber or (c-2) an incubation chamber and a centrifugation chamber.
  • the treatment chamber may comprise an input port and an output port coupled to a rotatable container having at least one sample chamber (which may also be referred to as an incubation chamber), and the treatment chamber may be configmed to rotate the container so as to apply a centrifugal force to a sample deposited in the chamber.
  • a centrifugal force may be used to separate a sample and/or for spinofection or spinoculation.
  • the apparatus may comprise a suction mechanism (e.g., a suction pump and optionally a suction tube or nozzle which may be inserted into the sample chamber(s)) which may be configured to apply suction to centrifuged samples or materials, allowing for removal of supernatant and/or collection of a cell compartment such as one comprising pelleted cells.
  • a suction mechanism e.g., a suction pump and optionally a suction tube or nozzle which may be inserted into the sample chamber(s)
  • a suction mechanism e.g., a suction pump and optionally a suction tube or nozzle which may be inserted into the sample chamber(s)
  • a suction mechanism e.g., a suction pump and optionally a suction tube or nozzle which may be inserted into the sample chamber(s)
  • the apparatus may comprise a suction mechanism (e.g., a suction pump and optionally a suction tube or nozzle which may be inserted into the sample chamber
  • the apparatus may further comprise a resuspension mechanism, e.g., one or more tubes or nozzles connected or connectable to a reservoir (e.g., containing a medium or buffer (such as wash buffer or MACS® buffer), which may be configured to add medium or buffer to a sample (e.g., pelleted cells obtained by removing supernatant from a centrifuged sample) in the sample chamber(s) and optionally resuspending the sample.
  • a resuspension mechanism e.g., one or more tubes or nozzles connected or connectable to a reservoir (e.g., containing a medium or buffer (such as wash buffer or MACS® buffer), which may be configured to add medium or buffer to a sample (e.g., pelleted cells obtained by removing supernatant from a centrifuged sample) in the sample chamber(s) and optionally resuspending the sample.
  • a resuspension mechanism e.g., one or more
  • the rotatable container is configured such that it is usable for culturing cells.
  • the rotable container preferably comprises at least one layer for growing cells thereon.
  • the at least one layer can be positioned perpendicular to a rotating axis. It is preferred to arrange a plurality' of layers for growing cells thereon in the rotatable container.
  • the rotatable container can be manufactured in a disposable form. It is also preferred that the rotatable container can be sterilized to allow cell processing without contamination.
  • Exemplary chambers that may be comprised in the selector and the mechanism of operations thereof include but are not limited to those described in US10705091 (see, e.g., Figures 9-12 and corresponding texts), US11339407 (see, e.g., Figures 1-7 and corresponding texts), or WO2013072288A1, (entire content of the patent application publications are incorporated herein) or one contained in CliniMACS® apparatuses such as CliniMACS Prodigy® and CliniMACS® Plus (Miltenyi).
  • the second connector may comprise a second mechanism (e.g., needle or aspirator) which allows for cell transfer/administration to a subject or is operably connectable to a second mechanism (e.g., needle or aspirator) which allows for cell transfer/administration to a subject or (see, e.g., FIG 4A).
  • a second mechanism e.g., needle or aspirator
  • an apparatus may further comprise a conditioning element configured to be operably and/or directly connectable to the subject, optionally via the second connector or a third connector, to allow for administration of a conditioning agent to the subject.
  • an apparatus may comprise a monitoring element configured to analyze one or more of: (i) the level of one or more molecules and/or cells of interest in the biological sample or any sample materials contained in the apparatus, (ii) one or more conditions in the apparatus, optionally temperature, pressure, pH, or any combination thereof; (iii) body temperature, breathing rate, blood pressure, heart rate, oxygen levels, or any combination thereof of the subject. [0441] In certain embodiments, at least part a monitoring element may be comprised within or outside the first connector, the selector, the treatment chamber, and/or the second connector.
  • the monitoring element may comprise a fourth connector configured to be operably and/or directly connectable to the subject to allow for collection of a monitoring sample from the subject through the fourth connector.
  • a monitoring element may be further configured to trigger an instruction to adjust operations of the apparatus based on the results obtained by the monitoring element.
  • an apparatus may further comprise a display.
  • the display may provide information on the progress of cell therapy, such as the number of cells acquired, the ratio of particular cell type(s) (e.g., CD4 T cell: CD8 T cell ratio), the progress of each step or substep, the amount of cell selected or successfully treated, the phenotype of cell selected or successfully treated.
  • the ratio of particular cell type(s) e.g., CD4 T cell: CD8 T cell ratio
  • the display may provide a user interface configured to accept one or more user-inputs pertaining to one or more of the operations of the apparatus.
  • the one or more user-inputs may identify a condition of the cell separation in the selector, optionally the at least one cell type of interest and/or a least one cell type not to be selected.
  • the one or more user-inputs may identify a condition of die cell treatment in the treatment chamber, optionally the duration of the treatment, a temperature used during the treatment, and/or a volume of a solution containing the cells during the treatment, [0448]
  • the user interface may be configured to allow for adjustment of at least one of the operations in accordance with the one or more user-inputs.
  • Non-target cell container/bag (optional)
  • an apparatus may further comprise or may be configured to be connected to a non-target cell container/bag, which may be configured to receive cells and/or elements that were not selected (or determined not to be used as cells for cell therapy) in step (B) and/or (C).
  • such a not-target container or bag may be operably connected to the selector and/or the treatment chamber.
  • the cells not selected may comprise plasma, platelet, erythrocytes, non-T cell leukocytes, and/or non-stem cells.
  • a non-target container or bag may comprise a mechanism (e.g., needle or aspirator) which allows for transfer/administration of elements and/or cells to a subject or is operably connectable to a mechanism (e.g., needle or aspirator) which allows for transfer/administration of elements and/or cells to a subject (see, connectability such as one shown in e.g., FIG 4A).
  • Waste container/bag (optional)
  • an apparatus may further comprise a waste container configured to receive one or materials not to be transferred to the patients (e.g., wash fluid, supernatant generated by centrifugation, eluant, used medium, used buffer, cxccss/uscd transduction/transfcction reagents, excess vectors, and/or excess beads).
  • a waste container configured to receive one or materials not to be transferred to the patients (e.g., wash fluid, supernatant generated by centrifugation, eluant, used medium, used buffer, cxccss/uscd transduction/transfcction reagents, excess vectors, and/or excess beads).
  • the waste container is operably connected to the selector and/or the treatment chamber.
  • an apparatus may comprise fluid circuitry configured to allow fluid flow between the first connector and the selector. In some embodiments, an apparatus may comprise fluid circuitry configured to allow fluid flow between the first connector and the treatment chamber. In some embodiments, an apparatus may comprise fluid circuitry configured to allow fluid flow between the selector and the treatment chamber. In some embodiments, an apparatus may comprise fluid circuitry configured to allow fluid flow between the selector and the second connector. In some embodiments, an apparatus may comprise fluid circuitry configured to allow fluid flow between the treatment chamber and the second connector. In some embodiments, such fluid circuitry may be disposable or single-use.
  • an apparatus may comprise a valve and/or pump configured to control fluid flow between the first connector and the treatment chamber. In some embodiments, an apparatus may comprise a valve and/or pump configured to control fluid flow between the selector and the treatment chamber. In some embodiments, an apparatus may comprise a valve and/or pump configured to control fluid flow between the selector and the second connector. In some embodiments, an apparatus may comprise a valve and/or pump configured to control fluid flow between the treatment chamber and the second connector.
  • an apparatus may comprise a valve and/or pump configured to control fluid flow between the selector and a reservoir (e.g., containing a medium or buffer), between the selector and a reagent bag (e.g., containing MACS® beads), and/or between the selector and a waste bag (e.g., which may collect unbound MACS® beads, unselected cells, used buffer, supernatant, etc).
  • a reservoir e.g., containing a medium or buffer
  • a reagent bag e.g., containing MACS® beads
  • a waste bag e.g., which may collect unbound MACS® beads, unselected cells, used buffer, supernatant, etc.
  • an apparatus may comprise a valve and/or pump configured to control fluid flow between the treatment chamber (e.g., a sample chamber therein) and a reservoir (e.g., containing a medium or buffer), between the treatment chamber (e.g., a sample chamber therein) and a reagent bag (e.g., containing transduction vectors, reagent, cytokines), and/or between the selector and a waste bag (e.g., which may collect unused vectors, used media, etc).
  • a valve and/or pump configured to control fluid flow between the treatment chamber (e.g., a sample chamber therein) and a reservoir (e.g., containing a medium or buffer), between the treatment chamber (e.g., a sample chamber therein) and a reagent bag (e.g., containing transduction vectors, reagent, cytokines), and/or between the selector and a waste bag (e.g., which may collect unused vectors, used media, etc).
  • components of an apparatus may be configured, constructed, and/or arranged so that a user of the apparatus can manufacture and administer cells for cell therapy according to the following procedures: (A) collecting a biological sample containing cells from a subject and transferring the biological sample through the first connector (i) to the treatment chamber or (ii) to the selector; (B) selecting cells of at least one cell ty pe of interest from a/thc biological sample (i) in the treatment chamber and then in the selector or (ii) in the selector and transferring the selected cells to (i) the treatment chamber; and (C) treating the selected cells (i) in the treatment chamber or (ii) in the treatment chamber and then in the selector and optionally then in the treatment chamber; and (D) transferring one or more of the cells treated in (C) to the subject.
  • the selector may comprise an input port and an output port.
  • input and output ports may comprise at least one sterile filter.
  • the treatment chamber may comprise an input port and an output port.
  • the biological sample in step (A), may be transferred to the selector through the input port of the selector.
  • the biological sample in step (A), may be transferred to the treatment chamber through the input port of the treatment chamber.
  • the biological in which optionally a first selecting, e.g., centrifugating cells optionally to isolate leukocytes from blood
  • all or part of the biological sample e.g., leukocytes
  • the selector in which optionally a second selecting, e.g., selecting T cells using a MACS® column.
  • step (B) and/or step (C) transfer of a material, i.e., a sample being process (e.g., a sample selected and/or treated or being selected and/or treated) from the treatment chamber to the selector may be by transferring the material from the treatment chamber through the output port of the treatment chamber to the selector through the input port of the selector.
  • a material i.e., a sample being process (e.g., a sample selected and/or treated or being selected and/or treated) from the treatment chamber to the selector may be by transferring the material from the treatment chamber through the output port of the treatment chamber to the selector through the input port of the selector.
  • step (B) and/or step (C) transfer of a material, i.e., a sample being process (e.g., a sample selected and/or treated or being selected and/or treated) from the selector to the treatment chamber may be by transferring the material from the selector through the output port of the selector to the treatment chamber through the input port of the treatment chamber.
  • a material i.e., a sample being process (e.g., a sample selected and/or treated or being selected and/or treated) from the selector to the treatment chamber may be by transferring the material from the selector through the output port of the selector to the treatment chamber through the input port of the treatment chamber.
  • step (D) the cells treated in step (C) may be transferred from the treatment chamber through the output port of the treatment chamber to the subject.
  • step (D) the cells treated in step (C) may be transferred from the selector through the output port of the selector to the subject.
  • transfer of a material e.g., a biological sample, cells, a solution of cells, a formulation
  • components e.g., the first connector, the selector, the treatment chamber, and/or the second connector
  • a component e.g., the first connector, the and/or the second connector
  • the operations of such a valve and/or a pump may be controlled by one or more instructions encoded on one or more CRMs, such as one or more CRMs according to the present invention.
  • transfer of a reagent e.g., medium, buffer (e.g., MACS® buffer, MACS® column priming buffer, eluant), wash fluid, transfection/transduction reagent, vector (e.g, viral vector such as lentivirus), cytokine, beads for separation (e.g., MACS® beads)) into one or more components (e.g., the first connector, the selector, the treatment chamber, and/or the second connector) of an apparatus may be through at least one open value optionally using a pump.
  • the operations of such a valve and/or a pump may be controlled by one or more instructions encoded on one or more CRMs, such as one or more CRMs according to the present.
  • an unselected elements of a sample e.g., plasma, platelet, non-T cell leukocyte, eluate
  • waste e.g., MACS® column priming buffer already used, wash fluid
  • one or more components e.g., non-target cell bag, waste bag, etc
  • such transfer may be through at least one open value optionally using a pump.
  • the operations of such a valve and/or a pump may be controlled by one or more instructions encoded on one or more CRMs, such as one or more CRMs according to the present.
  • an unselected elements of a sample may be transferred from one or more components (e.g., non-target cell bag) of an apparatus optionally through a connector (the second connector, or another connector, etc), or one or more components (e.g., non-target cell bag) attached to an apparatus the patient.
  • a connector the second connector, or another connector, etc
  • such transfer may be through at least one open value optionally using a pump.
  • the operations of such a valve and/or a pump may be controlled by one or more instructions encoded on one or more CRMs, such as one or more CRMs according to the present.
  • Exemplary circuitry, valves, and pumps or fluid/sample flow control mechanisms that may be comprised or used in the apparatus according to the present invention and the mechanism of operations thereof include but are not limited to those described in US10705091, US11339407, or WO2013072288A1, (entire content of the patent application publications are incorporated herein) or one contained in CliniMACS® apparatuses such as CliniMACS Prodigy® and CliniMACS® Plus (Miltenyi).
  • Example 1 Blood collection and white blood cell separation
  • a pre-defined amount range of blood sample is collected at a pre-determined speed range from a human patient into an apparatus for cell therapy according to an embodiment of the present disclosure. If none of unselected blood components are to be returned to the patient, about 100-1000 mL, about 200-750 m , or about 500 mL will be collected in about 0.5-10 minutes. If at least part of unselected blood components is to be returned to the patient (e.g., apheresis), about 0.5-5 blood volume in total, about 1-3 blood volume in total, or about 1.5-2 blood volume in total, while keeping the extracorporeal volume up to about 15% of blood volume, at a blood flow rate of up to 100 mL/minute.
  • the collection is initiated manually (e.g., by manually turning a valve and/or a pump to allow for transfer of blood from the patient’s vein) or semi-manually by manually instructing a computer to initiate a computer-readable instruction which instructs operations of a valve and/or a pump to allow for such blood collection and transfer to the apparatus).
  • the blood sample is transferred via a first connector to a treatment chamber (e.g., a sample chamber comprising an anticoagulant such as heparin or citrate) of the apparatus.
  • a treatment chamber e.g., a sample chamber comprising an anticoagulant such as heparin or citrate
  • a centrifugal force is applied to the blood sample in the treatment chamber to isolate white blood cells.
  • centrifugation at about 1500-2000 g for about 5-30 or about 10-20 minutes is applied to a whole blood sample, which separates the blood sample into an upper layer containing plasma, a lower layer containing red blood cells, and a thin interface between the upper and lower layers containing white blood cells.
  • the upper and/or lower layers is removed from the sample chamber, for example by suction.
  • a density gradient medium or reagent e.g., Ficoll®- Paque
  • Ficoll®- Paque may be added to a whole blood sample and incubated for example for about 20 minutes.
  • Centrifugation at about 200-800 g or about 300-600 g or about 400 g for about 10-50 minutes, about 20-40 minutes, or about 30 minutes may then be applied, which will separate the sample into an upper layer containing plasma, a lower layer containing red blood cells, a lower middle layer mostly comprised of the density gradient medium, and a thin interface between the upper and lower middle layers containing white blood cells.
  • the upper, lower middle, and/or lower layers will be removed from the sample chamber, for example by suction.
  • red blood cell depletion may be performed as described in Example 15 of US10705091.
  • a red blood cell depleting agent e g., hydroxy ethyl starch (HES), optionally added to the concentration of about 1-10%, or ammonium chloride
  • HES hydroxy ethyl starch
  • a red blood cell depleting agent may be added to a whole blood sample and incubated for example for about 5- 30 or about 10-20 minutes. Centrifugation at about 200-800 g or about 200-600 g or about 300-500 g for about 3-15 minutes or about 5-10 minutes may then be applied, which will separate the sample into an upper layer containing plasma and red blood cell lysis debris and a lower layer containing white blood cells. The upper layer will be removed from the sample chamber, for example by suction, to leave the cells.
  • red blood cell depletion may be performed as described in Example 4 of US11339407.
  • a compartment containing white blood cells are isolated, in some cases, all or part of non-whitc blood cell components optionally arc returned to the patient, for example via the second connector or another connector, at an appropriate speed.
  • the samples containing white blood cells typically are reconstituted with a medium or buffer.
  • the cell compartment containing white blood cells are reconstituted (“diluted”’) in buffer and added with and incubated at about 2-8°C for about 5-20 or about 15 minutes with CD3 -specific MACS® beads (if only CD8+ T or CD4+ T cells are desired, CD8-specific MACS® beads or CD4- specific MACS® beads, respectively, may instead be used for positive selection, or CD4-specific MACS® beads or CD8-specific MACS® beads, respectively, may instead be used for negative selection; alternatively, such CD8-based or CD4-based selection may be performed in tandem with CD3 -based selection) in the sample chamber.
  • CD3 -specific MACS® beads if only CD8+ T or CD4+ T cells are desired, CD8-specific MACS® beads or CD4- specific MACS® beads, respectively, may instead be used for positive selection, or CD4-specific MACS® beads or CD8-specific MACS® beads, respectively, may instead be used for negative selection; alternatively, such CD8-based or CD4-based selection may
  • Cells will be washed by (i) optionally adding buffer, (ii) applying centrifugation at about 200-500 g or about 300 g for about 5-20 or about 10 minutes, and (iii) removing the supernatant by e.g., aspiration, to remove unbound beads, and (i)-(iii) may optionally be repeated once or more.
  • Washed cells are reconstituted with buffer and transferred to the selector.
  • Cells will be applied to a pre-primed MACS® column in a magnetic field at up to about 2xl0 8 cells/mL.
  • An appropriate amount of MACS® buffer is applied to the column once or more to collect cells not bound to MACS® beads as part of the flow-through material(s) (or negative fraction(s)) optionally in a flow-through fraction container.
  • the magnetic field is removed from the column, and an appropriate amount of buffer and pressure is applied to the MACS® column to flush out cells as part of the elution material(s) (or positive fraction(s)) optionally in an elution fraction container.
  • the elution material(s) is transferred to the sample chamber of the treatment chamber.
  • Cells in the treatment chamber are washed by (i) optionally adding buffer, (ii) applying centrifugation at about 200-500 g or about 300 g for about 5-20 or about 10 minutes, and (iii) removing the supernatant by e.g., aspiration, to remove unbound beads, and (i)-(iii) may optionally be repeated one or more times.
  • Example 2 Cells isolated in Example 2 are transduced with a nucleic acid encoding a disease-specific chimeric antigen receptor, designed to elicit appropriate intracellular signaling in such cells upon binding of the CAR by its target antigen.
  • Washed cells from the elution material(s) are reconstituted in a medium and placed in a sample chamber with inner surfaces coated with RetroNectin®.
  • An appropriate amount of lentiviral or retroviral vectors will be placed in the sample chamber and centrifugation is applied at about 1000- 3000g or about 2000 g for about 15 minutes to about 3 hours or about 1-2 hours.
  • Cells in the treatment chamber will be washed by (i) optionally adding a medium or buffer, (ii) applying centrifugation at about 200-500 g or about 300 g for about 5-20 or about 10 minutes, and (iii) removing the supernatant by e.g., aspiration, to remove unused transduction materials and/or cy tokines released, and (i)-(iii) may optionally be repeated once or more.
  • viral transduction may be performed as described in Example 1 of US11339407.
  • transduced cells are rested or cultured in a medium to allow for expansion.
  • transduced cells may be rested or cultured for a short period of time, e.g., about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, or about 1 hour, or longer, optionally in the presence of one or more nutrients and/or stimulant(s).
  • Cells are then be washed by (i) optionally adding a medium or buffer, (ii) applying centrifugation at about 200-500 g or about 300 g for about 5- 20 or about 10 minutes, and (iii) removing the supernatant by e.g., aspiration, to remove unused transduction materials and/or cytokines released, and (i)-(iii) may optionally be repeated one or more times.
  • Example 4 Administration
  • a pharmaceutical composition comprising the cells transduced and optionally expanded in Example 3 is prepared and administered to the patient.
  • a desired number of the cells transduced and optionally expanded in Example 3 are reconstituted in an appropriate solvent comprising a desired pharmaceutically acceptable carrier to produce a pharmaceutical composition in the sample chamber.
  • a pharmaceutical composition is administered to the patient by transferring the composition at a pre -determined speed from the apparatus via the second connector to the patient.
  • Such transfer is generally initiated manually (e.g., by manually turning a valve and/or a pump to allow for transfer of the composition from the apparatus to the patient’s vein) or semi-manually by manually instructing a computer to initiate a computer- readable instruction which instructs operations of a valve and/or a pump to allow for such transfer to the patient).

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Abstract

La présente divulgation concerne des méthodes et des appareils pour une thérapie cellulaire améliorée et un médicament personnalisé. La présente divulgation concerne en outre des supports lisibles par ordinateur codant des instructions lisibles par ordinateur pour amener un ou plusieurs dispositifs de traitement à commander des opérations d'un appareil pour une thérapie cellulaire et un médicament personnalisé.
PCT/US2023/072432 2022-08-19 2023-08-18 Méthode et système améliorés de thérapie cellulaire WO2024040201A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
US20140236077A1 (en) * 2008-12-15 2014-08-21 Proteus Digital Health, Inc Body-Associated Receiver and Method
US20180002664A1 (en) * 2014-12-19 2018-01-04 Immusoft Corporation B cells for in vivo delivery of therapeutic agents
US11339407B2 (en) * 2011-11-18 2022-05-24 Miltenyi Biotec Gmbh Systems and methods for point/center-of-care immunotherapy
WO2022165260A1 (fr) * 2021-01-29 2022-08-04 Iovance Biotherapeutics, Inc. Procédés de fabrication de lymphocytes infiltrant les tumeurs modifiés et leur utilisation dans la thérapie cellulaire adoptive
US20230027004A1 (en) * 2021-07-01 2023-01-26 Kite Pharma, Inc. Closed-system and method for autologous and allogeneic cell therapy manufacturing

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140236077A1 (en) * 2008-12-15 2014-08-21 Proteus Digital Health, Inc Body-Associated Receiver and Method
US11339407B2 (en) * 2011-11-18 2022-05-24 Miltenyi Biotec Gmbh Systems and methods for point/center-of-care immunotherapy
US20180002664A1 (en) * 2014-12-19 2018-01-04 Immusoft Corporation B cells for in vivo delivery of therapeutic agents
WO2022165260A1 (fr) * 2021-01-29 2022-08-04 Iovance Biotherapeutics, Inc. Procédés de fabrication de lymphocytes infiltrant les tumeurs modifiés et leur utilisation dans la thérapie cellulaire adoptive
US20230027004A1 (en) * 2021-07-01 2023-01-26 Kite Pharma, Inc. Closed-system and method for autologous and allogeneic cell therapy manufacturing

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