WO2026020055A2 - Procédés d'évaluation d'exosomes dans une composition cellulaire et utilisations associées - Google Patents

Procédés d'évaluation d'exosomes dans une composition cellulaire et utilisations associées

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Publication number
WO2026020055A2
WO2026020055A2 PCT/US2025/038155 US2025038155W WO2026020055A2 WO 2026020055 A2 WO2026020055 A2 WO 2026020055A2 US 2025038155 W US2025038155 W US 2025038155W WO 2026020055 A2 WO2026020055 A2 WO 2026020055A2
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WIPO (PCT)
Prior art keywords
cell
composition
cells
exosomes
concentration
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Pending
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PCT/US2025/038155
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WO2026020055A3 (fr
Inventor
Ruby Mae LUNDE
Natalya Aleksandra GOLOVIZNINA
Miriam Verna GUTSCHOW
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Juno Therapeutics Inc
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Juno Therapeutics Inc
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Publication of WO2026020055A2 publication Critical patent/WO2026020055A2/fr
Publication of WO2026020055A3 publication Critical patent/WO2026020055A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4215—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present disclosure relates to methods for assessing exosomes produced from cells of a cell composition, such as a cell therapy composition, including compositions comprising chimeric antigen receptor (CAR) or T cell receptor (TCR) expressing T cells.
  • a cell therapy composition including compositions comprising chimeric antigen receptor (CAR) or T cell receptor (TCR) expressing T cells.
  • the methods can be used for predicting response to and/or efficacy of a cell therapy composition, for selecting patients for treatment, and in connection with T cell manufacturing methods.
  • an increase in the amount or concentration of the isolated exosomes compared to an amount or concentration from a control cell composition predicts a response to the cell therapy composition when it is administered to the subject.
  • introducing the polynucleotide encoding the recombinant receptor comprises transducing cells with a viral vector encoding the recombinant receptor.
  • the T cell stimulatory agent(s) comprises an anti-CD3 antibody and an anti-CD28 antibody, and optionally culturing cells of the population of input cells in a culture medium containing one or more recombinant cytokines selected from IL-2, IL- 15, IL-7 and IL-21.
  • the manufacturing process further comprises culturing cells introduced with the polynucleotide under conditions for expansion of T cells in the composition.
  • the population of input cells enriched from the biological sample comprises at or greater than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% T cells.
  • the human subject has a disease or condition.
  • the response comprises a partial response. In some of any of the provided embodiments, the response comprises a complete response. In some of any of the provided embodiments, the response does not comprise stable disease. In some of any of the provided embodiments, the response does not comprise progressive disease.
  • isolating is by centrifugation.
  • the centrifugation comprises at least one spin. In some of any of the provided embodiments, the centrifugation is performed for about 1 to 20 minutes. In some of any of the provided embodiments, the centrifugation comprises one spin at 300 g for 3 min. In some of any of the provided embodiments, the centrifugation comprises one spin at 2,500 g for 15 min. In some of any of the provided embodiments, the centrifugation comprises two spins at 2,500 g for 15 min. In some of any of the provided embodiments, the centrifugation comprises three spins comprising one spin at 300 g for 3 min and two spins at 2,500 g for 15 min.
  • detecting is by: (a) identifying cell particles that are surface positive for one or more exosome markers; and/or (b) identifying cell particles that have a size between about 30 nm to 150 nm. In some of any of the provided embodiments, detecting is by: (a) identifying cell particles that are surface positive for one or more exosome markers; and (b) identifying cell particles that have a size between about 30 nm to 150 nm. In some of any of the provided embodiments, the identifying cell particles is by identifying cell particles that have a size between about 30 nm to about 130 nm.
  • the identifying cell particles is by identifying cell particles that have a size between about 30 nm to about 100 nm.
  • the one or more exosome markers are identified by immunoaffinity-based capture.
  • the immunoaffinity-based capture comprises an antibody specific to the one or more exosome markers.
  • the one or more exosome markers are selected from CD63, CD81, CD9, and any combination thereof.
  • the one or more exosome markers is CD63.
  • the one or more exosome markers is CD81.
  • the one or more exosome markers is CD9.
  • the one or more exosome markers comprises CD63 and CD81.
  • the one or more exosome markers comprises CD63 and CD9.
  • the one or more exosome markers comprises CD81 and CD9.
  • the one or more exosome markers comprises CD63, CD81 and CD9.
  • the exosomes have a size between about 30 nm and 150 nm. In some of any of the provided embodiments, the exosomes have a size between about 30 nm and 130 nm. In some of any of the provided embodiments, the exosomes have a size between about 30 nm and 100 nm.
  • the size is between about 30 nm and about 40 nm, about 35 nm and about 45 nm, about 40 nm and about 50 nm, about 45 nm and about 55 nm, about 50 nm and about 60 nm, about 55 nm and about 65 nm, or about 60 nm and about 70 nm.
  • the recombinant receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • the CAR comprises an scFv specific for an antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta.
  • the antigen is expressed by the cells of the disease or condition.
  • the antigen is expressed by the cells of a cancer or tumor.
  • the cancer or tumor is a hematological malignancy.
  • the hematological malignancy is a myeloma, leukemia or lymphoma.
  • the hematological malignancy is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), and Diffuse Large B-Cell Lymphoma (DLBCL).
  • the antigen is a B cell antigen.
  • the B cell antigen is CD19. In some of any of the provided embodiments, the B cell antigen is BCMA. In some of any of the provided embodiments, the antigen is a plasma cell antigen. In some of any of the provided embodiments, the plasma cell antigen is GPRC5D.
  • the control cell composition comprises T cells of the cell therapy composition or T cell composition that have not been contacted with the recombinant receptor-stimulating agent.
  • the recombinant receptorstimulating agent comprises a target antigen or an extracellular domain binding portion thereof.
  • the target antigen is a recombinant antigen of the recombinant receptor.
  • the recombinant receptor-stimulating agent comprises an extracellular domain binding portion of the target antigen and the extracellular domain binding portion comprises an epitope recognized by the recombinant receptor.
  • the recombinant receptor-stimulating agent is an antibody specific to an extracellular binding domain of the recombinant receptor. In some of any of the provided embodiments, the recombinant receptor-stimulating agent is an anti-idiotypic antibody specific to an extracellular antigen binding domain of the recombinant receptor. In some of any of the provided embodiments, the recombinant receptor-stimulating agent is immobilized or attached to a solid support. In some of any of the provided embodiments, the solid support is a surface of a vessel, optionally a well of microwell plate, in which a plurality of incubations is performed. In some of any of the provided embodiments, the solid support is a bead.
  • the recombinant receptor-stimulating agent is an antigen-expressing cell, optionally wherein the antigen-expressing cell is a clone, from a cell line, or a primary cell taken from a subject.
  • the antigen-expressing cell is a cell line.
  • the cell line is a tumor cell line.
  • the antigen-expressing cell is a cell that has been introduced, optionally by transduction, to express an antigen of the recombinant receptor.
  • the other agent is a BTK inhibitor (e.g., ibrutinib or acalibrutinib), a BCL2 inhibitor (e.g., venetoclax), an immunomodulatory agent, a DGK inhibitor, an inhibitor of indoleamine 2, 3 -dioxygenase- 1 (IDO1) (e.g. epacadostat) or a checkpoint inhibitor.
  • a BTK inhibitor e.g., ibrutinib or acalibrutinib
  • BCL2 inhibitor e.g., venetoclax
  • an immunomodulatory agent e.g., a DGK inhibitor
  • IDO1 indoleamine 2, 3 -dioxygenase- 1
  • checkpoint inhibitor e.g. epacadostat
  • the immunomodulatory agent is an immunomodulatory imide drug (IMiD) or a cereblon E3 ligase modulator (CELMoD).
  • the immunomodulatory agent is thalidomide or a thalidomide derivative.
  • the immunomodulatory agent is selected from the group consisting of lenalidomide, pomalidomide, avadomide (CC-122), iberdomide (CC-220).
  • the checkpoint inhibitor is selected from a PD-1 inhibitor, LAG3 inhibitor and PD-L1 inhibitor.
  • the PD-1 inhibitor is an anti-PD-1 antibody (e.g, nivolumab).
  • the LAG3 inhibitor is an anti- LAG3 antibody (e.g., relatlimab).
  • the PD-L1 inhibitor is an anti-PD-Ll antibody (e.g. durvalumumab).
  • the BCL2 inhibitor is selected from the group consisting of venetoclax, navitoclax, ABT737, maritoclax, obatoclax and clitocine.
  • FIG. 1 shows detection of secreted CD63 in chimeric antigen receptor (CAR) T cell products comprising CD4+, CD8+, or CD4+ and CD8+ T cells obtained from patients who responded to treatment with the CAR-T cell product (responders or R) and patients who did not respond to treatment with the CAR-T cell product (non-responders or NR).
  • CAR chimeric antigen receptor
  • FIG. 2 shows the size (nm) and concentration (particles/mL) of extracellular vesicles (EVs) as measured using nanoparticle tracking analysis (NanoSight) in the total EV fraction of two representative T cell products comprising chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR) that were either stimulated or unstimulated.
  • FIG. 3 shows mean cytokine concentrations (pg/mL) in the lysed exosome fraction (“EV fraction”) and vesicle-free fraction (“EV-free fraction”) of T cell products comprising engineered T cell receptor (eTCR) that were stimulated or unstimulated.
  • FIG. 4 shows total area of red fluorescence emitted from spheroids (which served as a model for a solid-tumor), co-incubated with: the EV fraction of stimulated and unstimulated T cell products comprising engineered T cell receptor (eTCR); media alone; or stimulated and unstimulated eTCR cell products.
  • spheroids which served as a model for a solid-tumor
  • FIG. 5 shows the concentration (particles/mL) and size (nm) distribution of captured exosome particles derived from chimeric antigen receptor (CAR) T cell products in which the T cells were stimulated with an anti-idiotypic antibody against the CAR (alD) or unstimulated (Unstim).
  • the exosomes were captured with antibodies against CD63 (top panel), CD81 (middle panel) or CD9 (bottom panel).
  • Results are shown for CAR-T cell products that, when administered to the subject via autologous cell therapy, resulted in a complete response (CR) in the patient, also referred to as a responder, or progressive disease (PD) in the patient, also referred to as a non-responder, wherein the treatment effect was determined at 1 month post-treatment.
  • CR complete response
  • PD progressive disease
  • FIGS. 6A-6E show results of analysis of specified exosome particles derived from autologous chimeric antigen receptor (CAR) T cell products of patients that, one month after treatment with the autologous CAR-T cell product, experienced a complete response (CR) (also referred to as responders) or had progressive disease (PD) (also referred to as non-responder s).
  • the autologous CAR-T cells were stimulated with an anti-idiotypic antibody against the CAR (alD) or unstimulated (Unstim).
  • FIG. 6A shows concentration and size of CD63+, CD81+ and CD9+ exosomes derived from the autologous CAR-T cell products of patients.
  • FIG. 6B shows total concentration of extracellular vesicles positive for at least one tetraspanin (CD63, CD81, CD9) derived from the autologous CAR-T cell products of patients.
  • FIG. 6C shows concentration of CD63+ exosome particles derived from the autologous CAR-T cell products of patients.
  • FIG. 6D shows concentration of CD81+/CD9+ and CD81+/CD9+/CD63-I- exosome particles derived from the autologous CAR-T cell products of patients.
  • FIG. 6E shows concentration and size distribution of CD63+ exosome/EV particles derived the autologous CAR-T cell products of patients.
  • FIGS. 7A-7B show results of analyses of exosomes in chimeric antigen receptor (CAR) T cell products derived from multiple myeloma (MM) patients, wherein the product was stimulated with anti-idiotypic antibody against the CAR (alD) or unstimulated (Unstim).
  • FIG. 7A shows CD63+, CD81+ and CD9+ exosome size (nm) and concentration (particles/mL).
  • FIG. 7B shows median concentration of CD63+, CD81+ and CD9+ exosomes.
  • a cell composition such as a composition of cells enriched in, or containing, T cells engineered with a recombinant receptor (e.g., a chimeric antigen receptor (CAR) or T cell receptor (TCR)).
  • a recombinant receptor e.g., a chimeric antigen receptor (CAR) or T cell receptor (TCR)
  • the cell composition is a cell therapy such as a T cell therapy composition.
  • the cell composition is a T cell therapy composition.
  • the method for assessing exosomes is for predicting response to, and/or efficacy of, the T cell therapy composition.
  • a method of predicting response to, and/or efficacy of, a cell composition such as a composition of cells enriched in or containing T cells engineered with a recombinant receptor (e.g., CAR).
  • the cell composition is a cell therapy composition such as a T cell therapy composition.
  • the cell composition is a T cell therapy composition.
  • the immune cells are obtained from a subject to be treated (in this instance, the cells are referred to as “autologous cells” and the cell therapy composition is referred to as an autologous cell therapy).
  • the immune cells are obtained from a different subject from the subject to be treated, such as a healthy subject, and are made to be hypoimmune for administration to another subject as a therapy (in this instance, the cells are referred to as “allogeneic cells” and the cell therapy composition is referred to as an allogeneic cell therapy).
  • results provided herein demonstrate that exosomes produced from a cell therapy composition, particularly a cell therapy composition comprising T cells engineered with a recombinant receptor (also referred to as a “T cell therapy”), may serve as an indicator for patient response. In some cases, this association is observed more highly in compositions in which an engineered recombinant receptor, such as a CAR, has been stimulated in an antigen-dependent manner.
  • Embodiments provided herein are based on the observation that stimulation of T cell therapy compositions (e.g., autologous cell therapy compositions) results in increased production and secretion of exosomes compared to T cell therapy compositions that have not been stimulated, particularly in subjects that went on to achieve a response (e.g.
  • the increase in amount or concentration of exosomes from stimulated T cell therapy compositions serves as a biomarker to predict potency of the T cell therapy composition and subsequent responsiveness of a subject having a disease or condition to the T cell therapy composition.
  • the T cell therapy composition is a T cell therapy composition comprising T cells that have been engineered to express a recombinant receptor (e.g., chimeric antigen receptor (CAR) or T cell receptor (TCR)) and the exosomes are positive for an exosome marker disclosed herein (e.g., CD63).
  • a recombinant receptor e.g., chimeric antigen receptor (CAR) or T cell receptor (TCR)
  • the exosomes are cell-derived particles that are surface positive for CD63. In some embodiments, the exosomes are cell-derived particles that are greater than about 30 nm in diameter and less than about 100 nm in diameter, and that are surface positive for CD63.
  • Adoptive cell therapies can be effective in the treatment of cancer and other diseases and disorders compared to standard therapies (e.g., chemotherapy, radiation etc.).
  • standard therapies e.g., chemotherapy, radiation etc.
  • available approaches to adoptive cell therapy may not always be entirely satisfactory.
  • the ability of the administered cells to recognize and bind to a target to traffic localize to and successfully enter appropriate sites within the subject, tumors, and environments thereof, to become activated, expand, to exert various effector functions, including cytotoxic killing and secretion of various factors such as cytokines, to persist, including long-term, to differentiate, transition or engage in reprogramming into certain phenotypic states to provide effective and robust recall responses following clearance and re-exposure to target ligand or antigen, and avoid or reduce exhaustion, anergy, terminal differentiation, and/or differentiation into a suppressive state is not always guaranteed nor is it evident prior to administering the cells whether the aforementioned events will occur.
  • Exosomes are increasingly recognized as potent mediators of intercellular communication due to the ability of exosomes to transport a diverse array of bioactive molecules (e.g., membrane proteins, lipids, nucleic acids, cytosolic proteins, and other signaling molecules within their interior). Exosomes assume vital roles in a wide range of physiological and pathological processes and hold significant promise as emerging disease biomarkers, therapeutic agents, and carriers for drug delivery.
  • the exosomes detected as disclosed herein comprise the receptors expressed by an adoptive cell therapy, such as a CAR or TCR, and in some such embodiments, the CAR+ exosomes can induce effects on target cells (e.g., cancer and/or tumor cells).
  • Exosome effects on target cells may result at least in part from release of exosome content (which may also be referred to as cargo) that affects the target cells.
  • exosomes can carry cytokines that are cytotoxic to cancer and/or tumor cells.
  • CAR+ exosomes are less sterically hindered from penetrating solid-tumors.
  • the exosomes detected as disclosed herein are not only reflective of the performance of the cells of the adoptive cell therapies provided herein (e.g., CAR-T cell therapy), but the exosomes can also enhance the efficacy (e.g., the cancer and/or tumor cell killing ability) of the adoptive cell therapies provided herein.
  • assessing exosomes produced from cells of a cell composition such as a cell therapy composition, including compositions comprising CAR or TCR expressing T cells (e.g., a T cell therapy composition).
  • assessing exosomes produced from cells of a cell therapy composition has various downstream applications as described herein.
  • methods for predicting response to, and/or efficacy of, the cell composition can be determined.
  • methods for treatment, including adaptive treatment can be predicted or determined based the amount or concentration of exosomes assessed in the cell therapy compositions.
  • the cell composition is a T cell therapy composition.
  • exosomes are assessed in the T cell therapy composition by identifying cell particles that are surface positive for CD63.
  • exosomes are assessed in the T cell therapy composition by identifying cell particles that are greater than about 30 nm in diameter and less than about 100 nm in diameter, and identifying cell particles that are surface positive for CD63.
  • the provided methods allow for assessing exosome production from a cell composition, such as a cell therapy composition.
  • the method of assessing exosome production comprises contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent, detecting exosomes produced from cells of the cell therapy composition, and determining the amount or concentration of the isolated exosomes.
  • the cell therapy is a treatment or a candidate for a treatment to be administered to a subject.
  • the method of assessing exosome production is for predicting response to, and/or efficacy of, a cell therapy composition.
  • a method of assessing exosome production includes detecting an increased amount or concentration of exosomes produced by the stimulated cell composition compared to the amount or concentration of exosomes produced by the control cell composition. In some embodiments, a method of assessing exosome production includes detecting a decreased or an unchanged amount or concentration of exosomes produced by the stimulated cell composition compared to the amount or concentration of exosomes produced by the control cell composition.
  • the cell therapy composition comprises cells obtained from the subject (referred to as autologous cells). In some embodiments, the cell therapy composition comprises cells not obtained from the subject (referred to as allogeneic cells). In some embodiments, the method of assessing allows for detecting exosomes produced from a T cell therapy composition.
  • the method of assessing allows for detecting exosomes produced from a CAR-T cell therapy composition. In some embodiments, the method of assessing allows for detecting exosomes produced from a TCR cell therapy composition. In some embodiments, detecting the exosomes comprises identifying cell particles that are surface positive for CD63. In some embodiments, detecting the exosomes comprises identifying cell particles that are greater than about 30 nm in diameter and less than about 100 nm in diameter, and identifying cell particles that are surface positive for CD63.
  • the amount or concentration of the isolated exosomes in the stimulated cell composition is increased between about 1-fold and 3-fold, about 2-fold and 4-fold, about 3 -fold and 5 -fold, about 4-fold and 6-fold, about 5 -fold and 7-fold, about 6-fold and 8 -fold, about 7-fold and 9-fold, or about 8-fold and 10-fold compared to the amount or concentration of the isolated exosomes in the control cell composition.
  • the amount or concentration of the isolated exosomes in the stimulated cell composition is increased at least about 1-fold, at least about 2- fold, at least about 3 -fold, at least about 4-fold, at least about 5 -fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold or at least about 10-fold compared to the amount or concentration of the isolated exosomes in the control cell composition.
  • predicting response to and/or efficacy of the cell therapy composition does not occur when the amount or concentration of exosomes produced from the stimulated cell composition is decreased or is unchanged compared to the amount or concentration of exosomes produced by the control cell composition.
  • the methods provided herein allow for the development of treatment strategies that can include recommending a combination therapy or an adjuvant such as a T cell modulating agent to increase the likelihood of the subject having a positive or advantageous clinical response.
  • the provided methods include stimulating cells of a cell therapy composition to produce exosomes.
  • the cells of the cell therapy composition express a recombinant receptor.
  • the cells of the cell therapy composition express any of the recombinant receptors provided in Section III.
  • the cell therapy composition includes any of those described in Section III.
  • the cells of the cell therapy composition express a CAR or a TCR.
  • the cell therapy composition is a CAR-T cell therapy composition.
  • the cell therapy composition is a TCR cell therapy composition.
  • the method of stimulating cells of a cell therapy composition includes contacting the cells with an agent that binds to and stimulates the recombinant receptor.
  • purified or recombinant antigens) of the recombinant receptor also called a “target antigen”
  • antibodies such as anti-idiotype antibodies to the recombinant receptor
  • antigen-expressing cells also called “targetexpressing cells” that express an antigen target of the recombinant receptor on the cell surface.
  • a protein-based recombinant receptor-stimulating agent such as a surface immobilized binding molecule (e.g., target antigen or anti-idiotype antibody), is contacted with a sample of cells of the cell therapy.
  • the recombinant receptor-stimulating agent is in an amount of from 0.1 pg/mL to 100 pg/mL, such as from 0.5 pg/mL to 50 pg/mL, more generally 1 pg/mL to 10 pg/mL.
  • the recombinant receptor-stimulating agent is in an amount of from 1 pg/mL, 2 pg/mL, 3 pg/mL, 4 pg/mL, 5 pg/mL, 6 pg/mL, 7 pg/mL, 8 pg/mL, 9 pg/mL, 10 pg/mL, or any value between any of the foregoing.
  • the cells of the cell composition are present at a concentration of from 0.1 x 10 6 cells/mL to 100 x 10 6 cells/mL, such as from 0.5 x 10 6 cells/mL to 50 x 10 6 cells/mL, more generally 0.5 x 10 6 cells/mL to 10 x 10 6 cells/mL.
  • the cells of the cell composition are present at a concentration of from 0.5 x 10 6 cells/m, 1 x 10 6 cells/mL, 2 x 10 6 cells/mL, 3 x 10 6 cells/mL, 4 x 10 6 cells/mL, 5 x 10 6 cells/mL, 6 x 10 6 cells/mL, 7 x 10 6 cells/mL, 8 x 10 6 cells/mL, 9 x 10 6 cells/mL, 10 x 10 6 cells/mL, or any value between any of the foregoing.
  • target-expressing cells are used as the recombinant receptorstimulating agent
  • the target-expressing cells are present at a particular ratio relative to cells of the cell composition.
  • the ratio of target-expressing cells to cells of the cell composition is from 10:1 to 1:10, such as from 5:1 to 1:5, 3:1 to 1:3 or 2:1 to 1:2.
  • the recombinant-receptor stimulating agent is contacted with a sample of cells of the cell therapy and involves incubation under conditions suitable for activation of cells expressing the recombinant receptor.
  • a recombinant-receptor stimulating agent e.g., surface immobilized antigen of the recombinant receptor, e.g., CAR, for example, plate-bound antigen
  • the incubation is for 12 hours to 72 hours, such as 12 hours to 48 hours, for example at or about 24 hours.
  • the incubation is carried out at a temperature suitable for culture of the cells, such as a temperate of at or about 37°C + 4°C, for example, at or about 37°C.
  • a stable carbon dioxide is maintained, such as at or about 5% CO2.
  • the recombinant receptor-stimulating agent is composed of a binding molecule that is able to be bound by the recombinant receptor that is immobilized on a surface support.
  • the binding molecule may be an antigen or a portion of an antigen of the recombinant receptor (e.g. extracellular portion of an antigen) or an antibody (e.g., an anti-idiotypic antibody) specific to the recombinant receptor.
  • the recombinant receptorstimulating agent is immobilized or bound to a surface support, such as a microwell plate or a solid particle (e.g. bead).
  • the recombinant receptor-stimulating agent such as the binding molecule
  • the recombinant receptor-stimulating agent is immobilized to the surface of a plate. Any process that involves attaching binding molecules to a support material, such as a microtiter plate, so they can bind to contacted cells can be used.
  • a support material such as a microtiter plate
  • a culture plate such as a microwell plate
  • Suitable surfaces include, but are not limited to, polystyrene, polyvinylchloride, or polyethylene.
  • the surface such as one containing polystyrene
  • the surface can also be modified to have positively charged amine groups, which can ionically couple to small negatively charged binding molecules.
  • positively charged amine groups which can ionically couple to small negatively charged binding molecules.
  • microplates pre-coated with Protein A, G, L, or secondary antibodies, to facilitate IgG-specific binding of antibodies or immunoglobulin-containing molecules can be used. Microplates for different immobilization methods can be obtained commercially.
  • the binding molecule (e.g. antigen or binding portion thereof, or antibody) may be immobilized or bound to a surface support, such as a non-cell particle, wherein recombinant receptor-expressing cells (e.g. CAR-T cells) of the cell composition, are contacted with the surface support.
  • a particle described herein e.g., bead particle
  • an antigen or binding portion thereof, or an anti- idiotypic antibody can be bound or attached in a manner that permits an interaction between the binding molecule and a cell, in particular binding between the binding molecule and a recombinant receptor, e.g., a CAR, expressed on the surface of the cell.
  • a recombinant receptor e.g., a CAR
  • the interaction between the conjugated or attached binding molecule and the cell mediates stimulation of the recombinant receptor, including one or more recombinant receptor-dependent activity such as activation, expansion, cytokine production, cytotoxicity activity or other activity as described.
  • the surface support is a particle (e.g., a bead particle) to which the binding molecule (e.g. an antigen or binding portion thereof, or an anti-idiotypic antibody) is immobilized or attached.
  • the surface support is a solid support.
  • the solid support is a bead, and the antigen or portion is immobilized on the bead.
  • the solid support is the surface of a well or plate, e.g., a cell culture plate.
  • the surface support is a soluble oligomeric particle, and the antigen is immobilized on the surface of the soluble oligomeric particle.
  • the surface support is a particle that may include a colloidal particle, a microsphere, nanoparticle, a bead, such as a magnetic bead, or the like.
  • the particles or beads are biocompatible, i.e. non-toxic.
  • the particles or beads are non-toxic to cultured cells, e.g., cultured T cells.
  • the particles are monodisperse.
  • “monodisperse” encompasses particles (e.g., bead particles) with size dispersions having a standard deviation of less than 5%, e.g., having less than a 5% standard deviation in diameter.
  • the particles or beads have a diameter of between or between about 0.1 pm and 10 pm, 0.5 pm and 10 pm, 0.5 pm and 5 pm, 1 pm and 10 pm, or 1 pm and 5 pm, each inclusive.
  • the particles, e.g., beads may be any particles that can be modified, e.g., surface functionalized, to allow for the attachment of a binding molecule at the surface of the particle.
  • the particles e.g., beads
  • the particles are composed of glass, silica, polyesters of hydroxy carboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids.
  • the particles (e.g., bead particles) used in the methods described herein can be produced or obtained commercially.
  • Particles, e.g., beads, including methods of producing particles, e.g., beads, are well known in the art. See, for example, U.S. Pat. Nos. 6,074,884; 5,834,121; 5,395,688; 5,356,713; 5,318,797; 5,283,079; 5,232,782; 5,091,206; 4,774,265; 4,654,267; 4,554,088; 4,490,436; 4,452,773; U.S. Patent Application Publication No.
  • particles include, but are not limited to, ProMagTM (PolySciences, Inc.); COMPELTM (PolySciences, Inc.); BioMag® (Poly Sciences, Inc.), including BioMag® Plus (PolySciences, Inc.) and BioMag® Maxi (Bang Laboratories, Inc.); M-PVA (Cehmagen Biopolymer Technologic AG); SiMAG (Chemicell GmbH); beadMAG (Chemicell GmbH); MagaPhase® (Cortex Biochem); Dynabeads® (Invitrogen), including Dynabeads® M-280 Sheep Anti-rabbit IgG (Invitrogen), Dynabeads® FlowCompTM (e.g., Dynabeads® FlowCompTMHuman CD3, Invitrogen), Dynabead
  • the antigen or an extracellular domain portion thereof is bound to the particle (e.g. bead) via a covalent chemical bond.
  • a reactive group or moiety of an amino acid of the antigen or extracellular domain portion thereof is conjugated directly to a reactive group or moiety on the surface of the particle by a direct chemical reaction.
  • an amino acid carboxyl group e.g., a C-terminal carboxyl group
  • hydroxyl, thiol, or amine group such as an amino acid side chain group
  • a conjugating moiety conjugates, e.g., covalently binds, to both the binding molecule and the particle, thereby linking them together.
  • the surface of the particle comprises chemical moieties and/or functional groups that allow attachment (e.g., covalent, non-covalent) of the binding molecule (e.g., polypeptide antigen or antibody).
  • the particle surfaces contain exposed functional groups. Suitable surface exposed functional groups include, but are not limited to, carboxyl, amino, hydroxyl, sulfate groups, tosyl, epoxy, and chloromethyl groups.
  • the surface exposed functional group must be activated, i.e., it must undergo a chemical reaction to yield an intermediate product capable of directly binding a polypeptide.
  • a polypeptide binding molecule is covalently attached to the particle, e.g., a bead particle, at a surface exposed functional group that does not require activation by an agent prior to forming a covalent attachment.
  • functional groups include, but are not limited to, tosyl, epoxy, and chloromethyl groups.
  • a non-covalent bond between a ligand bound to the antigen peptide or protein and an anti-ligand attached to the surface support may conjugate the antigen to the support (e.g. bead).
  • a biotin ligase recognition sequence tag may be joined to the C-terminus of an antigen peptide or protein, and this tag may be biotinylated by biotin ligase. The biotin may then serve as a ligand to non-covalently conjugate the antigen peptide or protein to avidin or streptavidin which is adsorbed or otherwise bound to the surface of the carrier as an anti-ligand.
  • the binding molecule e.g. antigen
  • the Fc domain may act as a ligand
  • protein A either covalently or non-covalently bound to the surface of the surface support (e.g. bead)
  • protein A may serve as the anti-ligand to non-covalently conjugate the antigen peptide or protein to the carrier.
  • Other means are well known in the art which may be employed to non-covalently conjugate binding molecules (e.g. antigen or anti-idiotypic antibody) to a surface support (e.g.
  • beads including metal ion chelation techniques (e.g., using a poly-His tag at the C- terminus of the binding molecule, e.g. antigen, and a Ni -coated surface support), and these methods may be substituted for those described here.
  • metal ion chelation techniques e.g., using a poly-His tag at the C- terminus of the binding molecule, e.g. antigen, and a Ni -coated surface support
  • the recombinant receptor-stimulating agent is or includes a target, e.g., an antigen, a recombinant antigen, or fragment thereof.
  • the target is an antigen of the recombinant receptor.
  • the recombinant receptor-stimulating agent is or includes an antigen, e.g., a recombinant antigen or fragment thereof.
  • the recombinant receptor-stimulating agent may be target, such as an antigen, that is immobilized or bound to a surface support, such as a microwell plate, a solid particle (e.g. bead) or an oligomeric particle, e.g. as described above.
  • a surface support such as a microwell plate, a solid particle (e.g. bead) or an oligomeric particle, e.g. as described above.
  • the target e.g. antigen
  • the target is a polypeptide, or a variant or fragment of a polypeptide that is expressed on the surface of a cell that is associated with a disease, for example, a cancer cell and/or a tumor cell. It is understood that the target is any molecule that is recognized or bound by an extracellular domain of the recombinant receptor.
  • the target is an antibody that is recognized or bound by an extracellular domain of the recombinant receptor.
  • the target is an antigen and it is understood that the antigen is an antigen that is recognized or bound by an extracellular domain of the recombinant receptor.
  • a skilled artisan can determine the target, such as an antigen, and format of the target or antigen (e.g. cell expressed or immobilized on a solid surface) sufficient to stimulate the recombinant receptor.
  • the target is an antigen recognized by the extracellular domain of the recombinant receptor.
  • the antigen is or includes avP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer/testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type
  • avP6 integrin
  • Antigens targeted by the receptors include antigens associated with a B cell malignancy, such as any of a number of known B cell marker.
  • the antigen is or includes CD20, CD19, CD22, R0R1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.
  • the antigen is or comprises a portion of a polypeptide antigen that is recognized by or bound by a recombinant receptor, e.g. a CAR.
  • the portion of an antigen is a region that contains an epitope that is recognized by or bound by a recombinant receptor, e.g. a CAR.
  • a contiguous sequence of the extracellular domain of the antigen target that is recognized by or bound by a recombinant receptor and or a CAR can be incorporated as part of the binding molecule of the recombinant-receptor stimulating agent.
  • the recombinant receptor is an anti-BCMA CAR.
  • the extracellular domain of the recombinant receptor e.g. CAR
  • the antigen is BCMA or is an extracellular domain portion of BCMA.
  • the BCMA polypeptide is a mammalian BCMA polypeptide.
  • the BCMA polypeptide is a human BCMA polypeptide.
  • the BCMA antigen is or comprises an extracellular domain of BCMA or a portion thereof comprising an epitope recognized by an antigen receptor, e.g. CAR.
  • the BCMA antigen is or comprises a polypeptide with an amino acid sequence with at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 39 or a fragment thereof containing at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, or at least 180 contiguous amino acids of SEQ ID NO: 39.
  • the BCMA antigen is or includes the sequence set forth in SEQ ID NO: 39 or a portion thereof that is or contains an epitope recognized by an antigen receptor, e.g. CAR.
  • the recombinant receptor is an anti-RORl CAR.
  • the extracellular domain of the recombinant receptor e.g. CAR
  • the antigen is ROR1 or is an extracellular domain portion of ROR1.
  • the ROR1 polypeptide is mammalian.
  • the ROR1 polypeptide is human.
  • the antigen is an extracellular domain of ROR1 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g. CAR.
  • the antigen is a polypeptide with an amino acid sequence with at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 40 or a fragment thereof containing at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, or at least 180 contiguous amino acids of SEQ ID NO: 40.
  • the ROR1 antigen comprises the sequence set forth in SEQ ID NO: 40 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g. CAR.
  • the recombinant receptor is an anti-CD22 CAR.
  • the extracellular domain of the recombinant receptor e.g. CAR
  • the antigen is CD22 or is an extracellular domain portion of CD22.
  • the CD22 polypeptide is mammalian.
  • the CD22 polypeptide is human.
  • the antigen is an extracellular domain of CD22 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g. CAR.
  • the antigen is a polypeptide with an amino acid sequence with at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 41 or a fragment thereof containing at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, or at least 180 contiguous amino acids of SEQ ID NO: 41.
  • the CD22 antigen comprises the sequence set forth in SEQ ID NO: 41 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g. CAR.
  • the recombinant receptor is an anti-CD19 CAR.
  • the extracellular domain of the recombinant receptor e.g. CAR
  • the antigen is CD 19 or is an extracellular domain portion of CD 19.
  • the CD19 polypeptide is mammalian.
  • the CD19 polypeptide is human.
  • the antigen is an extracellular domain of CD 19 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g. CAR.
  • the antigen is a polypeptide with an amino acid sequence with at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 42 or a fragment thereof containing at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, or at least 180 contiguous amino acids of SEQ ID NO: 42.
  • the CD 19 antigen comprises the sequence set forth in SEQ ID NO: 42 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g. CAR.
  • the antigen or portion thereof may be formatted as a multimer, e.g. a dimer, comprising two or more polypeptide antigens, or portion or variant thereof, that is recognized and/or bound by a recombinant receptor, such as an antigen receptor (e.g. a CAR).
  • a recombinant receptor such as an antigen receptor (e.g. a CAR).
  • the polypeptide antigen, or portion thereof are identical.
  • the polypeptide antigen is linked, directly or indirectly, to a region or domain, e.g. a multimerization domain, that promotes or stabilizes interaction between two or more polypeptide antigens via complementary interactions between the domains or regions.
  • providing the polypeptide antigen as a multimer e.g.
  • dimer provides for a multivalent interaction between the antigen or extracellular domain portion thereof and the antigen-binding domain of the antigen receptor, e.g. CAR, which, in some aspects, can increase the avidity of the interaction.
  • an increased avidity may favor stimulatory or agonist activity of antigen receptor, e.g. CAR, by the antigen or extracellular domain portion thereof conjugated to the bead.
  • a polypeptide is joined directly or indirectly to a multimerization domain.
  • multimerization domains include the immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains.
  • the multimerization domain can be an immunoglobulin constant region or domain, such as, for example, the Fc domain or portions thereof from IgG, including IgGl, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD and IgM and modified forms thereof.
  • the polypeptide antigen is linked, directly or indirectly, to an Fc domain.
  • the polypeptide is a fusion polypeptide comprising the polypeptide antigen or portion thereof and the Fc domain.
  • an antigen or extracellular domain portion thereof is a fusion polypeptide that comprises an Fc domain.
  • the Fc domain is derived from an immunoglobulin (e.g., IgG, IgA, IgM, or IgE) of a suitable mammal (e.g., human, mouse, rat, goat, sheep, or monkey).
  • a suitable mammal e.g., human, mouse, rat, goat, sheep, or monkey.
  • the Fc domain is fused to the C-terminal of the polypeptide antigen.
  • the Fc domain is fused to the N-terminal of the polypeptide antigen.
  • the antigen or extracellular domain portion thereof is provided as a fusion polypeptide comprising an Fc domain, wherein the Fc domain is present at the C-terminus of the fusion polypeptide.
  • the Fc domain is an IgG Fc domain, or a portion or variant thereof.
  • the Fc domain is a human IgG Fc domain, or a portion or a variant thereof, that comprises an amino acid sequence set forth in SEQ ID NO: 43 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the sequence set forth in SEQ ID NO: 43.
  • the Fc domain is a wild-type human IgG Fc domain, or a portion or variant thereof.
  • the Fc domain is a variant of the wild-type human IgGl Fc domain.
  • the antigen and the multimerization domain, such as Fc domain are connected by a linker, such as an amino acid linker.
  • the antigen is fused to the N-terminus of an amino acid linker
  • the multimerization domain, such as Fc domain is fused to the C-terminus of the linker.
  • amino acid linkers can be any length and contain any combination of amino acids, the linker length may be relatively short (e.g., ten or fewer amino acids) to reduce interactions between the linked domains.
  • the amino acid composition of the linker also may be adjusted to reduce the number of amino acids with bulky side chains or amino acids likely to introduce secondary structure.
  • Suitable amino acid linkers include, but are not limited to, those up to 3, 4, 5, 6, 7, 10, 15, 20, or 25 amino acids in length.
  • Representative amino acid linker sequences include GGGGS (SEQ ID NO: 44), and linkers comprising 2, 3, 4, or 5 copies of GGGGS (SEQ ID NO: 44).
  • an amino acid linker comprises the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.
  • the antigen is provided as an extracellular domain of BCMA, e.g. human BCMA, fused to an Fc domain (BCMA-Fc).
  • BCMA-Fc Fc domain
  • the BCMA-Fc antigen contains all or a portion of the amino acid sequence set forth in SEQ ID NO: 45 or a sequence of amino acids that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to SEQ ID NO: 45, and that comprises an epitope recognize by an antigen receptor, e.g. CAR.
  • an antigen receptor e.g. CAR.
  • the antigen is provided as an extracellular domain of ROR1, e.g. human ROR1, fused to an Fc domain (RORl-Fc).
  • ROR-l-Fc antigen contains all or a portion of the amino acid sequence set forth in SEQ ID NO: 46 or a sequence of amino acids that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to SEQ ID NO: 46 and that comprises an epitope recognize by an antigen receptor, e.g. CAR.
  • the antigen is provided as an extracellular domain of CD22, e.g. human CD22, fused to an Fc domain (e.g. CD22-Fc).
  • the CD22-Fc antigen contains all or a portion of the amino acid sequence set forth in SEQ ID NO: 47 or a sequence of amino acids that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to SEQ ID NO: 47 and that comprises an epitope recognize by an antigen receptor, e.g. CAR.
  • an antigen receptor e.g. CAR.
  • the binding molecule is an antibody (e.g., an anti-idiotype antibody) or antigen-binding fragment thereof (“anti-IDs”) that specifically recognizes a recombinant receptor, for example a recombinant receptor, e.g., CAR.
  • an anti-idiotype antibody targets via binding an idiotype of an antibody, such as the antigen binding site of another antibody, such as the scFv of the extracellular antigen binding domain of a CAR.
  • the idiotype is any single antigenic determinant or epitope within the variable portion of an antibody.
  • the anti-ID is able to bind to the recombinant receptor to stimulate a recombinant receptor-dependent activity.
  • anti-idiotypic antibody can be made depending on the particular recombinant receptor, such as CAR.
  • exemplary anti-idiotype antibodies against antigen-specific CARs are known or can be generated by standard antibody technologies. These include, but are not limited to, anti-idiotypic antibodies directed against a CD22-directed CAR, see e.g. PCT Publication No. WO2013188864; CD19- directed CAR, such as directed against FMC63 scFv, see e.g. PCT Publication No. WO 2018/023100; Cat. No. REA1297 (Miltenyi Biotech); clone Y45 (Fisher Scientific, e.g., Cat. No.
  • the recombinant receptor-stimulating agent is a cell that expresses the target recognized by the antigen receptor, in this instance, the recombinant receptor-stimulating agent is a target-expressing cell.
  • the target is an antigen of the recombinant receptor and thus, in some cases, the target-expressing cells are antigen-expressing cells.
  • the recombinant receptor-stimulating agent is an antigen-expressing cell, such as a cell expressing a target or an antigen as described above.
  • the cells e.g., target-expressing cells, such as antigen-expressing cells are exogenous, heterologous, and/or autologous to a subject. In some embodiments, the cells are exogenous to the subject.
  • the target-expressing cells express a target that is bound by and/or recognized by the recombinant receptor.
  • the target is an antibody and the targetexpressing cells express the antibody.
  • the target-expressing cells are tumor cells.
  • the target-expressing cells are primary cells. 1 [0110]
  • the target is an antigen recognized by the recombinant receptor and the target-expressing cells are antigen-expressing cells.
  • the antigen-expressing cells express an antigen that is bound by and/or recognized by the recombinant receptor.
  • the antigen-expressing cells are tumor cells.
  • the antigenexpressing cells are primary cells.
  • the antigen-expressing cells are tumor cells.
  • Tumor cell lines are known and available. Tumor cell lines are known that express particular tumor antigens or surface expression of a tumor antigen can be readily determined or measured by as skilled artisan using any of a variety of techniques, such as by flow cytometry.
  • Exemplary tumor cell lines include, but are not limited to, lymphoma cells (Raji; Daudi; Jeko-1; BJAB; Ramos; NCI-H929; BCBL-1; DOHH-2, SC-1, WSU-NHL, JVM-2, Rec-1, SP-53, RL, Granta 519, NCEP-1, CL-01), leukemia cells (BALL-1, RCH-ACV, SUP-B15); cervical carcinoma cells (33A; CaSki; HeLa), lung carcinoma cells (NCI-H358; A549, H1355, H1975, Calu-1, H1650 and H727), breast cells, (Hs-578T; ZR-75-1; MCF-7; MCF-7/HER2; MCF10A; MDA-MB-231; SKBR-3, BT-474, MDA- MB-231); ovarian cells (ES-2; SKOV-3; OVCAR3; HEY1B); multiple myeloma cells (U266, NCI- 119
  • exemplary CD19-expressing cell lines include, but are not limited to, Raji, Daudi and BJAB; exemplary CD20-expressing cell lines include Daudi, Ramos and Raji; exemplary CD22-expressing cell lines include, but are not limited to, Ramos, Raji, A549, H727, and H1650; exemplary Her2-expressing cell lines include SKOV3, BT-474 and SKBR-3; exemplary BCMA- expressing cell lines include, but are not limited to, RPMI-8226, NCI-H929, MM1S, MM1R and KMS11; exemplary GPRC5D-expressing cell lines include, but are not limited to, AMO-1, EJM, NCI- 11929, MM.1S, MM1.R, MOEP-8, and OPM-2; exemplary ROR1 -expressing cell lines include, but are not limited to, A549, MDA-MB-231, H1975, BAEE-1 and RCH-ACV.
  • the target-expressing cell line is a cell line that has been transduced to express the target of the recombinant receptor.
  • the target is a tumor antigen.
  • the antigen-expressing cell line is a cell line that has been transduced to express the tumor antigen.
  • This cell line may be a mammalian cell line, including, but not limited to, human cell lines.
  • the human cell line may be K562, U937, 721.221, T2, and C1R cells.
  • the K562 chronic myeloid leukemia cell line may be introduced with a nucleic acid encoding the tumor antigen.
  • the exogenous nucleic acid may be integrated into the genome of the cell line (e.g. K562 cell) at a genomic safe harbor (GSH).
  • GSH is a site which supports stable integration and expression of exogenous nucleic acid while minimizing the risk of unwanted interactions with the host cell genome (see e.g. Sadelain et al., Nat Rev Cancer. (201 1 ) 12(1 ):51 -8).
  • GSHs for stable integration of exogenous nucleic acid in human cells have been identified, including AAVS1, a naturally occurring site of integration of AAV virus on chromosome 19; CCR5 gene a chemokine receptor gene also known as an HIV-1 coreceptor; and the human ortholog of the mouse Rosa26 locus (see e.g. Papapetrou and Schambach Mol Ther. (2016) 24(4): 678-684).
  • any of such target cells are non-dividing cels.
  • Cells may be rendered non-dividing while remaining metabolically active by exposing them to ionizing radiation, such as gamma or X-ray irradiation.
  • ionizing radiation such as gamma or X-ray irradiation.
  • Gamma irradiation typically delivered using a Cesium- 137 or Cobalt-60 source, or X-ray irradiation from a calibrated generator, may be applied at doses sufficient to abrogate proliferation without inducing immediate cytotoxicity.
  • Suitable irradiation doses generally range from approximately 20 to 50 gray (Gy), corresponding to 2000 to 5000 rad, where 1 Gy is equivalent to 100 rad.
  • cells may be exposed to about 30 to 36 Gy (i.e., 3000 to 3600 rad) to ensure mitotic arrest while preserving viability.
  • treatment with DNA crosslinking agents such as mitomycin C (e.g., 10-50 pg/mL for 2-3 hours) may be used to chemically inhibit cell division.
  • DNA crosslinking agents such as mitomycin C (e.g., 10-50 pg/mL for 2-3 hours) may be used to chemically inhibit cell division.
  • the absence of proliferation may be confirmed using one or more assays, including BrdU or EdU incorporation assays to assess DNA synthesis, Ki-67 staining to evaluate proliferation marker expression, and longitudinal cell counts to confirm a lack of expansion.
  • Cell viability and functionality may further be validated using standard metabolic activity assays (e.g., MTT or resazurin-based methods), ensuring the treated cells remain biologically competent for their intended use.
  • irradiated target cells are added to culture medium prior to the addition of a sample of cells of the cell therapy.
  • exosomes can be isolated or detected from the cell culture as part of the provided methods, such as after contacting cells of a cell composition with the recombinant receptor-stimulating agent.
  • the provided embodiments relate to observations herein that exosomes can serve as useful biomarkers of cell compositions manufactured to be used as a cell therapy, such as to predict likelihood of responsiveness of the cell therapy to the subject to whom it is administered. Further, the ability of a cell composition to produce exosomes also can indicate other desirable attributes of a cell composition since the produced exosomes also can have certain functions and activities that may potentiate the therapeutic utility of a composition.
  • the exosomes provided herein can express the recombinant receptor and can contact and communicate with a target cell.
  • the target cell is a cancer cell or a tumor cell.
  • An exosome is a cell-derived, lipid-bilayer encapsulated particle that contains bioactive molecules of the cell from which it originates.
  • an exosome contains a range of molecular cargo, including cellular proteins, metabolites, nucleic acids, and lipids.
  • the terminology in the field relating to exosomes including their size, surface markers, and biogenesis pathways — has evolved over time and continues to be refined.
  • MISEV2023 Minimal Information For Studies of Extracellular Vesicles 2023
  • MISEV2023 Minimal Information for studies of extracellular vesicles
  • EVs extracellular vesicles
  • MISEV2023 EVs are defined broadly as lipid bilayer-enclosed particles released from cells that cannot replicate, and are not necessarily defined by a specific biogenesis pathway.
  • exosomes are more specifically described as EVs of endosomal origin, typically characterized by small size and surface expression of one or more tetraspanins (e.g., CD63, CD81, CD9).
  • Exosome refers to vesicle defined by a particular size, by surface expression of exosome markers such as tetraspanins, or by both. Exosomes may be characterized by any one or more of the following properties: (i) a size described herein in Section I.B.l.a; (ii) surface expression of one or more exosome markers provided herein in Section I.B.l.b; or (iii) a combination of size described herein in Section I.B.l.a and surface marker expression described herein in Section I.B.l.b.
  • an exosome is any cell-derived particle that is greater than about 30 nm in diameter and less than about 200 nm, such as less than about 150 nm, less than about 130 nm, or less than about 100 nm in diameter.
  • an exosome is a cell-derived particle that is surface positive for one or more markers such as CD63, CD81, and CD9.
  • an exosome comprises both size characteristics and expression of one or more such surface markers.
  • an exosome is a cell-derived particle that is greater than about 30 nm in diameter and less than about 200 nm, such as less than about 150 nm, less than about 130 nm, or less than about 100 nm in diameter, and that is surface positive for one or more markers such as CD63, CD81, and CD9.
  • exosomes are characterized by both their size (e.g., between about 30 nm and 200 nm in diameter, such as between about 30 nm and 150 nm in diameter) and surface expression of one or more exosome markers.
  • exosome refers to a vesicle of endosomal origin. Size and surface marker expression may be concurrently evaluated using multiparameter detection instruments, such as the Leprechaun instrument (see, e.g., Example 4).
  • the exosome is understood to have originated from the endosomal compartment and to have undergone regulated biogenesis.
  • exosome biogenesis is a complex and highly regulated process that involves several stages starting with the initial formation of early endosomes and ending with the release of fully mature exosomes into the extracellular environment.
  • Exosome biogenesis begins with the formation of intraluminal vesicles (IL Vs) within an endosomal compartment known as a multi vesicular body (MVB).
  • IL Vs intraluminal vesicles
  • MVB multi vesicular body
  • the ILVs emerge through the inward budding of the endosome’s limiting membrane allowing for the encapsulation of cargoes destined for exosomes.
  • cargo destined for an exosome binds trafficking effectors that lead to membrane bending and scission processes that give rise to exosomes.
  • Trafficking effectors are known in the art.
  • a non-exhaustive list of exemplary trafficking effectors includes Hrs, TSG101, AARDC1, CD63, CD9, CD82, CD81, Syntenin, Syndecan-1, ALIX, Arf6, VPS4, CHMP4, Caveolin-1, hnRNPAl, hnRNPA2Bl, YBX1, FMRP, SYNCRIP, hnRNPU, MEX3C/AP-2, and La protein.
  • Exosomes play an important role in intercellular communication through the transmission of bioactive molecules to target organs, tissues, or cells.
  • the contents and functionality of an exosome can be tailored by manipulating its source cell.
  • the contents and functionality of an exosome can be altered when the source cell is engineered to express an exogenous molecule, such as a recombinant receptor as in the provided embodiments.
  • An exosome derived from a recombinant receptor engineered cell will not only contain the endogenous molecules of the source cell but also the recombinant receptor.
  • exosomes produced from the cells of the stimulated cell composition can be detected in a variety of ways including by methods that involve their isolation using methods known in the art.
  • exosomes are present in the supernatant of the cell therapy composition, which can be enriched and isolated in various ways.
  • exosomes are enriched and isolated from the supernatant of the cell therapy composition using centrifugation.
  • the centrifugation step removes cell debris.
  • centrifugation aids in the isolation of the exosomes.
  • exosomes are isolated without using centrifugation.
  • exosomes are enriched and isolated from the supernatant of the cell therapy composition using precipitation.
  • exosomes are isolated without using precipitation.
  • exosomes are enriched and isolated from the supernatant of the cell therapy composition using a combination of precipitation and centrifugation. In some embodiments, precipitation is performed prior to centrifugation. In some embodiments, precipitation is performed after centrifugation.
  • the exosomes are isolated by precipitation. Methods of precipitating exosomes are known in the art. In some embodiments, the exosomes are isolated by centrifugation. In some embodiments, the method of isolating exosomes by centrifugation comprises one or more spins at 100,000 g for 60 minutes. In some embodiments, the method of isolating exosomes by centrifugation comprises one or more spins at 100 g to 3,000 g for 1 minutes to 20 minutes. In some embodiments, the method of isolating exosomes by centrifugation comprises sequential spins, e.g., three sequential spins.
  • the first spin is at 100 g to 500 g (e.g., for 1 minute to 5 minutes and one or more subsequent spins are performed at a higher centrifugal force (e.g., at more than 500 g, e.g., at 1000 g to 3000 g)). In some embodiments, at least one of the one or more subsequent spins is performed for at least 5 minutes, at least 10 minutes or at least 15 minutes. In some embodiments, the first spin is at 300 g for 3 minutes. In some embodiments, the second spin is at 2,500 g for 15 minutes. In some embodiments, the third spin is at 2,500 g for 15 minutes.
  • exosomes such as those isolated in the supernatant from other cells, can be further detected and/or characterized using various methods known in the art.
  • the exosomes are detected based on size of the exosome.
  • the exosomes are detected based on markers present on the surface of the exosome.
  • exosomes are detected based on the size and presence of markers on the surface of the exosome.
  • the exosomes are detected based on any other parameter known in the art.
  • exosomes are detected and characterized based on surface markers using immunoaffinity-based detection methods.
  • immunoaffinity-based methods can be used using antibodies to stain for abundant tetraspanins that define exosomes (e.g., CD9, CD81 and/or CD63).
  • immunoaffinity-based methods are capable of simultaneously isolating, detecting and characterizing exosomes without the need for centrifugation or precipitation, such that prior purification of exosomes is not necessary.
  • the immunoaffinity-based methods include the use of fluorescent-based methods using fluorescently labeled anti-tetraspanin antibodies (e.g., anti-CD9, anti-CD81 and/or anti-CD63), involving fluorescent imaging or fluorescent microscopy.
  • fluorescently labeled anti-tetraspanin antibodies e.g., anti-CD9, anti-CD81 and/or anti-CD63
  • Such methods also can be combined with single particle interferometry to characterize exosomes by their size and allow determination of exosome concentration, size and phenotype (see e.g., Saftics et al., J Extracell Vesicles.
  • the immunoaffinitybased detection method is performed using the Leprechaun instrument from Unchained Labs (Pleasanton, CA).
  • exosomes are detected and characterized based on surface markers using the Leprechaun instrument.
  • Exosomes are typically characterized by their size, markers, cargo and/or function.
  • the methods provided herein characterize the exosome by its size.
  • the methods provided herein characterize the exosome by one or more of its markers.
  • the methods provided herein characterize the exosome by its cargo.
  • the methods provided herein characterize the exosome by its function.
  • the methods provided herein characterize the exosome by any combination of characteristics including size, marker(s), cargo, and function(s).
  • the methods provided herein characterize the exosome by its size and one or more of its markers.
  • the methods provided herein characterize the exosome by its size, one or more of its markers and its cargo. In some embodiments, the methods provided herein characterize the exosome by its size, one or more of its markers, its cargo and its function. a. Exosome Size
  • an exosome detected herein is between about 30 nm and 150 nm in diameter. In some embodiments, an exosome detected herein is between about 30 nm and 130 nm in diameter. In some embodiments, an exosome detected herein is between about 30 nm and 100 nm in diameter.
  • an exosome detected herein is between about 30 nm and 50 nm, 40 nm and 60 nm, 50 nm and 70 nm, 60 nm and 80 nm, 70 nm and 90 nm, 80 nm and 100 nm, 90 nm and 110 nm, 100 nm and 120 nm, 110 nm and 130 nm, or 120 nm and 150 nm in diameter.
  • an exosome detected herein is between about 30 nm and 40 nm, 35 nm and 45 nm, 40 nm and 50 nm, 45 nm and 55 nm, 50 nm and 60 nm, 55 nm and 65 nm, 60 nm and 70 nm, 65 nm and 75 nm, 70 nm and 80 nm, 75 nm and 85 nm, 80 nm and 90 nm, 85 nm and 95 nm, 90 nm and 100 nm, 95 nm and 105 nm, 100 nm and 110 nm, 105 nm and 115 nm, 110 nm and 120 nm, 115 nm and 125 nm, 120 nm and 130 nm, 125 nm and 135 nm, 130 nm and 140 nm, 135 nm and 145 nm, or 140 nm and 150 nm in
  • an exosome detected herein is between about 30 nm and 35 nm, 35 nm and 40 nm, 40 nm and 45 nm, 45 nm and 50 nm, 50 nm and 55 nm, 55 nm and 60 nm, 60 nm and 65 nm, 65 nm and 70 nm, 70 nm and 75 nm, 75 nm and 80 nm, 80 nm and 85 nm, 85 nm and 90 nm, 90 nm and 95 nm, 95 nm and 100 nm, 100 nm and 105 nm, 105 nm and 110 nm, 110 nm and 115 nm, 115 nm and 120 nm, 120 nm and 125 nm, 125 nm and 130 nm, 130 nm and 135 nm, 135 nm and 140 nm, 140 nm and 145 nm, or
  • exosomes of the present disclosure are detected by identifying cell particles that are surface positive for one or more exosome markers provided herein.
  • the one or more exosome markers comprise any one or more exosome markers known in the art.
  • the one or more exosome markers comprise scaffolding proteins (e.g., tetraspanins), transmembrane proteins, immunomodulatory proteins, adhesion molecules, lipids, glycoproteins, and/or glycolipids.
  • an exosome detected herein is characterized by an exosome marker that comprises one or more tetraspanins.
  • the one or more tetraspanins include one or a combination of any of CD63, CD81, CD9, CD37, and/or CD82.
  • characterizing an exosome based on the presence of one or more tetraspanins distinguishes exosomes of endosomal origin from non-exosome cell-derived particles that are not of endosomal origin.
  • the exosomes detected herein are distinguished from non-exosome cell-derived particles based on CD63. In some embodiments, the exosomes provided herein are distinguished from non-exosome cell-derived particles based on CD81. In some embodiments, the exosomes provided herein are distinguished from non-exosome cell-derived particles based on CD9. In some embodiments, the exosomes provided herein are distinguished from non-exosome cell-derived particles based on CD63 and CD81. In some embodiments, the exosomes provided herein are distinguished from non-exosome cell-derived particles based on CD63 and CD9.
  • the exosomes provided herein are distinguished from non-exosome cell-derived particles based on CD81 and CD9. In some embodiments, the exosomes provided herein are distinguished from non-exosome cell- derived particles based on CD63, CD81 and CD9.
  • the exosome marker is CD63. In some embodiments, the exosome marker is CD81. In some embodiments, the exosome marker is CD9. In some embodiments, the exosome marker comprises CD63 and CD81. In some embodiments, the exosome marker comprises CD63 and CD9. In some embodiments, the exosome marker comprises CD81 and CD9. In some embodiments, the exosome marker comprises CD63, CD81 and CD9.
  • the one or more exosome markers comprise Alix, actin, TSG101, tubulin, GAPDH, beta actin, and/or HSP70.
  • the exosomes comprise a surrogate marker of the recombinant receptor expressed by the cells of the T cell compositions provided herein.
  • the recombinant receptor or surrogate marker is localized to the surface of the exosomes provided herein.
  • the marker includes all or part (e.g., truncated form) of CD34, NGFR, or epidermal growth factor receptor (EGFR).
  • the marker is truncated EGFR (tEGFR).
  • the cargo comprises nucleic acids, such as messenger RNA (mRNA), microRNA (miRNA), or other non-coding RNAs, and/or proteins involved in intracellular signaling or immune regulation.
  • an exosome detected herein comprises GM-CSF, Granzyme A, Granzyme B, IFNy, IL- 13, IL-2, MIP-la, MIP-lb, Perforin and/or any combination of the foregoing. 2. Exosome Yield
  • the methods provided herein comprise comparing the amount or concentration of exosomes produced from a stimulated cell composition (e.g., a stimulated cell therapy composition) to the amount or concentration of exosomes produced from a control cell composition.
  • a stimulated cell composition e.g., a stimulated cell therapy composition
  • exosome yield (e.g., amount and/or concentration) is dependent on the number of cells in the cell therapy composition.
  • the number of cells in the cell therapy composition provided herein is between about 0.1 x 10 6 cells/mL and 1 x 10 6 cells/mL. In some embodiments, the number of cells in the cell therapy composition provided herein is between about 0.33 x 10 6 cells/mL and 1 x 10 6 cells/mL.
  • the number of cells in the cell therapy composition provided herein is between about 0.4 x 10 6 cells/mL and 0.6 x 10 6 cells/mL, 0.5 x 10 6 cells/mL and 0.7 x 10 6 cells/mL, 0.6 x 10 6 cells/mL and 0.8 x 10 6 cells/mL, 0.7 x 10 6 cells/mL and 0.9 x 10 6 cells/mL, or 0.8 x 10 6 cells/mL and 1 x 10 6 cells/mL.
  • the amount or concentration of exosomes detected in the stimulated cell composition is increased compared to the amount or concentration of the exosomes detected in the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased between about 20% and 100% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 20% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 25% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 30% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 35% compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 40% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 45% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 50% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 55% compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 60% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 65% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 70% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 75% compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 80% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 85% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 90% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 95% compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 100% compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased between about 1-fold and 10-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5- fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 1-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 1.5-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 2-fold compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 2.5-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 3 -fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 3.5-fold compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 4-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 4.5-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 5 -fold compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 5.5-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 6-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 6.5-fold compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 7-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 7.5-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 8 -fold compared to the amount or concentration of exosomes produced from the control cell composition.
  • the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 8.5-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 9-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 9.5-fold compared to the amount or concentration of exosomes produced from the control cell composition. In some embodiments, the amount or concentration of exosomes produced from the stimulated cell composition is increased by at least about 10-fold compared to the amount or concentration of exosomes produced from the control cell composition.
  • the concentration of exosomes produced from the stimulated cell composition comprises up to about 1 x IO 10 exosomes/mL. In some embodiments, the concentration of exosomes produced from the stimulated cell composition comprises up to about 1 x IO 10 exosomes/mL when the number of cells in the cell therapy composition is between about 0.33 x 10 6 cells/mL and 1 x 10 6 cells/mL. In some embodiments, the concentration of exosomes produced from the stimulated cell composition comprises greater than 1 x IO 10 exosomes/mL.
  • the concentration of exosomes produced from the stimulated cell composition comprises greater than 1 x IO 10 exosomes/mL when the number of cells in the cell therapy composition is between about 0.33 x 10 6 cells/mL and 1 x 10 6 cells/mL.
  • the concentration of exosomes produced by the stimulated cell composition comprises between about 1 x 10 6 exosomes/mL and 1 x 10 10 exosomes/mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises between about 1 x 10 6 exosomes/mL and 2 x 10 6 exosomes/mL, 1.5 x 10 6 exosomes/mL and 2.5 x 10 6 exosomes/mL, 2 x 10 6 exosomes/mL and 3 x 10 6 exosomes/mL, 2.5 x 10 6 exosomes/mL and 3.5 x 10 6 exosomes/mL, 3 x 10 6 exosomes/mL and 4 x 10 6 exosomes/mL, 3.5 x 10 6 exosomes/mL and 4.5 x 10 6 exosomes/mL, 4 x 10 6 exosomes/mL and 5 x 10 6 exosomes
  • the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 10 6 exosomes/mL, about 1.5 x 10 6 exosomes/mL, about 2 x 10 6 exosomes/mL, about 2.5 x 10 6 exosomes/mL, about 3 x 10 6 exosomes/mL, about 3.5 x 10 6 exosomes/mL, about 4 x 10 6 exosomes/mL, about 4.5 x 10 6 exosomes/mL, about 5 x 10 6 exosomes/mL, about 5.5 x 10 6 exosomes/mL, about 6 x 10 6 exosomes/mL, about 6.5 x 10 6 exosomes/mL, about 7 x 10 6 exosomes/mL, about 7.5 x 10 6 exosomes/mL, or about 8 x 10 6 exosomes/mL.
  • the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 10 7 exosomes/mL, about 2 x 10 7 exosomes/mL, about 3 x 10 7 exosomes/mL, about 4 x 10 7 exosomes/mL, about 5 x 10 7 exosomes/mL, or about 6 x 10 7 exosomes/mL.
  • the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 10 7 exosomes/mL.
  • the concentration of exosomes produced by the stimulated cell composition comprises about 2 x 10 7 exosomes/mL.
  • the concentration of exosomes produced by the stimulated cell composition comprises about 3 x 10 7 exosomes/mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 4 x 10 7 exosomes/mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 5 x 10 7 exosomes/mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 6 x 10 7 exosomes/mL.
  • the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 10 8 exosomes/mL, about 2 x 10 8 exosomes/mL, about 3 x 10 8 exosomes/mL, about 4 x 10 8 exosomes/mL, about 5 x 10 8 exosomes/mL, about 6 x 10 8 exosomes/mL, 7 x 10 8 exosomes/mL or 8 x 10 8 exosomes/mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 10 8 exosomes/mL.
  • the concentration of exosomes produced by the stimulated cell composition comprises about 7 x 10 8 exosomes/mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 8 x 10 8 exosomes/mL.
  • the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 10 9 exosomes/mL.
  • the total amount of exosomes produced by the stimulated cell composition can be derived from any one of the concentrations provided above.
  • the amount of exosomes produced by the stimulated composition comprises up to about 10,000,000,000 or 1 x 10 10 exosomes.
  • exosome function comprises cytotoxic capacity.
  • the cytotoxic capacity of exosomes produced from the stimulated cell composition is increased compared to the cytotoxic capacity of exosomes produced from the control cell composition.
  • cytotoxic capacity is determined by measuring the presence and/or levels of cytokines.
  • the cytotoxic cytokines comprise granzyme-A, granzyme-B, perforin, IFNy, GM-CSF, IL- 13, IL-2, MIP-la, and/or MIP-lb.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by about 20% to 100% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 20% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 25% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 30% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 35% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 40% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 45% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 50% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 55% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 60% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 65% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 70% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 75% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 80% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 85% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 90% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 95% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 100% compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased between about 1-fold and 10-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about: 1-fold, 1.5- fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 1-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 1.5-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 2-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 2.5-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 3-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 3.5-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 4-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 4.5-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 5-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 5.5-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 6-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 6.5-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 7-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 7.5-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 8 -fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 8.5-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 9-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 9.5-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by at least about 10-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.
  • exosome function comprises cytotoxic function.
  • cytotoxic function of exosomes produced from the stimulated cell composition is increased compared to the cytotoxic function of exosomes produced from the control cell composition.
  • cytotoxic function is determined by measuring cell killing ability (e.g., in a cell killing assay) of the exosomes.
  • exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased compared to the exosome-mediated cytotoxicity of the exosomes produced by the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased between about 20% and 100% compared to the exosome-mediated cytotoxicity of the exosomes produced by the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about: 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 20% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 25% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 30% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 35% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 40% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 45% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 50% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 55% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 60% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 65% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 70% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 75% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 80% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 85% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 90% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 95% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced by the stimulated cell composition is increased by at least about 100% compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased between about 1-fold and 10-fold compared to the exosome- mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 1-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 1.5-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome -mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 2-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 2.5-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 3-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 3.5-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 4-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 4.5-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 5 -fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 5.5-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 6-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 6.5-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 7-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 7.5-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 8 -fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 8.5-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 9-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 9.5-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced from the stimulated cell composition is increased by at least about 10-fold compared to the exosome-mediated cytotoxicity of the exosomes produced from the control cell composition.
  • Embodiments provided herein are based on the observation that stimulation of a cell therapy composition (e.g., T cell therapy compositions) resulted in increased production and secretion of exosomes from cells of the cell therapy composition, particularly in subjects that went on to achieve a response (e.g., complete response) to the cell therapy composition.
  • the provided methods include using exosomes as a biomarker to predict whether a T cell therapy composition will be efficacious after administration to a subject having a disease or condition treatable with the T cell therapy composition.
  • the exosome is a cell-derived particle that is surface positive for CD63.
  • the exosome is a cell-derived particle that is greater than about 30 nm in diameter and less than about 100 nm in diameter, and that is surface positive for CD63.
  • exosomes are assessed in the T cell therapy composition according to the methods described in Section I or by any methods known in the art.
  • exosomes are assessed in the T cell therapy composition by identifying cell particles that are surface positive for CD63.
  • exosomes are assessed in the T cell therapy composition by identifying cell particles that are greater than about 30 nm in diameter and less than about 100 nm in diameter, and identifying cell particles that are surface positive for CD63.
  • the subject if the amount or concentration of exosomes is increased, the subject is predicted as likely to respond to the T cell therapy composition. In some embodiments, if the amount or concentration of exosomes is decreased or unchanged, the subject is not predicted as likely to respond.
  • the exosomes detected herein are used as a biomarker to determine whether a subject will be administered the T cell therapy alone or in combination with another agent. Other agents are described in Section ILA.
  • the amount or concentration of exosomes if the amount or concentration of exosomes is increased, the subject is administered the T cell therapy composition. In some embodiments, if the amount or concentration of exosomes is decreased or unchanged, the subject is administered the T cell therapy in combination with another agent.
  • the exosomes detected herein are used as a biomarker to select a subject for treatment, including adaptive treatment, with a T cell therapy composition.
  • the subject if the amount or concentration of exosomes is increased, the subject is selected for administration of the T cell therapy composition. In some embodiments, if the amount or concentration of exosomes is decreased or unchanged, the subject is selected for administration of the T cell therapy in combination with another agent.
  • the provided methods allow for predicting a response to, and/or efficacy of, a cell composition, such as a cell therapy composition in a subject having a disease or condition prior to treatment with the cell therapy composition.
  • the method includes contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition, detecting the exosomes produced from the cells of the stimulated cell composition to provide isolated exosomes, and comparing the amount or concentration of the exosomes produced from the stimulated cell composition to the amount or concentration of exosomes produced from a control cell composition. In some embodiments, predicting response to the cell therapy composition occurs if the amount or concentration of the isolated exosomes in the stimulated cell composition is increased compared to the amount or concentration of the isolated exosomes in the control cell composition.
  • predicting response to the cell therapy composition does not occur if the amount or concentration of the isolated exosomes in the stimulated cell composition is decreased or unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition.
  • the method of predicting allows for detecting exosomes produced from a T cell therapy composition.
  • the method of predicting allows for detecting exosomes produced from a CAR-T cell therapy composition.
  • the method of predicting allows for detecting exosomes produced from a TCR cell therapy composition.
  • the method of predicting a response to, and/or efficacy of, the cell composition comprises detecting exosomes in a T cell therapy composition.
  • detecting the exosomes comprises identifying cell particles that are surface positive for CD63. In some embodiments, detecting the exosomes comprises identifying cell particles that are greater than about 30 nm in diameter and less than about 100 nm in diameter, and identifying cell particles that are surface positive for CD63.
  • a method of treating a subject having a disease or condition comprising contacting cells of a cell therapy composition with a recombinant receptor- stimulating agent to generate a stimulated cell composition, detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes, comparing the amount or concentration of the isolated exosomes produced from the stimulated cell composition to the amount or concentration of isolated exosomes produced from a control cell composition, and administering the treatment to the subject.
  • the subject is treated with the cell therapy composition if the amount or concentration of the isolated exosomes in the stimulated cell composition is increased compared to the amount or concentration of the isolated exosomes in the control cell composition.
  • the subject is treated with the cell therapy composition in combination with another agent if the amount or concentration of the isolated exosomes in the stimulated cell composition is decreased or is unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition.
  • the method of treating the subject with the cell composition comprises detecting exosomes in a T cell therapy composition.
  • detecting the exosomes comprises identifying cell particles that are surface positive for CD63.
  • detecting the exosomes comprises identifying cell particles that are greater than about 30 nm in diameter and less than about 100 nm in diameter, and identifying cell particles that are surface positive for CD63.
  • a method of selecting a subject having a disease or condition for treatment with a cell therapy composition comprising contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition, detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes, comparing the amount or concentration of the isolated exosomes produced from the stimulated cell composition to the amount or concentration of isolated exosomes produced from a control cell composition, and administering the treatment to the subject.
  • the subject is selected for treatment with the cell therapy composition if the amount or concentration of the isolated exosomes in the stimulated cell composition is increased compared to the amount or concentration of the isolated exosomes in the control cell composition. In some embodiments, the subject is selected for treatment with the cell therapy composition in combination with another agent if the amount or concentration of the isolated exosomes in the stimulated cell composition is decreased or is unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition. In some embodiments, the method of selecting the subject for treatment with the cell composition comprises detecting exosomes in a T cell therapy composition. In some embodiments, detecting the exosomes comprises identifying cell particles that are surface positive for CD63. In some embodiments, detecting the exosomes comprises identifying cell particles that are greater than about 30 nm in diameter and less than about 100 nm in diameter, and identifying cell particles that are surface positive for CD63.
  • a method of treatment comprising administering a T cell therapy to a subject a disease or condition.
  • the T cell therapy is a composition comprising T cells engineered to express a recombinant receptor.
  • the subject has an increased amount or concentration of exosomes produced from the composition after ex vivo stimulation of the T cells of the composition with a recombinant receptor-stimulating agent.
  • the exosomes are cell-derived particles that are surface positive for CD63.
  • the exosomes are cell-derived particles that are greater than about 30 nm in diameter and less than about 100 nm in diameter, and that are surface positive for CD63.
  • provided herein is a method of treatment comprising administering a T cell therapy to a subject having a disease or condition.
  • the T cell therapy comprises T cells engineered to express a recombinant receptor.
  • the subject is a subject that has been selected for treatment according to the methods provided herein.
  • a method of adaptive treatment with a T cell therapy in a subject having a disease or a condition comprises determining an amount or concentration of isolated exosomes produced from a T cell composition, predicting the likelihood that the subject will respond to the T cell therapy, and administering the adaptive treatment to the subject.
  • the isolated exosomes are cell-derived particles that are surface positive for CD63.
  • the isolated exosomes are cell-derived particles that are greater than about 30 nm in diameter and less than about 100 nm in diameter, and that are surface positive for CD63.
  • the T cell composition comprises T cells engineered to express a recombinant receptor and is a T cell therapy treatment or is a candidate for a T cell therapy treatment to be administered to the subject.
  • the amount or concentration of isolated exosomes are determined by a method comprising contacting T cells of the T cell composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition and isolating exosomes produced from cells of the stimulated cell composition.
  • the subject is predicted to respond to the T cell therapy if the amount or concentration of the isolated exosomes in the stimulated cell composition is increased compared to the amount or concentration of the isolated exosomes in a control cell composition.
  • the subject is not predicted to respond to the T cell therapy if the amount or concentration of the isolated exosomes in the stimulated cell composition is decreased or is unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition.
  • the adaptive treatment is selected from the T cell therapy if the subject is assessed as likely to be responsive to the T cell therapy and the T cell therapy in combination with another agent if the subject is assessed as not likely to be responsive to the T cell therapy.
  • the disease or condition is an autoimmune or inflammatory disease or disorder.
  • the subject has an autoimmune or inflammatory disease or disorder and is a candidate for treatment with a cell therapy composition (e.g., T cell therapy composition).
  • a cell therapy composition e.g., T cell therapy composition
  • the autoimmune or inflammatory disease or condition is arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave’s disease, Crohn’s disease multiple sclerosis, asthma, and/or a disease or condition associated with transplant.
  • arthritis e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave’s disease, Crohn’s disease multiple sclerosis, asthma, and/or a disease or condition associated with transplant.
  • the subject is a human, such as a subject who is a patient in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells in some embodiments are primary cells, e.g., primary human cells.
  • the subject has a cancer or tumor that is a hematological malignancy.
  • the subject has a cancer or tumor that is a lymphoma or a leukemia.
  • Lymphoma and leukemia are cancers of the blood that specifically affect lymphocytes. All leukocytes in the blood originate from a single type of multipotent hematopoietic stem cell found in the bone marrow. This stem cell produces both myeloid progenitor cells and lymphoid progenitor cell, which then give rise to the various types of leukocytes found in the body.
  • Leukocytes arising from the myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells.
  • Leukocytes arising from the lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphomas and leukemias can affect one or more of these cell types in a patient.
  • Lymphomas can be divided into at least two sub-groups: Hodgkin lymphoma and nonHodgkin lymphoma.
  • Non-Hodgkin Lymphoma (NHL) is a heterogeneous group of cancers originating in B lymphocytes, T lymphocytes or natural killer cells.
  • Diffuse large B cell lymphoma is the most common subtype of NHL, accounting for approximately 30% of NHL cases. It is classified as an aggressive lymphoma with the majority of patients cured with conventional chemotherapy (NCCN guidelines NHL 2014).
  • the cancer is a leukemia, lymphoma, e.g., acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), Anaplastic large cell lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM), a B cell malignancy is selected from among acute lymphoblastic leukemia (AML), chronic myeloid (
  • the subject has a lymphoma.
  • the lymphoma is selected from small cell lymphoma, lymphoplasmacytic lymphoma (e.g., Waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell neoplasms (e.g., plasma cell myeloma such as multiple myeloma, or plasmacytoma), extranodal marginal zone B cell lymphoma (e.g., MALT lymphoma), nodal marginal zone B cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneous follicle center lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), Epstein-Barr virus- positive DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B- cell lymphoma (PMBCL),
  • lymphoplasmacytic lymphoma
  • the lymphoma is large B cell lymphoma (LB CL). In some embodiments, the lymphoma is diffuse large B cell lymphoma (DLBCL).
  • LB CL large B cell lymphoma
  • DLBCL diffuse large B cell lymphoma
  • the cancer is a pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancers, CNS cancers, brain tumors, bone cancer, or soft tissue sarcoma.
  • the cancer is refractory to or the cancer has relapsed following one or more of chemotherapy, radiotherapy, immunotherapy (including a T cell therapy and/or treatment with an antibody or antibody-drug conjugate), an autologous stem cell transplant, or any combination thereof.
  • the cancer is refractory diffuse large B cell lymphoma.
  • the subject is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • the individual or subject is a human.
  • the subject is a human.
  • the subject is a patient, e.g., a human patient having a cancer described herein.
  • a treatment strategy that includes an additional treatment may be selected or administered.
  • the cell therapy composition is administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • another therapeutic intervention such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • the cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
  • the cell therapy composition is co-administered with another therapy sufficiently close in time such that the therapeutic cell compositions enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the therapeutic cell compositions are administered prior to the one or more additional therapeutic agents.
  • the therapeutic cell compositions are administered after the one or more additional therapeutic agents.
  • the other agent may be administered before, concurrently with or after the cell therapy composition.
  • the other agent may be initiated 15 to 90 days prior to administering the T cell therapy, such as at about 15 days, 30 days, 60 days or 90 days prior to administering the T cell therapy.
  • administration of the other agent is initiated 15 to 90 days, such as 15 to 60 days, 15 to 30 days or 30 to 60 days prior to administering the T cell therapy to the subject.
  • the administration of the other agent is initiated 15 to 60 days, such as 15 days to 30 days or 30 days to 60 days prior to administering the T cell therapy to the subject.
  • the administration of the other agent is initiated 15 to 30 days prior to administering the T cell therapy to the subject.
  • the other agent may be initiated concurrently with administering the T cell therapy, such as within about 1 to 24 hours, about 1 to 12 hours or about 1 to 6 hours. In some embodiments, the other agent may be initiated concurrently with administering the T cell therapy within about 1 to 12 hours, such as within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, or within 12 hours. In some embodiments, the other agent may be initiated concurrently within the same day as the administration of the T cell therapy.
  • administration of the other agent is initiated 15 days to 90 days after administering the T cell therapy, such at about 15 days, 30 days, 60 days or 90 days after administering the T cell therapy to the subject. In some embodiments, administration of the other agent is initiated 15 to 90 days, such as 15 to 60 days, 15 to 30 days or 30 to 60 days after administering the T cell therapy to the subject. In some embodiments, the administration of the other agent is initiated 15 to 60 days, such as 15 days to 30 days or 30 days to 60 days after administering the T cell therapy to the subject. In some embodiments, the administration of the other agent is initiated 15 to 30 days after administering the T cell therapy to the subject.
  • the administration of the other agent is initiated about 3 days after, about 7 days after, about 15 days after, or about 28 days after to administering the T cell therapy to the subject. In some embodiments, the administration of the other agent is initiated about 1 month, 2 months or 3 months after administering the T cell therapy to the subject.
  • the other agent includes administration of a kinase inhibitor, such as a BTK inhibitor (e.g. ibrutinib or acalibrutinib); an inhibitor or a tryptophan metabolism and/or kynurenine pathway, such as an inhibitor of indoleamine 2,3-dioxygenase-l (IDO1) (e.g.
  • a BTK inhibitor e.g. ibrutinib or acalibrutinib
  • IDO1 indoleamine 2,3-dioxygenase-l
  • BCL-2 B-cell lymphoma 2
  • an immunomodulatory agent such as an immunomodulatory imide drug (ImiD), including a thalidomide or thalidomide derivative (e.g. lenalidomide, pomalidomide, avadomide (CC-122), or iberdomide (CC- 220)) or a cereblon E3 ligase modulator (CELMoD); a DGK inhibitor; an inhibitor of or a checkpoint inhibitor, such as an anti-PD-Ll antibody (e.g. durvalumab), a PD-1 inhibitor such as an anti-PD-1 antibody (e.g. nivolumab), or a LAG3 inhibitor such as an anti-LAG3 antibody (e.g. relatlimab).
  • ImiD immunomodulatory imide drug
  • CELMoD cereblon E3 ligase modulator
  • DGK inhibitor an inhibitor of or a checkpoint inhibitor, such as an anti-PD-Ll antibody (
  • the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI- 32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.
  • the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-inducible kinase (ITK), and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.
  • the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (l-[(3R)-3-[4- Amino-3-(4-phenoxyphenyl)- 1 H-pyrazolo[3 ,4-d]pyrimidin- 1 -yl]piperidin- 1 -yl]prop-2-en- 1 -one ; also known as PCI-32765).
  • BTK inhibitor e.g., ibrutinib (l-[(3R)-3-[4- Amino-3-(4-phenoxyphenyl)- 1 H-pyrazolo[3 ,4-d]pyrimidin- 1 -yl]piperidin- 1 -yl]prop-2-en- 1 -one ; also known as PCI-32765).
  • the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765), and the ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time, e.g., daily for 21 day cycle, or daily for 28 day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib are administered.
  • the BTK inhibitor is a BTK inhibitor described in International Application WO 2015/079417.
  • the other agent is an agent that regulates pro- or anti-apoptotic proteins.
  • the other agent includes a B-cell lymphoma 2 (BCL-2) inhibitor (e.g., venetoclax, also called ABT-199 or GDC-0199; or ABT-737).
  • BCL-2 B-cell lymphoma 2
  • Venetoclax is a small molecule (4-(4- ⁇ [2- (4-Chlorophenyl)-4,4-dimethyl- 1 -cyclohexen- 1 -yl]methyl ⁇ - 1 -piperazinyl)-N-( ⁇ 3-nitro-4-[(tetrahydro- 2H-pyran-4-ylmethyl)amino]phenyl ⁇ sulfonyl)-2-(lH-pyrrolo[2,3-b]55yridine-5-yloxy)benzamide) that inhibits the anti-apoptotic protein, BCL-2.
  • agents that modulate pro- or anti-apoptotic protein include BCL-2 inhibitor ABT-737, navitoclax (ABT-263); Mcl-1 siRNA or Mcl-1 inhibitor retinoid N- (4-hydroxyphenyl) retinamide (4-HPR) for maximal efficacy.
  • the other agent provides a pro-apoptotic stimuli, such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which can activate the apoptosis pathway by binding to TRAIL death receptors DR-4 and DR-5 on tumor cell surface, or TRAIL-R2 agonistic antibodies.
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand
  • the other agent is an immunomodulatory agent.
  • the combination therapy includes an immunomodulatory agent that can stimulate, amplify and/or otherwise enhance an anti-tumor immune response, e.g., anti-tumor immune response from the administered engineered cells, such as by inhibiting immunosuppressive signaling or enhancing immunostimulant signaling.
  • the immunomodulatory agent is a peptide, protein or is a small molecule.
  • the protein can be a fusion protein or a recombinant protein.
  • the immunomodulatory agent binds to an immunologic target, such as a cell surface receptor expressed on immune cells, such a T cells, B cells or antigen-presenting cells.
  • the immunomodulatory agent is an antibody or antigen-binding antibody fragment, a fusion protein, a small molecule or a polypeptide.
  • the recombinant receptors, cells and/or compositions are administered in combination with another agent that is an antibody or an antigen-binding fragment thereof, such as a monoclonal antibody.
  • the immunomodulatory agent blocks, inhibits or counteracts a component of the immune checkpoint pathway.
  • the immune system has multiple inhibitory pathways that are involved in maintaining self-tolerance and for modulating immune responses.
  • Tumors can use certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens (Pardoll (2012) Nature Reviews Cancer 12:252-264), e.g., engineered cells such as CAR-expressing cells. Because many such immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies against the ligands and/or their receptors.
  • a cell therapy composition comprising, including compositions comprises CAR or TCR expressing T cells.
  • the cell therapy composition is a T cell therapy composition.
  • the T cell therapy compositions provided herein are designed to recognize and/or specifically bind to antigens associated with the disease or condition, such as a cancer or tumor, or an autoimmune or inflammatory disorder.
  • all of the cells in the cell therapy composition express the recombinant receptor.
  • less than all of the cells in the cell therapy composition express the recombinant receptor.
  • at or greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of cells in the cell therapy composition express the recombinant receptor.
  • the antigen that is bound or recognized by the recombinant receptor is any antigen described below in Section III.A.
  • binding to the antigen results in a response, such as an immune response against such antigens.
  • the cells express or are engineered to express the recombinant receptor, such as a chimeric antigen receptor (CAR).
  • the recombinant receptor such as a CAR, generally includes an extracellular antigen (or ligand) binding domain specific to the antigen that is linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s).
  • the engineered cells are provided as pharmaceutical compositions and formulations suitable for administration to a subjects, such as for adoptive cell therapy. Also provided are therapeutic methods for selecting subjects for administration of the cells and compositions to subjects, e.g., patients. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.
  • the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids.
  • gene transfer is accomplished by first stimulating the cells, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
  • the cell composition is for use as a cell therapy.
  • the cell composition is a T cell composition.
  • the T cell composition such as a T cell therapy composition, comprises at or greater than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more T cells.
  • the T cells are primary cells. In some embodiments, the T cells are autologous cells. In some embodiments, the T cells are allogeneic cells. In some embodiments, the T cells are positive for T cell markers. In some embodiments, the T cells are positive for CD3, CD4, and/or CD8. In some embodiments, the T cells of the T cell therapy composition are CD3+. In some embodiments, the T cells are positive for CD3, CD4, and/or CD8. In some embodiments, the T cells of the T cell therapy composition are CD4+. In some embodiments, the T cells are positive for CD3, CD4, and/or CD8. In some embodiments, the T cells of the T cell therapy composition are CD8+. In some embodiments, the T cells are positive for CD3, CD4, and/or CD8. In some embodiments, the T cells of the T cell therapy composition are CD4+ and CD8+.
  • the cell therapy composition provided herein is cryopreserved and thawed prior to detecting, isolating and characterizing the exosomes.
  • the engineered cells express a chimeric receptors, such as a chimeric antigen receptor (CAR), that contains one or more domains that combine a ligand-binding domain (e.g. antibody or antibody fragment) that provides specificity for a desired antigen (e.g., tumor antigen such as CD19) with intracellular signaling domains.
  • a ligand-binding domain e.g. antibody or antibody fragment
  • a desired antigen e.g., tumor antigen such as CD19
  • the intracellular signaling domain is a stimulating or an activating intracellular domain portion, such as a T cell stimulating or activating domain, providing a primary activation signal or primary signal.
  • the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions.
  • the receptor Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an IT AM- transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition.
  • an immunostimulatory signal such as an IT AM- transduced signal
  • chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival and/or persistence in vivo, such as for use in adoptive cell therapy methods.
  • Exemplary antigen receptors including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, W02013/123061, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S.
  • the antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 Al.
  • the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No.: 7,446,190, US Patent No.: 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No.: 7,446,190, and US Patent No.: 8,389,282.
  • the engineered cells such as T cells express a recombinant receptor such as a chimeric antigen receptor (CAR) with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type.
  • a recombinant receptor such as a chimeric antigen receptor (CAR) with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type.
  • the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule.
  • the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells.
  • the recombinant receptor e.g., a chimeric antigen receptor
  • the recombinant receptor includes an extracellular portion containing one or more antigen-binding domains, such as an antibody or fragment thereof, and one or more intracellular signaling region or domain (also interchangeably called a cytoplasmic signaling domain or region).
  • the recombinant receptor e.g., CAR
  • the spacer and/or transmembrane domain can link the extracellular portion containing the ligand- (e.g., antigen-) binding domain and the intracellular signaling region(s) or domain(s).
  • the chimeric receptors such as CARs, generally include an extracellular antigen binding domain that is an antigen-binding portion or portions of an antibody molecule.
  • the antigen-binding domain is a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.
  • the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAh).
  • scFv single-chain antibody fragment
  • the antigen-binding domain is a single domain antibody (sdAb), such as sdFv, nanobody, VHH and VNAR.
  • sdAb single domain antibody
  • an antigenbinding fragment comprises antibody variable regions joined by a flexible linker.
  • the antigen is a B cell antigen or a plasma cell antigen.
  • the antigen is or includes avP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer/testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor protein (EGFR), type III epi
  • Antigens targeted by the receptors include antigens associated with a B cell malignancy, such as any of a number of known B cell marker.
  • the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.
  • the antigen is or includes a pathogen-specific or pathogen-expressed antigen.
  • the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.
  • Antigens targeted by the receptors include antigens associated with a B cell malignancy, such as any of a number of known B cell marker.
  • the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.
  • the antigen is CD19.
  • any of such antigens are antigens expressed on human B cells.
  • the antigen that is bound or recognized by the recombinant receptor is CD19.
  • the antigen that is bound or recognized by the recombinant receptor is CD20.
  • the antigen targeted by the receptor is a polypeptide.
  • the antigen target is CD19.
  • the antigen is selectively expressed on B cells targeted for treating a cancer.
  • the CAR typically includes in its extracellular portion one or more antibody or antigen-binding fragment or portion that targets CD 19.
  • the antibody or an antigen-binding fragment e.g., scFv or VH domain
  • the antibody or antigen-binding fragment is derived from, or is a variant of, antibodies or antigen-binding fragment that specifically binds to CD19.
  • the antigen is CD19.
  • the antibody or an antigen-binding fragment contains a variable heavy chain and a variable light chain with six CDRs, CDRH1-3 and CDRL1-3, that confer binding to CD19.
  • the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19.
  • the extracellular binding domain of the CD19 CAR is derived from an antibody specific to CD19, including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol.
  • the extracellular binding domain of the CD 19 CAR can comprise or consist of the VH, the VL, and/or one or more CDRs of any of the antibodies.
  • the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1.
  • the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016/0152723.
  • the antigen-binding domain includes a VH and/or VL derived from FMC63, which, in some aspects, can be an scFv.
  • FMC63 generally refers to a mouse monoclonal IgGl antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing III. 302).
  • the FMC63 antibody comprises CDR-H1 and CDR-H2 set forth in SEQ ID NOS: 12 and 13, respectively, and CDR-H3 set forth in SEQ ID NOS: 14 or 3 and CDR-L1 set forth in SEQ ID NO: 15 and CDR-L2 set forth in SEQ ID NOS: 16 or 17 and CDR-L3 sequences set forth in SEQ ID NOS: 18 or 19.
  • the FMC63 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:20 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:21.
  • the scFv comprises a variable light chain containing the CDR-L1 sequence of SEQ ID NO: 15, a CDR-L2 sequence of SEQ ID NO: 16, and a CDR-L3 sequence of SEQ ID NO: 18 and/or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO: 12, a CDR-H2 sequence of SEQ ID NO: 13, and a CDR-H3 sequence of SEQ ID NO: 14, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the scFv comprises a variable heavy chain region of FMC63 set forth in SEQ ID NO:20 and a variable light chain region of FMC63 set forth in SEQ ID N0:21, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the FMC63 antibody comprises CDR-H1 and CDR-H2 set forth in SEQ ID NO: 12 and 13, respectively, and CDR-H3 set forth in SEQ ID NO: 14 or 3 and CDR-L1 set forth in SEQ ID NO: 15 and CDR-L2 set forth in SEQ ID NO: 16 or 17 and CDR-L3 sequences set forth in SEQ ID NO: 18 or 19.
  • the FMC63 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:20 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:21.
  • the scFv comprises a variable light chain containing the CDR-L1 sequence of SEQ ID NO: 15, a CDR-L2 sequence of SEQ ID NO: 16, and a CDR-L3 sequence of SEQ ID NO: 18 and/or a variable heavy chain containing a CDR- H1 sequence of SEQ ID NO: 12, a CDR-H2 sequence of SEQ ID NO: 13, and a CDR-H3 sequence of SEQ ID NO: 14, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the scFv comprises a variable heavy chain region of FMC63 set forth in SEQ ID NO:20 and a variable light chain region of FMC63 set forth in SEQ ID NO:21, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • variable heavy and variable light chains are connected by a linker.
  • the linker is set forth in SEQ ID NO:22.
  • the scFv comprises, in order, a VH, a linker, and a VL- In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH- In some embodiments, the scFv is encoded by a sequence of nucleotides set forth in SEQ ID NO:23 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:23.
  • the scFv comprises the sequence of amino acids set forth in SEQ ID NO:24 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24.
  • the antigen-binding domain includes a VH and/or VL derived from SJ25C1, which, in some aspects, can be an scFv.
  • SJ25C1 is a mouse monoclonal IgGl antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing III. 302).
  • the SJ25C1 antibody comprises CDR-H1, CDR-H2 and CDR-H3 set forth in SEQ ID NOS: 25, 8, 2, respectively, and CDR-L1, CDR-L2 and CDR-L3 sequences set forth in SEQ ID NOS: 26, 4, 5, respectively.
  • the SJ25C1 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11.
  • the scFv comprises a variable light chain containing a CDR-L1 sequence of SEQ ID NO:26, a CDR-L2 sequence of SEQ ID NO:4, and a CDR-L3 sequence of SEQ ID NO:5 and/or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO:25, a CDR-H2 sequence of SEQ ID NO:8, and a CDR- H3 sequence of SEQ ID N0:2, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the scFv comprises a variable heavy chain region of SJ25C1 set forth in SEQ ID NO:10 and a variable light chain region of SJ25C1 set forth in SEQ ID NO:11, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the variable heavy and variable light chains are connected by a linker.
  • the linker is set forth in SEQ ID NO:6.
  • the scFv comprises, in order, a VH, a linker, and a VL- In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH- In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:1 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.
  • the anti-CD19 CAR includes an antigen-binding domain described in PCT Pub. No. WO2015187528. In some embodiments, the anti-CD19 CAR is a CAR described in PCT Pub. No. WO2015187528.
  • the anti-CD19 CAR includes an antigen-binding domain that is a single chain antibody derived from a fully human antibody.
  • the single chain antibody is an scFv.
  • Exemplary fully human anti-CD19 antibodies are described in PCT Pub. No. W02016033570, PCT Pub. No. WO2020233589, U.S. Pub. No. US2010/0104509 and U.S. Pub. No. US20220220200.
  • the CAR targets CD19 and at least one other antigen expressed on B cells.
  • the antigen associated with the disease or disorder is selected from CD20, CD19, CD22, ROR1, BCMA, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.
  • the other antigen is CD20 and the CAR is a CD20/CD19 directed CAR product.
  • the CAR is a bispecific CAR in which the extracellular antigen-binding domain binds CD19 and the one other antigen (e.g. CD20).
  • the CD19 directed scFv comprises a variable heavy chain region and a variable light chain region of FMC63 (e.g. variable heavy chain region set forth in SEQ ID NO: 20 and a variable light chain region set forth in SEQ ID NO:21).
  • the CD19 scFv is Hul9 and comprises the variable heavy chain region set forth in SEQ ID NO:27 and the variable light chain region set forth in SEQ ID NO:28.
  • Exemplary CD19-directed antigen receptors e.g., CARs
  • CARs also include the CARs of FDA- approved products BREYANZI® (lisocabtagene maraleucel), TECARTUSTM (brexucabtagene autoleucel), KYMRIAHTM (tisagenlecleucel), and YESCARTATM (axicabtagene ciloleucel).
  • the CAR is the CAR of BREYANZI® (lisocabtagene maraleucel), TECARTUSTM (brexucabtagene autoleucel), KYMRIAHTM (tisagenlecleucel), YESCARTATM (axicabtagene ciloleucel).
  • the CAR is the CAR of BREYANZI® (lisocabtagene maraleucel, see Sehgal et al., 2020, Journal of Clinical Oncology 38:15_suppl, 8040; Teoh et al., 2019, Blood 134(Supplement_l):593; and Abramson et al., 2020, The Lancet 396(10254): 839-852).
  • the CAR is the CAR of TECARTUSTM (brexucabtagene autoleucel, see Mian and Hill, 2021, Expert Opin Biol Ther; 21(4):435- 441; and Wang et al., 2021, Blood 138(Supplement 1):744).
  • the CAR is the CAR of KYMRIAHTM (tisagenlecleucel, see Bishop et al., 2022, N Engl J Med 386:629:639; Schuster et al., 2019, N Engl J Med 380:45-56; Halford et al., 2021, Ann Pharmacother 55(4):466-479; Mueller et al., 2021, Blood Adv. 5(23):4980-4991; and Fowler et al., 2022, Nature Medicine 28:325-332).
  • KYMRIAHTM tisagenlecleucel, see Bishop et al., 2022, N Engl J Med 386:629:639; Schuster et al., 2019, N Engl J Med 380:45-56; Halford et al., 2021, Ann Pharmacother 55(4):466-479; Mueller et al., 2021, Blood Adv. 5(23):4980-4991; and Fowler et al.,
  • the CAR is the CAR of YESCARTATM (axicabtagene ciloleucel, see Neelapu et al., 2017, N Engl J Med 377(26):2531-2544; Jacobson et al., 2021, The Lancet 23(l):P91-103; and Locke et al., 2022, N Engl J Med 386:640-654).
  • the antigen is CD20.
  • the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD20.
  • the antibody or antibody fragment that binds CD20 is an antibody that is or is derived from Rituximab, such as is Rituximab scFv.
  • the antigen is CD22.
  • the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD22.
  • the antibody or antibody fragment that binds CD22 is an antibody that is or is derived from m971, such as is m971 scFv.
  • the antigen or antigen binding domain is GPRC5D.
  • the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to GPRC5D.
  • the antibody or antibody fragment that binds GPRC5D is or contains a VH and a VL from an antibody or antibody fragment set forth in International Patent Applications, Publication Number WO 2016/090329, WO 2016/090312, and WO 2020/092854.
  • the CAR is an anti-BCMA CAR that is specific for BCMA, e.g., human BCMA.
  • Chimeric antigen receptors containing anti-BCMA antibodies, including mouse antihuman BCMA antibodies and human anti-human antibodies, and cells expressing such chimeric receptors have been previously described. See Carpenter et al., Clin Cancer Res., 2013, 19(8):2048- 2060, WO 2016/090320, W02016090327, W02010104949A2 and WO2017173256.
  • the antigen or antigen binding domain is BCMA.
  • the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to BCMA.
  • the antibody or antibody fragment that binds BCMA is or contains a VH and a VL from an antibody or antibody fragment set forth in International Patent Applications, Publication Number WO 2016/090327 and WO 2016/090320.
  • CARs such as anti-BCMA CARs
  • CARs include the CARs of idecabtagene vicleucel, ABECMA®, BCMA02, JCARH125, JNJ-68284528 (LCAR-B38M; ciltacabtagene autoleucel; CARVYKTITM) (Janssen/Legend), P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA-Allol (Poseida), Allo-715 (Pfizer/Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), ARI-002 (Hospital Clinic Barcelona, IDIBAPS), and CTX120 (CRISPR Therapeutics).
  • CARs include the CARs of idecabtagene vicleucel, ABECMA®, BCMA02, JCARH125, JNJ-68284528 (LCAR-
  • the CAR is the CAR of idecabtagene vicleucel cells.
  • the CAR is the CAR of ABECMA® cells (cells used in ABECMA® immunotherapy).
  • the CAR is the CAR of ciltacabtagene autoleucel cells.
  • the CAR is the CAR of CARVYKTITM cells (cells used in CARVYKTITM immunotherapy ).
  • the recombinant receptor such as the CAR further includes a spacer between the antigen-recognition component, e.g., scFv, and transmembrane domain, which may be or include a hinge region.
  • the spacer is a CD8 hinge sequence.
  • the hinge region is a portion of the Fc of an immunoglobulin, such as an IgGl or IgG4.
  • the spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer.
  • Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain.
  • Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol Res. 3(2): 125-135 or international patent application publication number WO2014031687.
  • the CAR contains a hinge-containing immunoglobulin spacer between the scFv and the transmembrane domain.
  • the spacer is set forth in SEQ ID NO:29.
  • the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain.
  • the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain.
  • the intracellular signaling domain comprises an IT AM.
  • the antigen recognition domain e.g. extracellular domain
  • the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain.
  • the antigen-binding component e.g., antibody
  • the antigen-binding component is linked to one or more transmembrane and intracellular signaling domains.
  • a transmembrane domain that naturally is associated with one of the domains in the receptor e.g., CAR
  • the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (z.e.
  • the transmembrane domain in some embodiments is synthetic.
  • the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • the linkage is by linkers, spacers, and/or transmembrane domain(s).
  • the transmembrane domain contains a transmembrane portion of CD28 or a variant thereof.
  • the extracellular domain and transmembrane can be linked directly or indirectly.
  • the extracellular domain and transmembrane are linked by a spacer, such as any described herein.
  • the transmembrane domain of the receptor e.g., the CAR
  • the transmembrane domain of the receptor is a transmembrane domain of a human CD28.
  • the transmembrane domain has the sequence set forth in SEQ ID NO:30.
  • the transmembrane domain has a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:30.
  • the transmembrane domain has the sequence set forth in SEQ ID NO:31.
  • the transmembrane domain has a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:31.
  • the transmembrane domain of the receptor e.g., the CAR
  • the transmembrane domain of a human CD8a is a transmembrane domain of a human CD8a.
  • the recombinant receptor e.g., CAR
  • the recombinant receptor includes at least one intracellular signaling component or components, such as an intracellular signaling region or domain.
  • T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).
  • the CAR includes one or both of such signaling components.
  • a short oligo- or polypeptide linker for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.
  • IT AM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon.
  • cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
  • the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain.
  • the antigen-binding portion is linked to one or more cell signaling modules.
  • cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains.
  • the receptor e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor y, CD8alpha, CD8beta, CD4, CD25, or CD16.
  • the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-Q or Fc receptor y and CD8alpha, CD8beta, CD4, CD25 or CD 16.
  • the intracellular (or cytoplasmic) signaling region comprises a human CD3 chain, optionally a CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3 ⁇ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993.
  • the CD3-zeta domain has the sequence set forth in SEQ ID NO: 32.
  • the CD3zeta signaling domain has a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • full activation In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal.
  • a component for generating secondary or co-stimulatory signal is also included in the CAR.
  • the CAR does not include a component for generating a costimulatory signal.
  • an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
  • the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule.
  • the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40 (CD134), CD27, DAP10, DAP12, ICOS and/or other costimulatory receptors.
  • the CAR includes a costimulatory region or domain of CD28 or 4-1BB, such as of human CD28 or human 4-1BB.
  • the intracellular signaling region or domain comprises an intracellular costimulatory signaling domain of human CD28 or functional variant or portion thereof, such as a 41 amino acid domain thereof and/or such a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein.
  • the same CAR includes both the primary (or activating) cytoplasmic signaling regions and costimulatory signaling components.
  • the 4-1BB costimulatory signaling domain has the sequence set forth in SEQ ID NO:33. In some embodiments, the 4-1BB costimulatory signaling domain has a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:33.
  • nucleic acid molecules encoding such CAR constructs further includes a sequence encoding a 2A ribosomal skip element separating the CAR from a downstream sequence, such as another transgene sequence encoding a surrogate marker sequence (e.g., truncated EGFR sequence), an immunomodulatory or cytokine sequence or another CAR sequence.
  • a 2A ribosomal skip element separating the CAR from a downstream sequence, such as another transgene sequence encoding a surrogate marker sequence (e.g., truncated EGFR sequence), an immunomodulatory or cytokine sequence or another CAR sequence.
  • a surrogate marker sequence e.g., truncated EGFR sequence
  • an immunomodulatory or cytokine sequence e.g., cytokine sequence
  • Many 2A elements are known. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein, without limitation, 2A sequences from the foot-
  • 2A sequences that can be used in the methods and system disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO:34), equine rhinitis A virus (E2A, e.g., SEQ ID NO:9), Thosea asigna virus (T2A, e.g., SEQ ID NO: 35 or 36), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 37 or 38) as described in U.S. Patent Publication No. 20070116690.
  • F2A foot-and-mouth disease virus
  • E2A equine rhinitis A virus
  • T2A e.g., SEQ ID NO: 35 or 36
  • P2A porcine teschovirus-1
  • the cells are genetically engineered to express a recombinant receptor.
  • the engineering is carried out by introducing polynucleotides that encode the recombinant receptor.
  • polynucleotides encoding a recombinant receptor and vectors or constructs containing such nucleic acids and/or polynucleotides.
  • the nucleic acid sequence encoding the recombinant receptor contains a signal sequence that encodes a signal peptide.
  • the signal sequence may encode a signal peptide derived from a native polypeptide.
  • the nucleic acid sequence encoding the recombinant receptor e.g., chimeric antigen receptor (CAR) contains a signal sequence that encodes a signal peptide.
  • the polynucleotide encoding the recombinant receptor contains at least one promoter that is operatively linked to control expression of the recombinant receptor. In some examples, the polynucleotide contains two, three, or more promoters operatively linked to control expression of the recombinant receptor.
  • recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV).
  • recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy, 2014 Apr 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp.
  • the viral vector is an adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV) or spleen focus forming virus (SFFV).
  • LTR long terminal repeat sequence
  • MoMLV Moloney murine leukemia virus
  • MPSV myeloproliferative sarcoma virus
  • MSV murine embryonic stem cell virus
  • MSCV murine stem cell virus
  • SFFV spleen focus forming virus
  • retroviral vectors are derived from murine retroviruses.
  • the retroviruses include those derived from any avian or mammalian cell source.
  • the retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans.
  • the gene to be expressed replaces the retroviral gag, pol and/or env sequences.
  • retroviral systems A number of illustrative retroviral systems have been described e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
  • the engineered cells are produced by a process that generates an output composition of enriched cells (e.g., T cells) from one or more input compositions and/or from a single biological sample.
  • the output composition contains cells that express a recombinant receptor, e.g., a CAR.
  • the cells of the output compositions are suitable for administration to a subject as a therapy, e.g., an autologous cell therapy.
  • the output composition is a composition of enriched CD3+ T cells, or enriched CD4+ and CD8+ T cells.
  • the T cells are engineered by methods that involve introduction of a nucleic acid encoding the CAR into cells under conditions in which the nucleic acid is integrated into the genome of the cells.
  • the engineering methods include transduction with viral vectors, such as lenti viral vectors.
  • the process for generating or producing engineered cells is by a process that includes some or all of the steps of: collecting or obtaining a biological sample; isolating, selecting, or enriching input cells from the biological sample; cryopreserving and storing the input cells; thawing and/or incubating the input cells under stimulating conditions; engineering the stimulated cells to express or contain a recombinant polynucleotide, e.g., a polynucleotide encoding a recombinant receptor such as a CAR; cultivating the engineered cells, e.g., to a threshold amount, density, or expansion; formulating the cultivated cells in an output composition; and/or cry opreserving and storing the formulated output cells until the cells are released for infusion and/or are suitable to be administered to a subject.
  • a recombinant polynucleotide e.g., a polynucleotide encoding a recombinant receptor such as a CAR
  • engineered cells used in accord with the provided methods and uses are produced or generated by exemplary processes as described in, for example, PCT/US2019/046062, PCT/US2019/046048, PCT/EP2024/052653, PCT/US2024/026349, WO 2019/089855, WO 2015/164675, WO 2019/113557, WO 2020/033927, WO 2023/147515, and WO 2024/100604, each of which is incorporated by reference in their entirety.
  • the cells for introduction of the nucleic acid encoding the transgenic receptor such as the CAR may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • a sample such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells in some embodiments are primary cells, e.g., primary human cells.
  • the samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample is blood or a blood-derived sample, or is derived from an apheresis or leukapheresis product.
  • exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
  • Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis.
  • the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
  • the sample containing cells e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product
  • PBMC peripheral blood mononuclear cells
  • an unfractionated T cell sample e.g., a lymphocyte sample
  • a white blood cell sample e.g., an apheresis product, or a leukapheresis product
  • cryopreserved and/or cryoprotected e.g., frozen
  • an apheresis product or a leukapheresis product is cryopreserved and/or cryoprotected (e.g., frozen) and then thawed before being subject to a cell selection or isolation step (e.g., a T cell selection or isolation step).
  • a cell selection or isolation step e.g., a T cell selection or isolation step.
  • an apheresis product or a leukapheresis product is cryopreserved and/or cryoprotected (e.g., frozen).
  • the cry opreservation solution or buffer is or contains, for example, a DMSO solution optionally comprising human serum albumin (HSA), or other suitable cell freezing media.
  • HSA human serum albumin
  • selection, isolation, or enrichment of the cells or populations includes one or more preparation and/or non-affinity based cell separation steps.
  • cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents.
  • cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
  • the selection step includes incubation of cells with a selection reagent.
  • the incubation with a selection reagent or reagents e.g., as part of selection methods which may be performed using one or more selection reagents for selection of one or more different cell types based on the expression or presence in or on the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid.
  • surface markers e.g., surface proteins, intracellular markers, or nucleic acid.
  • any known method using a selection reagent or reagents for separation based on such markers may be used.
  • the selection reagent or reagents result in a separation that is affinity- or immunoaffinity-based separation.
  • the selection in some aspects includes incubation with a reagent or reagents for separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • a reagent or reagents for separation of cells and cell populations based on the cells’ expression or expression level of one or more markers typically cell surface markers
  • an antibody or binding partner that specifically binds to such markers
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation.
  • the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
  • the separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
  • positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.
  • negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
  • multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.
  • a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection.
  • multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
  • specific subpopulations of T cells such as cells positive or expressing high levels of one or more surface markers, e.g., CD3+, CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + , and/or CD45RO + T cells, are isolated by positive or negative selection techniques.
  • a CD4 + or CD8 + selection step is used to separate CD4 + helper and CD8 + cytotoxic T cells.
  • Such CD4 + and CD8 + populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
  • isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection.
  • positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker + ) at a relatively higher level (marker hlgh ) on the positively or negatively selected cells, respectively.
  • a biological sample e.g., a sample of PBMCs or other white blood cells
  • the selection results in an enriched composition of input cells in which at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in the composition are T cells.
  • a biological sample e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD3+ T cells.
  • the selection results in an enriched composition of input cells in which at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in the composition are CD3+ T cells.
  • a biological sample e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD4+ T cells and CD8+ T cells.
  • the selection results in an enriched composition of input cells in which at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in the composition are CD4+ and CD8+ T cells.
  • a volume of cells is mixed with an amount of a desired affinity-based selection reagent.
  • the immunoaffinity-based selection can be carried out using any system or method that results in a favorable energetic interaction between the cells being separated and the molecule specifically binding to the marker on the cell, e.g., the antibody or other binding partner on the solid surface, e.g., particle.
  • methods are carried out using particles such as beads, e.g., magnetic beads, that are coated with a selection agent (e.g., antibody) specific to the marker of the cells.
  • the particles e.g., beads
  • a container such as a tube or bag
  • shaking or mixing with a constant cell density-to-particle (e.g., bead) ratio to aid in promoting energetically favored interactions.
  • the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection.
  • a solid support or matrix such as a magnetic bead or paramagnetic bead
  • the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher ⁇ Humana Press Inc., Totowa, NJ).
  • the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads).
  • the magnetically responsive material, e.g., particle generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
  • a binding partner e.g., an antibody
  • the incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
  • the antibodies or binding partners, or molecules such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
  • the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells.
  • positive selection cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained.
  • a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
  • the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient.
  • the magnetizable or magnetically responsive particles are removed from the cells.
  • Methods for removing magnetizable particles from cells include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers.
  • the magnetizable particles are biodegradable.
  • the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto.
  • MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered.
  • the non-target cells are labelled and depleted from the heterogeneous population of cells.
  • the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods.
  • the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination.
  • the system is a system as described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al.
  • cells are isolated, selected, or enriched by chromatographic isolation, such as by column chromatography including affinity chromatography or gel permeations chromatography.
  • the method employs a receptor binding reagent that binds to a receptor molecule that is located on the surface of a target cell, e.g., the cell to be isolated, selected, or enriched.
  • a receptor binding reagent that binds to a receptor molecule that is located on the surface of a target cell, e.g., the cell to be isolated, selected, or enriched.
  • Such methods may be described as (traceless) cell affinity chromatography technology (CATCH).
  • CATCH cell affinity chromatography technology
  • methods, techniques, and reagents for selection, isolation, and enrichment are described, for example, in WO2013124474 and WO2015164675, which are hereby incorporated by reference in their entirety.
  • a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream.
  • a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting.
  • a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. l(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
  • MEMS microelectromechanical systems
  • compositions or cells such as those enriched by any of the abovedescribed selection methods are incubated in the presence of stimulating conditions or a stimulatory agent prior to their transduction.
  • stimulating conditions include those designed to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
  • the stimulating conditions or stimulatory reagents include one or more reagent, e.g., ligand, which is capable of stimulating or activating an intracellular signaling domain of a TCR complex.
  • the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell, such as agents suitable to deliver a primary signal, e.g., to initiate activation of an IT AM-induced signal, such as those specific for a TCR component, e.g., anti-CD3, and/or an agent that promotes a costimulatory signal, such as one specific for a T cell costimulatory receptor, e.g., anti-CD28, or anti-4-lBB, for example, bound to solid support such as a bead, and/or one or more cytokines.
  • the stimulatory reagents are anti-CD3/anti-CD28 beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander,
  • the stimulatory reagent is a streptavidin-based oligomer, such as a streptavidin mutein oligomer, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fabs.
  • the oligomeric particle reagent is any as described in WO2015/158868 or WO2018/197949.
  • the cells are engineered by introduction, delivery or transfer of nucleic acid sequences that encode the recombinant receptor and/or other molecules.
  • methods for producing engineered cells includes the introduction of a polynucleotide encoding a recombinant receptor (e.g., CAR) into a cell, e.g., such as a stimulated or activated cell.
  • a recombinant receptor e.g., CAR
  • the recombinant receptor is a CAR, such as any described in Section III.A.
  • Any method of introducing a heterologous or recombinant polynucleotide that would result in integration of the polynucleotide encoding the recombinant receptor into the genome of a cell such as a T cell may be used, including viral and non-viral methods of genetic engineering.
  • Introduction of the nucleic acid molecules encoding the recombinant receptor in the cell may be carried out using any of a number of known vectors.
  • Such vectors include viral and non-viral systems, including lenti viral and gammaretroviral systems, as well as transposon-based systems such as PiggyBac or Sleeping Beautybased gene transfer systems.
  • Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
  • the engineering produces one or more engineered compositions of enriched T cells.
  • genetically engineering the cells is or includes introducing the polynucleotide, e.g., the heterologous or recombinant polynucleotide, into the cells by transduction.
  • the cells are transduced or subjected to transduction with a viral vector.
  • the cells are transduced or subjected to transduction with a viral vector.
  • the virus is a retroviral vector, such as a gammare tro viral vector or a lenti viral vector. Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother.
  • the provided methods include genetically engineering the cells, e.g., introducing a heterologous or recombinant polynucleotide encoding a recombinant protein, using a non- viral method, such as electroporation, calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection, nanoparticles such as lipid nanoparticles, tungsten particle-facilitated microparticle bombardment, strontium phosphate DNA co-precipitation, and other approaches described in, e.g., WO 2014055668, and U.S. Patent No. 7,446,190. Transposon-based systems also are contemplated.
  • a non- viral method such as electroporation, calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection, nanoparticles such as lipid nanoparticles, tungsten particle-facilitated microparticle bombardment, strontium phosphate DNA co-precipitation, and other approaches described in, e
  • the cells are engineered in the presence of one or more cytokines.
  • the one or more cytokines are recombinant cytokines.
  • the one or more cytokines are human recombinant cytokines.
  • the one or more cytokines bind to and/or are capable of binding to receptors that are expressed by and/or are endogenous to T cells.
  • the one or more cytokines is or includes a member of the 4-alpha-helix bundle family of cytokines.
  • members of the 4-alpha-helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL-15), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • cells e.g., stimulated cells are engineered under stimulating conditions in the presence of IE-2, IE-7, and/or IL-15.
  • the transduction is carried out by contacting one or more cells of a population with a nucleic acid molecule encoding the recombinant protein, e.g., recombinant receptor.
  • the contacting can be effected with centrifugation, such as spinoculation (e.g., centrifugal inoculation).
  • centrifugation such as spinoculation (e.g., centrifugal inoculation).
  • centrifugation such as spinoculation (e.g., centrifugal inoculation).
  • Such methods include any of those as described in International Publication Number W02016/073602.
  • Exemplary centrifugal chambers include those produced and sold by Biosafe SA, including those for use with the Sepax® and Sepax® 2 system, including an A-200/F and A-200 centrifugal chambers and various kits for use with such systems.
  • Exemplary chambers, systems, and processing instrumentation and cabinets are described, for example, in US Patent No. 6,123,655, US Patent No. 6,733,433 and Published U.S. Patent Application, Publication No.: US 2008/0171951, and published international patent application, publication no. WO 00/38762, the contents of each of which are incorporated herein by reference in their entirety.
  • Exemplary kits for use with such systems include, but are not limited to, single -use kits sold by BioSafe SA under product names CS-430.1, CS-490.1, CS-
  • the methods for generating the engineered cells include one or more steps for incubating or cultivating the cells.
  • an incubation on engineered cells is carried out under conditions that do not promote proliferation and/or expansion.
  • the engineered cells are incubated or cultivated under conditions for expansion of cells.
  • the incubation is performed under static conditions, such as conditions that do not involve centrifugation, shaking, rotating, rocking, or perfusion, e.g., continuous or semi-continuous perfusion of the media.
  • the cells produced by the manufacturing method are harvested by collecting the cells, including optionally after multiple washings to remove any reagents used during the manufacturing process.
  • the cells that are harvested are formulated, such as in a pharmaceutically acceptable buffer for use as a therapeutic cell composition.
  • the cells that are harvested are formulated in the presence of a cryoprotectant for cryopreservation of the cells prior to their use or analysis in the provided methods.
  • cells can be formulated into a container, such as a bag or vial.
  • the vial may be an infusion vial.
  • the vial is formulated with a single unit dose of the engineered cells, such as including the number of cells for administration in a given dose or fraction thereof.
  • the cells are formulated in a pharmaceutically acceptable buffer, which may, in some aspects, include a pharmaceutically acceptable carrier or excipient.
  • the processing includes exchange of a medium into a medium or formulation buffer that is pharmaceutically acceptable or desired for administration to a subject.
  • the processing steps can involve washing the transduced and/or expanded cells to replace the cells in a pharmaceutically acceptable buffer that can include one or more optional pharmaceutically acceptable carriers or excipients.
  • Exemplary of such pharmaceutical forms, including pharmaceutically acceptable carriers or excipients can be any described below in conjunction with forms acceptable for administering the cells and compositions to a subject.
  • the pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations.
  • the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3 -pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • Buffering agents in some aspects are included in the compositions.
  • Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts.
  • a mixture of two or more buffering agents is used.
  • the buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition.
  • Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
  • the formulations can include aqueous solutions.
  • the formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • chemotherapeutic agents e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • the agents or cells are administered in the form of a salt, e.g., a pharmaceutically acceptable salt.
  • Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
  • the formulation buffer contains a cryopreservative.
  • such cells produced by the method, or a composition comprising such cells are administered to a subject for treating a disease or condition.
  • provided herein are methods comprising manufacturing or producing engineered cells (e.g., CAR-T cells) in the presence of a modulating agent, thereby improving the persistence, lack of exhaustion, and/or efficacy of the engineered cells manufactured or produced by the methods.
  • manufacturing or producing engineered cells (e.g., CAR-T cells) in the presence of the modulating agent increases exosome production and secretion by the engineered cells.
  • the modulating agent increases exosome production, wherein the produced exosomes are cell-derived particles that are surface positive for CD63.
  • the modulating agent increases exosome production, wherein the produced exosomes are cell-derived particles that are greater than about 30 nm in diameter and less than about 100 nm in diameter, and that are surface positive for CD63.
  • the provided methods produce compositions of cells that include primary T cells engineered to express a recombinant receptor, such as for use in cell therapy, that (i) contain fewer exhausted cells and/or fewer cells that display markers or phenotypes associated with exhaustion; (ii) contain an increased percentage of memory-like T cells, such as long-lived memory T cells; (iii) are less differentiated; (iv) exhibit improved or enhanced survival, expansion, persistence, and/or anti-tumor activity; (v) exhibit improved therapeutic efficacy; (vi) exhibit improved clinical durability of response, as compared to compositions of engineered cells that are produced by alternative methods, such as alternative methods that are not carried out in the presence of the modulating agent; and/or exhibit increased exosome production and secretion.
  • the modulating agent is in contact with the cells or cell populations (e.g., the modulating agent is in a cell or interacts with one or more cell surface molecule) prior to collecting, harvesting, or formulating the cells.
  • the modulating agent is present prior to, during, or after the cells are subjected to stimulation, e.g., T cell activation.
  • the modulating agent is in contact with the cells or cell populations (e.g., the modulating agent is in a cell or interacts with one or more cell surface molecule) prior to or during the stimulation, e.g., a stimulation described herein.
  • the modulating agent is present prior to, during, or after the cells are subjected to engineering, e.g., transduction.
  • the modulating agent is in contact with the cells or cell populations (e.g., the modulating agent is in a cell or interacts with one or more cell surface molecule) prior to or during the engineering, e.g., an engineering described herein.
  • the modulating agent is in contact with the cells or cell populations (e.g., the modulating agent is in a cell or interacts with one or more cell surface molecule) during or after the incubation, e.g., an incubation described herein.
  • the modulating agent is in contact with the cells or cell populations (e.g., the modulating agent is in a cell or interacts with one or more cell surface molecule) during the stimulation (e.g., a stimulation described herein.), during the engineering (an engineering described herein), and/or during the incubation (e.g., an incubation described herein such as in).
  • the cells or cell population undergoes a process, procedure, step, or technique in the presence of the modulating agent after the incubation but prior to steps for collecting, harvesting, or formulating the cells.
  • the cells or cell population undergoes a process, procedure, step, or technique in the presence of the modulating agent after the incubation.
  • cells to be engineered are contacted (e.g., incubated) with the modulating agent, e.g. in a culture media, prior to the engineering.
  • the cells are engineered in the presence of the modulating agent.
  • one or more engineered cells are contacted (e.g., incubated) with the modulating agent, e.g. in a culture media such as a basal medium without one or more recombinant cytokines or without any recombinant cytokine.
  • compositions during the manufacture or production of engineered cells that comprise (i) the modulating agent and (ii) cells to be engineered and/or cells that have been subjected to engineering (including engineered cells), such as primary immune cells (e.g., T cells).
  • engineered cells such as primary immune cells (e.g., T cells).
  • the modulating agent is selected from the group consisting of a PI3K inhibitor, an Akt pathway, an mTOR inhibitor, a Ras/ERK inhibitor, an NF-KB inhibitor, a BET inhibitor, a CDK inhibitor, a CRAC channel inhibitor, a Cox inhibitor, a dopamine antagonist, an ERK5 inhibitor, a glucocorticoid, an IGF-1R inhibitor, an IKK inhibitor, a JAK inhibitor, Lek inhibitor, a PDK1 inhibitor, a Raf inhibitor, and a Syk inhibitor.
  • the Src inhibitors include, but are not limited to dasatinib, saracatinib, bosutinib, KX01, and rebastinib (DCC-2036). In some embodiments, the Src inhibitor comprises rebastinib (DCC-2036). Certain agents useful as the modulating agent of the present disclosure are disclosed in W02019018603, WO2018106595, and PCT/US2018/058812, all of which are incorporated herein by reference in the entirety.
  • the modulating agent is or comprises a compound, a small molecule, e.g., small organic molecule, a polynucleotide, an oligonucleotide, an siRNA, or a polypeptide, or a fragment, isoform, variant, analog, or derivative thereof that inhibits, reduces, prevents, and/or is capable of inhibiting, reducing, or preventing, one or more activities of the target such as mTOR.
  • a small molecule e.g., small organic molecule, a polynucleotide, an oligonucleotide, an siRNA, or a polypeptide, or a fragment, isoform, variant, analog, or derivative thereof that inhibits, reduces, prevents, and/or is capable of inhibiting, reducing, or preventing, one or more activities of the target such as mTOR.
  • the agent is a small molecule with a molecular weight of less than 10 kD, less than 9 kD, less than 8 kD, less than 7 kD, less than 6 kD, less than 5 kD, less than 4 kD, less than 3 kD, less than 2 kD, less than 1 kD, less than 0.5 kD, or less than 0.1 kD.
  • the modulating agent is or comprises an agent that inhibits mTOR activity.
  • cells to be engineered e.g. transduced
  • the cells are engineered in the presence of an mTOR inhibitor.
  • one or more engineered cells are contacted (e.g., incubated) with an mTOR inhibitor, e.g. in a culture media such as a basal medium without one or more recombinant cytokines or without any recombinant cytokine.
  • compositions during the manufacture or production of engineered cells that comprise (i) an mTOR inhibitor and (ii) cells to be engineered and/or cells that have been subjected to engineering (including engineered cells).
  • an agent that inhibits mTOR activity inhibits, reduces, and/or decreases, and/or is capable of inhibiting, reducing, and/or decreasing at least one activity of mTOR.
  • an agent that inhibits mTOR activity inhibits, reduces, and/or decreases, and/or is capable of inhibiting, reducing, and/or decreasing an mTOR kinase activity.
  • an agent that inhibits mTOR activity inhibits, reduces, and/or decreases, and/or is capable of inhibiting, reducing, and/or decreasing an mTORCl activity, e.g., an mTORCl kinase activity, and/or an mTORC2 activity.
  • the agent that inhibits mTOR activity prevents the formation of and/or destabilizes the mTORCl complex. In particular embodiments, the agent that inhibits activity prevents the formation of and/or destabilizes the mTORC2 complex.
  • the agent that inhibits mTOR activity inhibits the activity of at least one additional kinase.
  • the at least one additional kinase is PI3K.
  • the agent that inhibits mTOR activity : (i) does not inhibit PI3K activity; (ii) does not detectably inhibit PI3K activity at the IC50 for mTOR activity; and/or (iii) does not detectably inhibit PI3K at all concentrations that detectably inhibit mTOR activity.
  • the agent that inhibits mTOR activity inhibits, e.g., selectively inhibits, mTORCl and mTORC2 kinase activity relative to PI3K activity.
  • the agent that inhibits mTOR activity inhibits mTORCl and mTORC2 kinase activity.
  • the agent that inhibits mTOR activity selectively inhibits mTORCl activity, such as the mTORCl kinase activity.
  • the agents that inhibit mTOR activity include, but are not limited to, CC214-1 (Celgene), CC214-2 (Celgene), CC0470324, GDC0980, , SAR245409, VS5584, PI-103, SF1126, BGT226, XL765, PF-04691502, Dactolisib (codenamed NVP-BEZ235 and BEZ-235), a pyrazolopyrimidine, Torin 1, Torkinib (PP242), PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth), DS3078a, rapamycin (sirolimus), temsirolimus (CC1779), everolimus (RAD001), deforolimus (AP23573), AZD8055 (AstraZeneca), and OSI-027 (OSI).
  • the agent include, but are not limited to, CC214
  • the agent comprises a formula set forth in Formula (I),
  • R 1 is substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl
  • R 2 is substituted or unsubstituted Ci-salkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl
  • R 3 and R 4 are independently H or Ci-8 alkyl.
  • the agent that inhibits mTOR activity is or comprises a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the agent that inhibits mTOR activity is or comprises a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the agent that inhibits mTOR activity is or comprises 2-(3-hydroxyphenyl)-9-(2-isopropylphenyl)-8-oxo-8,9- dihydro-7H-purine-6-carboxamide, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the agent that inhibits mTOR activity is or comprises ' , or a pharmaceutically acceptable salt thereof.
  • the agent comprises a formula set forth in Formula (II), Formula (II) wherein L is a direct bond, NH or O,Y is N or CR 3 , wherein R 1 is H, substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted C2-8 alkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl or substituted or unsubstituted heterocycloalkyl, R 2 is H, substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl, R 3 is H, substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted C2-8 alkenyl
  • the agent that inhibits mTOR activity is or comprises a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof.
  • the agent that inhibits mTOR activity is or comprises 6-(4-(2H-l,2,4-triazol-3-yl)phenyl)- l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-lH-imidazo [4,5-b]pyrazine-2(3H)-one, or a pharmaceutically acceptable salt or solvate thereof.
  • the agent that inhibits mTOR activity is or comprises pharmaceutically acceptable salt thereof.
  • the agent comprises a formula set forth in Formula (III),
  • R 1 is substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heterocyclylalkyl
  • R 2 is H, substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aralkyl, or substituted or unsubstituted cycloalkylalkyl
  • R 3 is H, or a substituted or unsubstituted Ci-8 alkyl.
  • R 1 is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In some embodiments, R 1 is pyridyl that is substituted. In some embodiments, the agent that inhibits mTOR activity is or comprises a compound of Formula (III), or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the agent that inhibits mTOR activity is or comprises a compound of Formula (III), or a pharmaceutically acceptable salt thereof.
  • the agent that inhibits mTOR activity is or comprises 7-(6-(2-hydroxypropan-2-yl)pyridin- 3-yl)-l-((lr,4r)-4-methoxycyclohexyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one, or a pharmaceutically acceptable salt or solvate thereof.
  • the agent that inhibits mTOR activity is or comprises pharmaceutically acceptable salt thereof.
  • analogues or derivatives of all other agents functionally categorized under their respective class based on their targets which analogues or derivatives include, but are not limited to, salt, ester, ether, solvate, hydrate, stereoisomer or prodrug.Definitions
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length.
  • Polypeptides including the provided receptors and other polypeptides, e.g., linkers or peptides, may include amino acid residues including natural and/or non-natural amino acid residues.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, and phosphorylation.
  • the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • rlgG recombinant IgG
  • scFv single chain variable fragments
  • single domain antibodies e.g., sdAb, sdFv, nanobody
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs.
  • FRs conserved framework regions
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a Vnor VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).
  • an “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
  • a “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all framework regions (FRs) amino acid residues are derived from human FRs.
  • the humanized forms of a non-human antibody e.g., a murine antibody, are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulin.
  • the humanized antibodies are antibodies from non-human species having one or more complementarity determining regions (CDRs) from the non- human species and a framework region (FR) from a human immunoglobulin molecule.
  • a humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, in this instance, the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen.
  • a monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.
  • treatment refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
  • delay development of a disease means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as severe refractory SLE). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. In some embodiments, sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
  • Preventing includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease.
  • the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.
  • to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, cells that suppress or reduce immune activity compared to the absence of the cells.
  • an “effective amount” of an agent refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
  • a “therapeutically effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment.
  • the therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered.
  • the provided methods involve administering the cells and/or compositions at effective amounts, e.g., therapeutically effective amounts.
  • composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, nonaqueous or any combination thereof.
  • enriching when referring to one or more particular cell type or cell population, refers to increasing the number or percentage of the cell type or population, e.g., compared to the total number of cells in or volume of the composition, or relative to other cell types, such as by positive selection based on markers expressed by the population or cell, or by negative selection based on a marker not present on the cell population or cell to be depleted.
  • the term does not require complete removal of other cells, cell type, or populations from the composition and does not require that the cells so enriched be present at or even near 100% in the enriched composition.
  • a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker.
  • a surface marker refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
  • FMO fluorescence minus one
  • a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker.
  • a surface marker refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
  • FMO fluorescence minus one
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • response vectorse or “responsiveness” refers to a beneficial response of a subject (for instance, a partial or complete response) to treatment with a cell therapy.
  • the term “partial response” is a decrease in disease burden (for instance, a decrease in the size of a tumor or in the amount of cancer in the body), such as at least 50% smaller than it was before treatment, but in which the cancer is still present.
  • the term “complete response” is the disappearance of all signs of the disease or condition (for instance, cancer) in the body.
  • the term “unresponsiveness” refers to a subject that has stable disease or progressive disease after receiving a cell therapy.
  • the term “stable disease” refers to a disease or condition that is neither decreasing nor increasing in extent or severity.
  • the term “progressive disease” refers to a disease or condition whose course is worsening, such as growing or metastasizing in the context of a cancer or tumor.
  • Embodiment 1 A method of assessing exosome production by a cell therapy composition, the method comprising:
  • Embodiment 2 The method of embodiment 1 , wherein the cell therapy composition is a treatment or a candidate for a treatment to be administered to a subject.
  • Embodiment 3 The method of embodiment 2, wherein the method is for predicting a response to or efficacy of the cell therapy composition in the subject to which it is administered.
  • Embodiment 4 The method of embodiment 2 or embodiment 3, wherein an increase in the amount or concentration of the isolated exosomes compared to an amount or concentration from a control cell composition predicts a response to the cell therapy composition when it is administered to the subject.
  • Embodiment 5. The method of any one of embodiments 2 to 4, wherein the cell therapy composition comprises cells obtained from the subject.
  • Embodiment 6 A method comprising:
  • Embodiment 7 A method comprising:
  • Embodiment 8 A method comprising :
  • the subject is selected for treatment with the cell therapy composition if the amount or concentration of the isolated exosomes in the stimulated cell composition is increased compared to the amount or concentration of the isolated exosomes in the control cell composition; or (ii) the subject is selected for treatment with the cell therapy composition in combination with an other agent if the amount or concentration of the isolated exosomes in the stimulated cell composition is decreased or unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition.
  • Embodiment 11 A method comprising:
  • Embodiment 12 A method of treatment comprising :
  • Embodiment 13 A method of treatment comprising :
  • Embodiment 14 A method of treatment comprising administering a cell therapy composition for treating a disease or condition in a subject, wherein the subject for treatment is selected by a method comprising:
  • Embodiment 15 A method of treatment comprising:
  • Embodiment 16 A method of treatment comprising administering a cell therapy composition in combination with an other agent for treating a disease or condition in a subject, wherein the subject for treatment is selected by a method comprising:
  • Embodiment 17 A method of treatment, the method comprising administering a cell therapy composition comprising a T cell therapy to a subject having a disease or condition, wherein the T cell therapy is a composition comprising T cells engineered to express a recombinant receptor, and wherein the subject has an increased amount or concentration of exosomes produced from the composition after ex vivo stimulation of the T cells of the composition with a recombinant receptor-stimulating agent.
  • the T cell therapy is a composition comprising T cells engineered to express a recombinant receptor
  • the subject has an increased amount or concentration of exosomes produced from the composition after ex vivo stimulation of the T cells of the composition with a recombinant receptor-stimulating agent.
  • a method of treatment comprising administering a cell therapy composition comprising a T cell therapy to a subject having a disease or condition, wherein the T cell therapy comprises T cells engineered to express a recombinant receptor, and wherein the subject is a subject that has been selected for treatment by the method of any one of embodiments 9-11.
  • Embodiment 19 A method of adaptive treatment with a cell therapy composition comprising a T cell therapy in a subject having a disease or condition, the method comprising:
  • T cell composition comprises T cells engineered to express a recombinant receptor and is a T cell therapy treatment or is a candidate for a T cell therapy treatment to be administered to the subject, wherein the amount or concentration of isolated exosomes is determined by a method comprising:
  • the subject is predicted to respond to the T cell therapy if the amount or concentration of the isolated exosomes in the stimulated cell composition is increased compared to the amount or concentration of the isolated exosomes in a control cell composition, or
  • the subject is not predicted to respond to the T cell therapy if the amount or concentration of the isolated exosomes in the stimulated cell composition is decreased or is unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition;
  • Embodiment 20 A method of adaptive treatment with a cell therapy composition comprising a T cell therapy in a subject having a disease or condition, the method comprising: (a) determining an amount or concentration of isolated exosomes produced from a T cell composition, wherein the T cell composition comprises T cells engineered to express a recombinant receptor and is a T cell therapy treatment or is a candidate for a T cell therapy treatment to be administered to the subject, wherein the amount or concentration of isolated exosomes is determined by a method comprising:
  • Embodiment 21 A method of adaptive treatment , comprising administering a cell therapy composition comprising a T cell therapy to a subject having a disease or condition predicted to respond with a response to the T cell therapy based on an amount of concentration of isolated exosomes being increased in a T cell composition compared to an amount or concentration of the isolated exosomes in a control cell composition, wherein the T cell composition comprises T cells engineered to express a recombinant receptor and is the T cell therapy or is a candidate for a T cell therapy treatment to be administered to the subject, and wherein the amount or concentration of the isolated exosomes produced from the T cell composition is determined by:
  • Embodiment 22 The method of any one of embodiments 1 to 16, wherein the cell therapy composition comprises T cells engineered to express a recombinant receptor.
  • Embodiment 23 The method of any one of embodiments 19 to 22, wherein the isolated exosomes are further characterized for presence of the recombinant receptor.
  • Embodiment 24 The method of any one of embodiments 1 to 16, wherein the cell therapy composition is a treatment or a candidate for a treatment to be administered to a subject.
  • Embodiment 25 The method of any one of embodiments 17 to 24, wherein the T cells are primary cells.
  • Embodiment 26 The method of any one of embodiments 17 to 25, wherein the T cells are autologous cells.
  • Embodiment 27 The method of any one of embodiments 17 to 25, wherein the T cells are allogeneic cells.
  • Embodiment 28 The method of any one of embodiments 17 to 27, wherein the T cells are CD3+.
  • Embodiment 29 The method of any of embodiments 17 to 28, wherein the T cells are CD4+, and/or CD8+ T cells.
  • Embodiment 30 The method of any one of embodiments 17 to 29, wherein the T cells of the cell therapy composition or T cell composition comprise CD4+ T cells and CD8+ T cells.
  • Embodiment 31 The method of any one of embodiments 1 to 30, wherein the cells of the cell therapy composition are at or greater than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% T cells.
  • Embodiment 32 The method of any one of embodiments 17 to 31, wherein at or greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the cells of the cell therapy composition express the recombinant receptor.
  • Embodiment 33 The method of any one of embodiments 1 to 32, wherein the cell therapy composition had been cryopreserved and is thawed prior to detecting exosomes produced from cells of the stimulated cell composition.
  • Embodiment 34 The method of any one of embodiments 17 to 33, wherein the cell therapy composition has been produced by a manufacturing process comprising: enriching primary T cells from a biological sample from a human subject to produce a population of input cells; activating the cells of the population of input cells with a stimulatory reagent; and during or subsequent to activating the cells of the population of input cells, introducing into cells a polynucleotide encoding the recombinant receptor.
  • Embodiment 35 The method of embodiment 34, wherein introducing the polynucleotide encoding the recombinant receptor comprises transducing cells with a viral vector encoding the recombinant receptor.
  • Embodiment 36 The method of embodiment 34 or embodiment 35, wherein the stimulatory reagent comprises an anti-CD3 antibody and an anti-CD28 antibody, and optionally culturing cells of the population of input cells in a culture medium containing one or more recombinant cytokines selected from IL-2, IL- 15, IL-7 and IL-21.
  • Embodiment 37 The method of any one of embodiments 34 to 36, wherein the manufacturing process further comprises culturing cells introduced with the polynucleotide under conditions for expansion of T cells in the composition.
  • Embodiment 38 The method of any one of embodiments 34 to 37, wherein the biological sample comprises a whole blood sample, a huffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.
  • the biological sample comprises a whole blood sample, a huffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.
  • PBMC peripheral blood mononuclear cell
  • Embodiment 39 The method of any one of embodiments 34 to 38 wherein the biological sample is an apheresis product or leukapheresis product.
  • Embodiment 40 The method of embodiment 39, wherein the apheresis product or leukapheresis product has been previously cryopreserved.
  • Embodiment 41 The method of any one of embodiments 34 to 40, wherein the population of input cells enriched from the biological sample comprises at or greater than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% T cells.
  • Embodiment 42 The method of any of embodiments 34 to 41, wherein the human subject has a disease or condition.
  • Embodiment 43 The method of any one of embodiments 34 to 41, further comprising harvesting cells produced by the manufacturing process and formulating the harvested cells in a pharmaceutically acceptable buffer.
  • Embodiment 44 The method of embodiment 43, wherein the harvested cells further comprises a cryoprotectant.
  • Embodiment 45 The method of any one of embodiments 6 to 44, wherein the disease or condition is a cancer or tumor.
  • Embodiment 46 The method of any one of embodiments 6 to 44, wherein the disease or condition is an autoimmune disease or condition.
  • Embodiment 47 The method of any one of embodiments 3 to 6 and 19-21, wherein the response comprises a partial response.
  • Embodiment 48 The method of any one of embodiments 3 to 6 and 19-21, wherein the response comprises a complete response.
  • Embodiment 49 The method of any one of embodiments 3 to 6 and 19-21, wherein the response does not comprise stable disease.
  • Embodiment 50 The method of any one of embodiments 3 to 6 and 19-21, wherein the response does not comprise progressive disease.
  • Embodiment 51 The method of any one of embodiments 4 to 50, wherein the amount or concentration of the isolated exosomes in the stimulated cell composition is increased about 1-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold compared to the amount or concentration of the isolated exosomes in the control cell composition.
  • Embodiment 52 The method of any one of embodiments 1 to 51 , wherein detecting comprises isolating the exosomes.
  • Embodiment 53 The method of embodiment 52, wherein isolating is by centrifugation.
  • Embodiment 54 The method of embodiment 53, wherein the centrifugation comprises at least one spin.
  • Embodiment 55 The method of embodiment 53 or embodiment 54, wherein the centrifugation is performed for about 1 to 20 minutes.
  • Embodiment 56 The method of any one of embodiments 53 to 55, wherein the centrifugation comprises one spin at 300 g for 3 min.
  • Embodiment 57 The method of any one of embodiments 53 to 56, wherein the centrifugation comprises one spin at 2,500 g for 15 min.
  • Embodiment 58 The method of any one of embodiments 53 to 57, wherein the centrifugation comprises two spins at 2,500 g for 15 min.
  • Embodiment 59 The method of any one of embodiments 53 to 58, wherein the centrifugation comprises three spins comprising one spin at 300 g for 3 min and two spins at 2,500 g for 15 min.
  • Embodiment 60 The method of any one of embodiments 1 to 59, wherein detecting is by:
  • Embodiment 61 The method of any one of embodiments 1 to 60, wherein detecting is by:
  • Embodiment 62 The method of embodiment 60 or embodiment 61, wherein the identifying cell particles is by identifying cell particles that have a size between about 30 nm to about 130 nm.
  • Embodiment 63 The method of any one of embodiments 60 to 62, wherein the identifying cell particles is by identifying cell particles that have a size between about 30 nm to about 100 nm.
  • Embodiment 64 The method of embodiment 60, wherein the one or more exosome markers are identified by immunoaffinity-based capture.
  • Embodiment 65 The method of embodiment 64, wherein the immunoaffinity-based capture comprises an antibody specific to the one or more exosome markers.
  • Embodiment 66 The method of any one of embodiments 60 to 65, wherein the one or more exosome markers are selected from CD63, CD81, CD9, and any combination thereof.
  • Embodiment 67 The method of embodiment 66, wherein the one or more exosome markers is CD63.
  • Embodiment 69 The method of embodiment 66, wherein the one or more exosome markers is CD9.
  • Embodiment 70 The method of embodiment 66, wherein the one or more exosome markers comprises CD63 and CD8L
  • Embodiment 71 The method of embodiment 66, wherein the one or more exosome markers comprises CD63 and CD9.
  • Embodiment 72 The method of embodiment 66, wherein the one or more exosome markers comprises CD81 and CD9.
  • Embodiment 73 The method of embodiment 66, wherein the one or more exosome markers comprises CD63, CD81 and CD9.
  • Embodiment 74 The method of any one of embodiments 60 to 73, wherein the exosomes have a size between about 30 nm and 150 nm.
  • Embodiment 75 The method of any one of embodiments 60 to 74, wherein the exosomes have a size between about 30 nm and 130 nm.
  • Embodiment 76 The method of any one of embodiments 60 to 75, wherein the exosomes have a size between about 30 nm and 100 nm.
  • Embodiment 77 The method of any one of embodiments 60 to 76, wherein the size is between about 30 nm and about 40 nm, about 35 nm and about 45 nm, about 40 nm and about 50 nm, about 45 nm and about 55 nm, about 50 nm and about 60 nm, about 55 nm and about 65 nm, or about 60 nm and about 70 nm.
  • Embodiment 78 The method of any one of embodiments 60 to 77, wherein the one or more exosome markers comprises CD63 and wherein the exosomes have a size between about 30 nm and about 100 nm.
  • Embodiment 79 The method of any one of embodiments 17 to 78, wherein a surface of the exosomes comprises the recombinant receptor expressed by the cells of the cell therapy composition or T cell composition, or a surrogate marker of the recombinant receptor expressed by the cells of the cell therapy composition.
  • Embodiment 80 The method of embodiment 79, wherein the surface of the exosomes comprises the recombinant receptor expressed by the cells of the cell therapy composition or T cell composition.
  • Embodiment 81 The method of embodiment 79, wherein the surface of the exosomes comprises the surrogate marker of the recombinant receptor expressed by the cells of the T cell composition.
  • Embodiment 82 The method of embodiment 79 or embodiment 80, wherein the surrogate marker is a truncated cell surface receptor, optionally a truncated epidermal growth factor receptor (tEGFR).
  • Embodiment 83 The method of any one of embodiments 17 to 82, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • Embodiment 84 The method of embodiment 83, wherein the CAR comprises an scFv specific for an antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta.
  • Embodiment 85 The method of embodiment 84, wherein the antigen is expressed by the cells of the disease or condition.
  • Embodiment 87 The method of embodiment 86, wherein the cancer or tumor is a hematological malignancy.
  • Embodiment 88 The method of embodiment 87, wherein the hematological malignancy is a myeloma, leukemia or lymphoma.
  • Embodiment 89 The method of embodiment 87 or embodiment 88, wherein the hematological malignancy is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), and Diffuse Large B-Cell Lymphoma (DLBCL).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • NHL non-Hodgkin lymphoma
  • Embodiment 90 The method of any one of embodiments 84 to 87, wherein the antigen is a B cell antigen.
  • Embodiment 91 The method of embodiment 90, wherein the B cell antigen is CD19.
  • Embodiment 92 The method of embodiment 90, wherein the B cell antigen is BCMA.
  • Embodiment 93 The method of any one of embodiments 84 to 87, wherein the antigen is a plasma cell antigen.
  • Embodiment 94 The method of embodiment 93, wherein the plasma cell antigen is GPRC5D.
  • Embodiment 95 The method of any one of embodiments 4 to 94, wherein the control cell composition comprises T cells of the cell therapy composition that have not been contacted with the recombinant receptor-stimulating agent.
  • Embodiment 96 The method of any one of embodiments 1 to 95, wherein the recombinant receptor-stimulating agent comprises a target antigen or an extracellular domain binding portion thereof, optionally a recombinant antigen of a recombinant receptor.
  • Embodiment 97 The method of embodiment 96, wherein the recombinant receptor-stimulating agent comprises an extracellular domain binding portion of the target antigen and the extracellular domain binding portion comprises an epitope recognized by the recombinant receptor.
  • Embodiment 98 The method of embodiment 96 or embodiment 97, wherein the recombinant receptor-stimulating agent is an antibody specific to an extracellular binding domain of the recombinant receptor.
  • Embodiment 99 The method of any one of embodiments 17 to 98, wherein the recombinant receptor-stimulating agent is an anti-idiotypic antibody specific to an extracellular antigen binding domain of the recombinant receptor.
  • Embodiment 100 The method of any one of embodiments 1 to 99, wherein the recombinant receptor-stimulating agent is immobilized or attached to a solid support.
  • Embodiment 101 The method of embodiment 100, wherein the solid support is a surface of a vessel, optionally a well of microwell plate, in which a plurality of incubations is performed.
  • Embodiment 102 The method of embodiment 100, wherein the solid support is a bead.
  • Embodiment 103 The method of any one of embodiments 1 to 102, wherein the recombinant receptor-stimulating agent is an antigen-expressing cell, optionally wherein the antigen-expressing cell is a clone, from a cell line, or a primary cell taken from a subject.
  • the recombinant receptor-stimulating agent is an antigen-expressing cell, optionally wherein the antigen-expressing cell is a clone, from a cell line, or a primary cell taken from a subject.
  • Embodiment 104 The method of embodiment 103, wherein the antigen-expressing cell is a cell line.
  • Embodiment 105 The method of embodiment 104, wherein the cell line is a tumor cell line.
  • Embodiment 106 The method of any one of embodiments 103 to 105, wherein the antigenexpressing cell is a cell that has been introduced, optionally by transduction, to express an antigen of the recombinant receptor.
  • Embodiment 107 The method of any one of embodiments 9, 11, 12 , 15, 16, and 19, wherein the other agent is a BTK inhibitor (e.g., ibrutinib or acalibrutinib), a BCL2 inhibitor (e.g., venetoclax), an immunomodulatory agent, a DGK inhibitor, an inhibitor of indoleamine 2,3-dioxygenase-l (IDO1) (e.g. epacadostat) or a checkpoint inhibitor.
  • a BTK inhibitor e.g., ibrutinib or acalibrutinib
  • BCL2 inhibitor e.g., venetoclax
  • an immunomodulatory agent e.g., a DGK inhibitor
  • IDO1 indoleamine 2,3-dioxygenase-l
  • checkpoint inhibitor e.g. epacadostat
  • Embodiment 108 The method of embodiment 107, wherein the immunomodulatory agent is an immunomodulatory imide drug (IMiD) or a cereblon E3 ligase modulator (CELMoD).
  • IMD immunomodulatory imide drug
  • CELMoD cereblon E3 ligase modulator
  • Embodiment 109 The method of embodiment 107 or embodiment 108, wherein the immunomodulatory agent is thalidomide or a thalidomide derivative.
  • Embodiment 110 The method of any one of embodiments 107 to 109, wherein the immunomodulatory agent is selected from the group consisting of lenalidomide, pomalidomide, avadomide (CC-122), and iberdomide (CC-220).
  • the immunomodulatory agent is selected from the group consisting of lenalidomide, pomalidomide, avadomide (CC-122), and iberdomide (CC-220).
  • Embodiment 111 The method of embodiment 107, wherein the checkpoint inhibitor is selected from a PD-1 inhibitor, LAG3 inhibitor and PD-L1 inhibitor.
  • Embodiment 112. The method of embodiment 111, wherein the PD-1 inhibitor is an anti-PD-1 antibody (e.g, nivolumab).
  • an anti-PD-1 antibody e.g, nivolumab
  • Embodiment 113 The method of embodiment 111, wherein the LAG3 inhibitor is an anti-LAG3 antibody (e.g., relatlimab).
  • an anti-LAG3 antibody e.g., relatlimab
  • Embodiment 114 The method of embodiment 111 or embodiment 112, wherein the PD-L1 inhibitor is an anti-PD-Ll antibody (e.g. durvalumumab).
  • Embodiment 115 The method of embodiment 107, wherein the BCE2 inhibitor is selected from the group consisting of venetoclax, navitoclax, ABT737, maritoclax, obatoclax and clitocine.
  • LBCL Large B-Cell Lymphoma
  • Eligible subjects were administered therapeutic T cell compositions containing engineered cells expressing anti-CD19 CARs.
  • the therapeutic T cell compositions administered had been generated by a process including immunoaffinity-based (e.g., immunomagnetic selection) enrichment of CD4+ and CD8+ cells from leukapheresis samples from the individual subjects to be treated.
  • Isolated CD4+ and CD8+ T cells were separately activated and independently transduced with a viral vector (e.g., lentiviral vector) encoding an anti-CD19 CAR, followed by separate expansion and cry opreservation of the engineered cell populations.
  • a viral vector e.g., lentiviral vector
  • the CAR contained an anti-CD19 scFv derived from a murine antibody (variable region derived from FMC63, VE-linker-VH orientation), an IgG4 immunoglobulin-derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4- IBB, and a CD3-zeta intracellular signaling domain.
  • the viral vector further contained sequences encoding a truncated receptor, which served as a surrogate marker for CAR expression; separated from the CAR sequence by a T2A ribosome skip sequence.
  • the cryopreserved therapeutic T cell compositions were thawed prior to intravenous administration.
  • the therapeutic T cell dose was administered as a defined cell composition by administering a formulated CD4+ CAR+ cell population and a formulated CD8+ CAR+ population administered at a target ratio of approximately 1:1.
  • Subjects were administered a single dose of CAR-expressing T cells (each single dose via separate infusions of CD4+ CAR-expressing T cells and CD8+ CAR-expressing T cells, respectively) as follows: a single dose of dose level 1 (DE-1) containing 5 x 10 7 total CAR-expressing T cells or a single dose of dose level 2 (DE-2) containing 1 x 10 8 total CAR-expressing T cells.
  • dose level 1 DE-1
  • DE-2 single dose level 2
  • CD63 Chimeric antigen receptor
  • CD63 which is a marker of exosomes, are reflective of performance of CAR-T cell products and therefore exosomes could be an indicator for patient success with CAR-T cell therapy.
  • the present example relates to the phenotypic and functional characterization of extracellular vesicles (EVs), which include exosomes, produced by CAR-T cell products.
  • EVs extracellular vesicles
  • An exemplary CAR-T cell product comprising a population of engineered CAR-T cells produced substantially as described in Example 1 was cultured in RPMI media + 1% exosome-free FBS (GibcoTM Fetal Bovine Serum, Cat. No. A2720803) and co-stimulated for 24 hours with plate bound anti- idiotypic antibody (anti-ID) specific to an extracellular antigen binding domain of the CD19-directed CAR (e.g., as described in PCT publication No. WO2018/023100) and plate bound anti-CD28 antibody (aCD28), both of which were immobilized to the plate each at 4.69 ug/cm 2 .
  • a media alone was incubated in anti-ID/anti-CD28 coated wells without CAR-T cell product.
  • EVs were isolated from supernatant media using the Total Exosome Isolation Isolation Kit (Invitrogen) which precipitates out the total EV fraction, which includes exosomes (Patel et al. Scientific Reports, vol. 9, Article 5335, 2019, doi:10.1038/s41598-019-41800-2). Specifically, cell-free supernatant was collected from the CAR-T cell product after overnight stimulation, and commercially available Total Exosome Isolation Reagent (Thermo Fisher Scientific, catalog no. 4478359) was added to isolate the exosomes and other EVs.
  • Thermo Fisher Scientific catalog no. 4478359
  • EV were precipitated from the CAR-T cell product as described above and were characterized using Nanoparticle Tracking Analysis (NTA) to determine size and concentration, and a multiplex assay to determine cargo.
  • NTA Nanoparticle Tracking Analysis
  • EV fraction particles were analyzed using nanoparticle tracking analysis (NTA). Exosome- sized particles (35-200nm) were detected in the total EV fraction of stimulated and unstimulated CAR-T cell product (data not shown). However, more exosome-sized particles that were between 30-130 nm in diameter were detected in the stimulated CAR-T cell product compared to the unstimulated CAR-T cell product.
  • FIG. 2 shows that the concentration of total EVs per mL increased two-fold in stimulated CAR- T cell product compared to unstimulated CAR-T cell product.
  • FIG. 3 shows the cytokines present in lysed EVs derived from stimulated or unstimulated CAR-T cell product and the cytokines present in EV-free fractions derived from stimulated or unstimulated CAR-T cell product.
  • Granzyme B was detected at the upper limit of quantitation (ULOQ) in the EV and EV-free fraction derived from stimulated CAR-T cell product.
  • Granzyme A and Perforin were detected in EVs above the lower limit of quantitation (LLOQ).
  • LLOQ lower limit of quantitation
  • the EV fraction derived from stimulated CAR-T cell product contained a higher amount of Granzyme A and Perforin.
  • IFNy was 3-fold higher
  • Granzyme A was 40-fold higher
  • Perforin was 30-fold higher in the EV fraction derived from the stimulated CAR-T cell product compared to the EV fraction derived from unstimulated CAR-T cell product, indicating that these cytokines are specifically contained in or on EVs when delivered out of the cells of the CAR-T cell product.
  • the cytolytic activity of CAR-T cell product derived EVs was assessed in tumor model spheroid cultures.
  • the tumor spheroids were formed by plating 30,000 SCC152 cells in a 96-well S-Bio Hydrophilic Spheroid plate (Cat: MS-9096UZ), centrifuging the cells briefly (200 x g for 2 minutes) to collect cells at the bottom of the well, and incubating in X-VIVO 15 media for 3 days at 37°C to form spheroids.
  • the SCC152 cells also were engineered to express a red fluorescent dye to permit monitoring of tumor cell lysis by microscopy.
  • spheroids were allowed to grow for three days, and then co-incubated with effectors provided as either (1) EV isolate or (2) the cell of origin (i.e., CAR- T cell product), each from stimulated and unstimulated conditions. A media alone condition was used as control. The co-incubation of the effectors with the spheroids was for 10 days. Cytolytic activity was determined by measuring changes in red fluorescence. FIG. 4 shows the total area of red fluorescence imaged in the tumor spheroids.
  • EVs produced by stimulated CAR-T cell product were able to kill tumor cells completely. Both stimulated and unstimulated CAR-T cell product were able to kill the tumor cells. However, the tumor spheroids incubated with stimulated CAR-T cell product showed a much more homogenous and immediate decrease in red fluorescence compared to unstimulated CAR-T cell product that exhibited a more varied and delayed killing effect. EVs derived from unstimulated CAR-T cell product killed the tumor cells similarly to the media control, and thus were unable to kill the tumor cells.
  • the present example demonstrates that EVs derived from CAR-T cell product, which predominantly include exosome-sized particles, can perform the same functions as CAR-T cell product and can therefore enhance CAR-T cell killing.
  • NTA Nanoparticle Tracking Analysis
  • serum i.e., FBS
  • FBS serum
  • serum contains a nominal amount of exosome-sized particles.
  • exosome-sized particles were removed from the serum by ultracentrifugation.
  • Cell health was measured by flow cytometry using the Sartorius iQue Human 4-Plex Apoptosis Kit (cat: 90053), which detects caspase 3/7, annexin V, viability, and mitochondrial damage.
  • 90053 Sartorius iQue Human 4-Plex Apoptosis Kit
  • NTA substantially as described in Example 2 was performed on raw media. NTA output images showed high background for media alone (data not shown). Table El provides background particles (particles/mL) and cell health data (i.e., caspase 3/7, annexin V+, viability, and mitochondrial damage) for the cells incubated in both stimulated and unstimulated conditions for 24 hours.
  • RPMI media supplemented with 1% vesicle-free FBS yielded the least number of background particles (6.6e+07 particles/mL) compared to X-VIVO-15 including X-VIVO-15 following ultracentrifugation (Table El). Further, RPMI media supplemented with 1% vesicle-free FBS showed comparable cell health as measured by the percentage of cells that were caspase 3/7+, annexin V+, viable, and had mitochondrial damage compared to both exosome-free X-VIV and standard X-VIVO media (Table El).
  • LB CL Large B-Cell Lymphoma
  • CAR anti-CD19 chimeric antigen receptor
  • Clinical results were determined after administration of the therapeutic CAR-T cell product to the subject (e.g., one month after infusion) and reported as complete response (CR) signifying an efficacious autologous CAR- T cell lot, progressive disease (PD) signifying an autologous CAR-T cell lot that did not effectively treat the patient’s cancer, or stable disease (SD).
  • CAR-T cell product e.g., one month after infusion
  • CR complete response
  • PD progressive disease
  • SD stable disease
  • CAR-T cell products also were generated by various manufacturing methods from subjects with multiple myeloma (MM) in which CD4+ T cells and CD8+ T cells were engineered with a CAR, either an anti-BCMA CAR, anti-GPRC5D CAR, or anti-BCMA and anti-GPRC5D CAR.
  • MM multiple myeloma
  • the CAR-T cells products from the three different CAR products were then administered as an autologous cell therapy and clinical results were reported as described above in which subjects were divided into responders (R) or non-responders (NR).
  • the engineered T cells were plated at 0.66e6 live CAR+ cells (for LBCL samples) or 0.5e6 (for MM samples) in 1.5 mL per well on 24-well plate culture well (Corning #351147), which was previously coated with 5 pg/mL of anti-ID or was uncoated. On the next day, samples were collected and the cells were removed using centrifugation (300 x g for 3 min). The supernatants were further cleared of cell debris by centrifugation twice at 2,500 x g for 15 min and the supernatant was transferred to a new tube following the first centrifugation step to avoid resuspending the debris pellet. This cleared supernatant is termed Exosome-Containing Media (ECM). The ECM was aliquoted into 8-strip tubes and immediately stored at -80°C and freeze-thaws were avoided.
  • ECM Exosome-Containing Media
  • the Leprechaun instrument In order to analyze the exosome content of the ECM, the Leprechaun instrument from Unchained Labs was used in conjunction with the Human Tetraspanin Kit (Unchained Labs #251-1044). Advantages of using the Leprechaun instrument and human tetraspanin kit are that total EVs, including exosomes, do not need to be precipitated from the CAR-T cell product as described in Example 2. Unlike other methods used in examples above, the Leprechaun instrument can simultaneously perform specific immunoaffinity-based capture and fluorescence-based analysis of exosomes .
  • the Human Tetraspanin kit uses Luni chips with 4 unique antibody capture spots (i.e., CD63, CD81, CD9, and IgG control spots, printed in triplicate) for specific isolation of exosomes.
  • the fluorescence readout is limited to three color channels (Lasers: LED: 470 nm (Blue), 567 nm (Green), 623 nm (Red)) and the phase imaging capacity allows the Leprechaun to measure particles ranging from 30 nm to 200 nm in diameter
  • the Leprechaun was used as per manufacturer protocol without deviation..
  • the ECM was incubated on the Luni chips for approximately 18 hours.
  • the antibody- captured exosomes from the ECM were then labeled with secondary anti-tetraspanin (CD63, CD81, and/or CD9) antibodies conjugated to different fluorophores for visualization. Since this detection method is immunoaffinity-based, exosomes were specifically captured by their membrane-bound tetraspanins, while all other non-specific and non-exosome particles were washed away.
  • the Leprechaun analytical software uses sizing data to measure the diameter of the particles, further ensuring that protein aggregates (particles ⁇ 30nm) or any kind of debris (particles >200 nm) are omitted from analysis. Therefore, to ensure specificity for exosome analysis, only the particles that were bound to a capture antibody, were labeled with a secondary antibody, and had a relevant overlapping phase contrast image were included in the total count.
  • chimeric antigen receptor (CAR) T cell products from LB CL patients that experienced CR and PD produced a large quantity of exosomes, with an enrichment in the exosome fraction.
  • CD63+ exosomes were produced at a higher concentration compared to exosome populations marked by exosome-specific tetraspanins CD9 and CD81. Further characterization of the CD63+ exosome compartment showed that CAR-T cell product from LBCL patients experiencing CR produced more CD63+ exosomes compared to CAR-T cell product from LBCL patients that had PD (FIG. 6A).
  • FIGS. 6B-6E show extracellular vesicles, including exosomes, produced by CAR-T cell product for patients experiencing CR or PD after one month of treatment. As shown in FIG.
  • FIG. 6B stimulated CAR-T cell product from patients experiencing a CR after one month of treatment secreted more total extracellular vesicles compared to stimulated CAR-T cell product from patients experiencing PD after one month of treatment, and unstimulated CAR-T cell product from patients experiencing PD and CR.
  • the data in FIG. 6B was further analyzed by selecting for extracellular vesicles comprising exosome markers CD63, CD81, and/or CD9.
  • FIG. 6C demonstrates that stimulated CAR-T cell product from patients experiencing a CR after one month of treatment secreted more CD63+ exosomes compared to stimulated CAR-T cell product from patients experiencing PD after one month of treatment, and unstimulated CAR-T cell product from patients experiencing PD and CR.
  • Analysis of additional exosome markers including CD81 and CD9 is shown in FIG. 6D. As shown in FIG.
  • 6D stimulated CAR-T cell product from patients experiencing a CR after one month of treatment secreted more CD81+/CD9+ and CD81+/CD9+/CD63-I- exosomes compared to stimulated CAR-T cell product from patients experiencing PD after one month of treatment, and unstimulated CAR-T cell product from patients experiencing PD and CR.
  • FIG. 6E demonstrates that the exosome compartment derived from the CAR-T cell product contains several distinct populations, which can be stratified by size, and may have functional differences (Willms, E. 2016. Sci Rep).
  • FIG. 6E shows that the majority of CD63+ exosomes were under 100 nm in diameter, without any major separation between small ( ⁇ 50 nm), medium (50 nm to 100 nm), and larger ( > 100 nm) exosomes.
  • FIGS. 7A-7B show exosome analysis in CAR-T cell product derived from MM patients, in which the engineered T cells expressed anti-CD19 CAR, anti-BCMA CAR, anti-GPRC5D CAR, or anti- BCMA and anti-GPRC5D CAR. These CAR-T cells expressing different CARs are depicted in FIGS.
  • CAR-T cell product 1 to 6 such that CAR-T cell products 1 , 5 and 6 correspond to CAR-T cell products comprising engineered T cells expressing anti-BCMA CAR, CAR-T cell product 2 corresponds to CAR-T cell products comprising engineered T cells expressing anti-BCMA and anti-GPRC5D CAR, CAR-T cell product 3 corresponds to CAR-T cell products comprising engineered T cells expressing anti-GPRC5D CAR, and CAR-T cell product 4 corresponds to CAR-T cell products comprising engineered T cells expressing anti-CD19 CAR.
  • CD63+ exosomes As shown in FIG. 7A, high levels of CD63+ exosomes were produced in stimulated and unstimulated CAR-T cell products compared to CD81+ or CD9+ exosomes. As shown in FIG. 7B, median concentration of CD63+ exosomes was higher in stimulated and unstimulated CAR-T cell products compared to the median concentration of CD81+ or CD9+ exosomes in stimulated or unstimulated CAR-T cell products.
  • CD63+ exosomes may serve as an indicator for patient response across various autologous CAR-T cell therapies.
  • Example 5 Exosomes as a Predictor of Treatment Outcome
  • CD63+ exosomes may be predictive of CR.
  • the results in Examples 4 and 5 show that in the cell therapy products from patients that showed better clinical response, more total and more diverse populations of exosomes were detected. These results suggest that the CAR-T cells from responders secrete more total and more diverse populations of exosomes.

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Abstract

L'invention concerne des procédés d'évaluation d'exosomes produits à partir de cellules d'une composition cellulaire, telle qu'une composition de thérapie cellulaire, comprenant des compositions contenant un récepteur antigénique chimérique (CAR) ou un récepteur des lymphocytes T (TCR) exprimant des lymphocytes T. Dans certains modes de réalisation, les procédés peuvent être utilisés pour prédire la réponse à une composition de thérapie cellulaire et/ou son efficacité, pour sélectionner des patients en vue d'un traitement et en relation avec des procédés de production de lymphocytes T.
PCT/US2025/038155 2024-07-18 2025-07-17 Procédés d'évaluation d'exosomes dans une composition cellulaire et utilisations associées Pending WO2026020055A2 (fr)

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Citations (82)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4452773A (en) 1982-04-05 1984-06-05 Canadian Patents And Development Limited Magnetic iron-dextran microspheres
US4490436A (en) 1981-10-30 1984-12-25 Japan Synthetic Rubber Co., Ltd. Polymer filler particles with filler free coating
US4554088A (en) 1983-05-12 1985-11-19 Advanced Magnetics Inc. Magnetic particles for use in separations
US4654267A (en) 1982-04-23 1987-03-31 Sintef Magnetic polymer particles and process for the preparation thereof
US5091206A (en) 1987-10-26 1992-02-25 Baxter Diagnostics Inc. Process for producing magnetically responsive polymer particles and application thereof
US5219740A (en) 1987-02-13 1993-06-15 Fred Hutchinson Cancer Research Center Retroviral gene transfer into diploid fibroblasts for gene therapy
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5232782A (en) 1989-12-27 1993-08-03 Rhone-Poulenc Chimie Magnetizable "core-shell" microspheres based on a cross-linked organopolysiloxane and a process for their preparation
US5318797A (en) 1990-06-20 1994-06-07 Clarkson University Coated particles, hollow particles, and process for manufacturing the same
US5356713A (en) 1989-03-31 1994-10-18 Rhone-Poulenc Chimie Magnetizable composite microspheres of hydrophobic crosslinked polymer, process for preparing them and their application in biology
US5395688A (en) 1987-10-26 1995-03-07 Baxter Diagnostics Inc. Magnetically responsive fluorescent polymer particles
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5834121A (en) 1996-01-16 1998-11-10 Solid Phase Sciences Corp. Composite magnetic beads
WO2000014257A1 (fr) 1998-09-04 2000-03-16 Sloan-Kettering Institute For Cancer Research Recepteurs de fusion specifiques a l'antigene prostatique specifique membranaire et ses utilisations
US6074884A (en) 1997-10-09 2000-06-13 Coulter International Corp. Stable protein-nickel particles and methods of production and use thereof
WO2000038762A1 (fr) 1998-12-24 2000-07-06 Biosafe S.A. Systeme de separation sanguine convenant en particulier pour la concentration de cellules souche hematopoietiques
US6123655A (en) 1996-04-24 2000-09-26 Fell; Claude Cell separation system with variable size chamber for the processing of biological fluids
US6207453B1 (en) 1996-03-06 2001-03-27 Medigene Ag Recombinant AAV vector-based transduction system and use of same
US6410319B1 (en) 1998-10-20 2002-06-25 City Of Hope CD20-specific redirected T cells and their use in cellular immunotherapy of CD20+ malignancies
US6451995B1 (en) 1996-03-20 2002-09-17 Sloan-Kettering Institute For Cancer Research Single chain FV polynucleotide or peptide constructs of anti-ganglioside GD2 antibodies, cells expressing same and related methods
US20020131960A1 (en) 2000-06-02 2002-09-19 Michel Sadelain Artificial antigen presenting cells and methods of use thereof
US7070995B2 (en) 2001-04-11 2006-07-04 City Of Hope CE7-specific redirected immune cells
US20070116690A1 (en) 2001-12-10 2007-05-24 Lili Yang Method for the generation of antigen-specific lymphocytes
US20080171951A1 (en) 2005-03-23 2008-07-17 Claude Fell Integrated System for Collecting, Processing and Transplanting Cell Subsets, Including Adult Stem Cells, for Regenerative Medicine
US7446190B2 (en) 2002-05-28 2008-11-04 Sloan-Kettering Institute For Cancer Research Nucleic acids encoding chimeric T cell receptors
US7446179B2 (en) 2000-11-07 2008-11-04 City Of Hope CD19-specific chimeric T cell receptor
WO2009072003A2 (fr) 2007-12-07 2009-06-11 Miltenyi Biotec Gmbh Système et procédés de traitement d'échantillons
WO2010033140A2 (fr) 2008-05-06 2010-03-25 Innovative Micro Technology Appareil amovible/jetable pour dispositif de tri de particules de mems
US20100104509A1 (en) 2006-12-13 2010-04-29 Medarex, Inc. Human antibodies that bind cd19 and uses thereof
US20100207051A1 (en) 2006-12-21 2010-08-19 Invitrogen Dynal As Particles and their use in a method for isolating nucleic acid or a method for isolating phosphoproteins
WO2010104949A2 (fr) 2009-03-10 2010-09-16 Biogen Idec Ma Inc. Anticorps anti-bcma
WO2012129514A1 (fr) 2011-03-23 2012-09-27 Fred Hutchinson Cancer Research Center Méthodes et compositions pour une immunothérapie cellulaire
US8324353B2 (en) 2001-04-30 2012-12-04 City Of Hope Chimeric immunoreceptor useful in treating human gliomas
US8339645B2 (en) 2008-05-27 2012-12-25 Canon Kabushiki Kaisha Managing apparatus, image processing apparatus, and processing method for the same, wherein a first user stores a temporary object having attribute information specified but not partial-area data, at a later time an object is received from a second user that includes both partial-area data and attribute information, the storage unit is searched for the temporary object that matches attribute information of the received object, and the first user is notified in response to a match
EP2537416A1 (fr) 2007-03-30 2012-12-26 Memorial Sloan-Kettering Cancer Center Expression constitutive de ligands costimulants sur des lymphocytes T transférés de manière adoptive
US8398282B2 (en) 2011-05-12 2013-03-19 Delphi Technologies, Inc. Vehicle front lighting assembly and systems having a variable tint electrowetting element
WO2013071154A1 (fr) 2011-11-11 2013-05-16 Fred Hutchinson Cancer Research Center Immunothérapie par des lymphocytes t ciblant la cycline a1 pour le traitement du cancer
US20130149337A1 (en) 2003-03-11 2013-06-13 City Of Hope Method of controlling administration of cancer antigen
US8479118B2 (en) 2007-12-10 2013-07-02 Microsoft Corporation Switching search providers within a browser search box
WO2013123061A1 (fr) 2012-02-13 2013-08-22 Seattle Children's Hospital D/B/A Seattle Children's Research Institute Récepteurs d'antigène chimères bispécifiques et utilisations thérapeutiques de ceux-ci
WO2013126726A1 (fr) 2012-02-22 2013-08-29 The Trustees Of The University Of Pennsylvania Lymphocytes t doubles transgéniques comportant un car et un tcr, et leurs procédés d'utilisation
WO2013124474A2 (fr) 2012-02-23 2013-08-29 Stage Cell Therapeutics Gmbh Isolement chromatographique de cellules et d'autres matériaux biologiques complexes
US20130287748A1 (en) 2010-12-09 2013-10-31 The Trustees Of The University Of Pennsylvania Use of Chimeric Antigen Receptor-Modified T-Cells to Treat Cancer
WO2013166321A1 (fr) 2012-05-03 2013-11-07 Fred Hutchinson Cancer Research Center Récepteurs de lymphocyte t à affinité augmentée et procédés pour fabriquer ceux-ci
WO2013188864A2 (fr) 2012-06-15 2013-12-19 Sinomab Bioscience Limited Anticorps anti-idiotypiques anti-cd22 et leurs utilisations
WO2014031687A1 (fr) 2012-08-20 2014-02-27 Jensen, Michael Procédé et compositions pour l'immunothérapie cellulaire
WO2014055668A1 (fr) 2012-10-02 2014-04-10 Memorial Sloan-Kettering Cancer Center Compositions et procédés d'immunothérapie
WO2015079417A1 (fr) 2013-11-29 2015-06-04 Novartis Ag Nouveaux dérivés d'aminopyrimidine
WO2015158868A2 (fr) 2014-04-16 2015-10-22 Juno Therapeutics Gmbh Méthodes, kits et appareil pour la multiplication d'une population de cellules
WO2015164675A1 (fr) 2014-04-23 2015-10-29 Juno Therapeutics, Inc. Procédés d'isolement, de culture et de manipulation génétique de populations de cellules immunitaires pour une thérapie adoptive
WO2015187528A1 (fr) 2014-06-02 2015-12-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs d'antigènes chimériques ciblant cd-19
WO2016033570A1 (fr) 2014-08-28 2016-03-03 Juno Therapeutics, Inc. Anticorps et récepteurs antigéniques chimériques spécifiques du cd19
WO2016073602A2 (fr) 2014-11-05 2016-05-12 Juno Therapeutics, Inc. Procédés de transduction et de traitement de cellules
WO2016090329A2 (fr) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Anticorps ciblant le récepteur couplé aux protéines g et procédés d'utilisation
WO2016090312A1 (fr) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Récepteurs d'antigènes chimériques ciblant un récepteur couplé à la protéine g et leurs utilisations
WO2016090327A2 (fr) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Anticorps ciblant l'antigène de maturation des lymphocytes b et procédés d'utilisation
WO2016090320A1 (fr) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Récepteurs antigéniques chimériques ciblant l'antigène de maturation des cellules b et leurs utilisations
WO2017173256A1 (fr) 2016-04-01 2017-10-05 Kite Pharma, Inc. Récepteurs antigéniques chimériques et récepteurs de lymphocytes t et leurs procédés d'utilisation
WO2018023100A2 (fr) 2016-07-29 2018-02-01 Juno Therapeutics, Inc. Anticorps anti-idiotypes et procédés associés
WO2018071873A2 (fr) 2016-10-13 2018-04-19 Juno Therapeutics, Inc. Méthodes et compositions d'immunothérapie impliquant des modulateurs de la voie métabolique du tryptophane
WO2018085731A2 (fr) 2016-11-03 2018-05-11 Juno Therapeutics, Inc. Polythérapie de type thérapie cellulaire t et inhibiteur de btk
WO2018102785A2 (fr) 2016-12-03 2018-06-07 Juno Therapeutics, Inc. Méthodes et compositions pour l'utilisation de lymphocytes t thérapeutiques en association avec des inhibiteurs de kinase
WO2018102786A1 (fr) 2016-12-03 2018-06-07 Juno Therapeutics, Inc. Procédés de modulation de lymphocytes t modifiés par car
WO2018106595A1 (fr) 2016-12-05 2018-06-14 Fate Therapeutics, Inc. Compositions et procédés pour la modulation de cellules immunitaires dans des immunothérapies adoptives
WO2018170188A2 (fr) 2017-03-14 2018-09-20 Juno Therapeutics, Inc. Procédés de stockage cryogénique
WO2018197949A1 (fr) 2017-04-27 2018-11-01 Juno Therapeutics Gmbh Reactifs particulaires oligomères et leurs méthodes d'utilisation
WO2018204427A1 (fr) 2017-05-01 2018-11-08 Juno Therapeutics, Inc. Combinaison d'une thérapie cellulaire et d'un composé immunomodulateur
WO2019018603A2 (fr) 2017-07-19 2019-01-24 Fate Therapeutics, Inc. Compositions et procédés pour la modulation de cellules immunitaires dans des immunothérapies adoptives
WO2019027850A1 (fr) 2017-07-29 2019-02-07 Juno Therapeutics, Inc. Réactifs d'expansion de cellules exprimant des récepteurs recombinants
WO2019089855A1 (fr) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Procédé de génération de compositions thérapeutiques de cellules modifiées
WO2019113557A1 (fr) 2017-12-08 2019-06-13 Juno Therapeutics, Inc. Procédé de production d'une compositions de lymphocytes t modifiés
WO2019152743A1 (fr) 2018-01-31 2019-08-08 Celgene Corporation Polythérapie utilisant une thérapie cellulaire adoptive et un inhibiteur de point de contrôle
WO2019213184A1 (fr) 2018-05-03 2019-11-07 Juno Therapeutics, Inc. Polythérapie d'une thérapie par lymphocytes t à récepteur antigénique chimérique (car) et d'un inhibiteur de btk
WO2020033927A2 (fr) 2018-08-09 2020-02-13 Juno Therapeutics, Inc. Procédés de génération de cellules modifiées et compositions associées
WO2020092854A2 (fr) 2018-11-01 2020-05-07 Juno Therapeutics, Inc. Récepteurs antigéniques chimériques spécifiques du gprc5d (élément d du groupe 5 de classe c des récepteurs couplés à la protéine g)
WO2020233589A1 (fr) 2019-05-20 2020-11-26 南京驯鹿医疗技术有限公司 Anticorps entièrement humain ciblant cd19 et son application
WO2020252218A1 (fr) 2019-06-12 2020-12-17 Juno Therapeutics, Inc. Combinaison thérapeutique d'une thérapie cytotoxique à médiation cellulaire et d'un inhibiteur d'une protéine de la famille bcl2 pro-survie
WO2022133030A1 (fr) 2020-12-16 2022-06-23 Juno Therapeutics, Inc. Polythérapie de thérapie cellulaire et d'inhibiteur de bcl2
WO2022187406A1 (fr) 2021-03-03 2022-09-09 Juno Therapeutics, Inc. Combinaison d'une thérapie par lymphocytes t et d'un inhibiteur de dgk
WO2022212400A1 (fr) 2021-03-29 2022-10-06 Juno Therapeutics, Inc. Méthodes de dosage et de traitement au moyen d'une combinaison d'une thérapie par inhibiteur de point de contrôle et d'une thérapie par lymphocytes car t
WO2023147515A1 (fr) 2022-01-28 2023-08-03 Juno Therapeutics, Inc. Procédés de fabrication de compositions cellulaires
WO2024100604A1 (fr) 2022-11-09 2024-05-16 Juno Therapeutics Gmbh Procédés de fabrication de cellules immunitaires modifiées

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018208670A1 (fr) * 2017-05-08 2018-11-15 Trustees Of Tufts College VÉSICULES EXTRACELLULAIRES COMPRENANT UN TGF-β ATTACHÉ À LA MEMBRANE, COMPOSITIONS ET MÉTHODES D'UTILISATION ASSOCIÉES
CN108315305B (zh) * 2017-12-26 2020-11-06 沣潮医药科技（上海）有限公司 携带嵌合抗原受体的免疫细胞外泌体的制备方法及其应用
KR102568745B1 (ko) * 2021-05-18 2023-08-24 주식회사 바이오솔루션 세포외소포체 분비능이 증진된 면역세포 및 이를 활용한 면역 항암요법
CA3257225A1 (fr) * 2022-05-25 2023-11-30 Thomas Malcolm Expression contrôlée de biomolécules thérapeutiquement pertinentes pour charges utiles localisées dans une lumière dans des nanovésicules biomimétiques et des exosomes

Patent Citations (93)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4490436A (en) 1981-10-30 1984-12-25 Japan Synthetic Rubber Co., Ltd. Polymer filler particles with filler free coating
US4452773A (en) 1982-04-05 1984-06-05 Canadian Patents And Development Limited Magnetic iron-dextran microspheres
US4654267A (en) 1982-04-23 1987-03-31 Sintef Magnetic polymer particles and process for the preparation thereof
US4774265A (en) 1982-04-23 1988-09-27 Sintef Process for preparing magnetic polymer particles
US4554088A (en) 1983-05-12 1985-11-19 Advanced Magnetics Inc. Magnetic particles for use in separations
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5219740A (en) 1987-02-13 1993-06-15 Fred Hutchinson Cancer Research Center Retroviral gene transfer into diploid fibroblasts for gene therapy
US5283079A (en) 1987-10-26 1994-02-01 Baxter Diagnostics Inc. Process to make magnetically responsive fluorescent polymer particles
US5091206A (en) 1987-10-26 1992-02-25 Baxter Diagnostics Inc. Process for producing magnetically responsive polymer particles and application thereof
US5395688A (en) 1987-10-26 1995-03-07 Baxter Diagnostics Inc. Magnetically responsive fluorescent polymer particles
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5356713A (en) 1989-03-31 1994-10-18 Rhone-Poulenc Chimie Magnetizable composite microspheres of hydrophobic crosslinked polymer, process for preparing them and their application in biology
US5232782A (en) 1989-12-27 1993-08-03 Rhone-Poulenc Chimie Magnetizable "core-shell" microspheres based on a cross-linked organopolysiloxane and a process for their preparation
US5318797A (en) 1990-06-20 1994-06-07 Clarkson University Coated particles, hollow particles, and process for manufacturing the same
US5834121A (en) 1996-01-16 1998-11-10 Solid Phase Sciences Corp. Composite magnetic beads
US6207453B1 (en) 1996-03-06 2001-03-27 Medigene Ag Recombinant AAV vector-based transduction system and use of same
US6451995B1 (en) 1996-03-20 2002-09-17 Sloan-Kettering Institute For Cancer Research Single chain FV polynucleotide or peptide constructs of anti-ganglioside GD2 antibodies, cells expressing same and related methods
US6123655A (en) 1996-04-24 2000-09-26 Fell; Claude Cell separation system with variable size chamber for the processing of biological fluids
US6074884A (en) 1997-10-09 2000-06-13 Coulter International Corp. Stable protein-nickel particles and methods of production and use thereof
WO2000014257A1 (fr) 1998-09-04 2000-03-16 Sloan-Kettering Institute For Cancer Research Recepteurs de fusion specifiques a l'antigene prostatique specifique membranaire et ses utilisations
US6410319B1 (en) 1998-10-20 2002-06-25 City Of Hope CD20-specific redirected T cells and their use in cellular immunotherapy of CD20+ malignancies
US6733433B1 (en) 1998-12-24 2004-05-11 Biosafe S.A. Blood separation system particularly for concentrating hematopoietic stem cells
WO2000038762A1 (fr) 1998-12-24 2000-07-06 Biosafe S.A. Systeme de separation sanguine convenant en particulier pour la concentration de cellules souche hematopoietiques
US20020131960A1 (en) 2000-06-02 2002-09-19 Michel Sadelain Artificial antigen presenting cells and methods of use thereof
US7446179B2 (en) 2000-11-07 2008-11-04 City Of Hope CD19-specific chimeric T cell receptor
US7446191B2 (en) 2001-04-11 2008-11-04 City Of Hope DNA construct encoding CE7-specific chimeric T cell receptor
US7070995B2 (en) 2001-04-11 2006-07-04 City Of Hope CE7-specific redirected immune cells
US7265209B2 (en) 2001-04-11 2007-09-04 City Of Hope CE7-specific chimeric T cell receptor
US7354762B2 (en) 2001-04-11 2008-04-08 City Of Hope Method for producing CE7-specific redirected immune cells
US8324353B2 (en) 2001-04-30 2012-12-04 City Of Hope Chimeric immunoreceptor useful in treating human gliomas
US20070116690A1 (en) 2001-12-10 2007-05-24 Lili Yang Method for the generation of antigen-specific lymphocytes
US7446190B2 (en) 2002-05-28 2008-11-04 Sloan-Kettering Institute For Cancer Research Nucleic acids encoding chimeric T cell receptors
US20130149337A1 (en) 2003-03-11 2013-06-13 City Of Hope Method of controlling administration of cancer antigen
US20080171951A1 (en) 2005-03-23 2008-07-17 Claude Fell Integrated System for Collecting, Processing and Transplanting Cell Subsets, Including Adult Stem Cells, for Regenerative Medicine
US20100104509A1 (en) 2006-12-13 2010-04-29 Medarex, Inc. Human antibodies that bind cd19 and uses thereof
US20100207051A1 (en) 2006-12-21 2010-08-19 Invitrogen Dynal As Particles and their use in a method for isolating nucleic acid or a method for isolating phosphoproteins
US8389282B2 (en) 2007-03-30 2013-03-05 Memorial Sloan-Kettering Cancer Center Constitutive expression of costimulatory ligands on adoptively transferred T lymphocytes
EP2537416A1 (fr) 2007-03-30 2012-12-26 Memorial Sloan-Kettering Cancer Center Expression constitutive de ligands costimulants sur des lymphocytes T transférés de manière adoptive
WO2009072003A2 (fr) 2007-12-07 2009-06-11 Miltenyi Biotec Gmbh Système et procédés de traitement d'échantillons
US20110003380A1 (en) 2007-12-07 2011-01-06 Stefan Miltenyi Sample Processing System and Methods
US8479118B2 (en) 2007-12-10 2013-07-02 Microsoft Corporation Switching search providers within a browser search box
WO2010033140A2 (fr) 2008-05-06 2010-03-25 Innovative Micro Technology Appareil amovible/jetable pour dispositif de tri de particules de mems
US8339645B2 (en) 2008-05-27 2012-12-25 Canon Kabushiki Kaisha Managing apparatus, image processing apparatus, and processing method for the same, wherein a first user stores a temporary object having attribute information specified but not partial-area data, at a later time an object is received from a second user that includes both partial-area data and attribute information, the storage unit is searched for the temporary object that matches attribute information of the received object, and the first user is notified in response to a match
WO2010104949A2 (fr) 2009-03-10 2010-09-16 Biogen Idec Ma Inc. Anticorps anti-bcma
US20130287748A1 (en) 2010-12-09 2013-10-31 The Trustees Of The University Of Pennsylvania Use of Chimeric Antigen Receptor-Modified T-Cells to Treat Cancer
US8911993B2 (en) 2010-12-09 2014-12-16 The Trustees Of The University Of Pennsylvania Compositions for treatment of cancer
WO2012129514A1 (fr) 2011-03-23 2012-09-27 Fred Hutchinson Cancer Research Center Méthodes et compositions pour une immunothérapie cellulaire
US8398282B2 (en) 2011-05-12 2013-03-19 Delphi Technologies, Inc. Vehicle front lighting assembly and systems having a variable tint electrowetting element
WO2013071154A1 (fr) 2011-11-11 2013-05-16 Fred Hutchinson Cancer Research Center Immunothérapie par des lymphocytes t ciblant la cycline a1 pour le traitement du cancer
WO2013123061A1 (fr) 2012-02-13 2013-08-22 Seattle Children's Hospital D/B/A Seattle Children's Research Institute Récepteurs d'antigène chimères bispécifiques et utilisations thérapeutiques de ceux-ci
WO2013126726A1 (fr) 2012-02-22 2013-08-29 The Trustees Of The University Of Pennsylvania Lymphocytes t doubles transgéniques comportant un car et un tcr, et leurs procédés d'utilisation
WO2013124474A2 (fr) 2012-02-23 2013-08-29 Stage Cell Therapeutics Gmbh Isolement chromatographique de cellules et d'autres matériaux biologiques complexes
WO2013166321A1 (fr) 2012-05-03 2013-11-07 Fred Hutchinson Cancer Research Center Récepteurs de lymphocyte t à affinité augmentée et procédés pour fabriquer ceux-ci
WO2013188864A2 (fr) 2012-06-15 2013-12-19 Sinomab Bioscience Limited Anticorps anti-idiotypiques anti-cd22 et leurs utilisations
WO2014031687A1 (fr) 2012-08-20 2014-02-27 Jensen, Michael Procédé et compositions pour l'immunothérapie cellulaire
WO2014055668A1 (fr) 2012-10-02 2014-04-10 Memorial Sloan-Kettering Cancer Center Compositions et procédés d'immunothérapie
WO2015079417A1 (fr) 2013-11-29 2015-06-04 Novartis Ag Nouveaux dérivés d'aminopyrimidine
WO2015158868A2 (fr) 2014-04-16 2015-10-22 Juno Therapeutics Gmbh Méthodes, kits et appareil pour la multiplication d'une population de cellules
WO2015164675A1 (fr) 2014-04-23 2015-10-29 Juno Therapeutics, Inc. Procédés d'isolement, de culture et de manipulation génétique de populations de cellules immunitaires pour une thérapie adoptive
WO2015187528A1 (fr) 2014-06-02 2015-12-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Récepteurs d'antigènes chimériques ciblant cd-19
WO2016033570A1 (fr) 2014-08-28 2016-03-03 Juno Therapeutics, Inc. Anticorps et récepteurs antigéniques chimériques spécifiques du cd19
US20160152723A1 (en) 2014-08-28 2016-06-02 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for cd19
WO2016073602A2 (fr) 2014-11-05 2016-05-12 Juno Therapeutics, Inc. Procédés de transduction et de traitement de cellules
WO2016090329A2 (fr) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Anticorps ciblant le récepteur couplé aux protéines g et procédés d'utilisation
WO2016090312A1 (fr) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Récepteurs d'antigènes chimériques ciblant un récepteur couplé à la protéine g et leurs utilisations
WO2016090327A2 (fr) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Anticorps ciblant l'antigène de maturation des lymphocytes b et procédés d'utilisation
WO2016090320A1 (fr) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Récepteurs antigéniques chimériques ciblant l'antigène de maturation des cellules b et leurs utilisations
WO2017173256A1 (fr) 2016-04-01 2017-10-05 Kite Pharma, Inc. Récepteurs antigéniques chimériques et récepteurs de lymphocytes t et leurs procédés d'utilisation
WO2018023100A2 (fr) 2016-07-29 2018-02-01 Juno Therapeutics, Inc. Anticorps anti-idiotypes et procédés associés
WO2018071873A2 (fr) 2016-10-13 2018-04-19 Juno Therapeutics, Inc. Méthodes et compositions d'immunothérapie impliquant des modulateurs de la voie métabolique du tryptophane
WO2018085731A2 (fr) 2016-11-03 2018-05-11 Juno Therapeutics, Inc. Polythérapie de type thérapie cellulaire t et inhibiteur de btk
WO2018102785A2 (fr) 2016-12-03 2018-06-07 Juno Therapeutics, Inc. Méthodes et compositions pour l'utilisation de lymphocytes t thérapeutiques en association avec des inhibiteurs de kinase
WO2018102786A1 (fr) 2016-12-03 2018-06-07 Juno Therapeutics, Inc. Procédés de modulation de lymphocytes t modifiés par car
WO2018106595A1 (fr) 2016-12-05 2018-06-14 Fate Therapeutics, Inc. Compositions et procédés pour la modulation de cellules immunitaires dans des immunothérapies adoptives
WO2018170188A2 (fr) 2017-03-14 2018-09-20 Juno Therapeutics, Inc. Procédés de stockage cryogénique
WO2018197949A1 (fr) 2017-04-27 2018-11-01 Juno Therapeutics Gmbh Reactifs particulaires oligomères et leurs méthodes d'utilisation
WO2018204427A1 (fr) 2017-05-01 2018-11-08 Juno Therapeutics, Inc. Combinaison d'une thérapie cellulaire et d'un composé immunomodulateur
WO2019018603A2 (fr) 2017-07-19 2019-01-24 Fate Therapeutics, Inc. Compositions et procédés pour la modulation de cellules immunitaires dans des immunothérapies adoptives
WO2019027850A1 (fr) 2017-07-29 2019-02-07 Juno Therapeutics, Inc. Réactifs d'expansion de cellules exprimant des récepteurs recombinants
WO2019089855A1 (fr) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Procédé de génération de compositions thérapeutiques de cellules modifiées
WO2019113557A1 (fr) 2017-12-08 2019-06-13 Juno Therapeutics, Inc. Procédé de production d'une compositions de lymphocytes t modifiés
WO2019152743A1 (fr) 2018-01-31 2019-08-08 Celgene Corporation Polythérapie utilisant une thérapie cellulaire adoptive et un inhibiteur de point de contrôle
WO2019213184A1 (fr) 2018-05-03 2019-11-07 Juno Therapeutics, Inc. Polythérapie d'une thérapie par lymphocytes t à récepteur antigénique chimérique (car) et d'un inhibiteur de btk
WO2020033927A2 (fr) 2018-08-09 2020-02-13 Juno Therapeutics, Inc. Procédés de génération de cellules modifiées et compositions associées
WO2020092854A2 (fr) 2018-11-01 2020-05-07 Juno Therapeutics, Inc. Récepteurs antigéniques chimériques spécifiques du gprc5d (élément d du groupe 5 de classe c des récepteurs couplés à la protéine g)
WO2020233589A1 (fr) 2019-05-20 2020-11-26 南京驯鹿医疗技术有限公司 Anticorps entièrement humain ciblant cd19 et son application
US20220220200A1 (en) 2019-05-20 2022-07-14 Nanjing Iaso Biotherapeutics Co., Ltd. Fully human antibody targeting cd19 and application thereof
WO2020252218A1 (fr) 2019-06-12 2020-12-17 Juno Therapeutics, Inc. Combinaison thérapeutique d'une thérapie cytotoxique à médiation cellulaire et d'un inhibiteur d'une protéine de la famille bcl2 pro-survie
WO2022133030A1 (fr) 2020-12-16 2022-06-23 Juno Therapeutics, Inc. Polythérapie de thérapie cellulaire et d'inhibiteur de bcl2
WO2022187406A1 (fr) 2021-03-03 2022-09-09 Juno Therapeutics, Inc. Combinaison d'une thérapie par lymphocytes t et d'un inhibiteur de dgk
WO2022212400A1 (fr) 2021-03-29 2022-10-06 Juno Therapeutics, Inc. Méthodes de dosage et de traitement au moyen d'une combinaison d'une thérapie par inhibiteur de point de contrôle et d'une thérapie par lymphocytes car t
WO2023147515A1 (fr) 2022-01-28 2023-08-03 Juno Therapeutics, Inc. Procédés de fabrication de compositions cellulaires
WO2024100604A1 (fr) 2022-11-09 2024-05-16 Juno Therapeutics Gmbh Procédés de fabrication de cellules immunitaires modifiées

Non-Patent Citations (60)

* Cited by examiner, † Cited by third party
Title
"Metastasis Research Protocols", vol. 2, HUMANA PRESS INC., article "Cell Behavior In Vitro and In Vivo", pages: 17 - 25
"Thermo Fisher Scientific"
ABRAMSON ET AL., THE LANCET, vol. 396, no. 10254, 2020, pages 839 - 852
ALONSO-CAMINO ET AL., MOL. THER. NUCL. ACIDS.,, vol. 2, 2013, pages 93
BEJCEK ET AL., CANCER RES., vol. 55, 1995, pages 2346 - 2351
BISHOP ET AL., N ENGL J MED, vol. 386, 2022, pages 640 - 654
BORIS-LAWRIETEMIN, CUR. OPIN. GENET. DEVELOP., vol. 3, 1993, pages 102 - 109
BRENTJENS ET AL., SCI TRANSL MED., vol. 5, no. 177, 2013
BURNS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 8033 - 8037
CALLARD ET AL., J. IMMUNOLOGY, vol. 148, no. 10, 1992, pages 2983 - 2987
CARLENS ET AL., EXP. HEMATOL., vol. 28, no. 10, 2000, pages 1137 - 46
CARPENTER ET AL., CLIN CANCER RES., vol. 19, no. 8, 2013, pages 2048 - 2060
CAVALIERI ET AL., BLOOD, vol. 101, no. 2, 2003, pages 1637 - 1644
CHO ET AL., LAB CHIP, vol. 10, 2010, pages 1567 - 1573
CLARKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
COOPER ET AL., BLOOD., vol. 101, 2003, pages 1637 - 1644
DAVILA ET AL., PLOS ONE, vol. 8, no. 4, 2013, pages 61338
DE RIE, CELL. IMMUNOL., vol. 118, 1989, pages 368 - 381
FOWLER ET AL., NATURE MEDICINE, vol. 28, 2022, pages 325 - 332
GODIN ET AL., J BIOPHOTON., vol. 1, no. 5, 2008, pages 355 - 376
HALFORD ET AL., ANN PHARMACOTHER, vol. 55, no. 4, 2021, pages 466 - 479
HERBST ET AL., J. PHARMACOL. EXP. THER., vol. 335, 2010, pages 213 - 222
HUDECEK ET AL., CANCER IMMUNOL RES., vol. 3, no. 2, 2015, pages 125 - 135
HUDECEK ET AL., CLIN. CANCER RES., vol. 19, 2013, pages 3153
HUMAN GENE THERAPY, vol. 1, 1990, pages 5 - 14
JACOBSON ET AL., THE LANCET, vol. 23, no. 1, 2021, pages 91 - 103
KANSASTEDDER, J. IMMUNOL., vol. 147, 1991, pages 4094 - 4102
KINDT ET AL.: "Kuby Immunology", 2007, W.H. FREEMAN AND CO., pages: 91
KOCHENDERFER ET AL., NATURE REVIEWS CLINICAL ONCOLOGY, vol. 10, 2013, pages 267 - 276
KOSTE ET AL., GENE THERAPY, 3 April 2014 (2014-04-03)
LING, N. R. ET AL., LEUCOCYTE TYPING III., 1987, pages 302
MEEKER ET AL., HYBRIDOMA, vol. 3, 1984, pages 305 - 320
METHODS IN MOLECULAR MEDICINE, vol. 58
MIANHILL, EXPERT OPIN BIOL THER;, vol. 21, no. 4, 2021, pages 435 - 441
MILLERROSMAN, BIOTECHNIQUES, vol. 7, 1989, pages 980 - 990
MUELLER ET AL., BLOOD ADV., vol. 5, no. 23, 2021, pages 4980 - 4991
NEELAPU ET AL., N ENGL J MED, vol. 377, no. 26, 2017, pages 2531 - 2544
PAPAPETROUSCHAMBACH, MOL THER., vol. 24, no. 4, 2016, pages 678 - 684
PARDOLL, NATURE REVIEWS CANCER, vol. 12, 2012, pages 252 - 264
PARK ET AL., TRENDS BIOTECHNOL., no. 11, 29 November 2011 (2011-11-29), pages 550 - 557
PATEL ET AL., SCIENTIFIC REPORTS, vol. 9, no. 5335, 2019
PEZUTTO ET AL., J. IMMUNOL., vol. 138, no. 9, 1987, pages 2793 - 2799
PORTOLANO ET AL., J. IMMUNOL., vol. 150, 1993, pages 880 - 887
REMINGTON: "Pharmaceutical Sciences", 1980
REMINGTON: "The Science and Practice of Pharmacy", 1 May 2005, LIPPINCOTT WILLIAMS & WILKINS
SADELAIN ET AL., CANCER DISCOV., vol. 3, no. 4, April 2013 (2013-04-01), pages 388 - 398
SADELAIN ET AL., NAT REV CANCER., vol. 12, no. 1, pages 51 - 8
SAFTICS ET AL., J EXTRACELL VESICLES, vol. 12, no. 7, July 2023 (2023-07-01), pages 12346
SCARPA ET AL., VIROLOGY, vol. 180, 1991, pages 849 - 852
SCHUSTER ET AL., N ENGL J MED, vol. 380, 2019, pages 45 - 56
SEHGAL ET AL., JOURNAL OF CLINICAL ONCOLOGY, vol. 38, no. 15, 2020
SHARPE, PAU T.: "Methods of Cell Separation", 1988, ELSEVIER
TEOH ET AL., BLOOD, vol. 134, 2019, pages 593
TURTLE ET AL., CURR. OPIN. IMMUNOL., vol. 24, no. 5, October 2012 (2012-10-01), pages 633 - 39
VERHOEYEN ET AL., METHODS MOL BIOL., vol. 506, 2009, pages 97 - 114
WANG ET AL., BLOOD, vol. 138, 2021, pages 744
WANG ET AL., J. IMMUNOTHER., vol. 35, no. 9, 2012, pages 689 - 701
WELSH, J. A. ET AL.: "Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches", JOURNAL OF EXTRACELLULAR VESICLES, vol. 13, pages 12404, Retrieved from the Internet <URL:https://doi.org/10.1002/jev2.12404>
WU ET AL., CANCER, no. 2, 18 March 2012 (2012-03-18), pages 160 - 75
YAZAWA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 102, 2005, pages 15178 - 15183

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