Methods for assessing exosomes in a cell composition and related uses

Abstract

Provided are 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. In some embodiments, 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.

Description

METHODS FOR ASSESSING EXOSOMES IN A CELL COMPOSITION AND RELATED USESCross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 673,147, filed July 18, 2024, entitled “METHODS FOR ASSESSING EXOSOMES IN A CELL COMPOSITION AND RELATED USES,” the contents of which is incorporated by reference in its entirety.Reference to An Electronic Sequence Listing

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a filed entitled 735042028040SEQLIST.xml, created on July 14, 2025, which is 47,427 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] 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. In some embodiments, 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.Background

[0004] Various immunotherapy and / or cell therapy methods are available for treating diseases and conditions. For example, adoptive cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and / or other recombinant antigen receptors, such as T cell receptors, as well as other adoptive immune cell and adoptive T cell therapies) can be beneficial in the treatment of cancer or other diseases or disorders. Improved approaches are needed for determining whether a treatment or manufactured cell therapy will result in a beneficial clinical response. Provided herein are methods that address such needs.Summary

[0005] In some aspects, provided herein is a method of assessing exosome production by a cell therapy composition, the method comprising: (a) contacting cells of the cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; and (c) determining an amount or concentration of the isolated exosomes produced from the stimulated cell composition.

[0006] In some of any of the provided embodiments, the cell therapy composition is a treatment or a candidate for a treatment to be administered to a subject.

[0007] In some of any of the provided embodiments, the method is for predicting response to or efficacy of the cell therapy composition in the subject to which it is administered.

[0008] In some of any of the provided embodiments, 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.

[0009] In some of any of the provided embodiments, the cell therapy composition comprises cells obtained from the subject.

[0010] In some aspects, provided herein is a method comprising: (a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) predicting a response to the cell therapy composition in a subject having a disease or condition, wherein (i) the subject is predicted to respond to 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 predicted to not respond to the cell therapy composition 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.

[0011] In some aspects, provided herein is a method comprising: (a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) predicting efficacy of the cell therapy composition in a subject having a disease or condition, wherein (i) efficacy is predicted 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) efficacy is not predicted if the amount orconcentration 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.

[0012] In some aspects, provided herein is a method comprising: (a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) predicting efficacy of the cell therapy composition in a subject having a disease or condition, wherein the predicting efficacy is based on the amount or concentration of the isolated exosomes in the stimulated cell composition being increased compared to the amount or concentration of the isolated exosomes in the control cell composition.

[0013] In some aspects, provided herein is a method comprising: (a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) selecting a subject having a disease or condition, wherein: (i) 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 another 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.

[0014] In some aspects, provided herein is a method comprising: (a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) selecting a subject having a disease or condition for treatment with the cell therapy composition based on the amount or concentration of the isolated exosomes in the stimulated cell composition being increased compared to the amount or concentration of the isolated exosomes in the control cell composition.

[0015] In some aspects, provided herein is a method comprising: (a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced fromthe stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) selecting a subject having a disease or condition for treatment with the cell therapy composition in combination with an other agent based on the amount or concentration of the isolated exosomes in the stimulated cell composition being decreased or unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition.

[0016] In some aspects, provided herein is a method of treatment comprising: (a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) administering a treatment to a subject having a disease or condition, wherein: (i) 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; or (ii) the subject is treated 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 is unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition.

[0017] In some aspects, provided herein is a method of treatment, the method comprising: (a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) administering the cell therapy composition to a subject having a disease or condition, wherein the subject has an increased amount or concentration of exosomes produced from the stimulated cell composition compared to the amount or concentration of the isolated exosomes in the control cell composition.

[0018] In some aspects, provided herein is 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: (a) contacting cells of the cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) selecting the subject for treatment based on the amount or concentration of the isolated exosomes in the stimulated cell composition being increased compared to the amount or concentration of the isolated exosomes in the control cell composition.

[0019] In some aspects, provided herein is a method of treatment comprising (a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) administering the cell therapy composition in combination with an other agent to a subject having a disease or condition, wherein the subject has a decreased or unchanged amount or concentration of exosomes produced from the stimulated cell composition compared to the amount or concentration of the isolated exosomes in the control cell composition.

[0020] In some aspects, provided herein is 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: (a) contacting cells of the cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; (c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and (d) selecting the subject for treatment based on the amount or concentration of the isolated exosomes in the stimulated cell composition being decreased or unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition.

[0021] In some aspects, provided herein is 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.

[0022] In some aspects, provided herein is 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 comprises T cells engineered to express a recombinant receptor, and wherein the subject is a subject that has been selected for treatment by any of the methods provided herein.

[0023] In some aspects, provided herein is 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 amethod comprising: (i) contacting T cells of the T cell composition with a recombinant receptorstimulating agent to generate a stimulated cell composition; (ii) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; and (iii) comparing the amount or concentration of the isolated exosomes produced from the stimulated cell composition to the amount or concentration of the isolated exosomes produced from a control cell composition; (b) predicting a response to the T cell therapy in the subject, wherein (i) 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 (ii) 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; and (c) administering the adaptive treatment to the subject, wherein the adaptive treatment is selected from: (i) the T cell therapy if the subject is predicted to respond to the T cell therapy; and (ii) the T cell therapy in combination with another agent if the subject is not predicted to respond to the T cell therapy.

[0024] In some aspects, provided herein is 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: (i) contacting T cells of the T cell composition with a recombinant receptorstimulating agent to generate a stimulated cell composition; (ii) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; and (iii) comparing the amount or concentration of the isolated exosomes produced from the stimulated cell composition to the amount or concentration of the isolated exosomes produced from a control cell composition; (b) predicting the subject will respond with a response to the T cell therapy based on the amount or concentration of the isolated exosomes in the stimulated cell composition being increased compared to the amount or concentration of the isolated exosomes in a control cell composition; and (c) administering the T cell therapy to the subject predicted to respond to the T cell therapy.

[0025] In some aspects, provided herein is 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 theisolated exosomes produced from the T cell composition is determined by: (i) contacting T cells of the T cell composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition; (ii) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; and (iii) comparing the amount or concentration of the isolated exosomes produced from the stimulated cell composition to the amount or concentration of the isolated exosomes produced from a control cell composition.

[0026] In some of any of the provided embodiments, the cell therapy composition comprises T cells engineered to express a recombinant receptor. In some of any of the provided embodiments, the isolated exosomes are further characterized for presence of the recombinant receptor.

[0027] In some of any of the provided embodiments, the cell therapy composition is a treatment or a candidate for a treatment to be administered to a subject.

[0028] In some of any of the provided embodiments, the T cells are primary cells. In some of any of the provided embodiments, the T cells are autologous cells. In some of any of the provided embodiments, the T cells are allogeneic cells. In some of any of the provided embodiments, the T cells are CD3+. In some of any of the provided embodiments, the T cells are CD4+, and / or CD8+ T cells. In some of any of the provided embodiments, the T cells of the cell therapy composition or T cell composition comprise CD4+ T cells and CD8+ T cells.

[0029] In some of any of the provided embodiments, 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. In some of any of the provided embodiments, 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.

[0030] In some of any of the provided embodiments, the cell therapy composition had been cryopreserved and is thawed prior to detecting exosomes produced from cells of the stimulated cell composition.

[0031] In some of any of the provided embodiments, 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(s); and during or subsequent to activating the cells of the population of input cells, introducing into cells a polynucleotide encoding the recombinant receptor.

[0032] In some of any of the provided embodiments, introducing the polynucleotide encoding the recombinant receptor comprises transducing cells with a viral vector encoding the recombinant receptor.

[0033] In some of any of the provided embodiments, 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.

[0034] In some of any of the provided embodiments, the manufacturing process further comprises culturing cells introduced with the polynucleotide under conditions for expansion of T cells in the composition.

[0035] In some of any of the provided embodiments, 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. In some of any of the provided embodiments, the biological sample is an apheresis product or leukapheresis product. In some of any of the provided embodiments, the apheresis product or leukapheresis product has been previously cryopreserved.

[0036] In some of any of the provided embodiments, 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.

[0037] In some of any of the provided embodiments, the human subject has a disease or condition.

[0038] In some of any of the provided embodiments, the method further comprises harvesting cells produced by the manufacturing process and formulating the harvested cells in a pharmaceutically acceptable buffer. In some of any of the provided embodiments, the harvested cells further comprises a cryoprotectant.

[0039] In some of any of the provided embodiments, the disease or condition is a cancer or tumor. In some of any of the provided embodiments, the disease or condition is an autoimmune disease or condition.

[0040] In some of any of the provided embodiments, 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.

[0041] In some of any of the provided embodiments, 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.

[0042] In some of any of the provided embodiments, detecting comprises isolating the exosomes.

[0043] In some of any of the provided embodiments, isolating is by centrifugation. In some of any of the provided embodiments, 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 anyof 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.

[0044] 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 / 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. 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 100 nm. In some of any of the provided embodiments, the one or more exosome markers are identified by immunoaffinity-based capture. In some of any of the provided embodiments, the immunoaffinity-based capture comprises an antibody specific to the one or more exosome markers.

[0045] In some of any of the provided embodiments, the one or more exosome markers are selected from CD63, CD81, CD9, and any combination thereof. In some of any of the provided embodiments, the one or more exosome markers is CD63. In some of any of the provided embodiments, the one or more exosome markers is CD81. In some of any of the provided embodiments, the one or more exosome markers is CD9. In some of any of the provided embodiments, the one or more exosome markers comprises CD63 and CD81. In some of any of the provided embodiments, the one or more exosome markers comprises CD63 and CD9. In some of any of the provided embodiments, the one or more exosome markers comprises CD81 and CD9. In some of any of the provided embodiments, the one or more exosome markers comprises CD63, CD81 and CD9.

[0046] In some of any of the provided embodiments, 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. In some of any of the provided embodiments, 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.

[0047] In some of any of the provided embodiments, the one or more exosome markers comprises CD63 and wherein the exosomes have a size between about 30 nm and about 100 nm.

[0048] In some of any of the provided embodiments, 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. In some of any of the provided embodiments, the surface of the exosomes comprises the recombinant receptor expressed by the cells of the cell therapy composition or T cell composition. In some of any of the provided embodiments, the surface of the exosomes comprises the surrogate marker of therecombinant receptor expressed by the cells of the T cell composition. In some of any of the provided embodiments, the surrogate marker is a truncated cell surface receptor, optionally a truncated epidermal growth factor receptor (tEGFR).

[0049] In some of any of the provided embodiments, the recombinant receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some of any of the provided embodiments, 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.

[0050] In some of any of the provided embodiments, the antigen is expressed by the cells of the disease or condition.

[0051] In some of any of the provided embodiments, the antigen is expressed by the cells of a cancer or tumor. In some of any of the provided embodiments, the cancer or tumor is a hematological malignancy. In some of any of the provided embodiments, the hematological malignancy is a myeloma, leukemia or lymphoma. In some of any of the provided embodiments, 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). In some of any of the provided embodiments, the antigen is a B cell antigen. In some of any of the provided embodiments, 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.

[0052] In some of any of the provided embodiments, 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. In some of any of the provided embodiments, the recombinant receptorstimulating agent comprises a target antigen or an extracellular domain binding portion thereof. In some of any of the provided embodiments, the target antigen is a recombinant antigen of the recombinant receptor. In some of any of the provided embodiments, 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. In some of any of the provided embodiments, 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.

[0053] In some of any of the provided embodiments, 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. In some of any of the provided embodiments, the antigen-expressing cell is a cell line. In some of any of the provided embodiments, the cell line is a tumor cell line. In some of any of the provided embodiments, the antigen-expressing cell is a cell that has been introduced, optionally by transduction, to express an antigen of the recombinant receptor.

[0054] In some of any of the provided embodiments, 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.

[0055] In some of any of the provided embodiments, the immunomodulatory agent is an immunomodulatory imide drug (IMiD) or a cereblon E3 ligase modulator (CELMoD). In some of any of the provided embodiments, the immunomodulatory agent is thalidomide or a thalidomide derivative. In some of any of the provided embodiments, the immunomodulatory agent is selected from the group consisting of lenalidomide, pomalidomide, avadomide (CC-122), iberdomide (CC-220). In some of any of the provided embodiments, the checkpoint inhibitor is selected from a PD-1 inhibitor, LAG3 inhibitor and PD-L1 inhibitor. In some of any of the provided embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g, nivolumab). In some of any of the provided embodiments, the LAG3 inhibitor is an anti- LAG3 antibody (e.g., relatlimab). In some of any of the provided embodiments, the PD-L1 inhibitor is an anti-PD-Ll antibody (e.g. durvalumumab). In some of any of the provided embodiments, the BCL2 inhibitor is selected from the group consisting of venetoclax, navitoclax, ABT737, maritoclax, obatoclax and clitocine.Brief Description of the Drawings

[0056] 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). The treatment response was determined at one month posttreatment; further analysis of CAR-T cell products from the same R and NR cohorts is shown in FIG. 5, FIGS. 6A-6E, and FIG. 8.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] FIG. 8 shows the concentration of CD63+ exosomes in 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 progressivedisease (PD) (also referred to as non-responders). The autologous CAR-T cells were stimulated using an anti-idiotypic antibody against the CAR (alD).Detailed Description

[0064] Among the provided embodiments are methods directed to assessing exosomes produced from 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)). In some embodiments, the cell composition is a cell therapy such as a T cell therapy composition. In some embodiments, the cell composition is a T cell therapy composition. In some embodiment, the method for assessing exosomes is for predicting response to, and / or efficacy of, the T cell therapy composition.

[0065] In some aspects, provided herein is 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). In some embodiments, the cell composition is a cell therapy composition such as a T cell therapy composition. In some embodiments, the cell composition is a T cell therapy composition. In some embodiments, the method comprises contacting the T 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, and comparing the amount or concentration of exosomes produced from the stimulated cell composition to the amount or concentration of exosomes produced from a control cell composition. In some embodiments, the control cell composition is a T cell therapy composition that has not been stimulated by contacting it with the recombinant receptor-stimulating agent. In some embodiments, response to, and / or efficacy of, the T cell therapy composition is predicted if the amount or concentration of isolated exosomes in the stimulated cell composition is increased compared to isolated exosomes in the control cell composition. In some embodiments, response to, and / or efficacy of, the cell therapy composition is not predicted if the amount or concentration of isolated exosomes in the stimulated cell composition is decreased or unchanged compared to isolated exosomes in the control cell composition.

[0066] Also provided herein are methods of treating a subject having a disease or condition, selecting a subject having a disease or condition for treatment, and / or methods of adaptive treatment in a subject having a disease or condition with a cell composition such as a composition of cells enriched in, or containing, T cells engineered with a recombinant receptor (e.g., CAR or TCR). In some embodiments, the cell composition is a cell therapy composition. In some embodiments, the cell composition is a T cell therapy composition. In some embodiments, the T cell therapy composition comprises immune cells engineered to express a recombinant receptor (e.g., CAR or TCR). In some embodiments, 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 celltherapy). In some embodiments, 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). In some embodiments, the methods of treating and selecting are based on a method comprising contacting the T 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 and comparing the amount or concentration of exosomes produced from the stimulated cell composition to the amount or concentration of exosomes produced from a control cell composition. In some embodiments, the disease or condition comprises a cancer or tumor. In some embodiments, the disease or condition comprises an autoimmune or inflammatory disease.

[0067] 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. complete response) to the T cell therapy. In some embodiments provided herein, 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. In some embodiments, 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).

[0068] In some embodiments, 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.

[0069] Adoptive cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and / or other recombinant antigen receptors, such as T cell receptors (TCRs), as well as other adoptive immune cell and adoptive T cell therapies) can be effective in the treatment of cancer and other diseases and disorders compared to standard therapies (e.g., chemotherapy, radiation etc.). However, in certain contexts, available approaches to adoptive cell therapy may not always be entirely satisfactory. In some aspects, the ability of the administered cells to recognize and bind to a target to traffic, localize toand 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.

[0070] 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. In some embodiments, 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. For instance, exosomes can carry cytokines that are cytotoxic to cancer and / or tumor cells. Additionally, CAR+ exosomes are less sterically hindered from penetrating solid-tumors. Thus, in some embodiments, 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.

[0071] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0072] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. METHODS OF ASSESSING EXOSOMES OF A CELL COMPOSITION

[0073] Provided are methods for 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). In some embodiments, assessing exosomes produced from cells of a cell therapy composition has various downstream applications as described herein. In someembodiments, once the amount or concentration of exosomes is assessed, methods for predicting response to, and / or efficacy of, the cell composition (e.g., a T cell therapy composition) can be determined. Further, in some embodiments, methods for treatment, including adaptive treatment, can be predicted or determined based the amount or concentration of exosomes assessed in the cell therapy compositions. In some embodiments, the cell composition is a T cell therapy composition. In some embodiments, exosomes are assessed in the T cell therapy composition by identifying cell particles that are surface positive for CD63. In some embodiments, 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.

[0074] In some aspects, the provided methods allow for assessing exosome production from a cell composition, such as a cell therapy composition. In some embodiments, 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. In some embodiments, the cell therapy is a treatment or a candidate for a treatment to be administered to a subject. In some embodiments, the method of assessing exosome production is for predicting response to, and / or efficacy of, a cell therapy composition. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0075] In some embodiments, 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 isolatedexosomes in the control cell composition. In some embodiments, 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.

[0076] In some embodiments, responsiveness is determined by a subject’s partial response to the cell therapy composition. That is, in some embodiments, responsiveness comprises a partial response to the cell therapy composition. In some embodiments, responsiveness consists of a partial response to the cell therapy composition. In some embodiments, responsiveness is determined by a subject’s complete response to the cell therapy composition. That is, in some embodiments, responsiveness comprises a complete response to the cell therapy composition. In some embodiments, responsiveness consists of a complete response to the cell therapy composition.

[0077] In other embodiments, 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. Thus, in some embodiments, 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.A. Methods of Stimulating a Cell Therapy Composition

[0078] In some aspects, the provided methods include stimulating cells of a cell therapy composition to produce exosomes. In some embodiments, the cells of the cell therapy composition express a recombinant receptor. In some embodiments, the cells of the cell therapy composition express any of the recombinant receptors provided in Section III. In some embodiments, the cell therapy composition includes any of those described in Section III. In some embodiments, the cells of the cell therapy composition express a CAR or a TCR. In some embodiments, the cell therapy composition is a CAR-T cell therapy composition. In some embodiments, the cell therapy composition is a TCR cell therapy composition. In some embodiments, 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.

[0079] In some embodiments, the method of stimulating cells of a cell therapy composition to produce exosomes include means of stimulating the recombinant receptor expressed by the cells (e.g., CARs, TCRs) of the cell therapy composition. It is contemplated that any means suitable for stimulating the recombinant receptor that is also capable of being quantified may be used. In some embodiments, the means of stimulation of the recombinant receptor is achieved by a recombinant receptor-stimulating agent able to bind to the recombinant receptor to stimulate an intracellular signal by the recombinantreceptor. Exemplary recombinant receptor-stimulating agents include antigens (e.g. purified or recombinant antigens) of the recombinant receptor (also called a “target antigen”), antibodies such as anti-idiotype antibodies to the recombinant receptor, and antigen-expressing cells (also called “targetexpressing cells”) that express an antigen target of the recombinant receptor on the cell surface.

[0080] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, for the contacting with the recombinant-receptor stimulating agent, the cells of the cell composition are present at a concentration of from 0.1 x 106cells / mL to 100 x 106cells / mL, such as from 0.5 x 106cells / mL to 50 x 106cells / mL, more generally 0.5 x 106cells / mL to 10 x 106cells / mL. In some embodiments, for the contacting with the recombinant-receptor stimulating agent, the cells of the cell composition are present at a concentration of from 0.5 x 106cells / m, 1 x 106cells / mL, 2 x 106cells / mL, 3 x 106cells / mL, 4 x 106cells / mL, 5 x 106cells / mL, 6 x 106cells / mL, 7 x 106cells / mL, 8 x 106cells / mL, 9 x 106cells / mL, 10 x 106cells / mL, or any value between any of the foregoing.

[0081] In some embodiments, where 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. In some embodiments, 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.

[0082] In some embodiments, 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. In some embodiments, a recombinant-receptor stimulating agent (e.g., surface immobilized antigen of the recombinant receptor, e.g., CAR, for example, plate-bound antigen) is incubated with the cells for 2 hours to 96 hours. In particular embodiments, the incubation is for 12 hours to 72 hours, such as 12 hours to 48 hours, for example at or about 24 hours. In some embodiments, 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. In some embodiments, a stable carbon dioxide is maintained, such as at or about 5% CO2.Z Surface Immobiiized Binding Moiecuie

[0083] In particular embodiments, 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. In provided embodiments, the binding molecule may be an antigen or a portion of an antigen ofthe recombinant receptor (e.g. extracellular portion of an antigen) or an antibody (e.g., an anti-idiotypic antibody) specific to the recombinant receptor. In some embodiments, the recombinant receptorstimulating agent is immobilized or bound to a surface support, such as a microwell plate or a solid particle (e.g. bead).

[0084] In some embodiments, the recombinant receptor-stimulating agent, such as the binding molecule, 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. In some embodiments, a culture plate, such as a microwell plate, can be used that is has a surface to allow for passive adsorption of binding molecules. Suitable surfaces include, but are not limited to, polystyrene, polyvinylchloride, or polyethylene. In some embodiments, the surface, such as one containing polystyrene, can also be modified to have positively charged amine groups, which can ionically couple to small negatively charged binding molecules. For antibody binding, such as to an Fc protein or an anti-idiotype antibody, 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.

[0085] In other embodiments, 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. In some embodiments, a particle described herein (e.g., bead particle) provides a solid support or matrix to which the binding molecule (e.g. 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. In particular embodiments, 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.

[0086] In certain embodiments, 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. In some embodiments, the surface support is a solid support. In some examples, the solid support is a bead, and the antigen or portion is immobilized on the bead. In some embodiments, the solid support is the surface of a well or plate, e.g., a cell culture plate. In some embodiments, the surface support is a soluble oligomeric particle, and the antigen is immobilized on the surface of the soluble oligomeric particle. Examples of surface supports for immobilization or attachment of an agent (e.g. binding molecule) for recognition or binding to a recombinant receptor may be found in published International application WO 2019 / 027850, which is incorporated herein by reference for all purposes.

[0087] In particular embodiments, 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. In some embodiments, the particles or beads are biocompatible, i.e. non-toxic. In certain embodiments the particles or beads are non-toxic to cultured cells, e.g., cultured T cells. In particular embodiments, the particles are monodisperse. In certain embodiments, “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. In some embodiments, 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. In certain embodiments, 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. In some embodiments, the particles, e.g., beads, are composed of glass, silica, polyesters of hydroxy carboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids.

[0088] 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. 20100207051; and Sharpe, Pau T., Methods of Cell Separation, Elsevier, 1988. Commercially available particles, e.g., beads, (e.g., bead 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), Dynabeads® M-450 (e.g., Dynabeads® M-450 Tosylactivated, Invitrogen), Dynabeads® UntouchedTM (e.g., Dynabeads® UntouchedTM Human CD8 T Cells, Invitrogen), and Dynabeads® that bind, expand and / or activate T cells (e.g., Dynabeads® Human T- Activator CD3 / CD28 for T Cell Expansion and Activation, Invitrogen); Estapor® M (Merk Chimie SAS); Estapor® EM (Merk Chimie SAS); MACSiBeadsTM Particles (e.g., anti-biotin MACSiBead Particles, Miltenyi Biotec, catalog #130-091-147); Streptamer® Magnetic Beads (IBA BioTAGnology); Strep-Tactin® Magnetic Beads (IBA BioTAGnology); Sicastar®-M (Micormod Partikeltechnologie GmbH) Micromer®-M (Micromod Partikeltechnologie); MagneSilTM (Promega GmbH); MGP (Roche Applied Science Inc.); Pierce™ Protein G Magnetic Beads (Thermo Fisher Scientific Inc.); Pierce™ Protein A Magnetic Beads (Thermo Fisher Scientific Inc.); Pierce™ Protein A / G Magnetic Beads (Thermo Fisher Scientific Inc.); Pierce™ NHS-Activated Magnetic Beads (Thermo Fisher Scientific Inc.); Pierce™ Protein L Magnetic Beads (Thermo Fisher Scientific Inc.); Pierce™ Anti-HA MagneticBeads (Thermo Fisher Scientific Inc.); Pierce™ Anti-c-Myc Magnetic Beads (Thermo Fisher Scientific Inc.); Pierce™ Glutathione Magnetic Beads (Thermo Fisher Scientific Inc.); Pierce™ Streptavidin Magnetic Beads (Thermo Fisher Scientific Inc.); MagnaBindTM Magnetic Beads (Thermo Fisher Scientific Inc.); Sera-MagTM Magnetic Beads (Thermo Fisher Scientific Inc.); Anti-FLAG® M2 Magnetic Beads (Sigma- Aldrich); SPHEROTM Magnetic Particles (Spherotech Inc.); and HisPurTM Ni- NTA Magnetic Beads (Thermo Fisher Scientific Inc.).

[0089] In certain embodiments, the antigen or an extracellular domain portion thereof is bound to the particle (e.g. bead) via a covalent chemical bond. In particular embodiments, 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. In certain embodiments, 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) of the antigen or extracellular binding portion thereof is conjugated directly to a hydroxyl or carboxyl group of a PLA or PGA polymer, a terminal amine or carboxyl group of a dendrimer, or a hydroxyl, carboxyl or phosphate group of a phospholipid on the surface of the particle by direct chemical reaction. In some embodiments, a conjugating moiety conjugates, e.g., covalently binds, to both the binding molecule and the particle, thereby linking them together. In certain embodiments, 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). In particular embodiments, 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. In some embodiments, 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. In still other particular embodiments, 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. Examples of such functional groups include, but are not limited to, tosyl, epoxy, and chloromethyl groups.

[0090] In some embodiments, a non-covalent bond between a ligand bound to the antigen peptide or protein and an anti-ligand attached to the surface support (e.g. bead) may conjugate the antigen to the support (e.g. bead). In some embodiments, 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. Alternatively, if the binding molecule (e.g. antigen) are fused to an immunoglobulin domain bearing an Fc region, the Fc domain may act as a ligand, and protein A, either covalently or non-covalently bound to the surface of the surface support (e.g. bead), may serve as the anti-ligand to non-covalently conjugatethe 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.

[0091] In some embodiments, the recombinant receptor-stimulating agent is or includes a target, e.g., an antigen, a recombinant antigen, or fragment thereof. In some embodiments, the target is an antigen of the recombinant receptor. In some embodiments, the recombinant receptor-stimulating agent is or includes an antigen, e.g., a recombinant antigen or fragment thereof.

[0092] For instance, 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. In some embodiments, the target, e.g. antigen, 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. In some embodiments, the target is an antibody that is recognized or bound by an extracellular domain of the recombinant receptor. In some embodiments, 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.

[0093] In some embodiments, the target is an antigen recognized by the extracellular domain of the recombinant receptor. In some embodiments, 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 receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), IL- 13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa lightchain, LI cell adhesion molecule (Ll-CAM), CE7 epitope of Ll-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (R0R1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomer ase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen is or includes CD20, CD19, CD22, R0R1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.

[0094] In some embodiments, 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. In particular embodiments, 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. Typically, 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.

[0095] For instance, in some embodiments, the recombinant receptor is an anti-BCMA CAR. In such embodiments, the extracellular domain of the recombinant receptor (e.g. CAR) is specific for or binds to BCMA and the antigen is BCMA or is an extracellular domain portion of BCMA. In some embodiments, the BCMA polypeptide is a mammalian BCMA polypeptide. In particular embodiments, the BCMA polypeptide is a human BCMA polypeptide. In some embodiments, 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. In certain embodiments, 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. In some embodiments,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.

[0096] In some embodiments, the recombinant receptor is an anti-RORl CAR. In certain embodiments, the extracellular domain of the recombinant receptor (e.g. CAR) is specific for or binds to ROR1 and the antigen is ROR1 or is an extracellular domain portion of ROR1. In certain embodiments, the ROR1 polypeptide is mammalian. In particular embodiments, the ROR1 polypeptide is human. In some embodiments, the antigen is an extracellular domain of ROR1 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g. CAR. In some embodiments, 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. In some embodiments, 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.

[0097] In some embodiments, the recombinant receptor is an anti-CD22 CAR. In certain embodiments, the extracellular domain of the recombinant receptor (e.g. CAR) is specific for or binds to CD22 and the antigen is CD22 or is an extracellular domain portion of CD22. In certain embodiments, the CD22 polypeptide is mammalian. In particular embodiments, the CD22 polypeptide is human. In some embodiments, the antigen is an extracellular domain of CD22 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g. CAR. In some embodiments, 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. In some embodiments, 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.

[0098] In some embodiments, the recombinant receptor is an anti-CD19 CAR. In certain embodiments, the extracellular domain of the recombinant receptor (e.g. CAR) is specific for or binds to CD 19 and the antigen is CD 19 or is an extracellular domain portion of CD 19. In certain embodiments, the CD19 polypeptide is mammalian. In particular embodiments, the CD19 polypeptide is human. In some embodiments, the antigen is an extracellular domain of CD 19 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g. CAR. In some embodiments, 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 fragmentthereof 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. In some embodiments, 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.

[0099] In some embodiments, 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). In some embodiments, the polypeptide antigen, or portion thereof, are identical. In certain embodiments, 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. In some embodiments, 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. In some embodiment, 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.

[0100] In some embodiments, a polypeptide is joined directly or indirectly to a multimerization domain. Exemplary multimerization domains include the immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain, for example, 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. In particular embodiments, the polypeptide antigen is linked, directly or indirectly, to an Fc domain. In some embodiments, the polypeptide is a fusion polypeptide comprising the polypeptide antigen or portion thereof and the Fc domain.

[0101] In particular embodiments, an antigen or extracellular domain portion thereof is a fusion polypeptide that comprises an Fc domain. In some embodiments, 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). In certain embodiments, the Fc domain is fused to the C-terminal of the polypeptide antigen. In particular embodiments, the Fc domain is fused to the N-terminal of the polypeptide antigen. In some embodiments, 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.

[0102] In some embodiments, the Fc domain is an IgG Fc domain, or a portion or variant thereof. In some embodiments, the Fc domain is a human IgG Fc domain, or a portion or a variant thereof, thatcomprises 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. In particular embodiments, the Fc domain is a wild-type human IgG Fc domain, or a portion or variant thereof. In particular embodiments, the Fc domain is a variant of the wild-type human IgGl Fc domain.

[0103] In some embodiments, the antigen and the multimerization domain, such as Fc domain, are connected by a linker, such as an amino acid linker. In certain embodiments, the antigen is fused to the N-terminus of an amino acid linker, and the multimerization domain, such as Fc domain, is fused to the C-terminus of the linker. Although 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). In some embodiments, an amino acid linker comprises the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

[0104] In some embodiments, the antigen is provided as an extracellular domain of BCMA, e.g. human BCMA, fused to an Fc domain (BCMA-Fc). In particular embodiments, 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. In some embodiments, the antigen is provided as an extracellular domain of ROR1, e.g. human ROR1, fused to an Fc domain (RORl-Fc). In certain embodiments, the 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. In particular embodiments, the antigen is provided as an extracellular domain of CD22, e.g. human CD22, fused to an Fc domain (e.g. CD22-Fc). In certain embodiments, 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. a. Antibodies

[0105] In some aspects, 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, forexample a recombinant receptor, e.g., CAR. In particular, 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. In some embodiments, the idiotype is any single antigenic determinant or epitope within the variable portion of an antibody. In some cases it can overlap the actual antigen-binding site of the antibody, and in some cases it may comprise variable region sequences outside of the antigen-binding site of the antibody. The set of individual idiotypes of an antibody is in some embodiments referred to as the “idiotype” of such antibody. In some embodiments, the anti-ID is able to bind to the recombinant receptor to stimulate a recombinant receptor-dependent activity.

[0106] The choice of 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. 16871936); a GPRC5D-directed CAR, see e.g. PCT Application No. PCT / US2020 / 063497; and a BCMA-directed CAR, see e.g. PCT Application No. PCT / US2020 / 063492. The anti-idiotypic antibody can be immobilized or attached to a surface support (e.g., bead) as described above for use as a recombinant receptor-stimulating agent against cells expressing the recombinant receptor (e.g., CAR) targeted by the anti-idiotypic antibody.A Target-expressing cells

[0107] In some embodiments, 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. In some embodiments, the target is an antigen of the recombinant receptor and thus, in some cases, the target-expressing cells are antigen-expressing cells. In some embodiments, the recombinant receptor-stimulating agent is an antigen-expressing cell, such as a cell expressing a target or an antigen as described above.

[0108] In certain embodiments, 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.

[0109] In certain embodiments, the target-expressing cells, express a target that is bound by and / or recognized by the recombinant receptor. In some embodiments, the target is an antibody and the targetexpressing cells express the antibody. In some embodiments, the target-expressing cells are tumor cells. In particular embodiments, the target-expressing cells are primary cells. 1

[0110] In some embodiments, the target is an antigen recognized by the recombinant receptor and the target-expressing cells are antigen-expressing cells. In certain embodiments, the antigen-expressing cells, express an antigen that is bound by and / or recognized by the recombinant receptor. In some embodiments, the antigen-expressing cells are tumor cells. In particular embodiments, the antigenexpressing cells are primary cells. In some embodiments, the cell line is an immortal cell line. In particular embodiments, the antigen expressing cells are cancerous cells and / or tumor cells. In some embodiments, the antigen-expressing cells are derived from a cancer cell and / or a tumor cells, e.g., human cancer cells and / or human tumor cells. In some embodiments, the antigen-expressing cells are cells from a cancer cell line, optionally a human cancer cell line. In some embodiments, the antigenexpressing cells are cells from a tumor cell line, optionally a human tumor cell line.

[0111] In particular embodiments, the antigen-expressing cells are tumor cells.

[0112] Any of a number of 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- 11929, RPMI-8226, OPM2, EP-1, L363, MM.1S, MM.1R, MC / CAR, JJN3, KMS11, AMO-1, EJM; MOLP-8). For instance, 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.

[0113] In some embodiments, the target-expressing cell line is a cell line that has been transduced to express the target of the recombinant receptor. In some embodiments, the target is a tumor antigen. In particular embodiments, 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. In some embodiments, the human cell line may be K562, U937, 721.221, T2, and C1R cells. For instance, the K562 chronic myeloid leukemia cell line may be introduced with a nucleic acid encoding the tumor antigen. In some embodiments, the cell line can be engineered with plasmid vectors ormessenger RNAs (mRNAs) that encode the tumor antigen of interest. In some embodiments, the introduction can be by lentiviral-based transduction. In some embodiments the cell line (e.g. K562 cells) stably expresses the exogenous nucleic acid encoding the tumor antigen. In some embodiments the exogenous nucleic acid may be integrated into the genome of the cell line (e.g. K562 cell). In some embodiments the exogenous nucleic acid may be integrated into the genome of the cell line (e.g. K562 cell) at a particular locus. In some embodiments, the exogenous nucleic acid may be integrated into the genome of the cell line (e.g. K562 cell) at a genomic safe harbor (GSH). A 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). Several safe 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).

[0114] In some embodiments, 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. 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. In certain embodiments, cells may be exposed to about 30 to 36 Gy (i.e., 3000 to 3600 rad) to ensure mitotic arrest while preserving viability. As an alternative to physical irradiation, 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. Following irradiation or chemical treatment, 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. In some embodiments, irradiated target cells are added to culture medium prior to the addition of a sample of cells of the cell therapy.B. Exosomes

[0115] In provided embodiments, 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. In some embodiments, the exosomes provided herein can express the recombinant receptor and can contact and communicate with a target cell. In some embodiments, the target cell is a cancer cell or a tumor cell.

[0116] An exosome is a cell-derived, lipid-bilayer encapsulated particle that contains bioactive molecules of the cell from which it originates. Thus, 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. For instance, the Minimal Information For Studies of Extracellular Vesicles 2023 (MISEV2023; see, e.g., Welsh, J. A. et al., Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. Journal of Extracellular Vesicles, 13, el2404. https: / / doi.org / 10.1002 / jev2.12404, which is incorporated herein by reference), sets forth recommendations for characterizing extracellular vesicles (EVs), including exosomes, using a combination of physical, biochemical, and functional attributes. According to 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. Within this broader category, 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).

[0117] Consistent with this evolving understanding, the present disclosure uses the term “exosome” to refer 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. For example, in some embodiments, 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. In some embodiments, an exosome is a cell-derived particle that is surface positive for one or more markers such as CD63, CD81, and CD9. In some embodiments, an exosome comprises both size characteristics and expression of one or more such surface markers. In some embodiments, 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.

[0118] In embodiments where the exosome is characterized solely by its size (e.g., between about 30 nm and 150 nm in diameter), and not by surface marker expression, it is understood that such particles may also be referred to more generally as extracellular vesicles (EVs). Size may be measured by variousestablished methods, such as Nanoparticle Tracking Analysis (NTA). Accordingly, in some embodiments where size alone is determined, such as by using NTA (see, e.g., Example 3), without further marker analysis, the term “exosome” is used in a broad sense to encompass EVs of that size but that may not be limited to endosomal origin.

[0119] In other embodiments, 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. In such cases, it is understood that the term “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). In such embodiments, the exosome is understood to have originated from the endosomal compartment and to have undergone regulated biogenesis. Specifically, 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). The ILVs emerge through the inward budding of the endosome’s limiting membrane allowing for the encapsulation of cargoes destined for exosomes. Specifically, cargo destined for an exosome binds trafficking effectors that lead to membrane bending and scission processes that give rise to exosomes. As the MVB is transported to the cell surface, cargo continues to be incorporated into the MVB and ILVs via trafficking effectors. 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. In some embodiments, an exosome is characterized by its size as described and an exosome marker that is a tetraspanin, such as CD63, CD81 or CD9. In connection with provided methods, the methods particularly characterize an exosome by its size as described and the surface expression of CD63. In some embodiments, the exosome is characterized as surface positive for CD63. In some embodiments, the exosome is characterized as greater than about 30 nm in diameter and less than about 100 nm in diameter, and is surface positive for CD63.

[0120] Exosomes play an important role in intercellular communication through the transmission of bioactive molecules to target organs, tissues, or cells. As such, the contents and functionality of an exosome can be tailored by manipulating its source cell. As an example, 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.

[0121] In some embodiments, 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.

[0122] In provided embodiments, exosomes are present in the supernatant of the cell therapy composition, which can be enriched and isolated in various ways. In some embodiments, exosomes are enriched and isolated from the supernatant of the cell therapy composition using centrifugation. In some embodiments, the centrifugation step removes cell debris. In some embodiments, centrifugation aids in the isolation of the exosomes. In some embodiments, exosomes are isolated without using centrifugation. In some embodiments, exosomes are enriched and isolated from the supernatant of the cell therapy composition using precipitation. In some embodiments, exosomes are isolated without using precipitation. In some embodiments, 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.

[0123] In some embodiments, 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. In some embodiments, 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.

[0124] In provided embodiments, 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. In some embodiments, the exosomes are detected based on size of the exosome. In some embodiments, the exosomes are detected based on markers present on the surface of the exosome. In some embodiments, exosomes are detected based on the size and presence of markers on the surface of the exosome. In some embodiments, the exosomes are detected based on any other parameter known in the art. Methods of detecting and / or characterizing exosomes based on size, surface markers or any other parameter are known in the art and include optical, electrochemical assays, immunoreaction assays or immunoaffinitybased, aptamer-based detection, fluorescence, surface plasma resonance (SPR), Surface-Enhanced Raman scattering (SERS), chromatography, and microfluidic detection methods.

[0125] In some embodiments, exosomes are detected and characterized based on surface markers using immunoaffinity-based detection methods. In some embodiments, immunoaffinity-based methods can be used using antibodies to stain for abundant tetraspanins that define exosomes (e.g., CD9, CD81 and / or CD63). In some embodiments, 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. In some embodiments, 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. 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. 2023 Jul; 12(7): 12346). In some embodiments, the immunoaffinitybased detection method is performed using the Leprechaun instrument from Unchained Labs (Pleasanton, CA). In provided embodiments, exosomes are detected and characterized based on surface markers using the Leprechaun instrument.Z Exosome Characterization

[0126] Exosomes are typically characterized by their size, markers, cargo and / or function. In some embodiments, the methods provided herein characterize the exosome by its size. In some embodiments, the methods provided herein characterize the exosome by one or more of its markers. In some embodiments, the methods provided herein characterize the exosome by its cargo. In some embodiments, the methods provided herein characterize the exosome by its function. In some embodiments, the methods provided herein characterize the exosome by any combination of characteristics including size, marker(s), cargo, and function(s). In some embodiments, the methods provided herein characterize the exosome by its size and one or more of its markers. In some embodiments, 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

[0127] In some embodiments, an exosome of the present disclosure is distinguished from non- exosome cell-derived particles based on size. In some embodiments, an exosome detected herein is greater than about 30 nm in diameter. In some embodiments, an exosome detected herein is less than about 200 nm in diameter. In some embodiments, an exosome detected herein is less than about 150 nm in diameter. In some embodiments, an exosome detected herein is less than about 130 nm in diameter. In some embodiments, an exosome detected herein is less than about 100 nm in diameter. In some embodiments, an exosome detected herein is between about 30 nm and 200 nm in diameter. In some embodiments, an exosome detected herein is between about 30 nm and 150 nm in diameter. In someembodiments, 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. In some embodiments, 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. In some embodiments, 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 diameter. In some embodiments, 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 145 nm and 150 nm. b. Exosome Markers

[0128] In some embodiments, exosomes of the present disclosure are detected by identifying cell particles that are surface positive for one or more exosome markers provided herein. In some embodiments, the one or more exosome markers comprise any one or more exosome markers known in the art. In some embodiments, the one or more exosome markers comprise scaffolding proteins (e.g., tetraspanins), transmembrane proteins, immunomodulatory proteins, adhesion molecules, lipids, glycoproteins, and / or glycolipids.

[0129] In some embodiments, an exosome detected herein is characterized by an exosome marker that comprises one or more tetraspanins. In some embodiments, the one or more tetraspanins include one or a combination of any of CD63, CD81, CD9, CD37, and / or CD82. In some embodiments, 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.

[0130] In some embodiments, 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. In some embodiments, the exosomes provided herein are distinguished from non-exosome cell-derived particles based on CD81and CD9. In some embodiments, the exosomes provided herein are distinguished from non-exosome cell- derived particles based on CD63, CD81 and CD9.

[0131] In some embodiments, 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.

[0132] In additional embodiments, the one or more exosome markers comprise Alix, actin, TSG101, tubulin, GAPDH, beta actin, and / or HSP70.

[0133] In some embodiments, the exosomes detected herein comprise the recombinant receptor expressed by the cells of the cell therapy composition provided herein. Thus, in some embodiments, the exosomes provided herein comprise a chimeric antigen receptor (CAR) expressed by its cell of origin (and the cell of origin may be a cell of cell therapy composition, e.g., a T cell of a cell therapy composition). In some embodiments, an exosome provided herein comprises a T cell receptor (TCR), which TCR is expressed by a T cell of a T cell composition that is the cell of origin of the exosome. Thus, in some embodiments, the exosomes isolated herein are further characterized for presence of the recombinant receptor from the cell from which it originates (e.g., the cell therapy composition).

[0134] In some embodiments, the exosomes comprise a surrogate marker of the recombinant receptor expressed by the cells of the T cell compositions provided herein. In some embodiments, the recombinant receptor or surrogate marker is localized to the surface of the exosomes provided herein. In some embodiments, the marker includes all or part (e.g., truncated form) of CD34, NGFR, or epidermal growth factor receptor (EGFR). In some embodiments, the marker is truncated EGFR (tEGFR). c. Exosome Cargo

[0135] In addition to surface markers, the exosomes detected herein contain various internal cargo. In some embodiments, an exosome detected herein is distinguished from non-exosome cell-derived particles based on its internal or luminal cargo. In some embodiments, an exosome detected herein comprises any encapsulated cargo known in the art. Such cargo may be derived from the cytoplasm and / or nucleus of the cell of origin, and may be selectively enriched during exosome biogenesis. In some embodiments, an exosome detected herein comprises signaling molecules such as cytokines, growth factors, small molecules and metabolites. In some embodiments, 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. In some embodiments, 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

[0136] In some aspects, 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.

[0137] In some embodiments, exosome yield (e.g., amount and / or concentration) is dependent on the number of cells in the cell therapy composition. In some embodiments, the number of cells in the cell therapy composition provided herein is between about 0.1 x 106cells / mL and 1 x 106cells / mL. In some embodiments, the number of cells in the cell therapy composition provided herein is between about 0.33 x 106cells / mL and 1 x 106cells / mL. In some embodiments, the number of cells in the cell therapy composition provided herein is between about 0.4 x 106cells / mL and 0.6 x 106cells / mL, 0.5 x 106cells / mL and 0.7 x 106cells / mL, 0.6 x 106cells / mL and 0.8 x 106cells / mL, 0.7 x 106cells / mL and 0.9 x 106cells / mL, or 0.8 x 106cells / mL and 1 x 106cells / mL.

[0138] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 atleast 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. In some embodiments, 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. In some embodiments, 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.

[0139] In some embodiments, 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. In some embodiments, 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 thestimulated 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 orconcentration 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.

[0140] In some embodiments, the concentration of exosomes produced from the stimulated cell composition comprises up to about 1 x IO10exosomes / mL. In some embodiments, the concentration of exosomes produced from the stimulated cell composition comprises up to about 1 x IO10exosomes / mL when the number of cells in the cell therapy composition is between about 0.33 x 106cells / mL and 1 x 106cells / mL. In some embodiments, the concentration of exosomes produced from the stimulated cell composition comprises greater than 1 x IO10exosomes / mL. In some embodiments, the concentration of exosomes produced from the stimulated cell composition comprises greater than 1 x IO10exosomes / mL when the number of cells in the cell therapy composition is between about 0.33 x 106cells / mL and 1 x 106cells / mL.

[0141] In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises between about 1 x 106exosomes / mL and 1 x 1010exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises between about 1 x 106exosomes / mL and 2 x 106exosomes / mL, 1.5 x 106exosomes / mL and 2.5 x 106exosomes / mL, 2 x 106exosomes / mL and 3 x 106exosomes / mL, 2.5 x 106exosomes / mL and 3.5 x 106exosomes / mL, 3 x 106exosomes / mL and 4 x 106exosomes / mL, 3.5 x 106exosomes / mL and 4.5 x 106exosomes / mL, 4 x 106exosomes / mL and 5 x 106exosomes / mL, 4.5 x 106exosomes / mL and 5.5 x 106exosomes / mL, 5 x 106exosomes / mL and 6 x 106exosomes / mL, 5.5 x 106exosomes / mL and 6.5 x 106exosomes / mL, 6 x 106exosomes / mL and 7 x 106exosomes / mL, 6.5 x 106exosomes / mL and 7.5 x 106exosomes / mL, 7 x 106exosomes / mL and 8 x 106exosomes / mL, 7.5 x 106exosomes / mL and 8.5 x 106exosomes / mL, 8 x 106exosomes / mL and 9 x 106exosomes / mL, 8.5 x 106exosomes / mL and 9.5 x 106exosomes / mL, 8.5 x 106exosomes / mL and 9.5 x 106exosomes / mL, 9 x 106exosomes / mL and 10 x 106exosomes / mL, 9.5 x 106exosomes / mL and 1.5 x 107exosomes / mL, 1 x 107exosomes / mL and 2 x 107exosomes / mL, 1.5 x 107exosomes / mL and 2.5 x 107exosomes / mL, 2 x 107exosomes / mL and 3 x 107exosomes / mL, 2.5 x 107exosomes / mL and 3.5 x 107exosomes / mL, 3 x 107exosomes / mL and 4 x 107exosomes / mL, 3.5 x 107exosomes / mL and 4.5 x 107exosomes / mL, 4 x 107exosomes / mL and 5 x 107exosomes / mL, 4.5 x 107exosomes / mL and 5.5 x 107exosomes / mL, 5 x 107exosomes / mL and 7 x 107exosomes / mL, 5.5 x 107exosomes / mL and 7.5 x 107exosomes / mL, 7 x 107exosomes / mL and 7 x 107exosomes / mL, 7.5 x 107exosomes / mL and 7.5 x 107exosomes / mL, 7 x 107exosomes / mL and 8 x 107exosomes / mL, 7.5 x 107exosomes / mL and 8.5 x 107exosomes / mL, 8 x 107exosomes / mL and 9 x 107exosomes / mL, 8.5 x 107exosomes / mL and 9.5 x 107exosomes / mL, 8.5 x 107exosomes / mL and 9.5 x 107exosomes / mL, 9 x 107exosomes / mL and 10 x 107exosomes / mL, 9.5 x 107exosomes / mL and 1.5 x 108exosomes / mL, 1 x 108exosomes / mL and 2 x 108exosomes / mL, 1.5 x 108exosomes / mL and 2.5 x 108exosomes / mL, 2 x 108exosomes / mL and 3 x 108exosomes / mL, 2.5 x 108exosomes / mL and 3.5 x 108exosomes / mL, 3 x 108exosomes / mL and 4 x 108exosomes / mL, 3.5 x 108exosomes / mL and 4.5 x 108exosomes / mL, 4 x 108exosomes / mL and 5 x 108exosomes / mL, 4.5 x 108exosomes / mL and 5.5 x 108exosomes / mL, 5 x 108exosomes / mL and 8 x 108exosomes / mL, 5.5 x 108exosomes / mL and 8.5 x 108exosomes / mL, 8 x 108exosomes / mL and 7 x 108exosomes / mL, 8.5 x 108exosomes / mL and 7.5 x 108exosomes / mL, 7 x 108exosomes / mL and 8 x 108exosomes / mL, 7.5 x 108exosomes / mL and 8.5 x 108exosomes / mL, 8 x 108exosomes / mL and 9 x 108exosomes / mL, 8.5 x 108exosomes / mL and 9.5 x 108exosomes / mL, 8.5 x 108exosomes / mL and 9.5 x 108exosomes / mL, 9 x 108exosomes / mL and 10 x 108exosomes / mL, 9.5 x 108exosomes / mL and 1.5 x 109exosomes / mL, 1 x 109exosomes / mL and 2 x 109exosomes / mL, 1.5 x 109exosomes / mL and 2.5 x 109exosomes / mL, 2 x 109exosomes / mL and 3 x 109exosomes / mL, 2.5 x 109exosomes / mL and 3.5 x 109exosomes / mL, 3 x 109exosomes / mL and 4 x 109exosomes / mL, 3.5 x 109exosomes / mL and 4.5 x 109exosomes / mL, 4 x 109exosomes / mL and 5 x 109exosomes / mL, 4.5 x 109exosomes / mL and 5.5 x 109exosomes / mL, 5 x 109exosomes / mL and 9 x 109exosomes / mL, 5.5 x 109exosomes / mL and 9.5 x 109exosomes / mL, 9 x 109exosomes / mL and 7 x 109exosomes / mL, 9.5 x 109exosomes / mL and 7.5 x 109exosomes / mL, 7 x 109exosomes / mL and 8 x 109exosomes / mL, 7.5 x 109exosomes / mL and 8.5 x 109exosomes / mL, 8 x 109exosomes / mL and 9 x 109exosomes / mL, 8.5 x 109exosomes / mL and 9.5 x 109exosomes / mL, 8.5 x 109exosomes / mL and 9.5 x 109exosomes / mL, 9 x 109exosomes / mL and 10 x 109, 9.5 x 109exosomes / mL and 1.5 x IO10exosomes / mL.

[0142] In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 106exosomes / mL, about 1.5 x 106exosomes / mL, about 2 x 106exosomes / mL, about 2.5 x 106exosomes / mL, about 3 x 106exosomes / mL, about 3.5 x 106exosomes / mL, about 4 x 106exosomes / mL, about 4.5 x 106exosomes / mL, about 5 x 106exosomes / mL, about 5.5 x 106exosomes / mL, about 6 x 106exosomes / mL, about 6.5 x 106exosomes / mL, about 7 x 106exosomes / mL, about 7.5 x 106exosomes / mL, or about 8 x 106exosomes / mL.

[0143] In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 107exosomes / mL, about 2 x 107exosomes / mL, about 3 x 107exosomes / mL, about 4 x 107exosomes / mL, about 5 x 107exosomes / mL, or about 6 x 107exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 107exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 2 x 107exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 3 x 107exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 4 x 107exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 5 x 107exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 6 x 107exosomes / mL.

[0144] In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 108exosomes / mL, about 2 x 108exosomes / mL, about 3 x 108exosomes / mL, about 4 x 108exosomes / mL, about 5 x 108exosomes / mL, about 6 x 108exosomes / mL, 7 x 108exosomes / mL or 8 x 108exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 108exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 2 x 108exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 3 x 108exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 4 x 108exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 5 x 108exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 6 x 108exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 7 x 108exosomes / mL. In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 8 x 108exosomes / mL.

[0145] In some embodiments, the concentration of exosomes produced by the stimulated cell composition comprises about 1 x 109exosomes / mL.

[0146] In some embodiments, the total amount of exosomes produced by the stimulated cell composition can be derived from any one of the concentrations provided above. Thus, in an embodiment where the exosomes are collected in 1 mL of a composition, the amount of exosomes produced by the stimulated composition comprises up to about 10,000,000,000 or 1 x 1010exosomes.3. Exosome Function

[0147] In some embodiments, an exosome detected herein is distinguished from non-exosome cell- derived particles based its function. In some aspects, the methods provided herein comprise comparing the function of exosomes produced from a stimulated cell composition (e.g., stimulated cell therapy composition) to the function of exosomes produced from a control cell composition. In some embodiments, the function of exosomes produced from the stimulated cell composition is increased compared to the function of exosomes produced from the control cell composition.

[0148] In some embodiments, exosome function comprises cytotoxic capacity. In some embodiments, the cytotoxic capacity of exosomes produced from the stimulated cell composition isincreased compared to the cytotoxic capacity of exosomes produced from the control cell composition. In some embodiments, cytotoxic capacity is determined by measuring the presence and / or levels of cytokines. In some embodiments, the cytotoxic cytokines comprise granzyme-A, granzyme-B, perforin, IFNy, GM-CSF, IL- 13, IL-2, MIP-la, and / or MIP-lb.

[0149] In some embodiments, 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. In some embodiments, 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%, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0150] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the cytotoxic cytokines of the exosomes produced from the stimulated cell composition is increased by atleast 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 thestimulated cell composition is increased by at least about 10-fold compared to the cytotoxic cytokines of the exosomes produced from the control cell composition.

[0151] In some embodiments, exosome function comprises cytotoxic function. In some embodiments, 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. In some embodiments, cytotoxic function is determined by measuring cell killing ability (e.g., in a cell killing assay) of the exosomes. In some embodiments, 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. In some embodiments, 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.

[0152] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 theexosomes 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. In some embodiments, 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. In some embodiments, 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.

[0153] In some embodiments, 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. In some embodiments, the exosome-mediated cytotoxicity 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 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-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 exosomesproduced 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the exosome-mediated cytotoxicity of the exosomes produced fromthe 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.II. METHODS OF TREATMENT AND USES OF CELL THERAPY WITH GENETICALLY ENGINEERED CELLS

[0154] Provided are methods and uses of engineered cells (e.g., T cells) and compositions thereof, for the treatment of subjects having a disease or condition. In some embodiments, the cell composition is a cell therapy composition. In some embodiments, the cell composition is a T cell therapy composition. In some aspects, provided herein are methods of selecting a subject having a disease or condition for treatment and treating the subject having the disease or condition. In some aspects, provided herein are methods of treating a subject having a disease or condition. In some aspects, the method of treatment comprises adaptive treatment.

[0155] 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. Thus, 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. In some embodiments, the exosome is a cell-derived particle that is surface positive for CD63. In some embodiments, 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. In some embodiments, 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. In some embodiments, exosomes are assessed in the T cell therapy composition by identifying cell particles that are surface positive for CD63. In some embodiments, 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. In some embodiments, if the amount or concentration of exosomes is increased, the subject is predicted as likely to respond to the T cell therapy composition. In someembodiments, if the amount or concentration of exosomes is decreased or unchanged, the subject is not predicted as likely to respond. In further embodiments, 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. In some embodiments, 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. In additional embodiments, the exosomes detected herein are used as a biomarker to select a subject for treatment, including adaptive treatment, with a T cell therapy composition. In some embodiments, 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. In some aspects, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the method of predicting allows for detecting exosomes produced from a T cell therapy composition. In some embodiments, the method of predicting allows for detecting exosomes produced from a CAR-T cell therapy composition. In some embodiments, the method of predicting allows for detecting exosomes produced from a TCR cell therapy composition. In some embodiments, the method of predicting a response to, and / or efficacy of, 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.

[0156] In some aspects, provided herein is 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the method of treating the subject 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.

[0157] In some aspects, provided herein is 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. In some embodiments, 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.

[0158] In some aspects, provided herein is a method of treatment comprising administering a T cell therapy to a subject a disease or condition. In some embodiments, the T cell therapy is a composition comprising T cells engineered to express a recombinant receptor. In some embodiments, the subject hasan 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. In some embodiments, 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. In some aspects, provided herein is a method of treatment comprising administering a T cell therapy to a subject having a disease or condition. In some embodiments, the T cell therapy comprises T cells engineered to express a recombinant receptor. In some embodiments, the subject is a subject that has been selected for treatment according to the methods provided herein.

[0159] In some aspects, provided herein is a method of adaptive treatment with a T cell therapy in a subject having a disease or a condition. In some embodiments, the method 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. In some embodiments, the isolated exosomes are cell-derived particles that are surface positive for CD63. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0160] In some embodiments, the disease or condition is an autoimmune or inflammatory disease or disorder. In some embodiments, 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).

[0161] In some embodiments, the autoimmune or inflammatory disease or condition is arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory boweldisease, psoriasis, scleroderma, autoimmune thyroid disease, Grave’s disease, Crohn’s disease multiple sclerosis, asthma, and / or a disease or condition associated with transplant.

[0162] In some embodiments, the disease or condition is a cancer or tumor. In some embodiments, the subject has a cancer or tumor and has been or is a candidate for treatment with a cell therapy composition (e.g., T cell therapy composition). In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a B cell malignancy.

[0163] In some aspects, 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. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells.

[0164] In some embodiments, the subject has a cancer or tumor that is a hematological malignancy. In some embodiments, 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.

[0165] 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.

[0166] Diffuse large B cell lymphoma (DLBCL) 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).

[0167] Accordingly, in some embodiments, 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 (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), and Diffuse Large B-Cell Lymphoma (DLBCL).

[0168] In some embodiments, the subject has a lymphoma. In some embodiments, 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), Intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, Burkitt lymphoma, adult T-cell lymphoma, extranodal K / T-cell lymphoma, enteropathy-associated T-cell lymphoma, Hepatosplenic T- cell lymphoma, blastic NK cell lymphoma, Mycosis fungoides / Sezary syndrome, Primary cutaneous anaplastic large cell lymphoma, Lymphomatoid papulosis, Peripheral T-cell lymphoma, Angioimmunoblastic T cell lymphoma, Anaplastic large cell lymphoma, B- lymphoblastic lymphoma, B- lymphoblastic lymphoma with recurrent genetic abnormalities, T-lymphoblastic lymphoma, and Hodgkin lymphoma. In some embodiments, the cancer is refractory to one or more prior treatments, and / or the cancer has relapsed after one more prior treatments.

[0169] In some embodiments, the lymphoma is large B cell lymphoma (LB CL). In some embodiments, the lymphoma is diffuse large B cell lymphoma (DLBCL).

[0170] In some embodiments, 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.

[0171] In some embodiments, 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. In one particular embodiment, the cancer is refractory diffuse large B cell lymphoma.

[0172] In some embodiments, 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). In certain embodiments, the individual or subject is a human. In some embodiments, the subject is a human. In other embodiments, the subject is a patient, e.g., a human patient having a cancer described herein.A. Combination Therapy

[0173] In some embodiments, if the subject is assessed as not likely to be responsive to the cell therapy (e.g., T cell therapy), a treatment strategy that includes an additional treatment may be selected or administered. In some embodiments, 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. 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. In some contexts, 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. In some embodiments, the therapeutic cell compositions are administered prior to the one or more additional therapeutic agents. In some embodiments, the therapeutic cell compositions are administered after the one or more additional therapeutic agents. In some embodiments, the other agent may be administered before, concurrently with or after the cell therapy composition.

[0174] In some embodiments, 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. 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 prior to 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 prior to administering the T cell therapy to the subject. In some embodiments, the administration of the other agent is initiated 15 to 30 days prior to administering the T cell therapy to the subject. In some embodiments, the administration of the other agent is initiated about 3 days prior, about 7 days prior, about 15 days prior, or about 28 days prior 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 prior to administering the T cell therapy to the subject.

[0175] In some embodiments, 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.

[0176] In some embodiments, 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 15to 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. In some embodiments, 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.

[0177] In some embodiments, 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. epacadostat); a B-cell lymphoma 2 (BCL-2) inhibitor, e.g., venetoclax, navitoclax, ABT737, maritoclax, obatoclax or clitocine; 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).

[0178] In some embodiments, 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. In some embodiments, 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.

[0179] In some embodiments, 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). In some embodiments, 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. In some embodiments, the BTK inhibitor is a BTK inhibitor described in International Application WO 2015 / 079417.

[0180] In some embodiments, the other agent is an agent that regulates pro- or anti-apoptotic proteins. In some embodiments, 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). 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) thatinhibits the anti-apoptotic protein, BCL-2. Other 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. In some embodiments, 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.

[0181] In some embodiments, the other agent is an immunomodulatory agent. In some embodiments, 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. In some embodiments, the immunomodulatory agent is a peptide, protein or is a small molecule. In some embodiments, the protein can be a fusion protein or a recombinant protein. In some embodiments, 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. For example, in some embodiments, the immunomodulatory agent is an antibody or antigen-binding antibody fragment, a fusion protein, a small molecule or a polypeptide. In some embodiments, 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.

[0182] In some embodiments, 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.

[0183] Exemplary combination therapies and methods are described in PCT application No. PCT / US2023 / 068844 and in published international applications WO 2018 / 085731, WO 2018 / 102785, WO 2019 / 213184, WO 2018 / 071873, WO 2018 / 102786, WO 2018 / 204427, WO 2019 / 152743, WO2022 / 187406; W 02022 / 212400; WO2020 / 252218 and WO2022 / 133030, which are incorporated by reference in their entirety.III. METHODS FOR GENERATING A CELL THERAPY COMPOSITION

[0184] In some aspects, provided herein are methods for generating cell compositions such as a cell therapy composition comprising, including compositions comprises CAR or TCR expressing T cells. In some embodiments, the cell therapy composition is a T cell therapy composition. In some embodiments,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. In some embodiments, all of the cells in the cell therapy composition express the recombinant receptor. In some embodiments, less than all of the cells in the cell therapy composition express the recombinant receptor. In some embodiments, at or greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of cells in the cell therapy composition express the recombinant receptor.

[0185] In particular embodiments, the antigen that is bound or recognized by the recombinant receptor (e.g., CAR) is any antigen described below in Section III.A. In some embodiments, binding to the antigen results in a response, such as an immune response against such antigens. In some embodiments, 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).

[0186] In some aspects, 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.

[0187] In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, 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.

[0188] In some embodiments, the cell composition is for use as a cell therapy. In some embodiments, the cell composition is a T cell composition. In some embodiments, 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.

[0189] In some embodiments, 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 someembodiments, 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+.

[0190] In some embodiments, the cell therapy composition provided herein is cryopreserved and thawed prior to detecting, isolating and characterizing the exosomes.A. Chimeric Antigen Receptors

[0191] In some embodiments of the provided methods and uses, the engineered cells, such as T 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. In some embodiments, 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. In some embodiments, the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions. 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. In some embodiments, 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.

[0192] 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. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and / or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PloS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, 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. Examples of 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 alsoWO2014031687, 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.

[0193] In some embodiments, 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. In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, 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. In some aspects, the recombinant receptor, e.g., a chimeric antigen 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). In some aspects, the recombinant receptor, e.g., CAR, further includes a spacer and / or a transmembrane domain or portion. In some aspects, 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).

[0194] 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. In some embodiments, 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. In some embodiments, 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). In some embodiments, the antigen-binding domain is a single domain antibody (sdAb), such as sdFv, nanobody, VHH and VNAR. In some embodiments, an antigenbinding fragment comprises antibody variable regions joined by a flexible linker.

[0195] In some embodiments, the antigen is a B cell antigen or a plasma cell antigen. In some embodiments, 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 receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2),ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), IL- 13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (Ll-CAM), CE7 epitope of Ll-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomer ase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen that comprises or is associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30. In some embodiments, the antigen is or includes a pathogen-specific or pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and / or parasitic antigens.

[0196] Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30. In particular aspects, the antigen is CD19. In some embodiments, any of such antigens are antigens expressed on human B cells.

[0197] In particular embodiments, the antigen that is bound or recognized by the recombinant receptor (e.g., CAR) is CD19. In other embodiments, the antigen that is bound or recognized by the recombinant receptor is CD20.

[0198] In some embodiments, the antigen targeted by the receptor is a polypeptide. In particular embodiments, the antigen target is CD19. In some embodiments, the antigen is selectively expressed on B cells targeted for treating a cancer. In some embodiments, the CAR typically includes in its extracellular portion one or more antibody or antigen-binding fragment or portion that targets CD 19.

[0199] In some embodiments, the antibody or an antigen-binding fragment (e.g., scFv or VH domain) specifically recognizes an antigen, such as CD19. In some embodiments, the antibody or antigen-binding fragment is derived from, or is a variant of, antibodies or antigen-binding fragment that specifically binds to CD19. In some embodiments, the antigen is CD19. In some embodiments, the antibody or an antigen-binding fragment (e.g., scFv) contains a variable heavy chain and a variable light chain with six CDRs, CDRH1-3 and CDRL1-3, that confer binding to CD19.

[0200] In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19. In some embodiments, 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. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther. 335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB- CD19 (De Rie Cell. Immunol. 118:368-381(1989)). In any of these embodiments, 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. In some embodiments, the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016 / 0152723.

[0201] In some embodiments 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). In some embodiments, 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. In some embodiments, 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.

[0202] In some embodiments, 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. In some embodiments, 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 setforth 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.

[0203] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0204] In some embodiments, the variable heavy and variable light chains are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO:22. In some embodiments, 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. In some embodiments, 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.

[0205] In some embodiments 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 chaincontaining 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. In some embodiments, 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. In some embodiments, the variable heavy and variable light chains are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO:6. In some embodiments, 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.

[0206] In some embodiments, 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.

[0207] In some embodiments, the anti-CD19 CAR includes an antigen-binding domain that is a single chain antibody derived from a fully human antibody. In some embodiments, 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.

[0208] In some embodiments, the CAR targets CD19 and at least one other antigen expressed on B cells. In some embodiments, 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. In some embodiments, the other antigen is CD20 and the CAR is a CD20 / CD19 directed CAR product. In some embodiments, the CAR is a bispecific CAR in which the extracellular antigen-binding domain binds CD19 and the one other antigen (e.g. CD20). In some embodiments, 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). In some embodiments, 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.

[0209] Exemplary CD19-directed antigen receptors, e.g., CARs, also include the CARs of FDA- approved products BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), and YESCARTA™ (axicabtagene ciloleucel). In some of any of the provided embodiments, the CAR is the CAR of BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), YESCARTA™(axicabtagene ciloleucel). In some of any of the provided embodiments, 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). In some of any of the provided embodiments, the CAR is the CAR of TECARTUS™ (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). In some of any of the provided embodiments, the CAR is the CAR of KYMRIAH™ (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). In some of any of the provided embodiments, the CAR is the CAR of YESCARTA™ (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).

[0210] In some embodiments, the antigen is CD20. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD20. In some embodiments, the antibody or antibody fragment that binds CD20 is an antibody that is or is derived from Rituximab, such as is Rituximab scFv. In some embodiments, the antigen is CD22. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD22. In some embodiments, the antibody or antibody fragment that binds CD22 is an antibody that is or is derived from m971, such as is m971 scFv. In some embodiments, the antigen or antigen binding domain is GPRC5D. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to GPRC5D. In some embodiments, 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.

[0211] In some embodiments, 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. In some embodiments, the antigen or antigen binding domain is BCMA. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to BCMA. In some embodiments, 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.

[0212] Further exemplary antigen receptors, e.g., CARs, such as anti-BCMA CARs, include the CARs of idecabtagene vicleucel, ABECMA®, BCMA02, JCARH125, JNJ-68284528 (LCAR-B38M; ciltacabtagene autoleucel; CARVYKTI™) (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). In a particular embodiment, the CAR is the CAR of idecabtagene vicleucel cells. In a particular embodiment, the CAR is the CAR of ABECMA® cells (cells used in ABECMA® immunotherapy). In a particular embodiment, the CAR is the CAR of ciltacabtagene autoleucel cells. In a particular embodiment, the CAR is the CAR of CARVYKTI™ cells (cells used in CARVYKTI™ immunotherapy ).In some embodiments, 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. In some embodiments, the spacer is a CD8 hinge sequence. In some embodiment, 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.

[0213] In some embodiments, the CAR contains a hinge-containing immunoglobulin spacer between the scFv and the transmembrane domain. In some embodiments, the spacer is set forth in SEQ ID NO:29.

[0214] In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an IT AM. For example, in some aspects, the antigen recognition domain (e.g. extracellular domain) generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and / or signal via another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0215] In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, 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.

[0216] 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. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (4-1BB), or CD154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and / or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28 or a variant thereof. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein.

[0217] In some embodiments, the transmembrane domain of the receptor, e.g., the CAR, is a transmembrane domain of human CD28 or variant thereof, e.g., a 27-amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1).

[0218] In some embodiments, the transmembrane domain of the receptor, e.g., the CAR, is a transmembrane domain of a human CD28. In some embodiments, the transmembrane domain has the sequence set forth in SEQ ID NO:30. In some embodiments, 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. In some embodiments, the transmembrane domain has the sequence set forth in SEQ ID NO:31. In some embodiments, 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.

[0219] In some embodiments, the transmembrane domain of the receptor, e.g., the CAR, is a transmembrane domain of a human CD8a.

[0220] In some embodiments, the recombinant receptor, e.g., CAR, 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). In some aspects, the CAR includes one or both of such signaling components. Among the intracellular signaling region are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and10 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.

[0221] In some aspects, 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. Examples of IT AM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.

[0222] In some embodiments, 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. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and / or other CD transmembrane domains. In some embodiments, 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. For example, in some aspects, 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.

[0223] In some embodiments, 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.

[0224] In some embodiments, the CD3-zeta domain has the sequence set forth in SEQ ID NO: 32. In some embodiments, 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.

[0225] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.

[0226] In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some embodiments, 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. In some embodiments, the CAR includes a costimulatory region or domain of CD28 or 4-1BB, such as of human CD28 or human 4-1BB.

[0227] In some embodiments, 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.

[0228] In some aspects, the same CAR includes both the primary (or activating) cytoplasmic signaling regions and costimulatory signaling components.

[0229] In some embodiments, 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.

[0230] In some embodiments, 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.. 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-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus, and porcine teschovirus-1 as described in U.S. Patent Publication No. 20070116690. Examples of 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.B. Nucleic Acids, Vectors and Methods for Genetic Engineering

[0231] In some embodiments, the cells, e.g., T cells, are genetically engineered to express a recombinant receptor. In some embodiments, the engineering is carried out by introducing polynucleotides that encode the recombinant receptor. Also provided are polynucleotides encoding a recombinant receptor, and vectors or constructs containing such nucleic acids and / or polynucleotides.

[0232] In some cases, the nucleic acid sequence encoding the recombinant receptor contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In some cases, the nucleic acid sequence encoding the recombinant receptor, e.g., chimeric antigen receptor (CAR) contains a signal sequence that encodes a signal peptide.

[0233] In some embodiments, 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.

[0234] In some embodiments, 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). In some embodiments, 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. Hematol., 28(10): 1137-46; Alonso-Camino et al. (2013) Mol. Ther. Nucl. Acids., 2, e93; Park et al., Trends Biotechnol., 2011 November 29(11): 550-557.

[0235] In some embodiments, the viral vector is an adeno-associated virus (AAV).

[0236] In some embodiments, 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). Most retroviral vectors are derived from murine retroviruses. In some embodiments, 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. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and / or env sequences. 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.

[0237] Methods of lentiviral transduction are known. Exemplary methods are described in, e.g. , Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497- 505.

[0238] Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Patent No. 7,446,190.C. Methods of Manufacturing Engineered Cells

[0239] In some embodiments, cells, such as T cells, used in connection with the provided methods, are cells that have been genetically engineered to express a recombinant receptor, e.g., a CAR. In some embodiments, the engineered cells are used in the context of cell therapy, e.g., adoptive celltherapy. In some embodiments, the engineered cells are immune cells. In some embodiments, the engineered cells are T cells, such as CD4+ or CD8+ T cells.

[0240] In particular embodiments, 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. In certain embodiments, the output composition contains cells that express a recombinant receptor, e.g., a CAR. In particular embodiments, the cells of the output compositions are suitable for administration to a subject as a therapy, e.g., an autologous cell therapy. In some embodiments, 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. In some embodiments, the engineering methods include transduction with viral vectors, such as lenti viral vectors.

[0241] In some embodiments, 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.

[0242] In certain embodiments, the process for producing engineered cells further can include one or more of: activating and / or stimulating a cells, e.g., cells of an input composition; genetically engineering the activated and / or stimulated cells, e.g., to introduce a polynucleotide encoding a recombinant protein by transduction or transfection; and / or cultivating the engineered cells, e.g., under conditions that promote proliferation and / or expansion. In particular embodiments, the provided methods may be used in connection with harvesting, collecting, and / or formulating output compositions produced after the cells have been incubated, activated, stimulated, engineered, transduced, transfected, and / or cultivated.

[0243] In some embodiments, 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.

[0244] 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 derivedfrom a subject. In some embodiments, 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.

[0245] Accordingly, 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.

[0246] In some aspects, 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.

[0247] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, 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.

[0248] In some embodiments, 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) is cryopreserved and / or cryoprotected (e.g., frozen) and then thawed and optionally washed prior to any steps for isolating, selecting, activating, stimulating, engineering, transducing, transfecting, incubating, culturing, harvesting, formulating a population of the cells, and / or administering the formulated cell population to a subject.

[0249] In particular embodiments, 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). In particular embodiments, an apheresis product or a leukapheresis product is cryopreserved and / or cryoprotected (e.g., frozen). In some embodiments, 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. Exemplary methods and systems forcryogenic storage and processing of cells from a sample, such as an apheresis sample, can include those described in W02018170188.

[0250] In some embodiments, selection, isolation, or enrichment of the cells or populations includes one or more preparation and / or non-affinity based cell separation steps. In some examples, 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. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0251] In some embodiments, at least a portion of 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. In some embodiments, any known method using a selection reagent or reagents for separation based on such markers may be used. In some embodiments, the selection reagent or reagents result in a separation that is affinity- or immunoaffinity-based separation. For example, 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.

[0252] In some embodiments, 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. For example, 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.

[0253] 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 thatspecifically 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.

[0254] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0255] In some examples, 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. In some examples, 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. Likewise, 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.

[0256] For example, in some aspects, 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. In some aspects, 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.

[0257] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, 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 (markerhlgh) on the positively or negatively selected cells, respectively.

[0258] In some embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, are subjected to selection of T cells. In some embodiments, 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. In some embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD3+ T cells. In some embodiments, 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. In some embodiments, abiological sample, e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD4+ T cells and CD8+ T cells. In some embodiments, 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.

[0259] In some aspects of such processes, 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. In some embodiments, 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) can be incubated or mixed with cells in a container, such as a tube or bag, while shaking or mixing, with a constant cell density-to-particle (e.g., bead) ratio to aid in promoting energetically favored interactions.

[0260] In some embodiments, 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. For example, in some embodiments, 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).

[0261] In some aspects, 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.

[0262] 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.

[0263] In some aspects, 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. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, 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.

[0264] In some embodiments, 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. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0265] In some embodiments, 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. In certain embodiments, 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. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0266] In certain embodiments, 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. In some aspects, 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. In one example, the system is a system as described in International Patent Application, Publication Number W02009 / 072003, or US 20110003380 Al.

[0267] In some embodiments, cells, e.g., T cells, are isolated, selected, or enriched by chromatographic isolation, such as by column chromatography including affinity chromatography or gel permeations chromatography. In some embodiments, 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. Such methods may be described as (traceless) cell affinity chromatography technology (CATCH). In certain embodiments, 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.

[0268] In some embodiments, 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. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, 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) LabChip 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.

[0269] In some embodiments, the 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. Such conditions include those designed to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.

[0270] In some embodiments, 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. In some aspects, 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. Among the stimulatory reagents are anti-CD3 / anti-CD28 beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander, and / or ExpACT® beads).

[0271] In particular embodiments, 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. In some embodiments, the oligomeric particle reagent is any as described in WO2015 / 158868 or WO2018 / 197949.

[0272] In some embodiments, the cells are engineered by introduction, delivery or transfer of nucleic acid sequences that encode the recombinant receptor and / or other molecules. In some embodiments, 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. In particular embodiments, 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. In some embodiments, the engineering produces one or more engineered compositions of enriched T cells.

[0273] In some embodiments, genetically engineering the cells is or includes introducing the polynucleotide, e.g., the heterologous or recombinant polynucleotide, into the cells by transduction. Insome embodiments, the cells are transduced or subjected to transduction with a viral vector. In particular embodiments, the cells are transduced or subjected to transduction with a viral vector. In some embodiments, 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. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0274] In some embodiments, 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.

[0275] In particular embodiments, the cells are engineered in the presence of one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In particular embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, 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. In particular embodiments, the one or more cytokines is or includes a member of the 4-alpha-helix bundle family of cytokines. In some embodiments, 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). In particular embodiments, cells, e.g., stimulated cells are engineered under stimulating conditions in the presence of IE-2, IE-7, and / or IL-15.

[0276] In some embodiments, 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. In some embodiments, the contacting can be effected with 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-600.1 or CS-900.2.

[0277] In some embodiments, the methods for generating the engineered cells, e.g., for cell therapy in accord with any of provided methods, uses, articles of manufacture or compositions, include one or more steps for incubating or cultivating the cells. In some embodiments, an incubation on engineered cells is carried out under conditions that do not promote proliferation and / or expansion. In some embodiments, the engineered cells are incubated or cultivated under conditions for expansion of cells. In certain embodiments, 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.

[0278] In provided embodiments, 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. In some embodiments, the cells that are harvested are formulated, such as in a pharmaceutically acceptable buffer for use as a therapeutic cell composition. In some embodiments, 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.

[0279] In some embodiments, cells can be formulated into a container, such as a bag or vial. In some embodiments, the vial may be an infusion vial. In some embodiments, 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.

[0280] In some embodiments, the cells are formulated in a pharmaceutically acceptable buffer, which may, in some aspects, include a pharmaceutically acceptable carrier or excipient. In some embodiments, the processing includes exchange of a medium into a medium or formulation buffer that is pharmaceutically acceptable or desired for administration to a subject. In some embodiments, 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.

[0281] 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.

[0282] In some aspects, 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. For example, thepharmaceutical 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, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0283] 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. In some aspects, 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).

[0284] 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. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, 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. In some embodiments, 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.

[0285] In some embodiments, the formulation buffer contains a cryopreservative.

[0286] In some embodiments, such cells produced by the method, or a composition comprising such cells, are administered to a subject for treating a disease or condition.Z Small Molecules in Process

[0287] In some embodiments, 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. In some embodiments, 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. In some embodiments, the modulating agent increases exosome production, wherein the produced exosomes are cell-derived particles that are surface positive for CD63. In some embodiments, 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. In some aspects, 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. In some embodiments, the comparison to an alternative process is made to the same process that differs only in that the alternative process is not carried out in the presence of the modulating agent.

[0288] In some embodiments, 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. In some embodiments, the modulating agent is present prior to, during, or after the cells are subjected to stimulation, e.g., T cell activation. In some embodiments, 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. In some embodiments, the modulating agent is present prior to, during, or after the cells are subjected to engineering, e.g., transduction. In some embodiments, 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. In some embodiments, the modulating agent is in contact with the cells or cellpopulations (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. In some embodiments, 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). In certain embodiments, 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. In particular embodiments, the cells or cell population undergoes a process, procedure, step, or technique in the presence of the modulating agent after the incubation.

[0289] In some embodiments, cells to be engineered (e.g. transduced) are contacted (e.g., incubated) with the modulating agent, e.g. in a culture media, prior to the engineering. In some embodiments, the cells are engineered in the presence of the modulating agent. In some embodiments, 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. Also provided in some embodiments are compositions during the manufacture or production of engineered cells, e.g., for cell-based therapies, 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).

[0290] In some embodiments, 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. In some embodiments, 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.

[0291] In some embodiments, 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. In particular embodiments, 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.

[0292] In some embodiments, the modulating agent is or comprises an agent that inhibits mTOR activity. In some embodiments, cells to be engineered (e.g. transduced) are contacted (e.g., incubated) with an mTOR inhibitor prior to the engineering. In some embodiments, the cells are engineered in the presence of an mTOR inhibitor. In some embodiments, 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. Also provided in some embodiments are 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).

[0293] In some embodiments, 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. In particular embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0294] In particular embodiments, the agent that inhibits mTOR activity inhibits the activity of at least one additional kinase. In certain embodiments, the at least one additional kinase is PI3K. In particular embodiments, 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. In some embodiments, the agent that inhibits mTOR activity inhibits, e.g., selectively inhibits, mTORCl and mTORC2 kinase activity relative to PI3K activity. In certain embodiments, the agent that inhibits mTOR activity inhibits mTORCl and mTORC2 kinase activity. In particular embodiments, the agent that inhibits mTOR activity selectively inhibits mTORCl activity, such as the mTORCl kinase activity.

[0295] In certain embodiments, the agent that inhibits mTOR activity: (i) does not inhibit mTORC2 activity; (ii) does not detectably inhibit mTORC2 activity at the IC50 for mTORCl activity; and / or (iii) does not detectably inhibit mTORC2 at all concentrations that detectably inhibit mTORCl activity.

[0296] In some embodiments, 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). In someembodiments, the agent that inhibits mTOR activity has or includes a formula that is provided in Formula (I), Formula (II), or Formula (III). In some embodiments, the agent is Compound 155, Compound 246, or Compound 63.

[0297] In particular embodiments, the agent comprises a formula set forth in Formula (I),Formula (I) wherein R1is substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl, R2is substituted or unsubstituted Ci-salkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl, and R3and R4are independently H or Ci-8 alkyl. In some embodiments, 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 someembodiments, the agent that inhibits mTOR activity is or comprises ' , or a pharmaceutically acceptable salt thereof.

[0298] In some embodiments, 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 CR3, wherein R1is 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, R2is H, substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl, R3is H, substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, -NHR4or -N(R4)2, and R4is at each occurrence independently substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, the agent that inhibits mTOR activity is or comprises a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, 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. In some embodiments, the agent that inhibits mTOR activity is or comprisespharmaceutically acceptable salt thereof.

[0299] In particular embodiments, the agent comprises a formula set forth in Formula (III),Formula (III) wherein R1is substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heterocyclylalkyl, R2is 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, and R3is H, or a substituted or unsubstituted Ci-8 alkyl. In certain embodiments, R1is substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl. In some embodiments, R1is 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. In some embodiments, 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. In some embodiments, the agent that inhibits mTOR activity is or comprisespharmaceutically acceptable salt thereof.

[0300] As understood by those skilled in the art, the scope of the present invention also includes 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

[0301] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art

[0302] The terms “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. In some aspects, 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.

[0303] The term “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 variablefragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. 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. Unless otherwise stated, the term “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.

[0304] The term “variable region” or “variable domain” 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. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, 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).

[0305] 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. Examples of 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. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and / or a variable light chain region, such as scFvs.

[0306] 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. In some embodiments, 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. In certain embodiments, 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. In some embodiments, 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. In some embodiments, 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 restoreor improve antibody specificity or affinity. (See, e.g., Queen, U.S. Pat. No. 5,585,089 and Winter, U.S. Pat. No. 5,225,539.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0307] 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. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. 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.

[0308] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject, e.g., patient, to whom the agent or agents, cells, cell populations, or compositions are administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0309] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) 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.

[0310] As used herein, “delaying 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.

[0311] “Preventing,” as used herein, 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. In some embodiments, the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.

[0312] As used herein, 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.

[0313] An “effective amount” of an agent, e.g., a pharmaceutical formulation, cells, or composition, in the context of administration, 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.

[0314] A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation or cells, 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. In some embodiments, the provided methods involve administering the cells and / or compositions at effective amounts, e.g., therapeutically effective amounts.

[0315] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0316] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0317] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0318] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of thelimits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0319] As used herein, a 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.

[0320] As used herein, “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.

[0321] As used herein, 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. When referring to a surface marker, the term 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.

[0322] As used herein, 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. When referring to a surface marker, the term 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.

[0323] The term “vector,” as used herein, 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.”

[0324] As used herein, the terms “response” or “responsiveness” refers to a beneficial response of a subject (for instance, a partial or complete response) to treatment with a cell therapy. As used herein, 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. As used herein, the term “complete response” is the disappearance of all signs of the disease or condition (for instance, cancer) in the body. Conversely, as used herein, the term “unresponsiveness” refers to a subject that has stable disease or progressive disease after receiving a cell therapy. As used herein, the term “stable disease” refers to a disease or condition that is neither decreasing nor increasing in extent or severity. As used herein, 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.IV. EXEMPLARY EMBODIMENTS

[0325] Among the provided embodiments are: Embodiment 1. A method of assessing exosome production by a cell therapy composition, the method comprising:(a) contacting cells of the cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; and(c) determining an amount or concentration of the isolated exosomes produced from the stimulated cell composition.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:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) predicting a response to the cell therapy composition in a subject having a disease or condition, wherein (i) the subject is predicted to respond to 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 predicted to not respond to the cell therapy composition 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 7. A method comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) predicting efficacy of the cell therapy composition in a subject having a disease or condition, wherein (i) efficacy is predicted 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) efficacy is not predicted 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 8. A method comprising :(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) predicting efficacy of the cell therapy composition in a subject having a disease or condition, wherein the predicting efficacy is based on the amount or concentration of the isolated exosomes in the stimulated cell composition being increased compared to the amount or concentration of the isolated exosomes in the control cell composition.Embodiment 9. A method comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) selecting a subject having a disease or condition, wherein: (i) 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 10. A method comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) selecting a subject having a disease or condition for treatment with the cell therapy composition based on the amount or concentration of the isolated exosomes in the stimulated cell composition being increased compared to the amount or concentration of the isolated exosomes in the control cell composition.Embodiment 11. A method comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detectin...

Claims

CLAIMS1. A method of assessing exosome production by a cell therapy composition, the method comprising:(a) contacting cells of the cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; and(c) determining an amount or concentration of the isolated exosomes produced from the stimulated cell composition.

2. The method of claim 1 , wherein the cell therapy composition is a treatment or a candidate for a treatment to be administered to a subject.

3. The method of claim 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.

4. The method of claim 2 or claim 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.

5. The method of any one of claims 2 to 4, wherein the cell therapy composition comprises cells obtained from the subject.

6. A method comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) predicting a response to the cell therapy composition in a subject having a disease or condition, wherein (i) the subject is predicted to respond to 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 ispredicted to not respond to the cell therapy composition 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.

7. A method comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) predicting efficacy of the cell therapy composition in a subject having a disease or condition, wherein (i) efficacy is predicted 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) efficacy is not predicted 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.

8. A method comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) predicting efficacy of the cell therapy composition in a subject having a disease or condition, wherein the predicting efficacy is based on the amount or concentration of the isolated exosomes in the stimulated cell composition being increased compared to the amount or concentration of the isolated exosomes in the control cell composition.

9. A method comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) selecting a subject having a disease or condition, wherein: (i) 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.

10. A method comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) selecting a subject having a disease or condition for treatment with the cell therapy composition based on the amount or concentration of the isolated exosomes in the stimulated cell composition being increased compared to the amount or concentration of the isolated exosomes in the control cell composition.

11. A method comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) selecting a subject having a disease or condition for treatment with the cell therapy composition in combination with an other agent based on the amount or concentration of the isolatedexosomes in the stimulated cell composition being decreased or unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition.

12. A method of treatment comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) administering a treatment to a subject having a disease or condition, wherein: (i) 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; or (ii) the subject is treated 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 is unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition.

13. A method of treatment comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) administering the cell therapy composition to a subject having a disease or condition, wherein the subject has an increased amount or concentration of exosomes produced from the stimulated cell composition compared to the amount or concentration of the isolated exosomes in the control cell composition.

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:(a) contacting cells of the cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) selecting the subject for treatment based on the amount or concentration of the isolated exosomes in the stimulated cell composition being increased compared to the amount or concentration of the isolated exosomes in the control cell composition.

15. A method of treatment comprising:(a) contacting cells of a cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) administering the cell therapy composition in combination with an other agent to a subject having a disease or condition, wherein the subject has a decreased or unchanged amount or concentration of exosomes produced from the stimulated cell composition compared to the amount or concentration of the isolated exosomes in the control cell composition.

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:(a) contacting cells of the cell therapy composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(b) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes;(c) comparing an amount or concentration of the isolated exosomes produced from the stimulated cell composition to an amount or concentration of the isolated exosomes produced from a control cell composition; and(d) selecting the subject for treatment based on the amount or concentration of the isolated exosomes in the stimulated cell composition being decreased or unchanged compared to the amount or concentration of the isolated exosomes in the control cell composition.

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.

18. 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 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 claims 9-11.

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:(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:(i) contacting T cells of the T cell composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(ii) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; and(iii) comparing the amount or concentration of the isolated exosomes produced from the stimulated cell composition to the amount or concentration of the isolated exosomes produced from a control cell composition;(b) predicting a response to the T cell therapy in the subject, wherein(i) 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(ii) 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; and(c) administering the adaptive treatment to the subject, wherein the adaptive treatment is selected from:(i) the T cell therapy if the subject is predicted to respond to the T cell therapy; and(ii) the T cell therapy in combination with an other agent if the subject is not predicted to respond to the T cell therapy.

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:(i) contacting T cells of the T cell composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(ii) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; and(iii) comparing the amount or concentration of the isolated exosomes produced from the stimulated cell composition to the amount or concentration of the isolated exosomes produced from a control cell composition;(b) predicting the subject will respond with a response to the T cell therapy based on the amount or concentration of the isolated exosomes in the stimulated cell composition being increased compared to the amount or concentration of the isolated exosomes in a control cell composition; and(c) administering the T cell therapy to the subject predicted to respond to the T cell therapy.

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:(i) contacting T cells of the T cell composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition;(ii) detecting exosomes produced from cells of the stimulated cell composition to provide isolated exosomes; and(iii) comparing the amount or concentration of the isolated exosomes produced from the stimulated cell composition to the amount or concentration of the isolated exosomes produced from a control cell composition.

22. The method of any one of claims 1 to 16, wherein the cell therapy composition comprises T cells engineered to express a recombinant receptor.

23. The method of any one of claims 19 to 22, wherein the isolated exosomes are further characterized for presence of the recombinant receptor.

24. The method of any one of claims 1 to 16, wherein the cell therapy composition is a treatment or a candidate for a treatment to be administered to a subject.

25. The method of any one of claims 17 to 24, wherein the T cells are primary cells.

26. The method of any one of claims 17 to 25, wherein the T cells are autologous cells.

27. The method of any one of claims 17 to 25, wherein the T cells are allogeneic cells.

28. The method of any one of claims 17 to 27, wherein the T cells are CD3+.

29. The method of any of claims 17 to 28, wherein the T cells are CD4+, and / or CD8+ T cells.

30. The method of any one of claims 17 to 29, wherein the T cells of the cell therapy composition or T cell composition comprise CD4+ T cells and CD8+ T cells.

31. The method of any one of claims 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.

32. The method of any one of claims 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.

33. The method of any one of claims 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.

34. The method of any one of claims 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.

35. The method of claim 34, wherein introducing the polynucleotide encoding the recombinant receptor comprises transducing cells with a viral vector encoding the recombinant receptor.

36. The method of claim 34 or claim 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.

37. The method of any one of claims 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.

38. The method of any one of claims 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.

39. The method of any one of claims 34 to 38 wherein the biological sample is an apheresis product or leukapheresis product.

40. The method of claim 39, wherein the apheresis product or leukapheresis product has been previously cryopreserved.

41. The method of any one of claims 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.

42. The method of any of claims 34 to 41, wherein the human subject has a disease or condition.

43. The method of any one of claims 34 to 41, further comprising harvesting cells produced by the manufacturing process and formulating the harvested cells in a pharmaceutically acceptable buffer.

44. The method of claim 43, wherein the harvested cells further comprises a cryoprotectant.

45. The method of any one of claims 6 to 44, wherein the disease or condition is a cancer or tumor.

46. The method of any one of claims 6 to 44, wherein the disease or condition is an autoimmune disease or condition.

47. The method of any one of claims 3 to 6 and 19-21, wherein the response comprises a partial response.

48. The method of any one of claims 3 to 6 and 19-21, wherein the response comprises a complete response.

49. The method of any one of claims 3 to 6 and 19-21, wherein the response does not comprise stable disease.

50. The method of any one of claims 3 to 6 and 19-21, wherein the response does not comprise progressive disease.

51. The method of any one of claims 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.

52. The method of any one of claims 1 to 51 , wherein detecting comprises isolating the exosomes.

53. The method of claim 52, wherein isolating is by centrifugation.

54. The method of claim 53, wherein the centrifugation comprises at least one spin.

55. The method of claim 53 or claim 54, wherein the centrifugation is performed for about 1 to 20 minutes.

56. The method of any one of claims 53 to 55, wherein the centrifugation comprises one spin at 300 g for 3 min.

57. The method of any one of claims 53 to 56, wherein the centrifugation comprises one spin at 2,500 g for 15 min.

58. The method of any one of claims 53 to 57, wherein the centrifugation comprises two spins at 2,500 g for 15 min.

59. The method of any one of claims 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.

60. The method of any one of claims 1 to 59, wherein 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.

61. The method of any one of claims 1 to 60, wherein 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 about 150 nm.

62. The method of claim 60 or claim 61, wherein the identifying cell particles is by identifying cell particles that have a size between about 30 nm to about 130 nm.

63. The method of any one of claims 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.

64. The method of claim 60, wherein the one or more exosome markers are identified by immunoaffinity-based capture.

65. The method of claim 64, wherein the immunoaffinity-based capture comprises an antibody specific to the one or more exosome markers.

66. The method of any one of claims 60 to 65, wherein the one or more exosome markers are selected from CD63, CD81, CD9, and any combination thereof.

67. The method of claim 66, wherein the one or more exosome markers is CD63.

68. The method of claim 66, wherein the one or more exosome markers is CD81.

69. The method of claim 66, wherein the one or more exosome markers is CD9.

70. The method of claim 66, wherein the one or more exosome markers comprises CD63 and CD81.

71. The method of claim 66, wherein the one or more exosome markers comprises CD63 and CD9.

72. The method of claim 66, wherein the one or more exosome markers comprises CD81 and CD9.

73. The method of claim 66, wherein the one or more exosome markers comprises CD63, CD81 and CD9.

74. The method of any one of claims 60 to 73, wherein the exosomes have a size between about 30 nm and 150 nm.

75. The method of any one of claims 60 to 74, wherein the exosomes have a size between about 30 nm and 130 nm.

76. The method of any one of claims 60 to 75, wherein the exosomes have a size between about 30 nm and 100 nm.

77. The method of any one of claims 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.

78. The method of any one of claims 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.

79. The method of any one of claims 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.

80. The method of claim 79, wherein the surface of the exosomes comprises the recombinant receptor expressed by the cells of the cell therapy composition or T cell composition.

81. The method of claim 79, wherein the surface of the exosomes comprises the surrogate marker of the recombinant receptor expressed by the cells of the T cell composition.

82. The method of claim 79 or claim 80, wherein the surrogate marker is a truncated cell surface receptor, optionally a truncated epidermal growth factor receptor (tEGFR).

83. The method of any one of claims 17 to 82, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

84. The method of claim 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.

85. The method of claim 84, wherein the antigen is expressed by the cells of the disease or condition.

86. The method of claim 84, wherein the antigen is expressed by the cells of a cancer or tumor.

87. The method of claim 86, wherein the cancer or tumor is a hematological malignancy.

88. The method of claim 87, wherein the hematological malignancy is a myeloma, leukemia or lymphoma.

89. The method of claim 87 or claim 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).

90. The method of any one of claims 84 to 87, wherein the antigen is a B cell antigen.

91. The method of claim 90, wherein the B cell antigen is CD19.

92. The method of claim 90, wherein the B cell antigen is BCMA.

93. The method of any one of claims 84 to 87, wherein the antigen is a plasma cell antigen.

94. The method of claim 93, wherein the plasma cell antigen is GPRC5D.

95. The method of any one of claims 4 to 94, wherein the control cell composition comprises T cells of the cell therapy composition that have not been contacted with the recombinant receptorstimulating agent.

96. The method of any one of claims 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.

97. The method of claim 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.

98. The method of claim 96 or claim 97, wherein the recombinant receptor-stimulating agent is an antibody specific to an extracellular binding domain of the recombinant receptor.

99. The method of any one of claims 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.

100. The method of any one of claims 1 to 99, wherein the recombinant receptor-stimulating agent is immobilized or attached to a solid support.

101. The method of claim 100, wherein the solid support is a surface of a vessel, optionally a well of micro well plate, in which a plurality of incubations is performed.

102. The method of claim 100, wherein the solid support is a bead.

103. The method of any one of claims 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.

104. The method of claim 103, wherein the antigen-expressing cell is a cell line.

105. The method of claim 104, wherein the cell line is a tumor cell line.

106. The method of any one of claims 103 to 105, wherein the antigen-expressing cell is a cell that has been introduced, optionally by transduction, to express an antigen of the recombinant receptor.

107. The method of any one of claims 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.

108. The method of claim 107, wherein the immunomodulatory agent is an immunomodulatory imide drug (IMiD) or a cereblon E3 ligase modulator (CELMoD).

109. The method of claim 107 or claim 108, wherein the immunomodulatory agent is thalidomide or a thalidomide derivative.

110. The method of any one of claims 107 to 109, wherein the immunomodulatory agent is selected from the group consisting of lenalidomide, pomalidomide, avadomide (CC-122), and iberdomide (CC-220).

111. The method of claim 107, wherein the checkpoint inhibitor is selected from a PD-1 inhibitor, LAG3 inhibitor and PD-L1 inhibitor.

112. The method of claim 111, wherein the PD-1 inhibitor is an anti-PD-1 antibody (e.g, nivolumab).

113. The method of claim 111, wherein the LAG3 inhibitor is an anti-LAG3 antibody (e.g., relatlimab).

114. The method of claim 111 or claim 112, wherein the PD-L1 inhibitor is an anti-PD-Ll antibody (e.g. durvalumumab).

115. The method of claim 107, wherein the BCL2 inhibitor is selected from the group consisting of venetoclax, navitoclax, ABT737, maritoclax, obatoclax and clitocine.

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