Methods for determining efficacy of therapeutic cell compositions

Abstract

The present disclosure relates to a method for determining the efficacy of a therapeutic cell composition associated with cell therapy. The cells of the therapeutic cell composition can express a recombinant receptor, e.g., a chimeric receptor, e.g., a chimeric antigen receptor (CAR) or other transgenic receptor, such as a T cell receptor (TCR). The method provides an assay for identifying the efficacy, including the relative efficacy, of the therapeutic cell composition.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 164,527, filed March 22, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] Incorporation by reference to sequence listing This application is submitted with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 73504_2023040_SEQLIST.TXT, created on March 21, 2022, and is 84,579 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] The present disclosure relates to methods for determining the efficacy of therapeutic cell compositions for use in connection with cell therapy. The cells of the therapeutic cell composition can express a recombinant receptor, e.g., a chimeric receptor, e.g., a chimeric antigen receptor (CAR) or other transgenic receptor, such as a T cell receptor (TCR). The method provides an assay for determining the efficacy, including relative efficacy, of the therapeutic cell composition. [Background technology]

[0004] A variety of immunotherapy and / or cell therapy methods are available for treating diseases and conditions.For example, adoptive cell therapy (including the administration of cells expressing chimeric receptors, such as chimeric antigen receptors (CAR) and / or other recombinant antigen receptors, specific to the disease or disorder of interest, as well as other adoptive immune cell and adoptive T cell therapies) can be effective in treating cancer or other diseases or disorders.There is a need for improved approaches to characterize effective therapeutic cell compositions and to treat subjects using cell therapy, including in connection with the method of ex vivo production of compositions.Methods that address this need are provided herein. Summary of the Invention

[0005] Provided herein is a method for determining the efficacy of a therapeutic cell composition, the method comprising: performing a plurality of incubations, each of which comprises culturing cells of a therapeutic cell composition comprising cells engineered to express a recombinant receptor with a recombinant receptor-stimulating agent, wherein binding of the recombinant receptor-stimulating agent to the recombinant receptor stimulates a recombinant receptor-dependent activity in the cells, and each of which comprises a different adjusted ratio of cells of the therapeutic cell composition to the recombinant receptor-stimulating agent; measuring the recombinant receptor-dependent activity from each of the plurality of incubations; and determining an adjusted ratio that results in a specific recombinant receptor-dependent activity, e.g., half-maximal recombinant receptor-dependent activity, based on the recombinant receptor-dependent activity measured from each of the plurality of incubations. In some of any of the provided embodiments, the method further comprises determining the relative efficacy of the therapeutic cell composition by comparing the adjusted ratio that results in a specific receptor-dependent activity (e.g., half-maximal recombinant receptor-dependent activity) of the therapeutic cell composition to the adjusted ratio that results in a specific receptor-dependent activity (e.g., half-maximal recombinant receptor-dependent activity) of a reference standard.

[0006] Also provided herein is a method of determining the efficacy of a therapeutic cell composition, the method comprising: performing a plurality of incubations, each of the plurality of incubations comprising culturing cells of a therapeutic cell composition comprising cells engineered to express a recombinant receptor with a recombinant receptor stimulating agent, wherein binding of the recombinant receptor stimulating agent to the recombinant receptor stimulates a recombinant receptor-dependent activity in the cells, and each of the plurality of incubations comprising a different adjusted ratio of cells of the therapeutic cell composition to the recombinant receptor stimulating agent; measuring the recombinant receptor-dependent activity from each of the plurality of incubations; and determining the relative efficacy of the therapeutic cell composition by comparing the specific recombinant receptor-dependent activity of the therapeutic cell composition (e.g., the half-maximal recombinant receptor-dependent activity of the therapeutic cell composition) to the specific recombinant receptor-dependent activity of a reference standard (e.g., the half-maximal recombinant receptor-dependent activity).

[0007] In some of any of the provided embodiments, each of the plurality of incubations comprises culturing a fixed number of cells of the therapeutic composition with a different amount of the recombinant receptor stimulating agent to generate a plurality of different adjusted ratios. In some of any of the provided embodiments, each of the plurality of incubations comprises culturing a fixed amount of the binding molecule, e.g., the recombinant receptor stimulating agent, with a different number of cells of the therapeutic composition to generate a plurality of different adjusted ratios.

[0008] In some of any of the provided embodiments, the multiple incubations are performed on two or more, optionally 3, 4, 5, 6, 7, 8, 9, 10, or more, therapeutic cell compositions. In some of any of the provided embodiments, the two or more therapeutic cell compositions each comprise the same recombinant receptor. In some of any of the provided embodiments, the two or more therapeutic cell compositions each comprise a different recombinant receptor. In some of any of the provided embodiments, at least one of the two or more therapeutic cell compositions comprises a different recombinant receptor than the other therapeutic compositions.

[0009] In some of any of the provided embodiments, the two or more therapeutic cell compositions are each manufactured using the same manufacturing process. In some of the provided embodiments, the two or more therapeutic cell compositions are each manufactured using a different manufacturing process. In some of the provided embodiments, at least one of the two or more therapeutic cell compositions is manufactured using a different manufacturing process than that used to manufacture the other therapeutic cell compositions.

[0010] In some of any of the provided embodiments, two or more therapeutic cell compositions are generated from cells obtained from a single subject. In some of the provided embodiments, two or more therapeutic cell compositions are generated from cells obtained from different subjects. In some of the provided embodiments, the subjects are healthy subjects or subjects with a disease or condition.

[0011] In some of the provided embodiments, each of the different subjects has the same disease or condition. In some of the provided embodiments, each of the different subjects is to be treated with the same therapeutic cell composition for treating the disease or condition in the subject.

[0012] In some of any of the provided embodiments, the plurality of incubations is at least 3 incubations. In some of the provided embodiments, the plurality of incubations is at least 5 incubations. In some of the provided embodiments, the plurality of incubations is at least 7 incubations. In some of the provided embodiments, the plurality of incubations is at least 10 incubations.

[0013] In some of any of the provided embodiments, the recombinant receptor-dependent activity includes one or more of cytokine expression, cytolytic activity, receptor upregulation, receptor downregulation, proliferation, gene upregulation, gene downregulation, or cell health. In some of the provided embodiments, the recombinant receptor-dependent activity includes or is cytokine expression or production. In some of the provided embodiments, the recombinant receptor-dependent activity includes or is cytokine expression or production, wherein the cytokine is TNF-alpha, IFN gamma (IFNg), or IL-2. In some of the provided embodiments, the recombinant receptor-dependent activity includes or is cytolytic activity. In some of the provided embodiments, the recombinant receptor-dependent activity includes or is receptor downregulation. In some of the provided embodiments, the recombinant receptor-dependent activity includes or is proliferation. In some of the provided embodiments, the recombinant receptor-dependent activity includes or is gene upregulation. In some of the provided embodiments, the recombinant receptor-dependent activity includes or is gene downregulation. In some of any of the provided embodiments, the recombinant receptor-dependent activity includes or is cell health. In some of the provided embodiments, the recombinant receptor-dependent activity includes or is cell health, and cell health includes one or more of cell death, cell diameter, viable cell concentration, and cell count.

[0014] In some of any of the provided embodiments, the recombinant receptor-dependent activity measured in each of the multiple incubations is normalized to the maximum receptor-dependent activity measured for the therapeutic cell composition.

[0015] In some of any of the provided embodiments, the reference standard is a therapeutic cell composition comprising a validated adjusted ratio that results in a particular (e.g., half-maximal) recombinant receptor-dependent activity, a commercially available therapeutic cell composition, a therapeutic cell composition manufactured using a manufacturing process that is identical to the manufacturing process used to manufacture the therapeutic cell composition, a therapeutic cell composition manufactured using a manufacturing process that is different from the manufacturing process used to manufacture the therapeutic cell composition, a therapeutic cell composition that includes the same recombinant receptor as the therapeutic cell composition, a therapeutic cell composition that includes a different recombinant receptor than the therapeutic cell composition, a therapeutic cell composition manufactured from the same subject, or a therapeutic cell composition manufactured from a different subject.

[0016] In some of any of the provided embodiments, the reference standard is one of two or more therapeutic compositions.

[0017] In some of any of the provided embodiments, the recombinant receptor stimulating agent comprises a target antigen of the recombinant receptor or an extracellular domain-binding portion thereof, optionally the recombinant antigen. In some of the provided embodiments, the recombinant receptor stimulating agent comprises an extracellular domain-binding portion of the antigen, the extracellular domain-binding portion comprising an epitope recognized by the recombinant receptor. In some of the provided embodiments, the recombinant receptor stimulating agent comprises an antibody specific for the extracellular domain of the recombinant receptor (e.g., an epitope on the extracellular domain). In some of the provided embodiments, the recombinant receptor stimulating agent is an anti-idiotypic antibody specific for the extracellular antigen-binding domain of the recombinant receptor.

[0018] In some of the provided embodiments, the recombinant receptor stimulator is immobilized or attached to a solid support. In some of the provided embodiments, the solid support is a vessel in which multiple incubations are performed, optionally the surface of a well of a microwell plate. In some of the provided embodiments, the solid support is a bead.

[0019] In some of the provided embodiments, the recombinant receptor stimulator is an antigen-expressing cell, and the cell may be a clone from a cell line, a cell, or a primary cell taken from a subject. In some of the provided embodiments, the antigen-expressing cell is a cell line. In some of the provided embodiments, the cell line is a tumor cell line. In some of the provided embodiments, the antigen-expressing cell is a cell that may have been transduced to express the antigen of the recombinant receptor.

[0020] In some of any of the provided embodiments, the adjusted ratio achieves a linear dose-response range of the recombinant receptor-dependent activity of the reference standard. In some of the provided embodiments, the adjusted ratio includes the lower asymptote (minimum) recombinant receptor-dependent activity and the upper asymptote (maximum) recombinant receptor-dependent activity of the reference standard.

[0021] In some of the provided embodiments, the therapeutic cell composition comprises a single cell subtype enriched or purified from a biological sample, or a population of mixed cell subtypes obtained by mixing cell subtypes enriched or purified from a biological sample, as appropriate. In some of the provided embodiments, the biological sample comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.

[0022] In some of the provided embodiments, the therapeutic cell composition comprises primary cells. In some of the provided embodiments, the therapeutic cell composition comprises autologous cells obtained from the subject to be treated. In some of the provided embodiments, the therapeutic cell composition comprises allogeneic cells. In some of the provided embodiments, the therapeutic cell composition comprises CD3+, CD4+, and / or CD8+ T cells. In some of the provided embodiments, the therapeutic cell composition comprises or is CD4+ T cells. In some of the provided embodiments, the therapeutic cell composition comprises or is CD8+ T cells. In some of the provided embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0023] In some of the provided embodiments, the therapeutic cell composition comprises CD4+ T cells and CD8+ T cells. In some of the provided embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0024] In some of the provided embodiments, the multiple incubations are performed in flasks, tubes, or multi-well plates. In some of the provided embodiments, each of the multiple incubations is performed individually in a well of a multi-well plate. In some of the provided embodiments, the multi-well plate is a 96-well plate, a 48-well plate, a 12-well plate, or a 6-well plate.

[0025] In some of any of the provided embodiments, the method further comprises determining a dose of the cells of the therapeutic composition to administer to a subject in need thereof based on the adjusted ratio that results in a particular (e.g., half-maximal) recombinant receptor-dependent activity. In some of any of the provided embodiments, the method further comprises determining a dose of the cells of the therapeutic composition to administer to a subject in need thereof based on the relative potency.

[0026] In some of the provided embodiments, the subject has a disease or condition. In some of the provided embodiments, the disease or condition is cancer.

[0027] In some of any of the provided embodiments, the method further includes determining a manufacturing process that produces an optimal therapeutic cell composition potency based on the relative potency, where the optimal therapeutic cell composition potency correlates with a complete response and / or a durable response and / or reduced toxicity.

[0028] In some of any of the provided embodiments, the method further includes determining a manufacturing process that produces a therapeutic cell composition with reduced or low variance in potency based on the relative potency, where the reduced or low variance is determined compared to the variance in a different manufacturing process. [Brief explanation of the drawings]

[0029] [Figure 1A] Figures 1A-1B show cytokine secretion response curves at various target to effector cell ratios (T:E) for three different donors. Figure 1A shows the raw cytokine secretion curves, while Figure 1B shows the same secretion curves normalized by the upper asymptote (Vmax). [Figure 1B] Figures 1A-1B show cytokine secretion response curves at various target to effector cell ratios (T:E) for three different donors. Figure 1A shows the raw cytokine secretion curves, while Figure 1B shows the same secretion curves normalized by the upper asymptote (Vmax). DETAILED DESCRIPTION OF THE INVENTION

[0030] Provided herein are methods for evaluating or determining the efficacy of therapeutic cell compositions for cell therapy, including engineered T cell therapy (e.g., therapeutic cell compositions), for use in connection with monitoring ex vivo processes for producing cell therapy, and for determining doses for the treatment of various diseases and conditions, including cancer. Provided embodiments relate to therapeutic T cell compositions containing engineered T cells, such as those expressing recombinant receptors, engineered to express recombinant proteins designed to recognize and / or specifically bind to molecules associated with a disease or condition, and to elicit a response, e.g., an immune response, against such molecules upon binding. Receptors can include chimeric receptors, e.g., chimeric antigen receptors (CARs), and other transgenic antigen receptors, including transgenic T cell receptors (TCRs).

[0031] This method for determining the efficacy of a therapeutic cell composition typically uses the maximum antigen stimulation of the engineered cells of the therapeutic cell composition. For example, various existing methods measure the antigen-specific activity of the cells of the therapeutic cell composition at maximum stimulation, such as cytokine expression, receptor upregulation, or downregulation. The activity measured in response to stimulation is then compared for all the therapeutic cell compositions tested to determine which therapeutic cell composition had the most activity, for example, recombinant receptor-dependent activity. The therapeutic cell composition with the highest activity can be considered the most potent therapeutic cell composition.

[0032] In many cases, the use of saturating levels of antigen may not be physiologically relevant. Furthermore, the use of saturating levels of antigen cannot capture the sensitivity of a therapeutic cell composition to recombinant receptor stimulation. For example, current assays cannot distinguish the sensitivity of a recombinant receptor to the stimulus (e.g., amount or concentration of antigen) required to elicit a detectable activity response. Maximal antigen stimulation does not allow for elucidation of the activity of the recombinant receptor to varying stimuli, e.g., recombinant receptor-dependent activity. In short, current methods for assessing efficacy in therapeutic cell compositions provide a one-dimensional view of the efficacy of a therapeutic cell composition but further lack the ability to establish a measure that can capture the sensitivity (e.g., behavior, activity) of a therapeutic cell composition.

[0033] The methods provided herein are designed to more comprehensively evaluate the sensitivity (e.g., behavior, activity) of a therapeutic cell composition. The methods provided herein are designed to provide a more biologically relevant measure of the efficacy of a therapeutic cell composition. In some embodiments, the efficacy of a therapeutic cell composition determined according to the methods described herein may more strongly correlate with the safety and efficacy of the therapeutic cell composition. In some embodiments, the efficacy of a therapeutic cell composition determined according to the methods described herein may provide an improved measure of manufacturing control and / or variability, which in turn may allow for improved assessment of the stability and activity (e.g., recombinant receptor-dependent activity) of manufactured therapeutic cell compositions.

[0034] The provided methods provide a direct method for comparing how therapeutic cell compositions respond to antigens. Unlike existing methods that often compare activity against a single target antigen stimulus, which is the maximum possible stimulus, the provided methods adjust the ratio of target (e.g., antigen- or antibody-expressing cells) to effector cells (cells of the therapeutic composition). For example, this ratio can be controlled by maintaining a constant number of effector cells in the assay and varying the number of target-expressing cells. For example, the provided methods make it possible to determine the number of target-expressing cells and / or the amount of target (e.g., antigen or antibody) required to achieve a specific receptor-dependent activity. In some embodiments, the target is an antigen of a recombinant receptor. Thus, in some cases, the target-expressing cells are antigen-expressing cells. In some embodiments, the specific receptor-dependent activity is 50% of the maximum activity. In some embodiments, the specific receptor-dependent activity is 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the maximum receptor-dependent activity. In some embodiments, the particular receptor-dependent activity is in a range as disclosed herein. In some embodiments, the provided methods allow for comparison of therapeutic cell compositions. For example, therapeutic cell compositions can be evaluated by the methods provided herein to determine, for example, relative potency as described herein.

[0035] Importantly, it was not known that it would be possible to compare activity between and among different therapeutic cell compositions. In particular, the ability to compare therapeutic cell compositions was not known because therapeutic cell compositions were thought to have different sensitivity profiles (e.g., response to recombinant receptor-specific stimulation), e.g., different minimum and maximum responses. The variables affecting therapeutic cell compositions are much greater than other drug products, such as biologics, due to differences in the donor from which the cells are derived or obtained, the differentiation state of the cells, the antigen-binding ability of a particular recombinant receptor (e.g., CAR), the intracellular signaling components of a particular recombinant receptor, the percentage or frequency of cells in the composition expressing the recombinant receptor (e.g., CAR), the process used to generate the therapeutic composition, and other factors. For example, as shown in Figure 1A, therapeutic compositions derived from different donors exhibit different responses to antigen stimulation, such as those shown in the response curves. Therefore, this result would indicate that it may not be possible to compare therapeutic cell compositions. It is discovered herein that by normalizing activity to the upper asymptote of the response curve (e.g., maximum stimulation), it may be possible to compare the sensitivity of therapeutic cell compositions. The provided methods allow for measuring sensitivity between and among different cell products, including those that may vary as a result of different methods, expressing different antigen receptors, being generated from different donors, and other variables. Existing methods assess activity at maximal stimulation, in part because that is believed to be the only way to account for possible variance.

[0036] In some embodiments, the methods provided herein reduce or eliminate the source of variability.For example, the methods provided herein are robust to the variability that may be caused by donor heterogeneity and / or sampling or testing on a daily basis.In some cases, eliminating variability such as donor heterogeneity and / or sampling or testing variability allows for comparison of therapeutic cell compositions.

[0037] The methods provided herein include assay formats that include a series of incubations in which cells of a therapeutic cell composition and a recombinant receptor stimulating agent are cultured at different adjusted ratios. In provided aspects, the recombinant receptor stimulating agent is an agent that induces or is capable of inducing a signal through the intracellular signaling domain of the recombinant receptor. For example, the recombinant receptor stimulating agent can include an anti-idiotypic antibody specific for an antigen of the recombinant receptor, e.g., a purified or recombinant antigen, an antigen-expressing cell, or the extracellular antigen-binding domain (e.g., scFv) of the recombinant receptor. In some embodiments, the methods provided herein, including assay formats, are designed to measure the sensitivity of a therapeutic cell composition by measuring or determining the amount or concentration of a recombinant receptor stimulating agent, e.g., as described in Section IB, required to stimulate a recombinant receptor-dependent activity in engineered cells of the therapeutic cell composition. For example, the methods provided herein can determine the level (e.g., amount, concentration) of antigen that stimulates a quantifiable and detectable activity (e.g., recombinant receptor-dependent activity) of the therapeutic cell composition. In some embodiments, the measure of sensitivity includes a measurement of the recombinant receptor-dependent activity stimulated by the recombinant receptor stimulating agent binding to the recombinant receptor over multiple adjusted ratios. The ability of the method to assess recombinant receptor-dependent activity at different adjusted ratios allows for the determination, estimation and / or extrapolation of the general activity or behavior of a therapeutic cell composition in response to recombinant receptor-specific stimulation.

[0038] The methods provided herein include assays that allow for the evaluation of the efficacy of a therapeutic cell composition by measuring the activity of cells of the therapeutic cell composition expressing a recombinant receptor, e.g., a recombinant receptor described herein, in response to recombinant receptor stimulation in a series of controlled incubations. For example, the series of incubations can include culturing engineered cells of the therapeutic cell composition expressing the recombinant receptor with a recombinant receptor stimulating agent, e.g., as described herein (e.g., in Section IB), at different, controlled ratios that stimulate the activity of the recombinant receptor expressed by the cells when bound to the recombinant receptor, e.g., recombinant receptor-dependent activity, with each incubation being at a different, controlled ratio. In some embodiments, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more incubations, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more incubations are performed, each containing a different ratio of cells of the therapeutic cell composition to recombinant receptor stimulating agent. In some embodiments, three or at least three incubations are performed, each containing a different adjusted ratio of cells of the therapeutic cell composition to recombinant receptor stimulating agent. In some embodiments, six or at least six incubations are performed, each containing a different adjusted ratio of cells of the therapeutic cell composition to recombinant receptor stimulating agent. In some embodiments, ten or at least ten incubations are performed, each containing a different adjusted ratio of cells of the therapeutic cell composition to recombinant receptor stimulating agent.

[0039] In some embodiments, a fixed number of cells of a therapeutic composition are cultured with different amounts of a recombinant receptor stimulating agent to produce a series (e.g., multiple) of different adjusted ratios. Alternatively, in some embodiments, a fixed amount or concentration of a recombinant receptor stimulating agent, e.g., as described in Section IB, may be incubated with different numbers of cells of a therapeutic cell composition to produce a series (e.g., multiple) of different adjusted ratios. Regardless of how the different adjusted ratios are achieved, for example, by varying the total number of cells in the therapeutic cell composition or the amount of recombinant receptor stimulating agent, a series (e.g., multiple) of adjusted ratios can be used to evaluate recombinant receptor-dependent activity over a range of stimulation conditions. In some embodiments, a range of measurements can be used to extract, extrapolate, and / or determine how the engineered cells of a particular therapeutic cell composition respond to different levels of recombinant receptor stimulation.

[0040] Any number of measures can be determined, extracted, extrapolated, estimated, and / or inferred from the measured recombinant receptor-dependent activity generated according to the methods described herein. Non-limiting examples of measures include adjusted ratios that result in maximum, minimum, and half-maximal (50%) recombinant receptor-dependent activity, adjusted ratios that result in a specific percentage of maximum recombinant receptor-dependent activity (e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90%), and adjusted ratios that encompass a range of recombinant receptor-dependent activity, e.g., 10%-90%, 20%-80%, 30%-70%, 40%-60% of maximum recombinant receptor-dependent activity. In some embodiments, the measured recombinant receptor-dependent activity of a therapeutic cell composition is subjected to curve fitting to generate a recombinant receptor-dependent activity curve. In some embodiments, the curve resembles a dose-response curve. In some embodiments, a measure of recombinant receptor-dependent activity and / or the rate at which a particular recombinant receptor-dependent activity occurs is extrapolated and / or estimated from the curve. In some embodiments, a recombinant receptor-dependent activity curve can be used to extrapolate a value or measure of a therapeutic cell composition and / or recombinant receptor stimulating agent at which a particular recombinant receptor-dependent activity occurs. In some embodiments, the amount (e.g., mass (e.g., in picograms) in the case of a purified or recombinant target, or number of cells in the case of target-expressing cells) or concentration (e.g., mass / volume (e.g., in pg / ml) in the case of a purified or recombinant target, or number of cells per unit volume (e.g., cells / mL) in the case of target-expressing cells) of the recombinant receptor stimulating agent is used to determine the maximum, minimum, half-maximum amount, and range at which recombinant receptor-dependent activity occurs, for example, when the therapeutic cell composition cell count is held constant and the amount of recombinant receptor stimulating agent is varied. In some embodiments, for example, when the therapeutic cell composition cell count is varied and the amount of recombinant receptor stimulating agent is held constant, the number of cells (e.g., count, total number) of the therapeutic cell composition is used to determine the maximum, minimum, half-maximum amount and range at which recombinant receptor-dependent activity occurs.In some embodiments, the adjusted ratio(s) are used to determine the maximum, minimum, half-maximal amount, and range over which recombinant receptor-dependent activity occurs. In some embodiments, the amount or concentration of recombinant receptor stimulating agent is used to determine half-maximal recombinant receptor-dependent activity. In some embodiments, the amount (e.g., count) of cells in the therapeutic cell composition is used to determine half-maximal recombinant receptor-dependent activity. In some embodiments, the adjusted ratio is used to determine half-maximal recombinant receptor-dependent activity. These exemplified measures, as well as others not listed, provide quantitative descriptions of therapeutic cell compositions that can be used to determine potency and / or relative potency (e.g., potency relative to a reference standard, such as those described herein (e.g., in Section ID) of a therapeutic cell composition).

[0041] In some embodiments, the potency of a therapeutic cell composition is expressed as an adjusted ratio value or measure, the amount of cells of the therapeutic cell composition, and / or the amount or concentration of recombinant receptor-stimulating agent determined based on recombinant receptor-dependent activity. In some embodiments, the potency of a therapeutic cell composition is the adjusted ratio value or measure, the amount of cells of the therapeutic cell composition, and / or the amount or concentration of recombinant receptor-stimulating agent at which half-maximal recombinant receptor-dependent activity (e.g., 50% of maximal activity) occurs. In some embodiments, the potency of a therapeutic cell composition is the adjusted ratio at which half-maximal recombinant receptor-dependent activity (e.g., 50% of maximal activity) occurs. In some embodiments, the potency of a therapeutic cell composition is the concentration of recombinant receptor-stimulating agent at which half-maximal recombinant receptor-dependent activity occurs. In some embodiments, the half-maximal recombinant receptor-dependent activity at which 50% effective stimulation (ES50) of the therapeutic cell composition occurs according to the measured recombinant receptor-dependent activity reflects the adjusted ratio, concentration of recombinant receptor-stimulating agent, and / or cell count.

[0042] In some embodiments, the potency of a therapeutic cell composition is relative potency. For example, the adjusted ratio at which half-maximal recombinant receptor-dependent activity is measured for a therapeutic cell composition can be compared to the adjusted ratio at which half-maximal recombinant receptor-dependent activity is measured for a reference standard. Of course, the concentration or amount of recombinant receptor stimulating agent or cell count can be used instead of the adjusted ratio, where appropriate. In some embodiments, the reference standard is a therapeutic cell composition having a known and / or validated adjusted ratio at which half-maximal recombinant receptor-dependent activity occurs. In some embodiments, the reference standard is a commercially available therapeutic cell composition for which the adjusted ratio at which half-maximal recombinant receptor-dependent activity occurs has been determined, e.g., using a method as described herein. In some embodiments, the reference standard is a different therapeutic cell composition for which the adjusted ratio at which half-maximal recombinant receptor-dependent activity occurs has been determined, e.g., using a method as described herein. In some embodiments, the different therapeutic cell composition contains cells that bind to the same antigen as the test therapeutic cell composition but express a recombinant receptor with a different receptor structure. In some embodiments, the different therapeutic cell composition expresses the same recombinant receptor as the test therapeutic cell composition, but the therapeutic cell composition contains cells manufactured using a different process than the process used to manufacture the test therapeutic cell composition. In some embodiments, the relative potency is a ratio determined by dividing the adjusted ratio resulting in the half-maximal value of the test therapeutic cell composition by the adjusted ratio resulting in the half-maximal value of the reference standard. In some embodiments, the relative potency is a percentage determined by dividing the adjusted ratio resulting in the half-maximal value of the test therapeutic cell composition by the adjusted ratio resulting in the half-maximal value of the reference standard and multiplying by 100.

[0043] In some cases, normalizing the recombinant receptor-dependent activity of a therapeutic cell composition is useful for determining whether the recombinant receptor-dependent activity of two or more therapeutic cell compositions can be compared. For example, when the recombinant receptor-dependent activity of two or more therapeutic cell compositions is determined, the maximum and / or minimum recombinant receptor-dependent activity of each tested therapeutic cell composition may be different, and normalizing the recombinant receptor-dependent activity of each composition to its own maximum value may allow for an assessment of the appropriateness of comparing the recombinant receptor-dependent activities.

[0044] In some embodiments, the recombinant receptor-dependent activity is normalized to the maximum recombinant receptor-dependent activity value measured. In some embodiments, the recombinant receptor-dependent activity curve is normalized to the maximum recombinant receptor-dependent activity measured. In some embodiments, when the recombinant receptor-dependent activity curve is normalized, the maximum activity value is the average over the upper asymptote of the curve. In some embodiments, normalizing the recombinant receptor-dependent activities of the therapeutic cell composition and the reference standard by their respective maximum values ​​facilitates comparison between the test therapeutic cell composition and the reference standard. In some embodiments, normalizing the recombinant receptor-dependent activities of the therapeutic cell composition and the reference standard by their respective maximum values ​​facilitates calculation of relative potency.

[0045] In some embodiments, normalizing the recombinant receptor-dependent activities of the therapeutic cell composition and the reference standard by their respective maximum values ​​allows a parallel line test to be performed, hi some embodiments, the results of the parallel line test demonstrate the ability to compare the therapeutic cell composition and the reference standard.

[0046] The method for evaluating the efficacy of therapeutic cell compositions, including the assays provided herein, allows different therapeutic cell compositions, including reference standards, to be compared.The ability to compare therapeutic cell compositions not only identifies therapeutic cell compositions with improved, optimal, and / or consistent efficacy, but also identifies candidate therapeutic cell compositions for further development and / or analysis; identifies manufacturing processes and procedures that result in therapeutic cell compositions with improved or optimal efficacy; identifies manufacturing procedures that result in therapeutic cell compositions with consistent efficacy; and / or estimates the inherent variability of manufacturing procedures; determines the dose of therapeutic cell composition that should be administered to a subject in need thereof, for example, the dose that results in a clinical response without the occurrence of toxicity; and / or provides a method for comparing the efficacy of allogeneic therapeutic cell compositions with autologous therapeutic cell compositions.The method provided herein is designed to be compatible with a relative efficacy format that is agnostic to whether the test therapeutic composition or reference standard is derived from a different donor (e.g., subject), manufacturing process, and / or therapeutic product.

[0047] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0048] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0049] I. Cellular Potency Assay Provided herein are methods of evaluating the efficacy of a therapeutic cell composition, e.g., a therapeutic T cell composition containing T cells (e.g., CD3+, CD4+, CD8+ T cells) engineered to express a recombinant receptor (e.g., a CAR), e.g., as described in Section IA, using an assay comprising multiple incubations, each of the multiple incubations comprising culturing cells of the therapeutic cell composition containing cells engineered to express a recombinant receptor with a recombinant receptor stimulator, e.g., an antigen, antigen-expressing cells, or an antibody (e.g., an anti-idiotypic antibody), e.g., as described in Section IB, that is capable of being recognized by or bound by the recombinant receptor to stimulate a recombinant receptor-dependent activity in the cell. In some embodiments, the recombinant receptor-dependent activity is an activity of a cell that is induced in response to stimulation of the recombinant receptor. In some embodiments, the recombinant receptor-dependent activity is an activity such as, e.g., cytokine expression, cytolytic activity, receptor upregulation or downregulation, gene upregulation or downregulation, cytolytic activity, proliferative activity, and / or a measure of cell health, e.g., as described in Section IC. In some embodiments, each of the plurality of incubations contains a different adjusted ratio of cells of the therapeutic cell composition to recombinant receptor stimulating agent. In some embodiments, each of the plurality of incubations comprises culturing a fixed number of cells of the therapeutic composition with a different amount of recombinant receptor stimulating agent to generate a plurality of different adjusted ratios. In some embodiments, each of the plurality of incubations comprises culturing a different number of cells of the therapeutic composition with a fixed amount or concentration of recombinant receptor stimulating agent to generate a plurality of different adjusted ratios. In some embodiments, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more incubations are performed, each containing a different ratio of cells of the therapeutic cell composition to recombinant receptor stimulating agent.In some embodiments, three or at least three incubations are performed, each containing a different ratio of cells of the therapeutic cell composition to recombinant receptor stimulating agent. In some embodiments, six or at least six incubations are performed, each containing a different ratio of cells of the therapeutic cell composition to recombinant receptor stimulating agent. In some embodiments, ten or at least ten incubations are performed, each containing a different ratio of cells of the therapeutic cell composition to recombinant receptor stimulating agent. In some embodiments, recombinant receptor-dependent activity is measured from each of the multiple incubations. In some embodiments, recombinant receptor-dependent activity from each of the multiple incubations is measured by, for example, fluorescence, flow cytometry, ELISA, as described in Section IC. In some embodiments, the measured recombinant receptor-dependent activity is fitted with a curve to generate a recombinant receptor-dependent activity curve, for example, as described above. In some embodiments, an adjusted ratio that results in half-maximal recombinant receptor-dependent activity is determined based on the recombinant receptor-dependent activity measured from each of the multiple incubations. In some embodiments, the adjusted ratio resulting in half-maximal recombinant receptor-dependent activity is estimated, extrapolated, or predicted from the recombinant receptor-dependent activity curve. In some embodiments, the recombinant receptor-dependent activity curve is normalized to the measured maximum recombinant receptor-dependent activity. In some embodiments, the adjusted ratio resulting in half-maximal recombinant receptor-dependent activity is the efficacy of the therapeutic cell composition. In some embodiments, the concentration or amount of recombinant receptor stimulating agent or cell count, if applicable, may be reported instead of the adjusted ratio.

[0050] In some embodiments, the adjusted ratio that results in half-maximal recombinant receptor-dependent activity is compared to the adjusted ratio that results in half-maximal recombinant receptor-dependent activity in a reference standard. For example, the adjusted ratio of the therapeutic cell composition that results in half-maximal recombinant receptor-dependent activity is divided by the adjusted ratio that results in half-maximal recombinant receptor-dependent activity in a reference standard, e.g., determined according to the methods described herein, to result in relative potency. In some embodiments, the relative potency is expressed as a ratio. In some embodiments, the relative potency is expressed as a percentage.

[0051] The methods provided herein for determining potency may be performed with replicates. For example, assays may be performed two, three, four, five, or more times. In some embodiments, replicates are used to confirm the accuracy and / or precision of the assay, including the consistency of measured recombinant receptor-dependent activity and / or determined potency and / or relative potency. In some embodiments, a single assay is performed by running the assay in duplicate or triplicate for a particular therapeutic cell composition. In some embodiments, the assay is performed in duplicate. In some embodiments, the assay is performed in triplicate. In some cases where an assay is performed, for example, in duplicate or triplicate, the measured recombinant receptor-dependent activity from each of the replicates is used to provide a statistical measure of recombinant receptor-dependent activity. For example, in some cases, the mean, median, standard deviation, and / or variance of each measure of recombinant receptor-dependent activity is determined. In some embodiments, the mean of each measure of recombinant receptor-dependent activity is determined. In some embodiments, the standard deviation of each measure of recombinant receptor-dependent activity is determined. In some embodiments, the average measures of recombinant receptor-dependent activity are fitted using a mathematical model to generate a recombinant receptor-dependent activity curve. In some embodiments, the curve is normalized to the mean maximum. In some embodiments, the average adjusted ratio resulting in half-maximal recombinant receptor-dependent activity is the potency of the therapeutic cell composition. In some embodiments, the average concentration or amount of recombinant receptor stimulating agent or cell count, if applicable, may be reported instead of the adjusted ratio. In some embodiments, the potency of the therapeutic cell composition is the relative potency determined by taking the average adjusted ratio resulting in half-maximal recombinant receptor-dependent activity and comparing the average adjusted ratio to the single or average adjusted ratio resulting in half-maximal recombinant receptor-dependent activity in a reference standard. In some embodiments, the relative potency is the average potency of the therapeutic cell composition divided by the single or average potency of the standard reference. In some embodiments, the relative potency is expressed as a ratio. In some embodiments, the relative potency is expressed as a percentage.

[0052] The assays provided herein can be performed in any container(s) suitable for multiple incubations. In some embodiments, the assay is performed in a flask. In some embodiments, the assay is performed in a tube, e.g., a microcentrifuge tube, a PCR tube, or a tube. In some embodiments, the assay is performed in a multi-well plate. For example, the multi-well plate is a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate. In certain embodiments, the assay is performed or carried out in a 12-well plate.

[0053] The conditions under which the therapeutic cell composition and the recombinant receptor-stimulating agent are cultured may include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, and / or agents, such as nutrients, amino acids, antibiotics, and ions. The duration of the multiple incubations is intended to be commensurate with at least the minimum amount of time for potential recombinant receptor-dependent activity to be detected (e.g., measured). For example, the amount of time required to accurately measure cytokine expression may be longer than for measuring gene expression. It is further contemplated that within a type of activity, e.g., cytokine expression, there may be differences in the time for a particular cytokine to be measured compared to another. In some embodiments, the multiple incubations are performed for 1, 2, or 3 days, about 1, 2, or 3 days, or at least 1, 2, or 3 days. In some embodiments, the multiple incubations are performed for 1 or 2 days, about 1 or 2 days, or at least 1 or 2 days. In some embodiments, the multiple incubations are carried out for 24, 36, 48, 60, or 72 hours, about 24, 36, 48, 60, or 72 hours, or at least 24, 36, 48, 60, or 72 hours. In some embodiments, the multiple incubations are carried out for 24 or 48 hours, about 24 or 48 hours, or at least 24 or 48 hours. In some embodiments, the multiple incubations are carried out for 24 to 72 hours, or about 24 to about 72 hours. In some embodiments, the multiple incubations are carried out for 24 to 48 hours, or about 24 to about 48 hours. In some embodiments, the multiple incubations are carried out for 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the multiple incubations are carried out for 30 minutes, about 30 minutes, or at least 30 minutes. In some embodiments, the multiple incubations are carried out for 60 minutes, about 60 minutes, or at least 60 minutes.In some embodiments, the multiple incubations are carried out for between or about 10 to 60, 20 to 60, 30 to 60, 40 to 60, 50 to 60 minutes.

[0054] In some embodiments, the multiple incubations are carried out at a temperature of about 25 to about 38°C, e.g., about 30 to about 37°C, e.g., 37°C ± 2°C or about 37°C ± 2°C. In some embodiments, the multiple incubations are carried out at a CO2 level of about 2.5% to about 7.5%, e.g., about 4% to about 6%, e.g., 5% ± 0.5% or about 5% ± 0.5%. In some embodiments, the multiple incubations are carried out at a temperature of 37°C or about 37°C and / or at a CO2 level of 5% or about 5%.

[0055] A. Therapeutic cell composition The methods provided herein are directed to evaluating the efficacy of therapeutic cell compositions, e.g., therapeutic T cell compositions, manufactured by any process. In some embodiments, the methods provided herein can be used to evaluate therapeutic cell compositions manufactured according to the processes described herein (e.g., Section II). In some embodiments, the efficacy and / or relative efficacy of multiple therapeutic cell compositions manufactured by any process can be evaluated according to the methods provided herein. In some embodiments, the multiple therapeutic cell compositions being evaluated are produced by the same manufacturing process. In some embodiments, the multiple therapeutic cell compositions are manufactured by the same manufacturing process but contain different recombinant receptors. In some embodiments, the target is an antigen of the recombinant receptor. Thus, in some cases, the target-expressing cells are antigen-expressing cells. In some embodiments, the different recombinant receptors all bind to the same target, e.g., target antigen. In some embodiments, the different recombinant receptors bind to different targets, e.g., target antigens. In some embodiments, the multiple therapeutic cell compositions being evaluated are produced by different manufacturing processes. In some embodiments, the multiple therapeutic cell compositions are manufactured by different manufacturing processes but contain the same recombinant receptor. In some embodiments, the multiple therapeutic cell compositions are produced by different manufacturing processes and contain different recombinant receptors. In some embodiments, the different recombinant receptors all bind to the same antigen. In some embodiments, the multiple therapeutic cell compositions are produced from a single subject. In some embodiments, the multiple therapeutic cell compositions are produced from different subjects. In some embodiments, the subject is a healthy donor. In some embodiments, the subject has a disease or condition, e.g., cancer. The methods provided herein can allow for comparison of potency and / or relative potency between therapeutic cell compositions, including a reference standard that is a therapeutic cell composition, regardless of method of manufacture.

[0056] In some embodiments, the therapeutic cell composition is produced or manufactured in connection with a process for producing or generating a therapeutic cell composition containing one or more input populations, e.g., selected or enriched, e.g., engineered T cells derived from an input population, obtained from a single biological sample (see, e.g., Section II-A). In certain embodiments, the therapeutic cell composition contains cells expressing a recombinant receptor, e.g., a CAR, a TCR. In certain embodiments, the cells of the therapeutic cell composition are suitable for administration to a subject as a therapy, e.g., autologous cell therapy, allogeneic cell therapy. The methods provided herein can be used to assess the efficacy and / or relative efficacy of therapeutic cell compositions for cell therapy.

[0057] In some embodiments, the process of making or generating a therapeutic cell composition of engineered T cells includes some or all of the following steps: collecting or obtaining a biological sample; isolating, selecting, or enriching input cells from the biological sample; cryo-freezing and storing the input cells, followed by thawing; selecting and stimulating input cells of interest, e.g., T cells, e.g., CD3+, CD4+, CD8+ T cells; genetically engineering the stimulated cells to express or contain a recombinant polynucleotide, e.g., a polynucleotide encoding a recombinant receptor such as a CAR; formulating the cultured cells into an output composition; and cryo-freezing and storing the formulated output cells until the cells are released for infusion and / or administration to a subject. In some embodiments, the method of producing a therapeutic cell composition does not include expanding or increasing the number of cells during the process, such as by culturing the cells in a bioreactor under conditions whereby the cells are expanded to a threshold amount, such as at least 2-, 3-, 4-, 5-, or greater amount, level, or concentration of cells compared to the input population. In some embodiments, methods of producing a therapeutic cell composition include expanding or increasing the number of cells during the process, such as by incubating or culturing the cells in a bioreactor under conditions whereby the cells expand to, such as a threshold amount, that is at least 2-, 3-, 4-, 5-fold or greater amount, level, or concentration of cells compared to the input population. In some embodiments, genetically engineering the cells is or includes transducing the cells with a viral vector, such as by spinoculating the cells in the presence of viral particles, and then incubating the cells under static conditions in the presence of the viral particles. See, e.g., Section II-C.

[0058] In certain embodiments, the total duration of the process of generating engineered cells, from the initiation of stimulation to the harvesting, recovery or formulation of the cells, is, is about, or is less than 36, 42, 48, 54, 60, 72, 84, 96, 108 or 120 hours. In some embodiments, the total duration of the provided process for generating engineered cells, from the start of stimulation to harvesting, collecting, or formulating the cells, is between 36 and 120 hours, 48 ​​and 96 hours, or 48 and 72 hours, inclusive. In certain embodiments, the amount of time to complete the provided process, as measured from the start of incubation to harvesting, collecting, or formulating the cells, is 48, 72, or 96 hours, is about 48, 72, or about 96 hours, or is less than 48, 72, or 96 hours. In certain embodiments, the amount of time to complete the provided process, as measured from the start of incubation to harvesting, collecting, or formulating the cells, is 48 hours ± 6 hours, 72 hours ± 6 hours, or 96 hours ± 6 hours.

[0059] In some embodiments, the entire manufacturing process is performed using a single population of enriched T cells, e.g., CD3+, CD4+, and CD8+ T cells. In certain embodiments, the manufacturing process is performed using two or more input populations of enriched T cells that are combined prior to and / or during the process to create or generate a single therapeutic cell composition of enriched T cells (e.g., a therapeutic cell composition containing CD4+ and CD8+ T cells). In some embodiments, the enriched T cells are or include engineered T cells, e.g., T cells transduced to express a recombinant receptor.

[0060] In some embodiments, the duration or amount of time required to complete the provided processes, as measured from isolation, enrichment, and / or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time the engineered cells of the therapeutic cell composition are collected, formulated, and / or cryoprotected, is, is about, or is less than 48, 72, 96, 120, 4, 5, 7, or 10 days. In some embodiments, the duration or amount of time required to complete the provided processes, as measured from isolation, enrichment, and / or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time the engineered cells are collected, formulated, and / or cryoprotected, is, is about, or is 4-5 days. In some embodiments, the duration or amount of time required to complete the provided processes, as measured from isolation, enrichment, and / or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time the engineered cells are collected, formulated, and / or cryoprotected, is 5 days or about 5 days. In some embodiments, the duration or amount of time required to complete the provided processes, as measured from isolation, enrichment, and / or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time the engineered cells are collected, formulated, and / or cryoprotected, is 5 days or less. In some embodiments, the duration or amount of time required to complete the provided processes, as measured from isolation, enrichment, and / or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time the engineered cells are collected, formulated, and / or cryoprotected, is 4 days or about 4 days.In some embodiments, the isolated, selected, or enriched cells are not cryoprotected prior to stimulation, and the duration or amount required to complete the provided process, as measured from isolation, enrichment, and / or selection of input cells to the time the engineered cells are collected, formulated, and / or cryoprotected, is, is about, or is less than 48, 72, 96, or 120 hours.

[0061] In certain embodiments, the therapeutic cell composition is produced from cells isolated, enriched, or selected from a biological sample, e.g., a population of CD4+ and CD8+ T cells or CD3+ T cells. In some aspects, the time from when the biological sample is collected from a subject to generating or producing the therapeutic cell composition is within a reduced amount of time compared to other methods or processes.

[0062] In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, or at least 95% of the cells of the therapeutic cell composition express the recombinant receptor. In certain embodiments, at least 50% of the cells of the therapeutic cell composition express the recombinant receptor. In certain embodiments, at least 30%, at least 40%, at least 45%, 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%, or at least 95% of the CD3+ T cells of the therapeutic cell composition express the recombinant receptor. In some embodiments, at least 50% of the CD3+ T cells of the therapeutic cell composition express the recombinant receptor. In certain embodiments, at least at least 30%, at least 40%, at least 45%, 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 97%, at least 99%, or more than 99% of the CD4+ T cells of the therapeutic cell composition express the recombinant receptor. In certain embodiments, at least 50% of the CD4+ T cells of the therapeutic cell composition express the recombinant receptor. In some embodiments, at least 30%, at least 40%, at least 45%, 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 97%, at least 99%, or more than 99% of the CD8+ T cells of the therapeutic cell composition express the recombinant receptor. In certain embodiments, at least 50% of the CD8+ T cells of the therapeutic cell composition express the recombinant receptor.

[0063] In certain embodiments, the majority of the cells of the therapeutic cell composition are naive-like, central memory, and / or effector memory cells. In certain embodiments, the majority of the cells of the therapeutic cell composition are naive-like or central memory cells. In some embodiments, the majority of the cells of the therapeutic cell composition are positive for one or more of CCR7 or CD27 expression. In certain embodiments, the cells of the therapeutic cell composition have a greater proportion of naive-like or central memory cells in output populations created from alternative processes, such as processes involving expansion.

[0064] In certain embodiments, the cells of the therapeutic cell composition have a low proportion and / or frequency of depleted and / or senescent cells. In certain embodiments, the cells of the output population have a low proportion and / or frequency of depleted and / or senescent cells. In some embodiments, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells of the therapeutic cell composition are depleted and / or senescent. In certain embodiments, less than 25% of the cells of the therapeutic cell composition are depleted and / or senescent. In certain embodiments, less than 10% of the cells of the output population are depleted and / or senescent. In certain embodiments, the cells have a low proportion.

[0065] In some embodiments, the cells of a therapeutic cell composition have a low proportion and / or frequency of cells that are negative for CD27 and CCR7 expression, e.g., surface expression. In certain embodiments, the cells of a therapeutic cell composition have a low proportion and / or frequency of CD27-CCR7- cells. In some embodiments, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells of a therapeutic cell composition are CD27-CCR7- cells. In certain embodiments, less than 25% of the cells of a therapeutic cell composition are CD27-CCR7- cells. In certain embodiments, less than 10% of the cells of a therapeutic cell composition are CD27-CCR7- cells. In embodiments, less than 5% of the cells of a therapeutic cell composition are CD27-CCR7- cells.

[0066] In some embodiments, the cells of the therapeutic cell composition have a high proportion and / or frequency of cells that are positive for one or both of CD27 and CCR7 expression, e.g., surface expression. In some embodiments, the cells of the therapeutic cell composition have a high proportion and / or frequency of cells that are positive for one or both of CD27 and CCR7. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the cells of the therapeutic cell composition are positive for one or both of CD27 and CCR7. In various embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the CD4+CAR+ cells of the therapeutic cell composition are positive for one or both of CD27 and CCR7. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the CD8+CAR+ cells of the therapeutic cell composition are positive for one or both of CD27 and CCR7.

[0067] In certain embodiments, the cells of a therapeutic cell composition have a high proportion and / or frequency of cells positive for CD27 and CCR7 expression, e.g., surface expression. In some embodiments, the cells of a therapeutic cell composition have a high proportion and / or frequency of CD27+CCR7+ cells. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the cells of a therapeutic cell composition are CD27+CCR7+ cells. In various embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the CD4+CAR+ cells of a therapeutic cell composition are CD27+CCR7+ cells. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more than 95% of the CD8+CAR+ cells of the therapeutic cell composition are CD27+CCR7+ cells.

[0068] In certain embodiments, the cells of a therapeutic cell composition have a low proportion and / or frequency of cells that are negative for CCR7 and positive for CD45RA expression, e.g., surface expression. In some embodiments, the cells of a therapeutic cell composition have a low proportion and / or frequency of CCR7-CD45RA+ cells. In certain embodiments, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells of a therapeutic cell composition are CCR7-CD45RA+ cells. In some embodiments, less than 25% of the cells of an output population (e.g., a therapeutic cell composition) are CCR7-CD45RA+ cells. In certain embodiments, less than 10% of the cells of an output population (e.g., a therapeutic cell composition) are CCR7-CD45RA+ cells. In certain embodiments, less than 5% of the cells of a therapeutic cell composition are CCR7-CD45RA+ cells.

[0069] In some embodiments, the therapeutic cell manufacturing processes are different, such that alternative manufacturing processes can be compared, for example, by comparing the potency of the differently manufactured therapeutic cell compositions. For example, in some embodiments, the alternative process may contain a step of cell expansion. In some embodiments, the alternative process may not contain a step of cell expansion. In some embodiments, the alternative process includes separate steps for cell selection and stimulation. In some embodiments, the alternative process includes a single step for cell selection and stimulation. In some embodiments, the alternative process may differ in one or more specific aspects, but otherwise contain similar or identical features, aspects, steps, stages, reagents, and / or conditions of the processes associated with the provided methods. In some embodiments, alternative processes differ in ways including, but not limited to, one or more of: the inclusion of different reagents and / or media formulations; the presence of serum during incubation, transduction, transfection and / or culture; different cellular composition of the input population, e.g., the ratio of CD4+ to CD8+ T cells; different stimulation conditions and / or different stimulating reagents; different ratios of stimulating reagents to cells; different vectors and / or transduction methods; different timing or order for incubating, transducing and / or transfecting cells; the absence or difference in one or more recombinant cytokines present during incubation or transduction (e.g., different cytokines or different concentrations), or different timing for harvesting or collecting the cells.

[0070] In some embodiments, the cells of the therapeutic cell composition are engineered to express a recombinant receptor, e.g., a CAR or TCR, that specifically binds to a ligand, e.g., one associated with or expressed on cells associated with a disease or condition, e.g., a tumor or cancer (see, e.g., Section III). In some embodiments, the recombinant receptor contains an extracellular ligand-binding domain that specifically binds to an antigen. In some embodiments, the recombinant receptor is a CAR that contains an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the ligand, e.g., the antigen, is a protein expressed on the surface of a cell. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, e.g., a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.

[0071] Exemplary recombinant receptors, including CARs and recombinant TCRs, and methods for engineering and introducing receptors into cells are described, for example, in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US201301493 37, those described in 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 No. EP 2537416, and / or 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 embodiments, engineered antigen receptors include CARs such as those described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668 A1.

[0072] In some embodiments, the engineered cells of the therapeutic cell composition contain a recombinant receptor (e.g., a CAR) that binds to a tumor antigen. In some embodiments, the recombinant receptor specifically recognizes and / or targets an antigen associated with cancer and / or present on a universal tag.In some embodiments, the antigen recognized or targeted by the recombinant receptor is B-cell maturation antigen (BCMA), ROR1, carbonic anhydrase 9 (CAIX), Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, preferentially expressed antigen in melanoma (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, avb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, dual antigen, cancer-testis antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen , ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms' tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, optionally a human antigen of any of the foregoing; or a pathogen-specific antigen.In some embodiments, the antigen recognized and / or targeted by the recombinant receptor is selected from the group consisting of Notch1, Notch2, Notch3, Notch4, cell surface-associated mucin 1 (MUC1), ephrin B2, betaglycan (TGFBR3), CD43, CD44, CSF1R, CX3CR1, CXCL16, Delta1, E-cadherin, N-cadherin, HLA-A2, IFNaR2, IL1R1, IL1R2, IL6R, and amyloid precursor protein (APP).

[0073] In some embodiments, the antigen recognized and targeted by the recombinant receptor is B cell maturation antigen (BCMA). Exemplary antigen-binding domains and CARs containing such antigen-binding domains that target or specifically bind to BCMA are known, see, for example, WO2016 / 090320, WO2016090327, WO2010104949A2, and WO2017173256. In some embodiments, the antigen-binding domain is an scFv containing a VH and a VL derived from an antibody or antibody fragment specific for BCMA. In some embodiments, the antibody or antibody fragment that binds to BCMA is or contains a VH and a VL derived from an antibody or antibody fragment described in International Patent Application Publication Nos. WO2016 / 090327 and WO2016 / 090320.

[0074] As described above, an assay may include multiple incubations, each containing a different adjusted ratio of engineered cells of the therapeutic cell composition to a recombinant receptor stimulating agent, or vice versa, which can stimulate the recombinant receptor of the engineered cells to stimulate recombinant receptor-dependent activity. It is within the skill of the art to empirically determine the exact range or amount of cells of the therapeutic cell composition and receptor stimulating agent to achieve a tailored response in an assay. For example, the number or amount will vary depending on the particular format of the assay, e.g., the size of the vessel in which the assay is performed. It is understood that the amount will be less if the assay is performed in a vessel with a smaller surface area than in a vessel with a larger surface area. Typically, the amount of cells is such that the cells are subconfluent, such as 25% confluent or 50% confluent or less. Furthermore, the specific range of ratios can be empirically determined depending on the particular antigen and target cells being utilized. For example, the selected ratio is one that includes a linear dose-response increase in recombinant receptor-dependent activity across multiple adjusted amounts of the reference standard. In some embodiments, the ratio is also selected to include the lower asymptote of receptor-dependent activity and the upper asymptote of receptor-dependent activity of the reference standard, representing the minimum and maximum response, respectively.

[0075] In some embodiments, the number of engineered cells in the therapeutic cell composition is varied to produce different ratios while the amount or concentration of binding molecules is held constant. In some embodiments, the number of cells in the therapeutic cell composition is varied over the course of incubation from 1 x 10 to 1 x 10. 4 ~1×10 6 Individual cells, 1×10 4 ~9×10 5 Individual cells, 1×10 4 ~8×10 5 Individual cells, 1×10 4 ~7×10 5 Individual cells, 1×10 4 ~6×10 5 Individual cells, 1×10 4 ~5×10 5 Individual cells, 1×104 ~4×10 5 Individual cells, 1×10 4 ~3×10 5 Individual cells, 1×10 4 ~2×10 5 Individual cells, 1×10 4 ~1×10 5 Individual cells, 1×10 4 ~9×10 4 Individual cells, 1×10 4 ~8×10 4 Individual cells, 1×10 4 ~7×10 4 Individual cells, 1×10 4 ~6×10 4 Individual cells, 1×10 4 ~5×10 4 Individual cells, 1×10 4 ~4×10 4 Individual cells, 1×10 4 ~3×10 4 Individual cells, 1×10 4 ~2×10 4 cells or approximately 1 x 10 4 ~Approx. 1×10 6 Individual cells, approximately 1 x 10 4 ~Approx. 9×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 8×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 7×10 5 Individual cells, approximately 1 x 10 4 ~about 6×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 5×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 4×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 3×10 5 Individual cells, approximately 1 x 10 4 ~about 2×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 1×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 9×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 8×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 7×10 4Individual cells, approximately 1 x 10 4 ~about 6×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 5×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 4×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 3×10 4 Individual cells, approximately 1 x 10 4 ~about 2×10 4 In some embodiments, the number of cells in the therapeutic cell composition is adjusted to 1 x 10 over multiple incubations. 4 ~1×10 5 Individual cells, 1×10 4 ~8×10 4 Individual cells, 1×10 4 ~6×10 4 Individual cells, 1×10 4 ~4×10 4 Individual cells, 1×10 4 ~2×10 4 cells or approximately 1 x 10 4 ~Approx. 1×10 5 Individual cells, 1×10 4 ~Approx. 8×10 4 Individual cells, 1×10 4 ~about 6×10 4 Individual cells, 1×10 4 ~Approx. 4×10 4 Individual cells, 1×10 4 ~about 2×10 4In some embodiments, the cell number of the therapeutic cell composition is adjusted to 10,000 or about 10,000 to 1,000,000 or about 1,000,000 cells over multiple incubations. In some embodiments, the cell number of the therapeutic cell composition is adjusted to 10,000 or about 10,000 to 500,000 or about 500,000 cells over multiple incubations. In some embodiments, the cell number of the therapeutic cell composition is adjusted to 10,000 or about 10,000 to 250,000 or about 250,000 cells over multiple incubations. In some embodiments, the cell number of the therapeutic cell composition is adjusted to 10,000 or about 10,000 to 200,000 or about 200,000 cells over multiple incubations. In some embodiments, the cell number of a therapeutic cell composition is adjusted between 10,000 or about 10,000 to 150,000 or about 150,000 cells over multiple incubations. In some embodiments, the cell number of a therapeutic cell composition is adjusted between 10,000 or about 10,000 to 100,000 or about 100,000 cells over multiple incubations. In some embodiments, the cell number of a therapeutic cell composition is adjusted between 10,000 or about 10,000 to 50,000 or about 50,000 cells over multiple incubations. In some embodiments, the cell number of any of the foregoing therapeutic cell compositions is the total number of cells, the total number of viable cells, the total number of CAR+ cells, the total number of CD8+ cells, the total number of CD4+ cells, the total number of CD3+ cells, the total number of CD8+ / CAR+ cells, the total number of CD4+ / CAR+ cells, or the total number of CD3+ / CAR+ cells.

[0076] In some embodiments, the number of cells in the therapeutic cell composition is kept constant and the amount of recombinant receptor stimulating agent is adjusted over multiple incubations. In some embodiments, the constant number of cells in the therapeutic cell composition is about 1 x 10 over multiple incubations. 4 ~Approx. 1×10 6 Individual pieces, approx. 1 x 10 4 ~Approx. 9×10 5 Individual cells, approximately 1 x 10 4~Approx. 8×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 7×10 5 Individual cells, approximately 1 x 10 4 ~about 6×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 5×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 4×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 3×10 5 Individual cells, approximately 1 x 10 4 ~about 2×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 1×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 9×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 8×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 7×10 4 Individual cells, approximately 1 x 10 4 ~about 6×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 5×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 4×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 3×10 4 Individual cells, approximately 1 x 10 4 ~about 2×10 4 In some embodiments, the constant number of cells in the therapeutic cell composition is between 1 x 10 and 1 x 10 over multiple incubations. 4 ~1×10 5 Individual cells, 1×10 4 ~8×10 4 Individual cells, 1×10 4 ~6×10 4 Individual cells, 1×10 4 ~4×10 4 Individual cells, 1×10 4 ~2×10 4 cells or approximately 1 x 10 4 ~Approx. 1×10 5 Individual cells, 1×10 4 ~Approx. 8×10 4 Individual cells, 1×10 4 ~about 6×10 41 cell, 1×10 4 ~approximately 4×10 4 1 cell, 1×10 4 ~approximately 2×10 4In some embodiments, the fixed number of cells in the therapeutic cell composition is 10,000 or about 10,000 to 1,000,000 cells or about 1,000,000 cells over multiple incubations. In some embodiments, the fixed number of cells in the therapeutic cell composition is 10,000 or about 10,000 to 500,000 or about 500,000 cells over multiple incubations. In some embodiments, the fixed number of cells in the therapeutic cell composition is 10,000 or about 10,000 to 250,000 or about 250,000 cells over multiple incubations. In some embodiments, the fixed number of cells in the therapeutic cell composition is 10,000 or about 10,000 to 150,000 or about 150,000 cells over multiple incubations. In some embodiments, the fixed number of cells in the therapeutic cell composition is 10,000 or about 10,000 to 100,000 or about 100,000 cells over multiple incubations, hi some embodiments, the fixed number of cells in the therapeutic cell composition is 10,000 or about 10,000 to 50,000 or about 50,000 cells over multiple incubations. In some embodiments, the fixed number of cells in the therapeutic cell composition is at or about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 cells over multiple incubations. In some embodiments, the fixed number of cells in the therapeutic cell composition is at or about 5,000, 10,000, 20,000, 30,000, 40,000, or 50,000 cells over multiple incubations.In some embodiments, the fixed number of cells in the therapeutic cell composition is at or about 20,000, 30,000, 40,000, or 50,000 cells over multiple incubations. In some embodiments, the fixed number of cells in the therapeutic cell composition is at or about 50,000 cells over multiple incubations. In some embodiments, the fixed number of cells in the therapeutic cell composition in any of the above is the total number of cells, the total number of live cells, the total number of CAR+ cells, the total number of CD8+ cells, the total number of CD4+ cells, the total number of CD3+ cells, the total number of CD8+ / CAR+ cells, the total number of CD4+ / CAR+ cells, or the total number of CD3+ / CAR+ cells.

[0077] B. Recombinant Receptor Stimulators The methods for assessing efficacy provided herein include stimulating recombinant receptors (e.g., CAR, TCR) of engineered cells of a therapeutic cell composition. It is contemplated that any suitable means for stimulating a recombinant receptor that can be quantified and delivered to generate different ratios of cells of a therapeutic cell composition to the stimulating means may be used. In some embodiments, stimulating a recombinant receptor is achieved by a recombinant receptor stimulating agent, such as those described in Section IC, that can bind to and stimulate an intracellular signal by the recombinant receptor to generate recombinant receptor-dependent activity. Exemplary recombinant receptor stimulating agents include antigens of the recombinant receptor (e.g., purified antigens or recombinant antigens), antibodies such as anti-idiotypic antibodies, and antigen-expressing cells.

[0078] As noted above, in some embodiments, multiple incubations of different, adjusted ratios of cells of a therapeutic cell composition to a recombinant receptor stimulating agent can be achieved by culturing a fixed number of cells of a therapeutic cell composition (e.g., live cells, CAR+, CD4+, CD8+, CD3+, CD4+ / CAR+, CD8+ / CAR+, CD3+ / CAR+ cells) with varying or adjusted amounts (e.g., concentration, mass) of a recombinant receptor stimulating agent. In some embodiments, the amount or concentration of the recombinant receptor stimulator is changed or adjusted by at or about 10,000-fold, 5,000-fold, 1,000-fold, 1,500-fold, 500-fold, 250-fold, 200-fold, 150-fold, 100-fold, 75-fold, 50-fold, 25-fold, or 10-fold over multiple incubations. In some embodiments, the amount or concentration of the recombinant receptor stimulating agent is changed or adjusted by 10,000-100 fold, 5,000-100 fold, or 1,000-100 fold, or about 10,000-100 fold, 5,000-100 fold, or 1,000-100 fold over multiple incubations. In some embodiments, the amount or concentration of the recombinant receptor stimulating agent is changed or adjusted by 5,000-fold, 1,000-fold, 1,500-fold, or 500-fold over multiple incubations. In some embodiments, the amount or concentration of the recombinant receptor stimulating agent is changed or adjusted by 5,000-fold or about 5,000-fold over multiple incubations. In some embodiments, the amount or concentration of the recombinant receptor stimulator is varied or adjusted by at or about 1,000-fold over multiple incubations, hi some embodiments, the amount or concentration of the recombinant receptor stimulator is varied or adjusted by at or about 500-fold over multiple incubations.

[0079] 1. Binding Molecules and Surface Immobilization In certain embodiments, the recombinant receptor comprises a binding molecule (or target) capable of being bound by the recombinant receptor. In some embodiments, the binding molecule is immobilized on a surface support. In provided embodiments, the binding molecule can be an antigen or portion of the antigen (e.g., the extracellular portion of the antigen) of the recombinant receptor, or an antibody (e.g., an anti-idiotypic antibody) specific for the recombinant receptor. For example, the binding molecule (e.g., an antigen or a binding portion thereof, or an anti-idiotypic antibody) can be immobilized or bound to a surface support, e.g., a non-cellular particle, and a regulated amount of cells (e.g., CAR-T cells) expressing the recombinant receptor of the therapeutic composition, e.g., cells, is contacted with the surface support. In some embodiments, the particles (e.g., bead particles) described herein provide a solid support or matrix to which the binding molecule (e.g., an antigen or a binding portion thereof, or an anti-idiotypic antibody) can be bound or attached in a manner that allows interaction between the binding molecule and the cell, particularly binding between the binding molecule and the recombinant receptor, e.g., CAR, expressed on the surface of the cell. In certain 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 activities, such as activation, expansion, cytokine production, cytotoxic activity, or other activities, e.g., as described in Section IC.

[0080] In certain embodiments, the surface support is a particle (e.g., a bead particle) to which a 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 moiety 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 immobilizing or attaching agents (e.g., binding molecules) for recognition or binding to recombinant receptors can be found in published international application WO2019 / 027850, which is incorporated by reference for all purposes.

[0081] In certain embodiments, the surface support is a particle, which may include a colloid particle, a microsphere, a nanoparticle, a bead, e.g., a magnetic bead, etc. In some embodiments, the particle or bead is biocompatible, i.e., non-toxic. In certain embodiments, the particle or bead is non-toxic to cultured cells, e.g., cultured T cells. In certain embodiments, the particle is monodisperse. In certain embodiments, "monodisperse" encompasses particles (e.g., bead particles) having a size distribution with a standard deviation of less than 5%, e.g., a standard deviation in diameter of less than 5%.

[0082] In some embodiments, the particles or beads are made of biocompatible materials, i.e., materials suitable for biological use. In some embodiments, the particles, e.g., beads, are non-toxic to cultured cells, e.g., cultured T cells. In some embodiments, the particles, e.g., beads, can be any particle to which a binding molecule can be attached in a manner that allows interaction between the binding molecule and cells. In certain embodiments, the particles, e.g., beads, can be any particle that can be modified, e.g., surface-functionalized, to allow attachment of a binding molecule to the surface of the particle. In some embodiments, the particles, e.g., beads, are made of glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids. In some embodiments, particles, e.g., beads, may be composed of, or at least partially composed of, polyesters of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or polyanhydrides of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids. Furthermore, particles, e.g., beads, may be quantum dots or may be composed of quantum dots, e.g., quantum dot polystyrene particles, e.g., beads. Particles, e.g., beads, containing a mixture of ester and anhydride bonds (e.g., copolymers of glycolic acid and sebacic acid) may also be employed. For example, particles, e.g., beads, can comprise materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic acid) copolymers (PLGA), [rho]poly(lactic-co-sebacic acid) copolymers (PLSA), poly(glycolic-co-sebacic acid) copolymers (PGSA), and the like.Other polymers from which particles, e.g., beads, can be constructed include polymers or copolymers of caprolactone, carbonate, amide, amino acid, orthoester, acetal, cyanoacrylate, and degradable urethane, and copolymers thereof with linear or branched, substituted or unsubstituted, alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or dicarboxylic acids. Additionally, biologically important amino acids with reactive side groups, e.g., lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, may be included in copolymers with any of the above materials to provide reactive groups for conjugation to binding molecules, e.g., polypeptide antigens or antibodies.

[0083] In some embodiments, the particles are beads having a diameter greater than 0.001 μm, greater than 0.01 μm, greater than 0.05 μm, greater than 0.1 μm, greater than 0.2 μm, greater than 0.3 μm, greater than 0.4 μm, greater than 0.5 μm, greater than 0.6 μm, greater than 0.7 μm, greater than 0.8 μm, greater than 0.9 μm, greater than 1 μm, greater than 2 μm, greater than 3 μm, greater than 4 μm, greater than 5 μm, greater than 6 μm, greater than 7 μm, greater than 8 μm, greater than 9 μm, greater than 10 μm, greater than 20 μm, greater than 30 μm, greater than 40 μm, greater than 50 μm, greater than 100 μm, greater than 500 μm, and / or greater than 1,000 μm. In some embodiments, the particles or beads are between or about 0.001 μm to 1,000 μm, 0.01 μm to 100 μm, 0.1 μm to 10 μm, 0.1 μm to 100 μm, 0.1 μm to 10 μm, 0.001 μm to 0.01 μm, 0.01 μm to 0.1 μm, 0.1 μm to 1 μm, 1 μm to 10 μm, 1 μm to 2 μm, 2 μm to 3 μm, 3 μm to 4 μm, 4 μm to 5 μm, 1 μm to 5 μm and / or 5 μm to 10 μm, each inclusive. The diameter is 0.001 μm to about 1,000 μm, about 0.01 μm to about 100 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 100 μm, about 0.1 μm to about 10 μm, about 0.001 μm to about 0.01 μm, about 0.01 μm to about 0.1 μm, about 0.1 μm to about 1 μm, about 1 μm to about 10 μm, about 1 μm to about 2 μm, about 2 μm to about 3 μm, about 3 μm to about 4 μm, about 4 μm to about 5 μm, about 1 μm to about 5 μm, and / or about 5 μm to about 10 μm. In certain embodiments, the particles or beads have an average diameter of 1 μm and 10 μm, inclusive. In certain embodiments, the particles, e.g., beads, have a diameter of 1 μm or about 1 μm. In certain embodiments, the particles, e.g., beads, have an average diameter of at or about 2.8 μm. In some embodiments, the particles, e.g., beads, have a diameter of at or about 4.8 μm.

[0084] The particles (e.g., bead particles) used in the methods described herein can be commercially produced and obtained. Particles, e.g., beads, including methods for producing particles, e.g., beads, are well known in the art. See, e.g., U.S. Patent 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, for example, beads (e.g., bead particles), include ProMag™ (PolySciences, Inc.); COMPELTM (PolySciences, Inc.); BioMag® (PolySciences, Inc.), including BioMag® Plus (PolySciences, Inc.) and BioMag® Maxi (Bang Laboratories, Inc.).M-PVA (Cehmagen Biopolymer Technologie AG); SiMAG (Chemicell GmbH); beadMAG (Chemicell GmbH); MagaPhase® (Cortex Biochem); Dynabeads® (Invitrogen), including Dynabeads® M-280 sheep anti-rabbit IgG (Invitrogen), Dynabeads® FlowComp™ (e.g., Dynabeads® FlowComp™ Human CD3, Invitrogen), Dynabeads® M-450 (e.g., Dynabeads® M-450 tosyl-activated, Invitrogen), Dynabeads® Untouched™ (e.g., Dynabeads® Untouched™ Human CD8 T Cell, 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); MACSiBeads™ particles (e.g., anti-biotin MACSiBead particles, Miltenyi Biotec, catalog number 130-091-147); Streptamer® magnetic beads (IBA BioTAGnology); Strep-Tactin® magnetic beads (IBA BioTAGnology); Sicastar®-M (Micromod Partikeltechnologie GmbH); Micromer®-M (Micromod Partikeltechnologie); MagneSil™ (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 Magnetic Beads (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.); MagnaBind™ Magnetic Beads (Thermo Fisher Scientific Inc.); Sera-Mag™ Magnetic Beads (Thermo Fisher Scientific Inc.); Anti-FLAG® M2 Magnetic Beads (Sigma-Aldrich); SPHEROTM Magnetic Particles (Spherotech Inc.); and HisPur™ Ni-NTA Magnetic Beads (Thermo Fisher Scientific Inc.)

[0085] In certain embodiments, the antigen or its extracellular domain portion is bound to the particle (e.g., bead) via a covalent chemical bond. In certain embodiments, the reactive group or moiety of the amino acid of the antigen or its extracellular domain portion is directly conjugated to the reactive group or moiety on the surface of the particle by direct chemical reaction. In certain embodiments, the amino acid carboxyl group (e.g., C-terminal carboxyl group), hydroxyl, thiol, or amine group (e.g., amino acid side chain group) of the antigen or its extracellular binding portion is directly conjugated to the hydroxyl or carboxyl group of the PLA or PGA polymer, the terminal amine or carboxyl group of the dendrimer, or the hydroxyl, carboxyl, or phosphate group of the phospholipid on the surface of the particle by direct chemical reaction. In some embodiments, the conjugated moiety is conjugated to both the binding molecule and the particle, for example, by a covalent bond, thereby linking them together.

[0086] In certain embodiments, the surface of the particle contains chemical moieties and / or functional groups that allow for the attachment (e.g., covalent or non-covalent) of a binding molecule (e.g., a polypeptide antigen or antibody). In certain embodiments, the particle surface contains exposed functional groups. Suitable surface-exposed functional groups include, but are not limited to, carboxyl, amino, hydroxyl, sulfate, tosyl, epoxy, and chloromethyl groups. In some embodiments, the binding molecule is a polypeptide and is conjugated to the surface-exposed functional group. In some embodiments, the surface-exposed functional group must be activated, i.e., undergo a chemical reaction to produce an intermediate product that allows for direct binding to the polypeptide. For example, the carboxyl group of a polypeptide molecule can be activated using the agents described above to generate an intermediate ester that can be directly bound to the amino group exposed on the surface of the particle. In another example, free amine groups on the surface of a support surface (e.g., beads) can be covalently bound to antigenic peptides and proteins or antigenic peptide or protein fusion proteins using sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB) chemistry. In yet other specific embodiments, the polypeptide binding molecules are covalently attached to the particles, e.g., bead particles, at surface-exposed functional groups that do not require drug activation prior to covalent attachment, examples of such functional groups include, but are not limited to, tosyl, epoxy, and chloromethyl groups.

[0087] In some embodiments, non-covalent binding between a ligand bound to an antigenic peptide or protein and an anti-ligand attached to a surface support (e.g., a bead) can conjugate the antigen to the support (e.g., a bead). In some embodiments, a biotin ligase recognition sequence tag can be attached to the C-terminus of the antigenic peptide or protein, and this tag can be biotinylated by biotin ligase. The biotin can then serve as a ligand and non-covalently conjugate the antigenic peptide or protein to avidin or streptavidin adsorbed or otherwise bound to the surface of the carrier as an anti-ligand. Alternatively, if a binding molecule (e.g., an antigen) is fused to an immunoglobulin domain having an Fc region as described herein, the Fc domain can act as a ligand, and protein A covalently or non-covalently bound to the surface of the surface support (e.g., a bead) can serve as an anti-ligand and non-covalently conjugate the antigenic peptide or protein to the carrier. Other means are well known in the art and may be employed to non-covalently conjugate binding molecules (e.g., antigens or anti-idiotypic antibodies) to surface supports (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 herein.

[0088] In some embodiments, the binding molecule (eg, an antigen or an anti-idiotypic antibody) is conjugated to the particle by a linker. In certain embodiments, the linker may include, but is not limited to, various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutareldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., l,5-difluoro-2,4-dinitrobenzene). Specific coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), which provide disulfide bonds.

[0089] a. Antigen In some embodiments, the recombinant receptor stimulating agent is or includes an antigen, e.g., a recombinant antigen or a fragment thereof. For example, the recombinant receptor stimulating agent can be an antigen immobilized or bound to a surface support, e.g., a microwell plate, a solid particle (e.g., a bead), or an oligomeric particle, e.g., as described above. In some embodiments, the antigen is a polypeptide or a variant or fragment of a polypeptide expressed on the surface of a cell associated with a disease, e.g., a cancer cell and / or a tumor cell. It is understood that an antigen is an antigen recognized or bound by the extracellular domain of the recombinant receptor. One of skill in the art can determine antigens and formats of antigens (e.g., cells expressed on or immobilized on a solid surface) sufficient to stimulate the recombinant receptor.

[0090] In some embodiments, the antigen is αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer testis antigen, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC 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), epidermal growth factor receptor type III mutant (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, 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), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erb B dimer, human high-molecular-weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16,Natural killer group 2 member D (NKG2D) ligand, Melan-A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein G (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 isomerase, 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 a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens. In some embodiments, the antigen targeted by the receptor comprises an antigen associated with a B-cell malignancy, e.g., any of several known B-cell markers. In some embodiments, the antigen is or comprises CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30.

[0091] In some embodiments, the antigen is or comprises a portion of a polypeptide antigen that is recognized or bound by a recombinant receptor, such as a CAR. In certain embodiments, the portion of the antigen is a region that contains the epitope that is recognized or bound by a recombinant receptor, such as a CAR. In certain embodiments, the portion of the polypeptide antigen is about 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500 In certain embodiments, the polypeptide portion contains at least 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, or 500 amino acids, or in some cases, contiguous amino acids. In certain embodiments, the polypeptide portion comprises the amino acid sequence of an epitope recognized by a recombinant receptor and / or CAR.

[0092] In certain embodiments, the antigen or portion contains about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity to the polypeptide bound by and / or recognized by the recombinant receptor and / or CAR, or is a polypeptide variant containing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity.

[0093] In certain embodiments, the extracellular domain of a recombinant receptor (e.g., a CAR) is specific for or binds to BCMA, and the antigen is BCMA or a portion of the extracellular domain of BCMA. In some embodiments, the BCMA polypeptide is a mammalian BCMA polypeptide. In certain embodiments, the BCMA polypeptide is a human BCMA polypeptide. In some embodiments, the BCMA antigen is or comprises the extracellular domain of BCMA, or a portion thereof, that includes an epitope recognized by an antigen receptor, e.g., a CAR. In certain embodiments, the BCMA antigen is or comprises a polypeptide having an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 13, 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 consecutive amino acids of SEQ ID NO: 13. In some embodiments, the BCMA antigen is or comprises the sequence set forth in SEQ ID NO: 13 or a portion thereof that is or contains an epitope recognized by an antigen receptor, e.g., a CAR.

[0094] In certain embodiments, the extracellular domain of a recombinant receptor (e.g., a CAR) is specific for or binds to ROR1, and the antigen is ROR1 or a portion of the extracellular domain of ROR1. In certain embodiments, the ROR1 polypeptide is mammalian. In certain embodiments, the ROR1 polypeptide is human. In some embodiments, the antigen is the extracellular domain of ROR1 or a portion thereof that contains an epitope recognized by an antigen receptor, e.g., a CAR. In some embodiments, the antigen is a polypeptide having an amino acid sequence having 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: 19, 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 consecutive amino acids of SEQ ID NO: 19. In some embodiments, the ROR1 antigen comprises the sequence set forth in SEQ ID NO: 19 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g., a CAR.

[0095] 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 a portion of the extracellular domain of CD22. In certain embodiments, the CD22 polypeptide is mammalian. In certain embodiments, the CD22 polypeptide is human. In some embodiments, the antigen is the extracellular domain of CD22 or a portion thereof that contains an epitope recognized by an antigen receptor, for example, a CAR. In some embodiments, the antigen is a polypeptide having an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 14, 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 consecutive amino acids of SEQ ID NO: 14. In some embodiments, the CD22 antigen comprises the sequence set forth in SEQ ID NO: 14 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g., a CAR.

[0096] In certain embodiments, the extracellular domain of the recombinant receptor (e.g., CAR) is specific for or binds to CD19, and the antigen is CD19 or a portion of the extracellular domain of CD19. In certain embodiments, the CD19 polypeptide is mammalian. In certain embodiments, the CD19 polypeptide is human. In some embodiments, the antigen is the extracellular domain of CD19 or a portion thereof that contains an epitope recognized by an antigen receptor, for example, a CAR. In some embodiments, the antigen is a polypeptide having an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 15, 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 consecutive amino acids of SEQ ID NO: 15. In some embodiments, the CD19 antigen comprises the sequence set forth in SEQ ID NO: 15 or a portion thereof comprising an epitope recognized by an antigen receptor, e.g., a CAR.

[0097] In some embodiments, the antigen or portion thereof is formatted as a multimer, e.g., a dimer, comprising two or more polypeptide antigens or portions or variants thereof that are recognized and / or bound by a recombinant receptor, e.g., an antigen receptor (e.g., a CAR). In some embodiments, the polypeptide antigens or portions thereof are identical. In certain embodiments, the polypeptide antigens are directly or indirectly linked to regions or domains, e.g., multimerization domains, which promote or stabilize the interaction between two or more polypeptide antigens through complementary interactions between the domains or regions. In some embodiments, providing the polypeptide antigen as a multimer, e.g., a dimer, provides a multivalent interaction between the antigen or its extracellular domain portion and the antigen-binding domain of the antigen receptor, e.g., a CAR, which may, in some aspects, increase the avidity of the interaction. In some embodiments, the increased avidity of the antigen or its extracellular domain portion conjugated to the bead may be favorable for the stimulatory or agonistic activity of the antigen receptor, e.g., a CAR.

[0098] In some embodiments, the polypeptide is directly or indirectly linked to the multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain can be, for example, an immunoglobulin constant region or domain, such as an Fc domain or portions thereof derived from IgG, including IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, and IgM, and modified forms thereof. In certain embodiments, the polypeptide antigen is directly or indirectly linked to the Fc domain. In some embodiments, the polypeptide is a fusion polypeptide comprising a polypeptide antigen or portions thereof and an Fc domain.

[0099] In certain embodiments, the antigen or its extracellular domain portion is a fusion polypeptide comprising an Fc domain. In some embodiments, the Fc domain is composed of the second and third constant domains (i.e., CH2 and CH3 domains) of the heavy chain of an IgA or IgD isotype, for example, CH2 or CH3 of an IgG, IgA, or IgD isotype. In some embodiments, the Fc domain is composed of three heavy chain constant domains (i.e., CH2, CH3, and CH4 domains) of an IgM or IgE isotype. In some embodiments, the Fc domain may further comprise a hinge sequence or a portion thereof. In certain aspects, the Fc domain contains part or all of the hinge domain and the CH2 and CH3 domains of an immunoglobulin molecule. In some cases, the Fc domain may form a dimer of two polypeptide chains joined by one or more disulfide bonds. 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 some embodiments, the Fc domain is derived from the C of IgG. H 2 and C H In certain embodiments, the Fc domain is fused to the C-terminus of the polypeptide antigen. In certain embodiments, the Fc domain is fused to the N-terminus of the polypeptide antigen.

[0100] 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 variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 16 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%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 16. In certain embodiments, the Fc domain is a wild-type human IgG Fc domain or a portion or variant thereof. In certain embodiments, the Fc domain is a variant of a wild-type human IgG1 Fc domain.

[0101] In some embodiments, the fusion polypeptide comprises a variant Fc domain. In certain embodiments, the variant human IgG Fc domain contains a mutation, e.g., a substitution, deletion, or insertion, that reduces, decreases, and / or decreases pairing between the Fc domain and the light chain. In some embodiments, the variant human IgG Fc domain contains a mutation that reduces the binding affinity between the Fc domain and an Fc receptor. In certain embodiments, the variant human IgG Fc domain contains a mutation that reduces, decreases, and / or decreases the interaction, or the probability or likelihood of an interaction, between the Fc domain and an Fc receptor. In some embodiments, the variant human IgG Fc domain contains a mutation that reduces the binding affinity between the Fc domain and a protein of the complement system. In certain embodiments, the variant human IgG Fc domain contains a mutation that reduces, decreases, and / or decreases the interaction, or the probability or likelihood of an interaction, between the Fc domain and a protein of the complement system.

[0102] In some embodiments, the antigen or portion thereof is linked to a mutant human IgG1 Fc domain. In some embodiments, the mutant human IgG Fc domain contains a cysteine ​​to serine substitution in the hinge region of the Fc domain. In some embodiments, the mutant human IgG Fc domain contains a leucine to alanine substitution in the hinge region of the Fc domain. In certain embodiments, the mutant human IgG Fc domain contains a glycine to alanine substitution in the hinge region. In certain embodiments, the mutant human IgG Fc domain contains an alanine to serine substitution in the CH2 region of the Fc domain. In some embodiments, the mutant human IgG Fc domain comprises a proline to serine substitution in the CH2 region of the Fc domain. In some embodiments, the mutant human IgG Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0103] 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 at the C-terminus of the fusion polypeptide.

[0104] In some embodiments, the antigen and the multimerization domain, e.g., the Fc domain, are connected by a linker, e.g., an amino acid linker. In certain embodiments, the antigen is fused to the N-terminus of the amino acid linker, and the multimerization domain, such as the Fc domain, is fused to the C-terminus of the linker. The amino acid linker can be of any length and contain any combination of amino acids, but the linker length can be relatively short (e.g., 10 or fewer amino acids) to reduce interactions between the linked domains. The amino acid composition of the linker can also 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. A representative amino acid linker sequence includes GGGGS (SEQ ID NO: 22), and the linker contains 2, 3, 4, or 5 copies of GGGGS (SEQ ID NO: 22).

[0105] In some embodiments, the antigen is provided as the extracellular domain of BCMA, e.g., human BCMA, fused to an Fc domain (BCMA-Fc). In certain embodiments, the BCMA-Fc antigen contains all or a portion of the amino acid sequence set forth in SEQ ID NO: 18, 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% sequence similar to SEQ ID NO: 18, and that includes an epitope recognized by an antigen receptor, e.g., a CAR.

[0106] In some embodiments, the antigen is provided as the extracellular domain of ROR1, e.g., human ROR1, fused to an Fc domain (ROR1-Fc). In certain embodiments, the ROR-1-Fc antigen contains all or a portion of the amino acid sequence set forth in SEQ ID NO: 20, 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% sequence similar to SEQ ID NO: 20, and that includes an epitope recognized by an antigen receptor, e.g., a CAR.

[0107] In certain embodiments, the antigen is provided as the 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: 21, 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% identity to SEQ ID NO: 21, and that includes an epitope recognized by an antigen receptor, e.g., a CAR.

[0108] In some embodiments, the Fc fusion of the antigen or its extracellular binding domain is linked to or attached to a surface support as a dimer formed by two Fc fusion polypeptides containing the polypeptide antigen or its partial Fc domains. In some embodiments, the resulting polypeptide antigen-Fc fusion protein, e.g., BCMA-Fc, ROR1-Fc, CD22-Fc, or CD19-Fc, may be expressed, e.g., in a host cell transformed with an expression vector, whereby assembly between the Fc domains occurs via intramolecular disulfide bonds formed between the Fc moieties, resulting in a dimeric, e.g., bivalent, polypeptide antigen fusion protein. In some embodiments, the host cell is a mammalian cell line. Exemplary mammalian cells for recombinant expression of proteins include HEK293 cells or CHO cells or their derivatives. In some aspects, the nucleic acid encoding the Fc fusion protein further comprises a signal peptide for secretion from the cell. In an exemplary embodiment, the signal peptide is CD33 (e.g., as set forth in SEQ ID NO: 12).

[0109] In some embodiments, cells of the therapeutic cell composition express a CAR that binds to or recognizes a universal tag, which may be fused to an antibody or a fragment or variant thereof. In certain embodiments, cells expressing such a CAR can specifically recognize and kill target cells, e.g., tumor cells, bound by an antibody fused to the universal tag. An example includes, but is not limited to, T cells expressing an anti-FITC CAR that can bind to and / or recognize various human cancer cells when those cells are bound by a cancer-reactive FITC-labeled antibody. Thus, in some embodiments, the same CAR that binds to a universal tag is useful for treating various cancers, provided there is an available antibody that recognizes an antigen associated with the cancer that contains the universal tag. In certain embodiments, particles (e.g., bead particles) contain surface-exposed binding molecules that include a universal tag-binding molecule that can be bound or recognized by a recombinant receptor, e.g., a CAR. In certain embodiments, the binding molecule is a universal tag or a portion thereof that is bound or recognized by an antigen receptor, e.g., a CAR. Certain embodiments contemplate that any polypeptide domain that can be fused to an antibody, or antigen-binding fragment or variant thereof, that does not prevent the antibody from binding to its reactive target is suitable for use as a universal tag. In some embodiments, the particles are conjugated to a binding molecule comprising a universal tag, or a portion thereof, selected from the group consisting of FITC, streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, PE, HRP, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, and maltose binding protein.

[0110] b.Antibodies In some aspects, the binding molecule is an antibody or antigen-binding fragment thereof that specifically recognizes a recombinant receptor, e.g., a CAR. In some embodiments of these aspects, the antibody or antigen-binding fragment specifically recognizes the extracellular portion of the recombinant receptor, e.g., a CAR (e.g., specifically binds to an epitope on the extracellular side of the recombinant receptor).

[0111] In some aspects, the binding molecule is an anti-idiotypic antibody or antigen-binding fragment thereof ("anti-ID") that specifically recognizes a recombinant receptor, e.g., a recombinant receptor as described in Section III, e.g., a CAR. In particular, the anti-idiotypic antibody targets the antigen-binding site of another antibody, e.g., the scFv of the extracellular antigen-binding domain of a CAR. In some embodiments, the anti-ID can bind to the recombinant receptor and stimulate recombinant receptor-dependent activity. Exemplary anti-idiotypic antibodies against antigen-specific CARs are known. These include, but are not limited to, anti-idiotypic antibodies directed against CD22-directed CARs (see, for example, PCT Publication No. WO2013188864); CD19-directed CARs (see, for example, PCT Publication No. WO2018 / 023100); GPRC5D-directed CARs (see, for example, PCT Application No. PCT / US2020 / 063497); and BCMA-directed CARs (see, for example, PCT Application No. PCT / US2020 / 063492). Anti-idiotypic antibodies can be immobilized or attached to a surface support (e.g., beads) as described above for use as a recombinant receptor stimulator for cells expressing the recombinant receptor (e.g., CAR) targeted by the anti-idiotypic antibody.

[0112] The term "antibody" is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, such as fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, such as single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term also encompasses 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, and tandem tri-scFv. The term "antibody," unless otherwise specified, should be understood to encompass functional antibody fragments thereof. The term also includes intact or full-length antibodies, including antibodies of any class or subclass, such as IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0113] The term "anti-idiotypic antibody" refers to an antibody that specifically recognizes, is specifically targeted to, and / or specifically binds to the idiotope of an antibody, e.g., an antigen-binding fragment thereof. An antibody idiotope may include, but is not necessarily limited to, the complementarity-determining regions (CDRs) of an antibody, the variable regions of an antibody and / or residues within one or more of such variable regions and / or partial portions or portions of such CDRs and / or any combination of the foregoing. The CDRs may be one or more selected from the group consisting of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The antibody variable region may be a heavy chain variable region, a light chain variable region, or a combination of a heavy chain variable region and a light chain variable region. Partial fragments or portions of the heavy and / or light chain variable regions of an antibody can be fragments comprising two or more, five or more, or ten or more contiguous amino acids within the heavy or light chain variable region of the antibody, e.g., about 2 to about 100, about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 contiguous amino acids; idiotopes can comprise multiple, non-contiguous stretches of amino acids. Partial fragments of the heavy and light chain variable regions of an antibody can be fragments comprising two or more, five or more, or ten or more contiguous amino acids within the variable region, e.g., about 2 to about 100, about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 contiguous amino acids, and in some embodiments, contain one or more CDRs or CDR fragments. A CDR fragment can be two or more, five or more, contiguous or non-contiguous amino acids within a CDR. Thus, an antibody idiotope can be about 2 to about 100, about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 contiguous amino acids comprising one or more CDRs or one or more CDR fragments within the heavy chain or light chain variable region of an antibody.In another embodiment, the idiotope can be a single amino acid located in the variable region of an antibody, for example, in a CDR site.

[0114] In some embodiments, an idiotope is any single antigenic determinant or epitope within the variable portion of an antibody. In some cases, it may overlap the actual antigen-binding site of an antibody, and in some cases, it may include variable region sequences outside the antigen-binding site of an antibody. The set of individual idiotopes of an antibody is, in some embodiments, referred to as the "idiotype" of such an antibody.

[0115] The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art. They refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms "framework region" and "FR" are known in the art and refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.

[0116] The precise amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745. ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 (the "IMGT" numbering scheme) and Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70, (the "Aho" numbering scheme).

[0117] The boundaries of a given CDR or FR can vary according to the scheme used for identification.For example, Kabat scheme is based on structural alignment, while Chothia scheme is based on structural information.The numbering of both Kabat and Chothia schemes is based on the most common antibody region sequence length, and insertion corresponds to the insertion letter, for example, "30a", and deletion appears in some antibodies.The two schemes place certain insertions and deletions ("insertion deletions") in different positions, resulting in differential numbering.Contact scheme is based on the analysis of complex crystal structure, and is similar in many respects to the Chothia numbering scheme.

[0118] Table 1 below lists exemplary alignment boundaries for CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, and CDR-H3, as identified by Kabat, Chothia, and contact schemes, respectively. For CDR-H1, residue numbers are listed using both the Kabat and Chothia numbering schemes. FRs are located between the CDRs, e.g., FR-L1 is located between CDR-L1 and CDR-L2. The Kabat numbering scheme shown places the insertion at H35A and H35B, so it is known that when the end of the Chothia CDR-H1 loop is numbered using the Kabat numbering scheme shown, the numbering scheme varies between H32 and H34 depending on the length of the loop.

[0119] [Table 1]

[0120] Thus, unless otherwise specified, the "CDR" or "complementarity determining region" of a given antibody or region thereof, e.g., its variable region, or each specific CDR (e.g., "CDR-H1, CDR-H2"), should be understood to encompass one (or specific) complementarity determining region as defined by any of the above schemes. For example, if a designated CDR (e.g., CDR-H3) is a CDR of a given V Hor V L When an amino acid sequence is described as containing the amino acid sequence of a corresponding CDR in the variable region, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the variable region as defined by any of the above schemes. In some embodiments, the specified CDR sequence is specified.

[0121] Similarly, unless otherwise specified, a given antibody or region thereof, e.g., a FR of its variable region or each specific FR(s) (e.g., FR-H1, FR-H2), should be understood to encompass one (or specific) framework region as defined by any known scheme. In some cases, a scheme for identifying a particular CDR, FR or FRs or CDRs is specified, such as CDRs as defined by Kabat, Chothia, or contact methods. In other examples, the specific amino acid sequence of a CDR or FR is provided.

[0122] 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 an antigen. The variable domains of the heavy and light chains of a naturally occurring antibody (V H and V L ) generally have a similar structure, with each domain containing 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 V H or V L The V domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be identified by isolating the V domain from the antibody that binds the antigen. H or V L Using the domains, isolate and differentiate each complementary V L or V HMethods for screening libraries of domains are available (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0123] Among the antibodies provided are antibody fragments. The term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to 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 certain embodiments, the antibody is a single-chain antibody fragment, e.g., scFv, that contains a heavy chain variable chain region and / or a light chain variable chain region.

[0124] Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies.

[0125] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as well as production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, e.g., a fragment that contains a non-naturally occurring arrangement, e.g., two or more antibody regions or chains joined by a synthetic linker, e.g., a peptide linker, and / or that could not be produced by enzymatic digestion of a naturally occurring intact antibody. In some aspects, the antibody fragment is an scFv.

[0126] 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 amino acid residues are derived from human FRs. In some embodiments, humanized forms of non-human antibodies, e.g., murine antibodies, are chimeric antibodies containing minimal sequence derived from non-human immunoglobulin. In certain embodiments, a humanized antibody is an antibody from a non-human species having one or more complementarity-determining regions (CDRs) from the non-human species and framework regions (FRs) from a human immunoglobulin molecule. In some embodiments, a humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody generally refers to a variant of a non-human antibody that has undergone humanization to reduce immunogenicity to humans while retaining the specificity and affinity of the parent 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 were derived), e.g., to restore or 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.

[0127] In certain embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a heavy chain variable region of the recipient are replaced by residues from a heavy chain variable region of a non-human species (donor antibody), e.g., mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capacity. In some cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. In some embodiments, the nucleic acid sequences encoding the human heavy and light variable chains are altered so that one or more CDR sequences of the human (acceptor) sequence are replaced by the sequences encoding the respective CDRs in the non-human antibody sequence (donor sequence). In some embodiments, the human acceptor sequence may contain FRs derived from a different gene. In certain embodiments, a humanized antibody will contain substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In some embodiments, a humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), where appropriate, that typically of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409, which are incorporated herein by reference.In some embodiments, provided herein are humanized anti-idiotype antibodies.

[0128] In certain embodiments, antibodies, such as anti-idiotype antibodies, are humanized. In certain embodiments, antibodies are humanized by any suitable known means. For example, in some embodiments, a humanized antibody can have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are usually taken from an "import" variable domain. In certain embodiments, humanization can be performed essentially according to the method of Winter and coworkers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), e.g., by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species (U.S. Patent No. 4,816,567). In certain embodiments, humanized antibodies are human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0129] A sequence encoding a full-length antibody can then be obtained by joining the given heavy and light chain variable sequences to human constant heavy and light chain regions. Suitable human constant light chain sequences include kappa and lambda constant light chain sequences. Suitable human constant heavy chain sequences include sequences encoding IgG1, IgG2, and IgG1 mutants endowed with immunostimulatory properties. Such mutants may have reduced ability to activate complement and / or antibody-dependent cellular cytotoxicity and are described in U.S. Patent No. 5,624,821; WO99 / 58572; and U.S. Patent No. 6,737,056. Suitable constant heavy chains also include IgG1s containing the substitutions E233P, L234V, L235A, A327G, A330S, P331S, and deletion of residue 236. In another embodiment, the full-length antibody comprises an IgA, IgD, IgE, IgM, IgY, or IgW sequence.

[0130] Suitable human donor sequences can be determined by sequence comparison of the peptide sequence encoded by the mouse donor sequence to a group of human sequences, preferably sequences encoded by human germline immunoglobulin genes or mature antibody genes. The human sequence with high sequence identity, preferably the highest determined identity, can serve as the acceptor sequence for the humanization process.

[0131] In addition to exchanging human CDRs for murine CDRs, further manipulations of the human donor sequence may be performed to obtain a sequence encoding a humanized antibody with optimized properties (e.g., antigen affinity).

[0132] Additionally, the altered human acceptor antibody variable domain sequence may also be adapted to encode one or more amino acids (according to the Kabat numbering system) at positions 4, 35, 38, 43, 44, 46, 58, 62, 64, 65, 66, 67, 68, 69, 73, 85, 98 in the light variable region and positions 2, 4, 36, 39, 43, 45, 69, 70, 74, 75, 76, 78, 92 in the heavy variable region corresponding to the non-human donor (Carter and Presta, U.S. Pat. No. 6,407,213).

[0133] In certain embodiments, it is generally desirable for antibodies to be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, in some embodiments, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues from the recipient and imported sequences can be selected and combined to achieve the desired antibody characteristic, e.g., increased affinity for the target antigen(s). In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0134] In certain embodiments, the selection of human variable domains, both light and heavy, to be used in the production of humanized antibodies can be important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework of the humanized antibody. See, for example, Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. In another method, a specific framework derived from the consensus sequence of all human antibodies of a particular light or heavy chain subgroup is used. The same framework may be used for several different humanized antibodies. See, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623.

[0135] Among the antibodies provided are human antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a non-human source using a human or human cell or other human antibody-encoding sequence, including a human antibody repertoire or human antibody library. This term excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions, e.g., those in which all or substantially all CDRs are non-human.

[0136] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals usually contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci or exist extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci are generally inactivated. Human antibodies can also be derived from human antibody libraries, including phage display and cell-free libraries containing sequences encoding antibodies from the human repertoire.

[0137] Among the antibodies provided are monoclonal antibodies, including monoclonal antibody fragments. As used herein, the term "monoclonal antibody" refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for potential variants that contain naturally occurring mutations or arise during the production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain various antibodies directed against different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on an antigen. This term should not be construed as requiring the production of the antibody by any particular method. Monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, production from hybridomas, recombinant DNA methods, phage display, and other antibody display methods.

[0138] 2. Target-expressing cells In some embodiments, the recombinant receptor stimulator is a cell that expresses a target recognized by an antigen receptor, i.e., the recombinant receptor stimulator is a target-expressing cell. In some embodiments, the target is an antigen of the recombinant receptor, and therefore, in some cases, the target-expressing cell is an antigen-expressing cell. In some embodiments, the recombinant receptor stimulator is an antigen-expressing cell, for example, a cell that expresses an antigen as described above.

[0139] In certain embodiments, the cells, e.g., target-expressing cells, e.g., antigen-expressing cells, are exogenous, xenogeneic, and / or autologous to the subject. In some embodiments, the cells are exogenous to the subject.

[0140] In certain embodiments, the target-expressing cells express a target that is bound and / or recognized by the recombinant receptor. In some embodiments, the target is an antibody, and the target-expressing cells express the antibody. In some embodiments, the target-expressing cells are tumor cells. In certain embodiments, the target-expressing cells are primary cells.

[0141] In some embodiments, the target is an antigen recognized by the recombinant receptor, and the target-expressing cell is an antigen-expressing cell. In certain embodiments, the antigen-expressing cell expresses an antigen bound and / or recognized by the recombinant receptor. In some embodiments, the antigen-expressing cell is a tumor cell. In certain embodiments, the antigen-expressing cell is a primary cell. In some embodiments, the cell line is an immortal cell line. In certain embodiments, the antigen-expressing cell is a cancerous cell and / or tumor cell. In some embodiments, the antigen-expressing cell is derived from a cancer cell and / or tumor cell, e.g., a human cancer cell and / or human tumor cell. In some embodiments, the antigen-expressing cell is a cell derived from a cancer cell line, optionally a human cancer cell line. In some embodiments, the antigen-expressing cell is a cell derived from a tumor cell line, optionally a human tumor cell line.

[0142] In certain embodiments, the antigen-expressing cell is a tumor cell. In some embodiments, the antigen-expressing cell is a circulating tumor cell, e.g., a tumor immune cell, e.g., a tumor B cell (or a cell derived from a tumor B cell).

[0143] In certain embodiments, the antigen-expressing cells are selected from the group consisting of integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (also known as CA9, CAIX, or G250), cancer-testis antigen, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC 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), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutant (EGFR), and EGFR-specific EGFR. vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, 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), folate binding protein (FBP), folate receptor alpha, fetal acetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erb-B2) ), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-AIA1), 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 (LI-CAM), CE7 epitope of LI-CAM, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin(MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (KG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, The antigen-expressing cells express 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 isomerase, or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), or a combination thereof. In some embodiments, the antigen-expressing cells express pathogen-specific antigens or antigens expressed by pathogens or antigens associated with universal tags and / or biotinylated molecules and / or molecules expressed by HIV, HCV, HBV, or other pathogens. In certain embodiments, the antigen-expressing cells express one or more antigens associated with B-cell malignancies, e.g., any of several known B-cell markers. In certain embodiments, the antigen-expressing cells express CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, CD30, or a combination thereof. In some embodiments, the antigen-expressing cells express CD19, e.g., human CD19.

[0144] In some embodiments, the antigen is or includes a pathogen-specific antigen or an antigen expressed by a pathogen. In some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen. In certain embodiments, the antigen-expressing cell is or is derived from a tumor cell. In some embodiments, the tumor cell is cancerous. In certain embodiments, the tumor cell is non-cancerous. In some embodiments, the tumor cell is or is derived from a circulating B cell, e.g., a circulating B cell that is capable of forming a tumor in vivo. In some embodiments, the tumor cell is or is derived from a circulating B cell that is a neoplastic, tumorigenic, or cancerous B cell.

[0145] In certain embodiments, the tumor cells are or are derived from human cancer cells. In some embodiments, the tumor cells are from AID-related cancer, breast cancer, cancer of the digestive / gastrointestinal tract, anal cancer, appendix cancer, bile duct cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, islet cell tumor, pancreatic neuroendocrine tumor, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, stomach (gastric) cancer, endocrine system cancer, adrenocortical carcinoma, parathyroid cancer, pheochromocytoma, pituitary tumor, thyroid cancer, eye cancer, intraocular melanoma, retinoblastoma, bladder cancer, kidney (renal cell) cancer, penile cancer, prostate cancer, transitional cell renal pelvis and ureter cancer, testicular cancer, urethral cancer, Wilms' tumor or other pediatric kidney tumors, germ cell cancer, central nervous system cancer, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, gynecological cancer, cervical cancer, endometrial cancer, gestational trophoblastic tumor, ovarian epithelial cancer, uterine sarcoma, vaginal cancer, vulvar cancer. , head and neck cancer, hypopharyngeal cancer, pharyngeal cancer, lip and oral cavity cancer, metastatic squamous neck cancer, nasopharyngeal cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, throat cancer, musculoskeletal cancer, bone cancer, Ewing's sarcoma, gastrointestinal stromal tumor (GIST), osteosarcoma, malignant fibrous histiocytoma of bone, rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, neurological cancer, brain tumor, astrocytoma, brain stem glioma, central nervous system non-cancer derived from cells of typical teratoma / rhabdoid tumor, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharyngioma, ependymoma, medulloblastoma, spinal cord tumor, supratentorial primitive neuroectodermal tumor and pineoblastoma, neuroblastoma, respiratory cancer, thoracic cancer, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, thymoma, thymic carcinoma, skin cancer, Kaposi's sarcoma, melanoma or Merkel cell carcinoma or any equivalent human cancer.

[0146] In certain embodiments, the tumor cells are derived from a non-hematopoietic cancer, e.g., a solid tumor. In certain embodiments, the tumor cells are derived from a hematopoietic cancer. In certain embodiments, the tumor cells are derived from a cancer that is a B-cell malignancy or a hematopoietic malignancy. In certain embodiments, the tumor cells are derived from non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML), or a myeloma, e.g., multiple myeloma (MM), or any equivalent human cancer thereof. In some embodiments, the antigen-expressing cells are neoplastic, cancerous, and / or tumorigenic B cells. Multiple tumor cell lines are known and available and can be selected depending on the antigen recognized by a particular recombinant receptor (e.g., CAR).

[0147] Any of several tumor cell lines are known and available. Tumor cell lines that express particular tumor antigens are known, or surface expression of tumor antigens can be readily determined or measured by those skilled in the art using any of a variety of techniques, for example, by flow cytometry. Exemplary tumor cell lines include 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 cancer cells (33A; CaSki; HeLa), lung cancer cells (NCI-H358; A5 49, 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-H929, RPMI-8226, OPM2, LP-1, L363, MM.1S, MM.1R, MC / CAR, JJN3, KMS11, AMO-1, EJM; MOLP-8).For example, exemplary CD19-expressing cell lines include, but are not limited to, Raji, Daudi, and BJAB; exemplary CD20-expressing cell lines include, but are not limited to, 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, but are not limited to, SKOV3, BT-474, and SKBR-3; exemplary BCMA-expressing cell lines include, but are not limited to, Raji, 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, but are not limited to, SKOV3, BT-474, and SKBR-3; Exemplary 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-H929, MM.1S, MM1.R, MOLP-8, and OPM-2; exemplary ROR1-expressing cell lines include, but are not limited to, A549, MDA-MB-231, H1975, BALL-1, and RCH-ACV.

[0148] In some embodiments, the target-expressing cell line is a cell line that has been transduced to express a target of the recombinant receptor. In some embodiments, the target is a tumor antigen. In certain embodiments, the antigen-expressing cell line is a cell line that has been transduced to express a tumor antigen. The cell line may be a mammalian cell line, including, but not limited to, a human cell line. In some embodiments, the human cell line may be K562, U937, 721.221, T2, or C1R cells. For example, the K562 chronic myeloid leukemia cell line may be transduced with a nucleic acid encoding a tumor antigen. In some embodiments, the cell line may be engineered with a plasmid vector or messenger RNA (mRNA) encoding the tumor antigen of interest. In some embodiments, the transduction may be lentiviral-based transduction. In some embodiments, the cell line (e.g., K562 cells) stably expresses an 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 cells). In some embodiments, exogenous nucleic acids can be integrated into the genome of a cell line (e.g., K562 cells) at a specific locus. In some embodiments, exogenous nucleic acids can be integrated into the genome of a cell line (e.g., K562 cells) at a genomic safe harbor (GSH). GSHs support stable integration and expression of exogenous nucleic acids while minimizing the risk of unwanted interactions with the host cell genome (see, e.g., Sadelain et al., Nat Rev Cancer. (2011) 12(1):51-8). Several safe GSHs for stable integration of exogenous nucleic acids in human cells have been identified, including AAVS1, a naturally occurring site of AAV viral integration on chromosome 19; the CCR5 gene, a chemokine receptor gene also known as the HIV-1 co-receptor; and the human ortholog of the mouse Rosa26 locus (see, e.g., Papapetrou and Schambach Mol Ther. (2016) 24(4):678-684).

[0149] In some embodiments, the target-expressing cells are varied, or adjusted, over multiple incubations at an altered ratio relative to a fixed amount of cells (effector cells) expressing the recombinant receptor in the therapeutic composition. In some embodiments, the adjusted amount is a target-expressing target cell to effector T cell (T:E) ratio of 100:1 to 0.001, e.g., a T:E ratio of 50:1 to 0.050, a T:E ratio of 25:1 to 0.025, a T:E ratio of 12:1 to 0.012:1, a T:E ratio of 10:1 to 0.010, or a T:E ratio of 5:1 to 0.5. In some embodiments, the ratio is 12:1 to 0.012:1, or a T:E ratio of about 12:1 to about 0.012:1. The specific range of ratios can be empirically determined depending on the particular target and target cells being employed. For example, the selected ratio is one that includes a linear dose-response increase in recombinant receptor-dependent activity across multiple adjusted amounts. In some embodiments, the ratio is also selected to include a lower asymptote of receptor-dependent activity and an upper asymptote of receptor-dependent activity, representing a minimum and maximum response, respectively.

[0150] For example, the target is an antigen of a recombinant receptor. In some embodiments, the antigen-expressing cells are varied, or adjusted, over multiple incubations at an altered ratio compared to a fixed amount of cells (effector cells) of the therapeutic composition expressing the recombinant receptor. In some embodiments, the adjusted amount is an antigen-expressing target cell to effector T cell (T:E) ratio of 100:1 to 0.001, e.g., a T:E ratio of 50:1 to 0.050, a T:E ratio of 25:1 to 0.025, a T:E ratio of 12:1 to 0.012:1, a T:E ratio of 10:1 to 0.010, or a T:E ratio of 5:1 to 0.5. In some embodiments, the ratio is 12:1 to 0.012:1, or a T:E ratio of about 12:1 to about 0.012:1. The specific range of ratios can be determined empirically depending on the particular antigen and target cells being employed. For example, the selected ratio is one that includes a linear dose-response increase in recombinant receptor-dependent activity across multiple adjusted amounts. In some embodiments, the ratio is also selected to include a lower asymptote of receptor-dependent activity and an upper asymptote of receptor-dependent activity, representing a minimum and maximum response, respectively.

[0151] C. Measurement of Recombinant Receptor-Dependent Activity The methods of assessing efficacy provided herein include measuring the activity of a therapeutic cell composition in response to stimulation of a recombinant receptor of engineered cells of the therapeutic cell composition. As noted above, the provided assays allow for the measurement of recombinant receptor-dependent activity in response to a recombinant receptor stimulating agent, such as those described in Section IB, from multiple incubation conditions, each incubation containing a different, adjusted ratio of cells of the therapeutic cell composition to the recombinant receptor stimulating agent.

[0152] In certain embodiments, recombinant receptor-dependent activity, e.g., CAR-dependent activity, is intended to be an activity occurring in engineered cells expressing a recombinant receptor that does not occur and / or cannot occur in cells that do not express the recombinant receptor. In some embodiments, recombinant receptor-dependent activity is an activity that depends on the activity or presence of the recombinant receptor. Recombinant receptor-dependent activity can be any cellular process that is directly or indirectly affected by the expression and / or presence of the recombinant receptor, or by changes in the activity of the recombinant receptor, e.g., receptor stimulation. In some embodiments, recombinant receptor-dependent activity can include, but is not limited to, cellular processes such as cell division, DNA replication, transcription, protein synthesis, membrane trafficking, protein translocation and / or secretion, or immune cell function, e.g., cytolytic activity. In certain embodiments, recombinant receptor-dependent activity can be measured by changes in CAR receptor recognition, phosphorylation of intracellular signaling molecules, protein degradation, transcription, translation, protein translocation, and / or the production and secretion of proteins or factors such as growth factors and cytokines. In certain embodiments, the recombinant receptor is a CAR. In certain embodiments, the recombinant receptor is a TCR.

[0153] In some embodiments, recombinant receptor-dependent activity, for example, CAR-dependent activity, is the measured value of a factor, for example, the amount or concentration, or the change in the amount or concentration after stimulating therapeutic cell composition with recombinant receptor stimulating agent.In certain embodiments, the factor can be a protein, a phosphorylated protein, a cleaved protein, a translocated protein, a protein undergoing activity confirmation, a polynucleotide, an RNA polynucleotide, an mRNA and / or an shRNA.In some embodiments, the measured value can include, but is not limited to, kinase activity, protease activity, phosphatase activity, cAMP production, ATP metabolism, translocation, for example, the increase or decrease of nuclear localization of protein, the increase in transcription activity, translocation activity, the production and / or secretion of soluble factors, cellular uptake, ubiquitination and / or the increase in protein degradation.

[0154] In some embodiments, the factor is a secreted soluble factor, for example, a hormone, growth factor, chemokine and / or cytokine.

[0155] In some embodiments, the recombinant receptor-dependent activity, for example, CAR-dependent activity, is a response to stimulation with a recombinant receptor stimulating agent. In certain embodiments, cells are incubated in the presence of a recombinant receptor stimulating agent capable of stimulating the recombinant receptor-dependent activity, and the activity is or includes at least one aspect of the response to the stimulation. The response can include, but is not limited to, intracellular signaling events, such as increased activity of receptor molecules, increased kinase activity of one or more kinases, increased transcription of one or more genes, increased protein synthesis of one or more proteins, and / or increased intracellular signaling molecules, for example, increased kinase activity of proteins. In some embodiments, the response (e.g., recombinant receptor-dependent activity) is associated with immune activity, and can include, but is not limited to, the production and / or secretion of soluble factors, for example, cytokines, increased antibody production, and / or increased cytolytic activity.

[0156] In some embodiments, recombinant receptor-dependent activity is assessed by measuring, detecting, or quantifying recombinant receptor-dependent activity in response to a stimulus (e.g., a recombinant receptor stimulating agent), i.e., at least one activity initiated, induced, supported, prolonged, and / or caused by the stimulus (e.g., a recombinant receptor stimulating agent). In certain embodiments, cells of the therapeutic cell composition are cultured with a recombinant receptor stimulating agent, and interaction or binding of the recombinant receptor stimulating agent to the recombinant receptor stimulates, e.g., induces, a recombinant receptor-dependent activity that is specific to cells expressing the recombinant receptor. In certain embodiments, the recombinant receptor-dependent activity occurs in cells expressing the recombinant receptor, but does not occur, or occurs only minimally, in cells that do not express the receptor. In certain embodiments, the recombinant receptor is a CAR. In some embodiments, the activity is a CAR-dependent activity.

[0157] Conditions for stimulation of engineered cells, e.g., recombinant receptors of immune cells or T cells, with recombinant receptor stimulating agents can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, and ions. In some embodiments, recombinant receptor-dependent activity is determined by whether a soluble factor, e.g., a cytokine or chemokine, is produced or secreted.

[0158] In some embodiments, the recombinant receptor-dependent activity is specific to cells expressing the recombinant receptor. In some embodiments, the recombinant receptor-dependent activity is specific to cells expressing the recombinant receptor and does not occur in cells lacking expression of the recombinant receptor. In certain embodiments, the recombinant receptor is a CAR, and the activity is a CAR-dependent activity. In certain embodiments, the recombinant receptor-dependent activity is not present in cells lacking expression of the recombinant receptor under the same conditions in which the activity is present in cells expressing the recombinant receptor. In certain embodiments, the CAR-dependent activity is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or less than about 99% of the CAR-dependent activity in CAR cells under the same conditions.

[0159] In some embodiments, the recombinant receptor-dependent activity is specific for cells expressing the recombinant receptor, e.g., a CAR, and the activity is generated by stimulation with a recombinant receptor stimulating agent that is specific for cells of the therapeutic cell composition that express the recombinant receptor. In some embodiments, the recombinant receptor is a CAR, and the CAR-specific stimulation stimulates, induces, initiates, induces, and / or prolongs activity in CAR+ cells but does not stimulate, induce, initiate, induce, and / or prolong activity in CAR- cells. In some embodiments, the CAR-dependent activity is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or less than about 99% in CAR- cells than in CAR+ cells after stimulation with the CAR-specific stimulation.

[0160] In certain embodiments, the activity is a recombinant receptor-dependent, e.g., CAR-dependent, activity stimulated by a recombinant receptor stimulating agent, such as those described in Section IB, that is specific for the recombinant receptor. In some embodiments, the recombinant receptor stimulating agent, e.g., a CAR-specific agent, includes an antigen or epitope thereof that is bound and / or recognized by the recombinant receptor, e.g., a CAR. In some embodiments, the recombinant receptor stimulating agent includes an antibody, e.g., an anti-idiotypic antibody (anti-ID), or an active fragment, variant, or portion thereof, that binds to the recombinant receptor. In certain embodiments, the recombinant receptor stimulating agent is a cell that expresses an antigen on its surface. In certain embodiments, the recombinant receptor stimulating agent is a cell that expresses an antibody on its surface. In some embodiments, the cell is derived from a cell line, such as those described in Section IB-2. In some embodiments, the cell line is a tumor cell line. In some embodiments, the cell expresses a tumor antigen.

[0161] In some embodiments, recombinant receptor-dependent activity is measured in a therapeutic cell composition containing cells expressing a recombinant receptor, e.g., a CAR, and the measurement is compared to one or more controls. In certain embodiments, the control is a similar or identical composition of cells that has not been stimulated. For example, in some embodiments, recombinant receptor-dependent activity is measured in a cell composition after or during incubation with a recombinant receptor stimulating agent, and the resulting measurement is compared to a control measurement of activity from a similar or identical cell composition that has not been incubated with the recombinant receptor stimulating agent. In some embodiments, both the therapeutic cell composition and the control cell composition contain cells expressing a recombinant receptor. In some embodiments, the control is taken from a similar cell composition that does not contain cells expressing a recombinant receptor, e.g., CAR+ cells. Thus, in some embodiments, a therapeutic cell composition containing recombinant receptor-expressing cells and a control cell composition that does not contain recombinant receptor-expressing cells are contacted with a recombinant receptor stimulating agent. In certain embodiments, the control is a measurement from the same cell composition expressing a recombinant receptor taken before any stimulation. In certain embodiments, a control measurement is taken to determine background signal, and the control measurement is subtracted from the activity measurement. In some embodiments, the measured value of activity in the cell composition is divided by the control measurement to obtain a value that is the percentage of activity above the control level. In some embodiments, all recombinant receptor-dependent activity measurements are adjusted or normalized to a control measurement, for example, in which a recombinant receptor stimulating agent was not cultured with the cells of the therapeutic cell composition. In some embodiments, adjusting or normalizing measurements to the control condition provides a more accurate measure of recombinant receptor-dependent activity.

[0162] In certain embodiments, the recombinant receptor-dependent activity is or includes the production and / or secretion of a soluble factor. In some embodiments, the recombinant receptor-dependent activity, for example, CAR-dependent activity, is or includes the production and / or secretion of a soluble factor. In certain embodiments, the soluble factor is a cytokine or chemokine.

[0163] Suitable techniques for measuring the production or secretion of soluble factors are known in the art. The production and / or secretion of soluble factors can be measured by determining the concentration or amount of the extracellular amount of the factor or by determining the amount of transcriptional activity of the gene encoding the factor. Suitable techniques include, but are not limited to, immunoassays, immunoassays, aptamer-based assays, histological or cytological assays, mRNA expression level assays, enzyme-linked immunosorbent assays (ELISAs), immunoblotting, immunoprecipitation, radioimmunoassays (RIAs), immunostaining, flow cytometry assays, surface plasmon resonance (SPR), chemiluminescence assays, lateral flow immunoassays, inhibition or avidity assays, protein microarrays, high-performance liquid chromatography (HPLC), mesoscale discovery (MSD) electrochemiluminescence, and bead-based multiplex immunoassays (MIAs). In some embodiments, suitable techniques may employ detectable binding reagents that specifically bind to the soluble factor.

[0164] In certain embodiments, the measurement of soluble factors, e.g., cytokines, is measured by ELISA (enzyme-linked immunosorbent assay). ELISA is a plate-based assay technology designed to detect and quantify substances such as peptides, cytokines, antibodies, and hormones. In ELISA, the soluble factor must be immobilized on a solid surface and then complexed with an antibody, which is linked to an enzyme. Detection is achieved by evaluating the conjugated enzyme activity by incubation with a substrate that produces a detectable signal. In some embodiments, recombinant receptor-dependent activity is measured using an ELISA assay.

[0165] In some embodiments, the recombinant receptor-dependent activity is the secretion or production of a soluble factor (e.g., a cytokine). In certain embodiments, the production or secretion is stimulated in a therapeutic cell composition containing a recombinant receptor-expressing cell, e.g., a CAR-expressing cell, by a recombinant receptor stimulating agent capable of binding to the recombinant receptor, which stimulates the recombinant receptor-dependent activity, e.g., CAR-dependent activity. In some embodiments, the recombinant receptor stimulating agent comprises an antigen or an epitope thereof specific for the recombinant receptor; is a cell expressing the antigen; or comprises an antibody or a portion or variant thereof that binds to and / or recognizes the recombinant receptor; or a combination thereof (see, e.g., section IB above). In certain embodiments, the recombinant receptor stimulating agent is a recombinant protein comprising an antigen or an epitope thereof that is bound by or recognized by the recombinant receptor.

[0166] In certain embodiments, recombinant receptor-dependent activity is soluble factor production and / or secretion, and is measured by incubating a therapeutic cell composition containing cells expressing a recombinant receptor, e.g., a CAR, with a recombinant receptor stimulating agent, such as those described in Section IB. In certain embodiments, the soluble factor is a cytokine or chemokine. In some embodiments, cells of a therapeutic cell composition containing recombinant receptor-expressing cells are incubated in the presence of a recombinant receptor stimulating agent for a fixed amount of time, and soluble factor production and / or secretion is measured at one or more time points during the incubation. In some embodiments, the cells are incubated with the recombinant receptor stimulating agent for up to or about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 48 hours, or between 1 hour and 4 hours, between 1 hour and 12 hours, between 12 hours and 24 hours, or more than 24 hours, inclusive, and the amount of soluble factor, e.g., cytokine, is detected.

[0167] In some embodiments, the recombinant receptor stimulator is a particle (e.g., bead) to which an antibody, e.g., an anti-idiotypic antibody, specific for an antigen or portion thereof recognized by the recombinant receptor, or an extracellular domain of the recombinant receptor (e.g., an extracellular antigen-binding domain (e.g., scFv)) is attached or immobilized. In some embodiments, the recombinant receptor (e.g., CAR) and a number of cells of the therapeutic cell composition, together with the particle, are mixed at or about 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. The therapeutic cell composition may be incubated at a number of ratios of cells to particles, including 1:30, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1, or at ranges between any of the foregoing, for example, between 1:1 and 1:10 or between 1:0.2 and 1:12, inclusive. In some embodiments, the ratios include any and all ratios provided herein. In some embodiments, the recombinant receptor stimulator is a cell expressing an antigen recognized by the recombinant receptor.In some embodiments, the recombinant receptor is a CAR, and a controlled number of cells of the therapeutic cell composition are administered along with a number of such particles at multiple ratios of cells of the therapeutic cell composition to particles, for example, 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2, and the like. or 1:0.1 or about 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1, or a range between any of the foregoing, for example, between 1:1 and 1:10 or 1:0.2-1:12, inclusive. In some embodiments, the multiple ratios include any and all ratios provided herein.

[0168] In some embodiments, the recombinant receptor stimulator is a cell expressing a target (e.g., an antigen or antibody) recognized by the recombinant receptor. In some embodiments, the recombinant receptor (e.g., CAR) and a number of cells of the therapeutic cell composition are administered together with the cells at a concentration of 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1 or about 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, The cells of the therapeutic cell composition may be incubated at multiple ratios to cells expressing a target (e.g., an antigen or antibody), including 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1, or at ranges between any of the foregoing, e.g., between 1:1 and 1:10 or between 1:0.2 and 1:12, inclusive. In some embodiments, the multiple ratios include any and all ratios provided herein. In some embodiments, the recombinant receptor is a CAR, and the adjusted number of cells of the therapeutic cell composition is mixed with a number of cells expressing a target (e.g., an antigen or antibody) at or about 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. The cells of the therapeutic cell composition may be incubated at a number of ratios of cells of the therapeutic cell composition to cells expressing the antigen, including 1:50, 1:40, 1:30, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1, or at ranges between any of the foregoing, for example, between 1:1 and 1:10 or 1:0.2-1:12, inclusive.In some embodiments, the plurality of ratios includes any and all ratios provided herein.

[0169] In some embodiments, about 1 x 10 of the cell composition, each comprising a boundary. 2 to approximately 1 × 10 4 Between, about 1 × 10 3 to approximately 1 × 10 5 Between, about 1 × 10 4 to approximately 1 × 10 6 Between, about 1 × 10 5 to approximately 1 × 10 7 Between, about 1 × 10 6 to approximately 1 × 10 8 Between, about 1 × 10 7 to approximately 1 × 10 9 Between and about 1 x 10 8 to approximately 1 × 10 10 The cells between are incubated with a fixed amount or concentration of recombinant receptor stimulating agent.

[0170] In some embodiments, the cells of the therapeutic cell composition are incubated with the recombinant receptor stimulating agent in a volume of cell culture medium. The exact volume can be determined empirically, and it is understood that it is a function of the surface area of ​​the vessel (e.g., a multi-well plate) in which the assay is being performed. In certain embodiments, the cells are incubated with the recombinant receptor stimulating agent in a volume of at least or about 1 μL, at least or about 10 μL, at least or about 25 μL, at least or about 50 μL, at least or about 100 μL, at least or about 500 μL, at least or about 1 mL, at least or about 1.5 mL, at least or about 2 mL, at least or about 2.5 mL, at least or about 5 mL, at least or about 10 mL, at least or about 20 mL, at least or about 25 mL, at least or about 50 mL, at least or about 100 mL, or more than 100 mL. In certain embodiments, cells are incubated with a recombinant receptor stimulating agent in a volume between about 1 μL and about 100 μL, between about 100 μL and about 500 μL, between about 500 μL and about 1 mL, between about 500 μL and about 1 mL, between about 1 mL and about 10 mL, between about 10 mL and about 50 mL, or between about 10 mL and about 100 mL, each inclusive. In certain embodiments, cells are incubated with a recombinant receptor stimulating agent in a volume between about 100 μL and about 1 mL, inclusive. In certain embodiments, cells are incubated with a recombinant receptor stimulating agent in a volume of about 500 μL. In some embodiments, the multiwell plate is a 6-well plate and the volume is 1 mL, or about 1 mL to 3 mL, or about 3 mL. In some embodiments, the multiwell plate is a 12-well plate and the volume is 1 mL, or about 1 mL to 2 mL, or about 2 mL. In some embodiments, the multiwell plate is a 24-well plate and has a volume of 0.5 mL, or about 0.5 mL to 1 mL, or about 1 mL, hi some embodiments, the multiwell plate is a 48-well plate and has a volume of 0.2 mL, or about 0.2 mL to 0.4 mL, or about 0.4 mL.In some embodiments, the multi-well plate is a 96-well plate and has a volume of at or about 0.1 mL to at or about 0.2 mL.

[0171] In some embodiments, a number of cells of a therapeutic cell composition are incubated with a recombinant receptor stimulating agent at a concentration that varies between about 1 fmol and about 1 pmol, between about 1 pmol and about 1 nmol, between about 1 nmol and about 1 μmol, between about 1 μmol and about 1 mmol, or between about 1 mmol and 1 mol, each inclusive. In specific embodiments, a number of cells of a therapeutic cell composition are incubated with a recombinant receptor stimulating agent at a concentration that varies between about 1 fM and about 1 pM, between about 1 pM and about 1 nM, between about 1 nM and about 1 μM, between about 1 μM and about 1 mM, or between about 1 mM and 1 mol, each inclusive. Exemplary units include pg / mL, pg / (mL / hr), pg(mL x cells), pg / (mL x hr x cells), and pg / (mL x hr x 10 6 Examples of cells include, but are not limited to, cells.

[0172] In certain embodiments, the measured value of recombinant receptor-dependent activity, e.g., CAR-dependent activity, is the amount or concentration or relative amount or concentration of the soluble factor in the therapeutic cell composition at each time point during or at the end of incubation at each of the multiple ratios tested. In certain embodiments, the measured value is subtracted from or normalized to a control measurement. In some embodiments, the control measurement is a measurement from the same cell composition taken before incubation. In certain embodiments, the control measurement is a measurement from the same control cell composition that has not been incubated with the binding molecule. In certain embodiments, the control is a measurement taken at the same time point during incubation with the binding molecule from a cell composition that does not contain recombinant receptor-positive cells.

[0173] In some embodiments, the measured value is the normalized ratio of the amount or concentration compared to the control. In certain embodiments, the measured value is the amount or concentration of soluble factor per time amount, for example, per minute or per hour. In some embodiments, the measured value is per cell, or per set or reference number of cells, for example, per 100 cells, per 10 3 per cell, 10 4 per cell, 10 5 per cell, 10 6 The measurement may be the amount or concentration of a soluble factor, such as per cell. In certain embodiments, the measurement is the amount or concentration of a soluble factor per cell or per reference number of cells. In some embodiments, the measurement is the amount or concentration of a soluble factor per cell expressing a recombinant receptor. In certain embodiments, the measurement is the amount or concentration of a soluble factor per amount of time (e.g., per minute or per hour) per cell expressing a recombinant receptor, CAR+ cell of a therapeutic cell composition. In some embodiments, the measurement is the amount or concentration of a soluble factor per amount of time per amount or concentration of a recombinant receptor or recombinant receptor stimulating agent. In some embodiments, the measurement is the amount or concentration of a soluble factor per cell, or per set or reference number of cells, per amount or concentration of a recombinant receptor or recombinant receptor stimulating agent. In some embodiments, the measurement is the amount or concentration of a soluble factor per cell, or per reference number of cells, per amount of time per cell, or per amount or concentration of a recombinant receptor or recombinant receptor stimulating agent. In some embodiments, the measurement is the amount or concentration of a soluble factor per cell, or per reference number of cells, per amount of time. In some embodiments, the measurement is the amount or concentration of soluble factor per amount or concentration of recombinant receptor or recombinant receptor stimulating agent per cell expressing the recombinant receptor. In certain embodiments, the measurement is the amount or concentration of soluble factor per amount of recombinant receptor or recombinant receptor stimulating agent per amount of CAR+ cells of the therapeutic cell composition, per amount or concentration of recombinant receptor or recombinant receptor stimulating agent, per amount of time.

[0174] In certain embodiments, the recombinant receptor- or CAR-dependent activity is the production or secretion of two or more soluble factors. In certain embodiments, the recombinant receptor- or CAR-dependent activity is the production or secretion of more than 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 soluble factors. In some embodiments, measurements of more than 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 soluble factors are combined into an arithmetic or geometric mean. In certain measurements, the measurement of recombinant receptor-dependent activity is the secretion or mixture of more than 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 soluble factors.

[0175] In certain embodiments, recombinant receptor-dependent activity measurements are transformed, for example, by logarithmic transformation. In certain embodiments, recombinant receptor activity measurements are expressed in logarithmic terms (log 10 (x)), natural logarithm (ln(x)), or logarithm base 2 (log2(x)). In some embodiments, the measure of recombinant receptor-dependent activity is a composite of more than two soluble factor production or secretion measurements. In some embodiments, more than two soluble factor production or secretion measurements are transformed before being combined into a composite measurement. In certain embodiments, the measure of recombinant receptor-dependent activity is transformed before normalization to a reference measurement. In certain embodiments, the measure of recombinant receptor-dependent activity is transformed before normalization to a reference measurement. In some embodiments, the normalization of recombinant receptor-dependent activity is to the maximum recombinant receptor-dependent activity measured from multiple incubations.

[0176] In certain embodiments, the soluble factor is a cytokine. Cytokines are a large group of small signaling molecules that function widely in cell communication. Cytokines are most often associated with various immune regulatory molecules, including interleukins, chemokines, and interferons. Alternatively, cytokines may be characterized by their structure, which is classified into four families: four alpha-helical families, including the IL-2 subfamily, the IFN subfamily, and the IL-10 subfamily; the IL-1 family, including members of the transforming growth factor beta family, the IL-17 family, and cysteine-knot cytokines. In some embodiments, the recombinant receptor-dependent activity is the production or secretion of one or more soluble factors, including interleukins, interferons, and chemokines. In certain embodiments, the recombinant receptor-dependent activity, e.g., CAR-dependent activity, is the production or secretion of one or more of an IL-2 family member, an IFN subfamily member, an IL-10 subfamily member, an IL-1 family member, an IL-17 family member, a cysteine-knot cytokine, and / or a member of the transforming growth factor beta family.

[0177] In certain embodiments, the recombinant receptor-dependent or CAR-dependent activity is the production and / or secretion of one or more of IL-1, IL-1β, IL-2, sIL-2Ra, IL-3, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-27, IL-33, IL-35, TNF, TNFalpha, CXCL2, CCL2, CCL3, CCL5, CCL17, CCL24, PGD2, LTB4, interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1a, MIP-1b, Flt-3L, fractalkine, and / or IL-5. In certain embodiments, the CAR-dependent activity is the production or secretion of Th17 cytokines. In some embodiments, the Th17 cytokine is GMCSF. In some embodiments, the CAR-dependent activity comprises the production or secretion of a Th2 cytokine, wherein the Th2 cytokine is IL-4, IL-5, IL-10, or IL-13.

[0178] In certain embodiments, the recombinant receptor- or CAR-dependent activity is the production or secretion of proinflammatory cytokines, which play a role in initiating inflammatory responses and mediate innate immune responses to regulate host defense against pathogens. Inflammatory cytokines include, but are not limited to, interleukin (IL), interleukin-1-beta (IL-1), interleukin-3 (IL-3), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-13 (IL-13), tumor necrosis factor (TNF), C-X-C-chemokine ligand 2 (CXCL2), CC-chemokine ligand 2 (CCL2), CC-chemokine ligand 3 (CCL3), CC-chemokine ligand 5 (CCL5), CC-chemokine ligand 17 (CCL17), CC-chemokine ligand 24 (CCL24), prostaglandin D2 (PGD2), and leukotriene B4 (LTB4), as well as IL-33. In some embodiments, the recombinant receptor- or CAR-dependent activity is the production and / or secretion of interleukin and / or TNF family members. In certain embodiments, the recombinant receptor- or CAR-dependent activity is the production and / or secretion of IL-1, IL-6, IL-8 and IL-18, TNF-alpha, or combinations thereof.

[0179] In certain embodiments, the recombinant receptor activity (e.g., CAR-dependent activity) is the secretion of IL-2, IFN-gamma, TNF-alpha, or a combination thereof. In some embodiments, the recombinant receptor activity (e.g., CAR-dependent activity) is the secretion of IL-2. In some embodiments, the recombinant receptor activity (e.g., CAR-dependent activity) is the secretion of IFN-gamma. In some embodiments, the recombinant receptor activity (e.g., CAR-dependent activity) is the secretion of TNF-alpha.

[0180] In certain embodiments, the recombinant receptor-dependent activity is the cytolytic (cytotoxic) activity of a therapeutic cell composition. In some embodiments, recombinant receptor-dependent cytolytic activity is assessed by exposing, incubating, and / or contacting cells expressing the recombinant receptor or a cell composition containing cells expressing the recombinant receptor with varying amounts of target cells expressing the antigen and / or epitope bound and / or recognized by the recombinant receptor. Cytolytic activity can be measured by directly or indirectly measuring the number of target cells over time. For example, target cells may be incubated with a detectable marker, such as a detectable marker that allows the target cells to be subsequently lysed, or a detectable marker that is detectable in viable target cells, and then incubated with cells expressing the recombinant receptor. These readouts provide target cell number and / or target cell death, directly or indirectly, and can be measured at various time points during the assay. A reduction in target cell number and / or an increase in target cell death indicates cytolytic activity of the cells. Suitable methods for performing cytolytic assays are known in the art and include, but are not limited to, chromium-51 release assays, non-radioactive chromium assays, and flow cytometry assays using fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE), PKH-2, and PKH-26.

[0181] In certain embodiments, recombinant receptor-, e.g., CAR-dependent cytolytic activity is measured by incubating a cell composition containing cells expressing the recombinant receptor with target cells expressing an antigen or epitope thereof that is bound by or recognized by the recombinant receptor. In certain embodiments, the recombinant receptor is a CAR. In some embodiments, the cells of the therapeutic cell composition are incubated with the cells expressing the antigen at a ratio including 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10, or at a ratio between 10:1 and 1:1, 3:1 and 1:3, or 1:1 and 1:10, inclusive. In some embodiments, the cells of the cell composition are incubated with the target cells at a ratio of CAR+ cells of the therapeutic cell composition to target cells that includes a ratio of about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10, or at a ratio between 10:1 and 1:1, 3:1 and 1:3, or 1:1 and 1:10, each inclusive.

[0182] In certain embodiments, the cells of the therapeutic cell composition are incubated with the target cells for up to or about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours, about 48 hours, or more than 48 hours. In some embodiments, a number of cells of the therapeutic cell composition are incubated with the antigen-expressing cells for about 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, a number of cells of the therapeutic cell composition, each of which is about 1 x 10, inclusive, are incubated with the antigen-expressing cells. 2 to approximately 1 × 10 4 Between, about 1 × 10 3 to approximately 1 × 10 5 Between, about 1 × 10 4 to approximately 1 × 10 6 Between, about 1 × 10 5 to approximately 1 × 10 7 Between, about 1 × 106 to approximately 1 × 10 8 Between, about 1 × 10 7 to approximately 1 × 10 9 Between or about 1 x 10 8 to approximately 1 × 10 10 In certain embodiments, a fixed amount of cells of the therapeutic cell composition, each containing between about 1 x 10 cells, is incubated with varying numbers of antigen-expressing cells to produce multiple ratios. 2 to approximately 1 × 10 4 Between, about 1 × 10 3 to approximately 1 × 10 5 Between, about 1 × 10 4 to approximately 1 × 10 6 Between, about 1 × 10 5 to approximately 1 × 10 7 Between, about 1 × 10 6 to approximately 1 × 10 8 Between, about 1 × 10 7 to approximately 1 × 10 9 Between or about 1 x 10 8 to approximately 1 × 10 10 Between CAR+ cells are incubated with varying numbers of antigen-expressing cells to yield multiple ratios.

[0183] In some embodiments, the activity measurements are compared to a control. In certain embodiments, the control is a culture of antigen-expressing cells that has not been incubated with the cell composition. In some embodiments, the control is a measurement from a control cell composition that does not contain CAR+ cells that have been incubated with the antigen-expressing cells at the same ratio.

[0184] In certain embodiments, the measurement of the cytolytic activity assay is the number of viable antigen-expressing cells at or at the end of incubation for each ratio tested. In certain embodiments, the measurement is the amount of a target cell death marker, such as chromium-51, released during incubation. In some embodiments, the measurement is the amount of target cell death determined by subtracting the amount of target cells in a co-incubation at a given time point from the amount of control target cells incubated alone. In some embodiments, the measurement is the percentage of target cells remaining at a time point compared to the starting amount of target cells. In certain embodiments, the measurement is the amount of cells killed over a certain amount of time. In certain embodiments, the measurement is the amount of cells killed per cell of the cell composition. In some embodiments, the measurement is an amount. The amount of cells killed per cell, or the amount of cells killed per set number or reference of cells, for example, but not limited to, 10 per 100 cells of the composition. 3 per cell, 10 4 per cell, 10 5 per cell, 10 6 per cell, 10 7 per cell, 10 8 per cell, 10 9 per cell, or 10 10 In certain embodiments, the measured value is the amount of killed target cells per cell.In certain embodiments, the measured value is the amount of killed cells per each CAR+ cell of the cell composition or its reference number or set number.In certain embodiments, the measured value is the amount of killed cells per cell of the cell composition per amount of time.In certain embodiments, the measured value is the amount of killed cells per CAR+ cell of the therapeutic cell composition over an amount of time.

[0185] In some embodiments, recombinant receptor-dependent activity is the upregulation of genes in cells of a therapeutic cell composition. In some embodiments, recombinant receptor-dependent gene upregulation activity is assessed by exposing, incubating, and / or contacting cells expressing a recombinant receptor or a cell composition containing cells expressing a recombinant receptor with varying amounts of a recombinant receptor stimulating agent that binds to and stimulates the recombinant receptor. The upregulation of gene activity can be measured directly or indirectly by number over time.

[0186] In some embodiments, recombinant receptor-dependent activity is the downregulation of genes in cells of therapeutic cell compositions. In some embodiments, recombinant receptor-dependent gene downregulation activity is assessed by exposing, incubating, and / or contacting cells expressing recombinant receptors or cell compositions containing cells expressing recombinant receptors with varying amounts of recombinant receptor stimulating agents that bind to and stimulate recombinant receptors. Downregulation of gene activity can be measured directly or indirectly by number over time.

[0187] In some embodiments, recombinant receptor-dependent activity is the upregulation of receptors in cells of a therapeutic cell composition. In some embodiments, recombinant receptor-dependent receptor upregulation activity is assessed by exposing, incubating, and / or contacting cells expressing recombinant receptors or cell compositions containing cells expressing recombinant receptors with varying amounts of recombinant receptor stimulating agents that bind to and stimulate the recombinant receptor. Upregulation of receptor activity can be measured directly or indirectly by number over time.

[0188] In some embodiments, recombinant receptor-dependent activity is the downregulation of receptors in cells of therapeutic cell compositions. In some embodiments, recombinant receptor-dependent receptor downregulation activity is assessed by exposing, incubating, and / or contacting cells expressing recombinant receptors or cell compositions containing cells expressing recombinant receptors with varying amounts of recombinant receptor stimulating agents that bind to and stimulate the recombinant receptor. The downregulation of receptor activity can be measured directly or indirectly by number over time.

[0189] In some embodiments, measurements of recombinant receptor-dependent activity are fitted using a mathematical model to obtain a recombinant receptor-dependent activity curve. Curve fitting may, in some cases, allow for prediction or extrapolation of the behavior of a therapeutic cell composition, e.g., recombinant receptor-dependent activity. It is contemplated that any method known in the art for performing curve fitting may be used. In some embodiments, the curve is sigmoidal. In some embodiments, an adjusted ratio resulting in half-maximal recombinant receptor-dependent activity is determined based on the recombinant receptor-dependent activity measured from each of multiple incubations. In some embodiments, the adjusted ratio resulting in half-maximal recombinant receptor-dependent activity is predicted, extrapolated, or estimated from the recombinant receptor-dependent activity curve. In some embodiments, the recombinant receptor-dependent activity curve is normalized to the measured maximum recombinant receptor-dependent activity. In some embodiments, the recombinant receptor-dependent activity curve is normalized to the upper asymptote of the curve, optionally to the range of values ​​of the upper asymptote.

[0190] In some embodiments, methods including assays as described herein may be performed in duplicate, triplicate, or more to validate measurements of recombinant receptor-dependent activity. In some cases, e.g., when assays are performed in duplicate, triplicate, or more, the recombinant receptor-dependent activity measured from each of the replicates is used to provide a descriptive statistical measure of recombinant receptor-dependent activity. For example, in some cases, the mean (e.g., arithmetic mean), median, standard deviation, and / or variance of each measure of recombinant receptor-dependent activity is determined for each of multiple ratio tests. In some embodiments, the mean of each measure of recombinant receptor-dependent activity is determined. In some embodiments, the standard deviation of each measure of recombinant receptor-dependent activity is determined. In some embodiments, the mean measures of recombinant receptor-dependent activity are fitted using a mathematical model to generate or estimate a recombinant receptor-dependent activity curve. In some embodiments, the curve is normalized to the mean maximum value. In some embodiments, the curve is normalized to the upper asymptote, optionally the mean of the range of values ​​of the upper asymptote.

[0191] The scales described herein may be used with reference to a reference standard, such as the reference standards described herein, for example, in Section ID-1.

[0192] D. Determining the Efficacy of Therapeutic Cell Compositions The method provided herein allows for determining the efficacy of therapeutic cell compositions.It is contemplated that the assay described herein can be used to evaluate the efficacy of therapeutic cell compositions produced by processes such as those described herein (e.g., Section II) as well as any other manufacturing process that allows the cells of the produced therapeutic cell composition to be cultured in an assay that includes multiple incubations, each incubation including culturing different ratios of the cells of the therapeutic composition with a recombinant receptor stimulating agent that can stimulate the recombinant receptor-dependent activity in the therapeutic cell composition.In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more therapeutic cell compositions can be evaluated according to the method provided herein.

[0193] The efficacy of a therapeutic cell composition can be determined by taking a measurement of recombinant receptor-dependent activity at each of multiple ratios tested. In some embodiments, the measurement is a composite determined by taking the average or median across duplicate, triplicate, or more replicates. In some embodiments, the standard deviation and / or variance of the measurements can be determined. In some embodiments, one or more measurements, including a composite measurement, of the recombinant receptor-dependent activity of a therapeutic cell composition in response to a recombinant receptor stimulating agent can be used to determine the efficacy of the therapeutic cell composition. In some embodiments, the recombinant receptor-dependent activity can be any of those described in Section IC. In some embodiments, the recombinant receptor stimulating agent can be any of those described in Section IB.

[0194] In some embodiments, multiple incubations at different ratios result in multiple measurements to which curve fitting methods can be applied. In some embodiments, the multiple measurements include composite measurements (e.g., mean or median values). For example, recombinant receptor-dependent activity measurements can be fitted with a curve, e.g., a sigmoid, to allow for inference, extrapolation, or estimation of the behavior (e.g., sensitivity) of a therapeutic cell composition. In some embodiments, the curve fitted to the measurements can be used to estimate the behavior (e.g., sensitivity) of a therapeutic composition that was not directly examined during the assay. For example, the curves can be used to estimate a lower asymptote; a minimum value; loss of detection of recombinant receptor-dependent activity; a particular percentage (10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90%) of half-maximal activity; a half-maximal value (e.g., 50% recombinant receptor-dependent activity); a range of 10%-90%, 20%-80%, 30%-70%, or 40%-60% of maximum recombinant receptor-dependent activity (i.e., maximum activity as described below); an upper asymptote; and the rate at which each of the maximum values ​​and ranges occurs.

[0195] It is contemplated that any scale (e.g., ratio at half maximum, range, maximum, minimum, asymptote, and composite scales thereof) can be used to determine the potency of a therapeutic cell composition. In some embodiments, the potency is relative potency.

[0196] 1.Effectiveness In some embodiments, the efficacy of a therapeutic cell composition is defined as the ratio at which one or more or ranges of recombinant receptor-dependent activity measurements occur. In some embodiments, one or more or ranges of measurements are composite measurements, e.g., mean or median, determined from replicate experiments. In some embodiments, the measurements and ratios are determined from a recombinant receptor-dependent activity curve of the measured recombinant receptor-dependent activity. In some embodiments, the measured recombinant receptor-dependent activity is normalized to the maximum activity measured for the therapeutic composition. In some embodiments, the recombinant receptor-dependent activity curve is normalized to the maximum recombinant receptor-dependent activity measured for the therapeutic cell composition. In some embodiments, the recombinant receptor-dependent activity curve is normalized to the upper asymptote of the recombinant receptor-dependent activity measured for the therapeutic cell composition, optionally the average of the values ​​measured across the asymptote.

[0197] In some embodiments, the efficacy of a therapeutic cell composition is the ratio range at which 10% to 90% of the recombinant receptor-dependent activity occurs, or vice versa. In some embodiments, the ratio range at which 10% to 90% of the recombinant receptor-dependent activity occurs is estimated from a recombinant receptor-dependent activity curve. In some embodiments, for example, when the recombinant receptor-dependent activity scale or recombinant receptor-dependent activity curve is normalized, the recombinant receptor-dependent activity value range is 0.1 to 0.9 or 10% to 90%.

[0198] In some embodiments, the efficacy of a therapeutic cell composition is the ratio range at which 20% to 80% of the recombinant receptor-dependent activity occurs, or vice versa. In some embodiments, the ratio range at which 20% to 80% of the recombinant receptor-dependent activity occurs is estimated from a recombinant receptor-dependent activity curve. In some embodiments, for example, when the recombinant receptor-dependent activity scale or recombinant receptor-dependent activity curve is normalized, the recombinant receptor-dependent activity value range is 0.2 to 0.8 or 20% to 80%.

[0199] In some embodiments, the efficacy of a therapeutic cell composition is the ratio range at which 30% to 70% recombinant receptor-dependent activity occurs, or vice versa. In some embodiments, the ratio range at which 30% to 70% recombinant receptor-dependent activity occurs is estimated from a recombinant receptor-dependent activity curve. In some embodiments, for example, when the recombinant receptor-dependent activity scale or recombinant receptor-dependent activity curve is normalized, the recombinant receptor-dependent activity value range is 0.3 to 0.7 or 30% to 70%.

[0200] In some embodiments, the efficacy of a therapeutic cell composition is the ratio range at which 40% to 60% recombinant receptor-dependent activity occurs, or vice versa. In some embodiments, the ratio range at which 40% to 60% recombinant receptor-dependent activity occurs is estimated from a recombinant receptor-dependent activity curve. In some embodiments, for example, when the recombinant receptor-dependent activity scale or recombinant receptor-dependent activity curve is normalized, the recombinant receptor-dependent activity value range is 0.4 to 0.6 or 40% to 60%.

[0201] In some embodiments, the efficacy of a therapeutic cell composition is the range of ratios at which half-maximal recombinant receptor-dependent activity occurs. In some embodiments, the half-maximal value and the ratio at which the half-maximal value occurs are estimated from a recombinant receptor-dependent activity curve. In some embodiments, for example, when the recombinant receptor-dependent activity scale or recombinant receptor-dependent activity curve is normalized, the half-maximal recombinant receptor-dependent activity value is 0.5 or 50%.

[0202] In some embodiments, for example, when recombinant receptor-dependent activity curve is fitted by sigmoid, the linear part of the curve is determined.In some embodiments, efficacy is the measured value and the corresponding ratio from the linear part of the curve.In some embodiments, the half-maximal measured value and ratio are determined from the linear part of the curve.

[0203] 2. Relative potency The methods provided herein allow for the determination of the potency of a therapeutic cell composition relative to a different therapeutic cell composition, e.g., a reference standard. This type of potency is sometimes referred to as relative potency. For example, a therapeutic cell composition evaluated according to the methods provided herein can be compared to a different therapeutic cell composition (e.g., a reference standard, e.g., as described below), e.g., also evaluated according to the methods provided herein, to determine how the potencies of the therapeutic cell compositions relate to one another. This provides the advantage of being able to compare multiple therapeutic cell compositions to determine which composition has the highest or optimal potency. In some embodiments, optimal potency is potency that is capable of inducing a therapeutic effect, e.g., a durable response, progression-free survival, in a subject. In some embodiments, optimal potency is potency that does not result in toxicity in a subject. In some embodiments, optimal potency is potency that is capable of inducing a therapeutic effect, e.g., a durable response, progression-free survival, and does not result in toxicity in a subject.

[0204] In some embodiments, the relative potency of a therapeutic cell composition is defined as the ratio(s) at which one or more or ranges of recombinant receptor-dependent activity measurements of the therapeutic cell composition occur compared to the ratio(s) at which one or more or ranges of recombinant receptor-dependent activity measurements of the reference standard occur. In some embodiments, one or more or ranges of measurements of one or both of the therapeutic cell composition and the reference standard are combined measurements, e.g., mean or median, determined from replicate experiments. In some embodiments, the measurements and ratios of the therapeutic cell composition and the reference standard, respectively, are determined from recombinant receptor-dependent activity curves of the measured recombinant receptor-dependent activity of the composition. In some embodiments, the measured recombinant receptor-dependent activity of the therapeutic cell composition and the reference standard is normalized to the maximum activity measured for the therapeutic composition and the reference standard, respectively. In some embodiments, the recombinant receptor-dependent activity curves of the therapeutic cell composition and the reference standard are normalized to the maximum recombinant receptor-dependent activity measured for the therapeutic cell composition and the reference standard, respectively. In some embodiments, the recombinant receptor-dependent activity curves for the therapeutic cell composition and reference standard are normalized to the upper asymptote of the recombinant receptor-dependent activity measured for the therapeutic cell composition and reference standard, respectively, and, where appropriate, the average of the measured values ​​across the asymptote.

[0205] In some embodiments, the relative potency of a therapeutic cell composition is the ratio range in which 10% to 90% recombinant receptor-dependent activity occurs compared to the range in which 10% to 90% recombinant receptor-dependent activity occurs for a standard reference, or vice versa. In some embodiments, the ratio range in which 10% to 90% recombinant receptor-dependent activity occurs for the therapeutic cell composition and reference standard is estimated from the recombinant receptor-dependent activity curves of the therapeutic cell composition and reference standard, respectively. In some embodiments, for example, when the recombinant receptor-dependent activity scales or recombinant receptor-dependent activity curves of the therapeutic cell composition and reference standard are normalized, the range of recombinant receptor-dependent activity values ​​is 0.1 to 0.9 or 10% to 90%.

[0206] In some embodiments, the relative potency of a therapeutic cell composition is the ratio range in which 20%-80% recombinant receptor-dependent activity occurs compared to the range in which 20%-80% recombinant receptor-dependent activity occurs for the standard reference, or vice versa. In some embodiments, the ratio range in which 20%-80% recombinant receptor-dependent activity occurs for the therapeutic cell composition and the reference standard is estimated from the recombinant receptor-dependent activity curves of the therapeutic cell composition and the reference standard, respectively. In some embodiments, for example, when the recombinant receptor-dependent activity scales or recombinant receptor-dependent activity curves of the therapeutic cell composition and the reference standard are normalized, the range of recombinant receptor-dependent activity values ​​is 0.2-0.8 or 20%-80%.

[0207] In some embodiments, the relative potency of a therapeutic cell composition is the ratio range in which 30%-70% recombinant receptor-dependent activity occurs compared to the range in which 30%-70% recombinant receptor-dependent activity occurs for a standard reference, or vice versa. In some embodiments, the ratio range in which 30%-70% recombinant receptor-dependent activity occurs for a therapeutic cell composition and a reference standard is estimated from the recombinant receptor-dependent activity curves of the therapeutic cell composition and the reference standard, respectively. In some embodiments, for example, when the recombinant receptor-dependent activity scales or recombinant receptor-dependent activity curves of the therapeutic cell composition and the reference standard are normalized, the range of recombinant receptor-dependent activity values ​​is 0.3-0.7 or 30%-70%.

[0208] In some embodiments, the relative potency of a therapeutic cell composition is the ratio range in which 40%-60% recombinant receptor-dependent activity occurs compared to the range in which 30%-70% recombinant receptor-dependent activity occurs for the standard reference, or vice versa. In some embodiments, the ratio range in which 40%-60% recombinant receptor-dependent activity occurs for the therapeutic cell composition and reference standard is estimated from the recombinant receptor-dependent activity curves of the therapeutic cell composition and reference standard, respectively. In some embodiments, for example, when the recombinant receptor-dependent activity scales or recombinant receptor-dependent activity curves of the therapeutic cell composition and reference standard are normalized, the range of recombinant receptor-dependent activity values ​​is 0.4-0.6 or 40%-60%.

[0209] In some embodiments, the relative potency of a therapeutic cell composition is the ratio range at which half-maximal recombinant receptor-dependent activity occurs compared to the range at which 30% to 70% of recombinant receptor-dependent activity occurs for a standard reference. In some embodiments, the half-maximal values ​​and ratios at which half-maximal values ​​occur for the therapeutic cell composition and reference standard are estimated from the recombinant receptor-dependent activity curves of the therapeutic cell composition and reference standard, respectively. In some embodiments, for example, when the recombinant receptor-dependent activity scales or recombinant receptor-dependent activity curves of the therapeutic cell composition and reference standard are normalized, the half-maximal recombinant receptor-dependent activity value is 0.5 or 50%.

[0210] In some embodiments, for example, when the recombinant receptor-dependent activity curves of the therapeutic cell composition and the reference standard are fitted by a sigmoid, the linear portion of the curve is determined. In some embodiments, the relative potency is a comparison of the measured values ​​of the therapeutic cell composition and the corresponding ratio from the linear portion of the curve with the measured values ​​of the reference standard and the corresponding ratio from the linear portion of the curve. In some embodiments, the measured values ​​and ratio of the half-maximal values ​​of the therapeutic cell composition and the reference standard are determined from the linear portion of the curve.

[0211] In some embodiments, the comparison between measurements such as those described above of a therapeutic cell composition and a reference composition is a division. For example, the rate at which half-maximal recombinant receptor-dependent activity of the therapeutic cell composition occurs is divided by the rate at which half-maximal recombinant receptor-dependent activity of the reference standard occurs. In some embodiments, the relative potency is expressed as a ratio. In some embodiments, the relative potency is expressed as a percentage.

[0212] In some embodiments, for example, when the recombinant receptor-dependent activity curves of the therapeutic cell composition and the reference standard are fitted by a sigmoid and normalized as described above, the relative potency is the difference between the curves. In some embodiments, the difference between the curves is measured over the linear portion of the normalized curve. In some embodiments, the recombinant receptor-dependent activity curves of the therapeutic cell composition and the reference standard, e.g., normalized to a sigmoid curve, can be used to directly compare the recombinant receptor-dependent activity curves of the therapeutic cell composition and the reference standard.

[0213] a.Reference standard Certain embodiments contemplate that a measured value of recombinant receptor-dependent activity (e.g., CAR+-dependent activity) of a therapeutic cell composition can be compared to a reference measurement (i.e., a reference scale) of a reference standard to, for example, determine relative potency. In certain embodiments, the reference measurement is a predetermined measurement or value of the recombinant receptor-dependent activity of the reference standard. In some embodiments, the recombinant receptor-dependent activity of the reference standard is assessed according to the methods disclosed herein. In some embodiments, the reference standard is a therapeutic cell composition in which adjusted ratios that result in recombinant receptor-dependent activity have been verified. In some embodiments, the reference standard is a therapeutic cell composition in which adjusted ratios that result in recombinant receptor-dependent activity have been verified, and a curve, e.g., a sigmoid, is fitted to the measured activity to generate a recombinant receptor-dependent activity curve. In some embodiments, the recombinant receptor-dependent activity curve of the reference standard is normalized. In some embodiments, the recombinant receptor-dependent activity curve is normalized to the maximum measured recombinant receptor-dependent activity. In some embodiments, the recombinant receptor-dependent activity curve is normalized to the upper asymptote of the recombinant receptor-dependent activity curve. In some embodiments, the recombinant receptor-dependent activity curve is normalized. The recombinant receptor-dependent activity curve is normalized to a mean value calculated across the upper asymptote of the recombinant receptor-dependent activity curve. In some embodiments, the reference standard is a therapeutic cell composition comprising a validated adjusted ratio that results in half-maximal recombinant receptor-dependent activity. In some embodiments, the validated adjusted ratio that results in half-maximal recombinant receptor-dependent activity is determined from the recombinant receptor-dependent activity curve.

[0214] In some embodiments, the reference standard is a commercially available therapeutic cell composition. In some embodiments, the reference standard is a therapeutic cell composition manufactured using a manufacturing process that is identical to the manufacturing process used to manufacture the therapeutic cell composition being compared. In some embodiments, the reference standard is a therapeutic cell composition manufactured using a manufacturing process that is different from the manufacturing process used to manufacture the therapeutic cell composition being compared. In some embodiments, the reference standard is a therapeutic cell composition that includes the same recombinant receptor as the therapeutic cell composition being compared. In some embodiments, the reference standard is a therapeutic cell composition that includes a different recombinant receptor than the therapeutic cell composition being compared. In some embodiments, the reference standard is a therapeutic cell composition manufactured from the same subject being compared. In some embodiments, the reference standard is a therapeutic cell composition manufactured from a different subject than the therapeutic cell composition being compared. In some embodiments, the reference standard is a therapeutic cell composition derived from a healthy subject. In some embodiments, the reference standard is derived from a subject with a disease or condition. In some embodiments, the reference standard is derived from a subject with cancer. In some embodiments, the reference standard can be a combination of one or more of the above.

[0215] In some embodiments, a reference standard has been administered to a subject. In certain embodiments, administration of the reference standard to a subject has been observed or determined to result in an acceptable safety profile following administration to the subject. In certain embodiments, administration of the reference standard did not result in any severe toxicity. In certain embodiments, administration of the reference standard did not result in any severe neurotoxicity. In certain embodiments, the reference standard is a therapeutic cell composition that has been associated with a neurotoxicity score of Grade 4 or less, Grade 3 or less, Grade 2 or less, Grade 1 or less, or Grade 0. In some embodiments, the reference standard has been associated with an acceptable safety profile. In certain embodiments, an acceptable safety profile is the absence of observed Grade 1 or greater, observed Grade 2 or greater, observed Grade 3 or greater, or Grade 4 or greater neurotoxicity. In certain embodiments, the reference standard is associated with an acceptable safety profile of the absence of observed Grade 3 or greater neurotoxicity. In certain embodiments, the reference standard is associated with an acceptable safety profile of the absence of Grade 3 or greater neurotoxicity.

[0216] In certain embodiments, the reference standard has been observed or determined to produce desired efficacy after administration to a subject.In certain embodiments, the subject expresses the antigen or has a disease or condition related to the antigen, similar to the subject who is administered the reference standard.In certain embodiments, the reference standard has been observed or determined to produce complete response (CR).In certain embodiments, the reference standard has been observed or determined to produce sustained response.

[0217] II. Methods for generating engineered T cells In some embodiments, the therapeutic cell composition efficacy methods provided herein can be used in connection with generating therapeutic compositions (e.g., output compositions) of engineered cells, e.g., engineered CD4+ T cells and / or engineered CD8+ T cells expressing recombinant proteins, e.g., recombinant receptors, such as T cell receptors (TCRs) or chimeric antigen receptors (CARs). In some embodiments, the methods provided herein are used in connection with manufacturing, producing, or generating cell therapies and can be used in connection with further processing steps, such as steps for isolating, separating, selecting, activating or stimulating, transducing, washing, suspending, diluting, concentrating, and / or formulating cells. In some embodiments, methods of making or generating engineered cells, e.g., engineered CD4+ T cells and / or engineered CD8+ T cells, include one or more of isolating cells from a subject, preparing under stimulatory conditions, treating, incubating, and / or manipulating (e.g., transducing) the cells. In some embodiments, the method includes processing steps performed in the following order: first isolating, e.g., selecting or separating, input cells, e.g., primary cells, from a biological sample; incubating the input cells under stimulatory conditions and manipulating them with vector particles, e.g., viral vector particles, e.g., by transduction or transfection to introduce a recombinant polynucleotide into the cells; culturing the manipulated, e.g., transduced, cells, e.g., to expand the cells; and harvesting, collecting, and / or loading all or a portion of the cells into a container to formulate the cells into an output composition. In some embodiments, CD4+ and CD8+ T cells are produced independently of each other, e.g., in separate input compositions, but the manufacturing process includes the same processing steps. In some embodiments, CD4+ and CD8+ T cells are produced together, e.g., in the same input composition. In some embodiments, the cells of the generated output composition (e.g., a therapeutic cell composition) are reintroduced into the same subject before or after cryopreservation.In some embodiments, the engineered cell output composition (e.g., a therapeutic cell composition) is suitable for use in therapy, e.g., autologous cell therapy, allogeneic cell therapy. Exemplary manufacturing methods are described in published international patent application, publication number WO2019 / 089855, the disclosure of which is incorporated herein by reference in its entirety.

[0218] A. Sample and Cell Preparation In certain embodiments, the provided methods are used in connection with isolating, selecting, and / or enriching cells from a biological sample to generate one or more input compositions of enriched cells, e.g., T cells. In some embodiments, the provided methods include isolating cells or compositions thereof from a sample, e.g., obtained from or derived from a subject, e.g., one with a particular disease or condition, or one in need of or to which cell therapy is administered. In some aspects, the subject is a human, e.g., a patient in need of a particular therapeutic intervention, e.g., adoptive cell therapy, from which the cells are isolated, processed, and / or manipulated. Thus, the cells, in some embodiments, are primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject. Biological samples can be samples obtained directly from a biological source or samples to be processed. Biological samples include, but are not limited to, bodily fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue, and organ samples, including processed samples derived therefrom.

[0219] In some embodiments, the sample is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils, or other organ and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0220] In some examples, cells are obtained from the subject's circulating blood, for example, by apheresis or leukapheresis. The sample, in some embodiments, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and / or platelets, and in some embodiments, cells other than red blood cells and platelets.

[0221] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or most or all divalent cations. In some aspects, the wash step is accomplished by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 Cell Processor, Baxter) according to the manufacturer's instructions. In some aspects, the wash step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, after washing, the cells are washed in various biocompatible buffers, e.g., Ca ++ / Mg ++ In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0222] In some embodiments, the preparation method includes a step of freezing, e.g., cryoprotecting, the cells either before or after incubation for isolation, selection, and / or enrichment, and / or transduction and manipulation, and / or after culturing and / or recovery of the manipulated cells. In some embodiments, the freezing and subsequent thawing step removes granulocytes and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters may be used in some aspects. In some embodiments, cells are frozen, e.g., cryofrozen or cryopreserved, in medium and / or solution with a final concentration of at or about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO or between 1% and 15%, between 6% and 12%, between 5% and 10%, or between 6% and 8% DMSO. In certain embodiments, cells are frozen, e.g., cryofrozen or cryopreserved, in a medium and / or solution having a final concentration of at or about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25% HSA, or between 0.1% and -5%, 0.25% and 4%, 0.5% and 2%, or 1% and 2% HSA. One example includes using PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HSA). This is then diluted 1:1 with medium, resulting in final concentrations of DMSO and HAS of 10% and 4%, respectively.The cells are then typically frozen to or about -80°C at a rate of at or about 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0223] In some embodiments, isolation of a cell or population involves one or more preparative and / or non-affinity-based cell separation steps. In some instances, 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, or lyse or remove cells sensitive to a particular reagent. In some instances, cells are separated based on one or more properties, such as density, adhesive properties, size, sensitivity and / or resistance to a particular component. In some embodiments, the method involves a density-based cell separation method, such as preparing white blood cells from peripheral blood by lysis of red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0224] In some embodiments, at least a portion of the selection step involves incubation of cells with a selection reagent. For example, incubation with a selection reagent(s) as part of the selection method can be performed using one or more selection reagents for selection of one or more different cell types based on the expression or presence of one or more specific molecules, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, in or on the cells. In some embodiments, any known method using a selection reagent(s) for separation based on such markers can be used. In some embodiments, the selection reagent(s) results in a separation that is affinity- or immunoaffinity-based separation. For example, selection, in some aspects, involves incubation with a reagent(s) for separating cells and cell populations based on the cellular expression or expression level of one or more markers, usually cell surface markers, e.g., by incubation with an antibody or binding partner that specifically binds to such marker, typically followed by a washing step and separation of cells bound to the antibody or binding partner from cells not bound to the antibody or binding partner.

[0225] In some aspects of such processes, a fixed volume of cells is mixed with a fixed amount of a desired affinity-based selection reagent. Immunoaffinity-based selection can be performed using any system or method that results in favorable energy interactions between the cells being separated and molecules that specifically bind to markers on the cells, e.g., antibodies or other binding partners on a solid surface, e.g., particles. In some embodiments, the method is performed using particles, e.g., beads, e.g., magnetic beads, that are coated with a selection agent (e.g., antibody) specific for a marker on the cells. The particles (e.g., beads) can be incubated or mixed with the cells in a container, such as a tube or bag, with shaking or mixing, at a constant cell density-to-particle (e.g., bead) ratio to help promote energetically favorable interactions. In other cases, the method includes cell selection, and all or a portion of the selection is performed, for example, in the internal cavity of a centrifuge chamber under centrifugal rotation. In some embodiments, incubation of the cells with a selection reagent, e.g., an immunoaffinity-based selection reagent, is performed in the centrifuge chamber. In certain embodiments, the isolation or separation is performed using a system, device, or apparatus described in International Patent Application Publication No. WO2009 / 072003 or US20110003380. In one example, the system is a system such as that described in International Publication No. WO2016 / 073602.

[0226] In some embodiments, performing such selection steps, or portions thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads), in the cavities of a centrifuge chamber allows the user to control certain parameters, such as the volumes of various solutions, the addition of solutions during processing, and their timing, thereby providing advantages over other available methods. For example, the ability to reduce the liquid volume in the cavity during incubation can increase the concentration of the particles (e.g., bead reagents) used for selection, and therefore the chemical potential of the solution, without affecting the total number of cells in the cavity. This, in turn, can enhance pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, performing the incubation step in the chamber, e.g., when associated with the systems, circuits, and controls described herein, allows the user to agitate the solution at desired time(s) during incubation, which may also improve interactions.

[0227] In some embodiments, at least a portion of the selection step, including incubation of cells with a selection reagent, is performed in a centrifuge chamber. In some aspects of such processes, a fixed volume of cells is mixed with a fixed amount of the desired affinity-based selection reagent that is significantly less than that normally used when performing a similar selection in a tube or container to select the same number of cells and / or volume of cells according to the manufacturer's instructions. In some embodiments, an amount of selection reagent(s) is used that is 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 50% or less, 60% or less, 70% or less, or 80% or less of the same selection reagent(s) used to select cells in a tube- or solution-based incubation of the same number of cells and / or volume of cells according to the manufacturer's instructions.

[0228] In some embodiments, for cell selection, e.g., immunoaffinity-based selection, cells are incubated in a chamber cavity in a composition that also contains a selection buffer with a selection reagent, e.g., a molecule that specifically binds to a surface marker on cells desired to be enriched and / or depleted, but not on other cells in the composition, e.g., an antibody that may be coupled to a polymer or surface, e.g., a scaffold, such as beads, e.g., magnetic beads, e.g., magnetic beads coupled to monoclonal antibodies specific for CD4 and CD8. In some embodiments as described, the selection reagent is added to the cells in the chamber cavity in a substantially reduced amount (e.g., no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the amount) compared to the amount of selection reagent that would normally be used or required to achieve approximately the same or similar efficiency of selection of the same number of cells or the same volume of cells when selection is performed in a tube with shaking or rotation. In some embodiments, incubation is performed by adding a selection buffer to the cells and selection reagent to achieve a target volume, e.g., 10 mL to 200 mL, e.g., at least or about at least or about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL of reagent. In some embodiments, the selection buffer and selection reagent are premixed prior to addition of the cells. In some embodiments, the selection buffer and selection reagent are added separately to the cells. In some embodiments, the selection incubation is performed using periodic gentle mixing conditions, which can help promote energetically favorable interactions, thereby allowing the use of less overall selection reagent while achieving high selection efficiency.

[0229] In some embodiments, the total duration of incubation with the selection reagent is from 5 minutes to 6 hours or from about 5 minutes to about 6 hours, such as from 30 minutes to 3 hours, for example, at least or about at least 30 minutes, 60 minutes, 120 minutes or 180 minutes.

[0230] In some embodiments, incubation is generally carried out under mixing conditions, such as in the presence of rotation, generally at a relatively low force or speed, e.g., a speed lower than that used to pellet the cells, e.g., at or about 600 rpm to 1700 rpm (e.g., 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or about 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm), e.g., at an RCF of 80 g to 100 g, or about 80 g to about 100 g (e.g., 80 g, 85 g, 90 g, 95 g, or 100 g, or about 80 g, 85 g, 90 g, 95 g, or 100 g, or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, rotation is performed using a repeat interval of rotation at a slow speed followed by a rest period, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds of rotation and / or rest, e.g., approximately 1 or 2 seconds of rotation followed by approximately 5, 6, 7, or 8 seconds of rest.

[0231] In some embodiments, such a process is carried out in a completely closed system in which the chamber is essential. In some embodiments, this process (and in some aspects, one or more additional steps, such as a washing step before washing a cell-containing sample, such as an apheresis sample) is carried out in an automated manner using an automated program to draw cells, reagents, and other components into and push them out of the chamber at the appropriate time in a single closed system, and centrifugation is performed to complete the washing and binding steps.

[0232] In some embodiments, after incubation and / or mixing of the cells and selection reagent(s), the incubated cells are subjected to separation to select for cells based on the presence or absence of specific reagent(s). In some embodiments, the separation is performed in the same closed system in which the incubation of the cells with the selection reagent was performed. In some embodiments, after incubation with the selection reagent, the incubated cells, including the selection reagent-bound cells, are transferred into a system for immunoaffinity-based separation of cells. In some embodiments, the system for immunoaffinity-based separation is or contains a magnetic separation column.

[0233] Such separation steps may be based on positive selection, in which cells that bind a reagent, e.g., an antibody or binding partner, are retained for further use, and / or negative selection, in which cells that do not bind a reagent, e.g., an antibody or binding partner, are retained. In some instances, both fractions are retained for further use. In some embodiments, negative selection may be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, such that separation is best performed based on markers expressed by cells other than the desired population.

[0234] In some embodiments, the process steps further comprise negative and / or positive selection of the incubated cells, e.g., using a system or device capable of performing affinity-based selection. In some embodiments, isolation is performed by enriching for a particular cell population by positive selection, or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection selects cells for a marker expressed at or relatively high levels on the positively or negatively selected cells, respectively. high) is achieved by incubation with one or more antibodies or other binding agents that specifically bind to the expressed (marker+) one or more surface markers. Multiple rounds of the same selection step, e.g., positive or negative selection step, can be performed. In certain embodiments, the positively or negatively selected fraction is subjected to a selection process, such as by repeating the positive or negative selection step. In some embodiments, the selection is repeated two, three, four, five, six, seven, eight, nine, or more than nine times. In certain embodiments, the same selection is performed up to five times. In certain embodiments, the same selection step is performed three times.

[0235] Separation does not necessarily result in the enrichment or removal of 100% of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as one expressing a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as one expressing a marker, refers to reducing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0236] In some cases, multiple rounds of separation steps are performed, and 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 cases, a single separation step can simultaneously deplete cells expressing multiple markers by incubating cells with multiple antibodies or binding partners, each specific for a marker targeted for negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed in various cell types. In certain embodiments, one or more separation steps are repeated and / or performed more than once. In some embodiments, by repeating the positive or negative selection step, the positively or negatively selected fraction resulting from the separation step is subjected to the same separation step. In some embodiments, a single separation step is repeated and / or performed more than once to increase the yield of positively selected cells, increase the purity of negatively selected cells, and / or further remove positively selected cells from the negatively selected fraction. In certain embodiments, one or more separation steps are performed and / or repeated 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In certain embodiments, one or more selection steps are performed and / or repeated between 1 and 10 times, between 1 and 5 times, or between 4 and 5 times. In certain embodiments, one or more selection steps are repeated 3 times.

[0237] For example, in some aspects, specific subpopulations of T cells, e.g., cells expressing positive or high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more antibodies or binding partners that specifically bind to such markers. In some embodiments, selection can be achieved by conjugating the antibodies or binding partners, directly or indirectly, to a solid support or matrix, such as magnetic or paramagnetic beads. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28-conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander and / or ExpACT® beads).

[0238] In some embodiments, T cells are isolated from PBMC samples by negative selection for markers expressed on non-T cells, e.g., B cells, monocytes, or other leukocytes, e.g., CD14. 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 subpopulations by positive or negative selection for markers expressed on, or relatively highly expressed in, one or more naive, memory, and / or effector T cell subpopulations.

[0239] In some embodiments, CD8+ T cells are further enriched or depleted for naive stem cells, central memory stem cells, effector memory stem cells, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to enhance efficacy, e.g., to improve long-term survival, expansion, and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, efficacy is further enhanced by combining TCM-enriched CD8+ T cells and CD4+ T cells.

[0240] In embodiments, memory T cells reside in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for the CD62L-CD8+ and / or CD62L+CD8+ fractions using, for example, anti-CD8 and anti-CD62L antibodies.

[0241] In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127, and in some aspects is based on negative selection for cells that express or highly express CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA, positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is performed starting with a negative fraction of cells selected on the basis of CD4 expression, which is subjected to negative selection on the basis of CD14 and CD45RA expression and positive selection on the basis of CD62L.

[0242] In some embodiments, such selection is performed simultaneously, and in other embodiments, sequentially in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ T cell population or subpopulation is also used to generate the CD4+ T cell population or subpopulation, such that both the positive and negative fractions obtained from the CD4-based separation are retained and used in subsequent steps of the method, optionally in one or more additional positive or negative selection steps below. In some embodiments, the selection of the CD4+ T cell population and the selection of the CD8+ T cell population are performed simultaneously. In some embodiments, the selection of the CD4+ T cell population and the CD8+ T cell population are performed sequentially in either order. In some embodiments, methods of selecting cells may include those described in published U.S. application number US20170037369. In some embodiments, the selected CD4+ T cell population and the selected CD8+ T cell population may be combined after selection. In some aspects, the selected CD4+ T cell population and the selected CD8+ T cell population may be combined in a bioreactor bag as described herein. In some embodiments, the selected CD4+ T cell population and the selected CD8+ T cell population are treated separately, whereby the selected CD4+ T cell population is enriched for CD4+ T cells, incubated with stimulatory reagents (e.g., anti-CD3 / anti-CD28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g., a CAR), and cultured under conditions that expand T cells, and the selected CD8+ T cell population is enriched for CD8+ T cells, incubated with stimulatory reagents (e.g., anti-CD3 / anti-CD28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g., a CAR), e.g., the same recombinant protein as for engineering CD4+ T cells from the same donor, and cultured under conditions that expand T cells, such as consistent with the methods provided.

[0243] In certain embodiments, a biological sample, for example, a sample of PBMCs or other white blood cells, is subjected to selection of CD4+ T cells, and both negative and positive fractions are retained. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to selection of CD8+ T cells, and both negative and positive fractions are retained. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0244] In a specific example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ T cells, and both negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on markers characteristic of central memory T cells, such as CD62L or CCR7, with the positive and negative selections being performed in either order.

[0245] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, or CD4+ T cells. In some embodiments, central memory CD4+ T cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ T cells are CD62L- and CD45RO-.

[0246] In one example, to enrich CD4+ T cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic or paramagnetic bead, to allow for cell separation for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) 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: SA Brooks and U. Schumacher (c) Humana Press Inc., Totowa, NJ).

[0247] In some embodiments, the incubated sample or composition of cells to be separated is incubated with a selection reagent containing a small, magnetizable, or magnetically responsive material, e.g., magnetically responsive particles or microparticles, e.g., paramagnetic beads (e.g., Dynalbeads or MACS® beads, etc.). The magnetically responsive material, e.g., particles, are generally attached, directly or indirectly, to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on the cell, cells, or population of cells that is desired to be separated, e.g., negatively or positively selected.

[0248] In some embodiments, magnetic particles or beads comprise a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner.Many well-known magnetically responsive materials are known for use in magnetic separation methods, such as those described in Molday, U.S. Patent No. 4,452,773, and European Patent Specification EP452342B, which are incorporated herein by reference.Colloidal-sized particles can also be used, such as those described in Owen, U.S. Patent No. 4,795,698, and Liberti et al., U.S. Patent No. 5,200,084.

[0249] Incubation is generally carried out under conditions in which the antibody or binding partner attached to the magnetic particle or bead, or a molecule that specifically binds to such an antibody or binding partner, e.g., a secondary antibody or other reagent, specifically binds to the cell surface molecule if present on cells in the sample.

[0250] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are attached to cells via coating with a primary antibody specific to one or more markers. In certain embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell-type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used together with biotinylated primary or secondary antibodies.

[0251] In some embodiments, separation is achieved by placing a sample in a magnetic field, and cells with magnetically responsive or magnetizable particles attached are attracted to the magnet and separated from unlabeled cells. For positive selection, cells that are attracted to the magnet are retained, and for negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed during the same selection step, and positive and negative fractions are retained and further processed or subjected to additional separation steps.

[0252] In some embodiments, affinity-based selection is via magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetically activated cell sorting (MACS), for example, the CliniMACS system, allows for high-purity selection of cells attached to magnetized particles. In certain embodiments, MACS operates in a manner in which non-target and target species are sequentially eluted after applying an external magnetic field. That is, cells attached to magnetized particles remain in place, while unattached species are eluted. Then, after this first elution step is completed, species trapped in the magnetic field and prevented from elution are released in some manner so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous population of cells.

[0253] In some embodiments, the magnetically responsive particles remain attached to the cells that are then incubated, cultured, and / or manipulated, and in some aspects, the particles remain 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, for example, the use of competitive unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0254] In some embodiments, the isolation and / or selection results in one or more input compositions of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, two or more separate input compositions are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, the separate input compositions are isolated, selected, enriched, and / or obtained from separate biological samples collected, harvested, and / or obtained from the same subject.

[0255] In certain embodiments, the one or more input compositions are or comprise a composition of enriched T cells that comprises 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 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD3+ T cells. In certain embodiments, the enriched T cell input composition consists essentially of CD3+ T cells.

[0256] In certain embodiments, one or more input compositions are or comprise an enriched CD4+ T cell composition comprising 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 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD4+ T cells. In certain embodiments, the input CD4+ T cell composition comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or contains no CD8+ T cells and / or is free or substantially free of CD8+ T cells. In some embodiments, the enriched T cell composition consists essentially of CD4+ T cells.

[0257] In certain embodiments, one or more compositions are or comprise compositions of CD8+ T cells that are or comprise 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 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD8+ T cells. In certain embodiments, the composition of CD8+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or no CD4+ T cells, and / or is free or substantially free of CD4+ T cells. In some embodiments, the enriched T cell composition consists essentially of CD8+ T cells.

[0258] In some embodiments, one or more input compositions of enriched T cells are frozen, e.g., cryopreserved and / or cryofrozen, after isolation, selection, and / or enrichment. In some embodiments, one or more input compositions are frozen, e.g., cryopreserved and / or cryofrozen, before any step, including incubating, activating, stimulating, manipulating, transducing, transfecting, culturing, expanding, recovering, and / or formulating the composition of cells. In certain embodiments, one or more cryopreserved input compositions are stored, e.g., at or about -80°C, for between 12 hours and 7 days, between 24 hours and 120 hours, or between 2 and 5 days. In certain embodiments, one or more cryopreserved input compositions are stored at or about -80°C for an amount of time less than 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the one or more cryo-frozen input compositions are stored at or about -80°C for 1, 2, 3, 4, 5, or 6 days or for about 1, 2, 3, 4, 5, or 6 days.

[0259] B. Cell Activation and Stimulation In some embodiments, the provided methods are used in conjunction with incubating cells under stimulatory conditions. In some embodiments, the stimulatory conditions include conditions that activate or stimulate and / or are capable of activating or stimulating signals in cells, e.g., CD4+ T cells or CD8+ T cells, e.g., signals arising from the TCR and / or co-receptor. In some embodiments, the stimulatory conditions include one or more steps of culturing, cultivating, incubating, activating, or expanding cells with and / or in the presence of a stimulatory reagent, e.g., a reagent that activates or stimulates and / or is capable of activating or stimulating signals in the cells. In some embodiments, the stimulatory reagent stimulates and / or activates the TCR and / or co-receptor. In certain embodiments, the stimulatory reagent is a reagent described in Section II-B-1.

[0260] In certain embodiments, one or more compositions of enriched T cells are incubated under stimulatory conditions before genetically engineering the cells, e.g., transfecting and / or transducing the cells, such as by the techniques provided in Section II-C. In certain embodiments, one or more compositions of enriched T cells are incubated under stimulatory conditions after the one or more compositions have been isolated, selected, enriched, or obtained from a biological sample. In certain embodiments, the one or more compositions are input compositions. In certain embodiments, the one or more input compositions have been previously cryo-frozen, stored, and thawed prior to incubation.

[0261] In certain embodiments, the one or more compositions of enriched T cells are or comprise two separate compositions of enriched T cells, e.g., separate input compositions. In certain embodiments, the two separate compositions of enriched T cells, e.g., two separate compositions of enriched T cells selected, isolated, and / or enriched from the same biological sample, are incubated separately under stimulatory conditions. In certain embodiments, the two separate compositions comprise a composition of enriched CD4+ T cells. In certain embodiments, the two separate compositions comprise a composition of enriched CD8+ T cells. In some embodiments, the two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells are incubated separately under stimulatory conditions.

[0262] In some embodiments, the single composition of enriched T cells is incubated under stimulatory conditions. In certain embodiments, the single composition is a composition of enriched CD4+ T cells. In some embodiments, the single composition is a composition of enriched CD4+ and CD8+ T cells that are combined from separate compositions before incubation.

[0263] In some embodiments, the composition of enriched CD4+ T cells incubated under stimulatory conditions comprises 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 98%, at least 99%, at least 99.5%, at least 99.9%, or 100%, or about 100%, CD4+ T cells. In certain embodiments, the composition of enriched CD4+ T cells incubated under stimulatory conditions comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or is free and / or substantially free of CD8+ T cells.

[0264] In some embodiments, the composition of enriched CD8+ T cells incubated under stimulatory conditions comprises 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 98%, at least 99%, at least 99.5%, at least 99.9%, or 100%, or about 100%, CD8+ T cells. In certain embodiments, the composition of enriched CD8+ T cells incubated under stimulatory conditions comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or is free and / or substantially free of CD4+ T cells.

[0265] In some embodiments, the separate compositions of enriched CD4+ and CD8+ T cells are combined into a single composition and incubated under stimulatory conditions. In certain embodiments, the separate stimulated compositions of enriched CD4+ and enriched CD8+ T cells are combined into a single composition after incubation has been performed and / or completed. In some embodiments, the separate stimulated compositions of stimulated CD4+ and stimulated CD8+ T cells are processed separately after incubation is performed and / or completed, whereby the stimulated CD4+ T cell population (e.g., incubated with a stimulatory anti-CD3 / anti-CD28 magnetic bead stimulation reagent) is transduced with a viral vector encoding a recombinant protein (e.g., a CAR) and cultured under conditions that expand T cells, and the stimulated CD8+ T cell population (e.g., incubated with a stimulatory anti-CD3 / anti-CD28 magnetic bead stimulation reagent) is transduced with a viral vector encoding a recombinant protein (e.g., a CAR), e.g., the same recombinant protein as for engineering CD4+ T cells from the same donor, and cultured under conditions that expand T cells, such as consistent with the methods provided.

[0266] In some embodiments, incubation under stimulatory conditions can include culturing, culturing, stimulating, activating, and growing, including by incubation in the presence of stimulatory conditions, e.g., conditions designed to stimulate cells to induce proliferation, expansion, activation, and / or survival of cells in a population, to mimic antigen exposure, and / or for genetic manipulation, e.g., introduction of a recombinant antigen receptor. In certain embodiments, stimulatory conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0267] In some embodiments, stimulation and / or incubation under stimulatory conditions is performed according to techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0268] In some embodiments, cells, e.g., T cells, compositions of cells and / or cell populations, e.g., CD4 + and CD8 +T cells or compositions, populations, or subpopulations thereof, are expanded by adding the culture starting composition to feeder cells, e.g., non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., so that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded) and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with gamma rays in the range of about 3000-3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the population of T cells.

[0269] In some embodiments, the stimulatory conditions include a temperature suitable for the proliferation of human T lymphocytes, e.g., at least about 25°C, typically at least about 30°C, typically 37 or about 37°C. In some embodiments, a temperature shift, e.g., from 37°C to 35°C, is achieved during culture. Optionally, incubation may include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be gamma-irradiated in the range of about 6,000 to 10,000 rads. The LCL feeder cells, in some aspects, are provided in any suitable amount, e.g., at a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

[0270] In embodiments, antigen-specific CD4 + and CD8 + A population of cytomegalovirus antigens can be obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones against cytomegalovirus antigens can be generated by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen. Naive T cells can also be used.

[0271] In certain embodiments, the stimulatory conditions include incubating, culturing, and / or culturing the cells with a stimulatory reagent. In certain embodiments, the stimulatory reagent is a reagent described in Section II-B-1. In certain embodiments, the stimulatory reagent contains or comprises beads. An exemplary stimulatory reagent is or comprises anti-CD3 / anti-CD28 magnetic beads. In certain embodiments, the beginning and / or initiation of incubating, culturing, and / or culturing the cells under stimulatory conditions occurs upon contacting and / or incubating the cells with the stimulatory reagent. In certain embodiments, the cells are incubated before, during, and / or after genetic manipulation of the cells, e.g., introduction of a recombinant polynucleotide into the cells, such as by transduction or transfection.

[0272] In some embodiments, the enriched T cell composition is incubated with a stimulatory reagent and / or beads, e.g., anti-CD3 / anti-CD28 magnetic beads, to cell ratio of at or about 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1. In certain embodiments, the ratio of stimulation reagent and / or beads to cells is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, between 1.1:1 and 0.9:1, In certain embodiments, the ratio of stimulation reagent to cells is about 1:1 or is 1:1.

[0273] In certain embodiments, incubating cells at a ratio of stimulating reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, of 3:1 or less than 3 per cell, e.g., a 1:1 ratio, reduces the amount of cell death that occurs during incubation, such as by activation-induced cell death. In some embodiments, cells are incubated with stimulating reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, at a ratio of beads to cells of less than 3 (or 3:1 or less than 3 beads per cell). In certain embodiments, incubating cells at a ratio of less than 3:1 or less than 3 beads per cell, e.g., a 1:1 ratio, reduces the amount of cell death that occurs during incubation, such as by activation-induced cell death.

[0274] In certain embodiments, the enriched T cell composition is incubated with a stimulating reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, at a ratio of stimulating reagent and / or beads per cell of less than 3:1, such as a 1:1 ratio, and at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the surviving T cells are viable, e.g., viable and / or do not undergo necrosis, programmed cell death, or apoptosis, for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days after the incubation is completed. In certain embodiments, the enriched T cell composition is incubated with a stimulating reagent at a ratio of stimulating reagent and / or beads per cell of less than 3:1, e.g., a 1:1 ratio, and less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the cells undergo activation-induced cell death upon incubation.

[0275] In certain embodiments, the composition of enriched T cells is incubated with a stimulatory reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, at a ratio of beads per cells of less than 3:1, e.g., a 1:1 ratio, and the cells of the composition have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold greater survival compared to cells undergoing exemplary and / or alternative processes in which the composition of enriched T cells is incubated with a stimulatory reagent at a ratio of 3:1 or greater.

[0276] In some embodiments, the composition of enriched T cells incubated with a stimulatory reagent comprises 1.0 x 10 5 Individual cells / mL~1.0×10 8 cells / mL or approximately 1.0 x 10 5 Individual cells / mL ~ approx. 1.0×10 8 cells / mL, e.g., at least or about at least or about 1.0 x 10 5 Individual cells / mL, 5×10 5 Individual cells / mL, 1×10 6 Individual cells / mL, 5×10 6 Individual cells / mL, 1×10 7 Individual cells / mL, 5×10 7 cells / mL or 1 x 10 8 In some embodiments, the composition of enriched T cells incubated with a stimulatory reagent comprises about 0.5 x 10 cells / mL. 6 Individual cells / mL, 1×10 6 Individual cells / mL, 1.5×10 6 Individual cells / mL, 2×10 6 Individual cells / mL, 2.5×10 6 individual cells / mL, 3×10 6 Individual cells / mL, 3.5×10 6 Individual cells / mL, 4×10 6Individual cells / mL, 4.5×10 6 Individual cells / mL, 5×10 6 Individual cells / mL, 5.5×10 6 Individual cells / mL, 6×10 6 Individual cells / mL, 6.5×10 6 Individual cells / mL, 7×10 6 Individual cells / mL, 7.5×10 6 individual cells / mL, 8×10 6 Individual cells / mL, 8.5×10 6 Individual cells / mL, 9×10 6 Individual cells / mL, 9.5×10 6 cells / mL or 10 x 10 6 cells / mL, e.g., approximately 2.4 x 10 6 Contains 10 cells / mL.

[0277] In some embodiments, the enriched T cell composition is incubated with the stimulatory reagent at a temperature of about 25 to about 38°C, e.g., about 30 to about 37°C, e.g., about 37°C ± 2°C. In some embodiments, the enriched T cell composition is incubated with the stimulatory reagent at a CO2 level of about 2.5% to about 7.5%, e.g., about 4% to about 6%, e.g., 5% ± 0.5% or about 5% ± 0.5%. In some embodiments, the enriched T cell composition is incubated with the stimulatory reagent at a temperature of at or about 37°C and / or a CO2 level of 5% or about 5%.

[0278] In certain embodiments, the stimulatory conditions include incubating, culturing, and / or cultivating the enriched T cell composition with and / or in the presence of one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In some 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 a receptor expressed by and / or endogenous to T cells. In certain embodiments, the one or more cytokines are or include members of the four-alpha-helical bundle family of cytokines. In some embodiments, members of the four-alpha-helical 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 some embodiments, the one or more cytokines are or include IL-15. In certain embodiments, the one or more cytokines are or include IL-7. In certain embodiments, the one or more cytokines are or include IL-2. In some embodiments, the stimulatory conditions include incubating a composition of enriched T cells, e.g., enriched CD4+ T cells or enriched CD8+ T cells, in the presence of a stimulatory reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, and in the presence of one or more recombinant cytokines, as described.

[0279] In certain embodiments, the enriched CD4+ T cell composition is incubated with IL-2, e.g., recombinant IL-2. Without wishing to be bound by theory, certain embodiments contemplate that CD4+ T cells obtained from some subjects do not produce, or do not produce enough, IL-2 in amounts that allow for growth, division, and expansion throughout the process of generating a composition of output cells, e.g., engineered cells, suitable for use in cell therapy. In some embodiments, incubating the enriched CD4+ T cell composition under stimulatory conditions in the presence of recombinant IL-2 increases the probability or likelihood that the CD4+ T cells of the composition will continue to survive, grow, expand, and / or be activated during the incubation step and throughout the process. In some embodiments, incubating the composition of enriched CD4+ T cells in the presence of recombinant IL-2 reduces the probability and / or likelihood that an enriched CD4+ T cell output composition suitable for cell therapy, e.g., engineered CD4+ T cells, will be generated from the composition of enriched CD4+ T cells by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or more, compared to an alternative and / or exemplary method that does not incubate the composition of enriched CD4+ T cells in the presence of recombinant IL-2. at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 25 fold, at least 50 fold or at least 100 fold increase.

[0280] In certain embodiments, the amount or concentration of one or more cytokines is measured and / or quantified using International Units (IU). International units can be used to quantify vitamins, hormones, cytokines, vaccines, blood products, and similar biologically active substances. In some embodiments, an IU is or includes a unit of measurement of the potency of a biological preparation by comparison to an international reference standard of specific gravity and strength, e.g., the WHO First International Standard for Human IL-2, 86 / 504. International units are the only recognized and standardized method for reporting biological activity units, which is published and derived from international collaborative research efforts. In certain embodiments, the IU of a cytokine composition, sample, or source can be obtained by product comparison testing using a similar WHO standard product. For example, in some embodiments, the IU / mg of a composition, sample, or source of human recombinant IL-2, IL-7, or IL-15 is compared to a WHO standard IL-2 product (NIBSC code: 86 / 500), a WHO standard IL-17 product (NIBSC code: 90 / 530), and a WHO standard IL-15 product (NIBSC code: 95 / 554), respectively.

[0281] In some embodiments, the biological activity in IU / mg is expressed as the ED 50 ) -1 x10 6 In certain embodiments, the ED of recombinant human IL-2 or IL-15 is equivalent to 50 is equivalent to the concentration required for half-maximal stimulation of cell proliferation (XTT cleavage) using CTLL-2 cells. In certain embodiments, the ED of recombinant human IL-7 50is equivalent to the concentration required for half-maximal stimulation of PHA-activated human peripheral blood lymphocyte proliferation. Details relating to IL-2 assays and IU calculations are discussed in Wadhwa et al., Journal of Immunological Methods (2013), 379 (1-2): 1-7; and Gearing and Thorpe, Journal of Immunological Methods (1988), 114 (1-2): 3-9, and details relating to IL-15 assays and IU calculations are discussed in Soman et al., Journal of Immunological Methods (2009) 348 (1-2): 83-94, which are incorporated herein by reference in their entirety.

[0282] In certain embodiments, the enriched CD8+ T cell composition is incubated under stimulatory conditions in the presence of IL-2 and / or IL-15. In certain embodiments, the enriched CD4+ T cell composition is incubated under stimulatory conditions in the presence of IL-2, IL-7, and / or IL-15. In some embodiments, the IL-2, IL-7, and / or IL-15 are recombinant. In certain embodiments, the IL-2, IL-7, and / or IL-15 are human. In certain embodiments, the one or more cytokines are or comprise human recombinant IL-2, IL-7, and / or IL-15. In some aspects, incubation of the enriched T cell composition also includes the presence of a stimulatory reagent, e.g., anti-CD3 / anti-CD28 magnetic beads.

[0283] In some embodiments, the cells are incubated with a cytokine, e.g., a recombinant human cytokine, at a concentration of between 1 IU / ml and 1,000 IU / ml, between 10 IU / ml and 50 IU / ml, between 50 IU / ml and 100 IU / ml, between 100 IU / ml and 200 IU / ml, between 100 IU / ml and 500 IU / ml, between 250 IU / ml and 500 IU / ml, or between 500 IU / ml and 1,000 IU / ml.

[0284] In some embodiments, the enriched T cell composition is incubated with IL-2, e.g., human recombinant IL-2, at a concentration of between 1 IU / ml and 200 IU / ml, between 10 IU / ml and 200 IU / ml, between 10 IU / ml and 100 IU / ml, between 50 IU / ml and 150 IU / ml, between 80 IU / ml and 120 IU / ml, between 60 IU / ml and 90 IU / ml, or between 70 IU / ml and 90 IU / ml. In certain embodiments, the enriched T cell composition comprises 50 IU / ml, 55 IU / ml, 60 IU / ml, 65 IU / ml, 70 IU / ml, 75 IU / ml, 80 IU / ml, 85 IU / ml, 90 IU / ml, 95 IU / ml, 100 IU / ml, 110 IU / ml, 120 IU / ml, 130 IU / ml, 140 IU / ml or 150 IU / ml or In some embodiments, the composition of enriched T cells is incubated with recombinant IL-2 at a concentration of about 50 IU / ml, 55 IU / ml, 60 IU / ml, 65 IU / ml, 70 IU / ml, 75 IU / ml, 80 IU / ml, 85 IU / ml, 90 IU / ml, 95 IU / ml, 100 IU / ml, 110 IU / ml, 120 IU / ml, 130 IU / ml, 140 IU / ml, or 150 IU / ml. In some embodiments, the composition of enriched T cells is incubated in the presence of 85 IU / ml or about 85 IU / ml of recombinant IL-2. In some embodiments, the composition incubated with recombinant IL-2 is enriched for a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the population of T cells is a population of CD4+ T cells. In some embodiments, the composition of enriched T cells is an enriched CD8+ T cell composition. In certain embodiments, the enriched T cell composition is enriched for CD8+ T cells and not enriched for CD4+ T cells, and / or CD4+ T cells have been negatively selected or depleted from the composition. In some embodiments, the enriched T cell composition is an enriched CD4+ T cell composition. In certain embodiments, the enriched T cell composition is enriched for CD4+ T cells and not enriched for CD8+ T cells, and / or CD8+ T cells have been negatively selected or depleted from the composition.In some embodiments, an enriched CD4+ T cell composition incubated with recombinant IL-2 may also be incubated with recombinant IL-7 and / or recombinant IL-15, such as in the amounts described. In some embodiments, an enriched CD8+ T cell composition incubated with recombinant IL-2 may also be incubated with recombinant IL-15, such as in the amounts described.

[0285] In some embodiments, the enriched T cell composition is incubated with recombinant IL-7, e.g., human recombinant IL-7, at a concentration of between 100 IU / ml and 2,000 IU / ml, between 500 IU / ml and 1,000 IU / ml, between 100 IU / ml and 500 IU / ml, between 500 IU / ml and 750 IU / ml, between 750 IU / ml and 1,000 IU / ml, or between 550 IU / ml and 650 IU / ml. In certain embodiments, the enriched T cell composition comprises 50 IU / ml, 100 IU / ml, 150 IU / ml, 200 IU / ml, 250 IU / ml, 300 IU / ml, 350 IU / ml, 400 IU / ml, 450 IU / ml, 500 IU / ml, 550 IU / ml, 600 IU / ml, 650 IU / ml, 700 IU / ml, 750 IU / ml, 800 IU / ml, 750 IU / ml, 750 IU / ml, or 1,000 IU / ml or are incubated with recombinant IL-7 at a concentration of about 50 IU / ml, 100 IU / ml, 150 IU / ml, 200 IU / ml, 250 IU / ml, 300 IU / ml, 350 IU / ml, 400 IU / ml, 450 IU / ml, 500 IU / ml, 550 IU / ml, 600 IU / ml, 650 IU / ml, 700 IU / ml, 750 IU / ml, 800 IU / ml, 750 IU / ml, 750 IU / ml, or 1,000 IU / ml. In certain embodiments, the enriched T cell composition is incubated in the presence of 600 IU / ml or about 600 IU / ml of recombinant IL-7. In some embodiments, the composition incubated with recombinant IL-7 is enriched for a population of T cells, e.g., CD4+ T cells. In some embodiments, the enriched CD4+ T cell composition incubated with recombinant IL-7 may also be incubated with recombinant IL-2 and / or recombinant IL-15, such as the amounts described. In certain embodiments, the enriched T cell composition is enriched for CD4+ T cells and not enriched for CD8+ T cells, and / or CD8+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched CD8+ T cell composition is not incubated with recombinant IL-7.

[0286] In some embodiments, the enriched T cell composition is incubated with recombinant IL-15, e.g., human recombinant IL-15, at a concentration of between 0.1 IU / ml and 100 IU / ml, between 1 IU / ml and 100 IU / ml, between 1 IU / ml and 50 IU / ml, between 5 IU / ml and 25 IU / ml, between 25 IU / ml and 50 IU / ml, between 5 IU / ml and 15 IU / ml, or between 10 IU / ml and 100 IU / ml. In certain embodiments, the enriched T cell composition comprises 1 IU / ml, 2 IU / ml, 3 IU / ml, 4 IU / ml, 5 IU / ml, 6 IU / ml, 7 IU / ml, 8 IU / ml, 9 IU / ml, 10 IU / ml, 11 IU / ml, 12 IU / ml, 13 IU / ml, 14 IU / ml, 15 IU / ml, 20 IU / ml, 25 IU / ml, 30 IU / ml, 40 IU / ml or 50 IU / ml or In some embodiments, the composition is incubated with recombinant IL-15 at a concentration of about 1 IU / ml, 2 IU / ml, 3 IU / ml, 4 IU / ml, 5 IU / ml, 6 IU / ml, 7 IU / ml, 8 IU / ml, 9 IU / ml, 10 IU / ml, 11 IU / ml, 12 IU / ml, 13 IU / ml, 14 IU / ml, 15 IU / ml, 20 IU / ml, 25 IU / ml, 30 IU / ml, 40 IU / ml, or 50 IU / ml. In some embodiments, the enriched T cell composition is incubated at 10 IU / ml or about 10 IU / ml of recombinant IL-15. In some embodiments, the composition incubated with recombinant IL-15 is enriched for a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the population of T cells is a population of CD4+ T cells. In some embodiments, the enriched T cell composition is an enriched CD8+ T cell composition. In certain embodiments, the enriched T cell composition is enriched for CD8+ T cells and is not enriched for CD4+ T cells, and / or CD4+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched T cell composition is an enriched CD4+ T cell composition.In certain embodiments, the enriched T cell composition is enriched for CD4+ T cells and not enriched for CD8+ T cells, and / or CD8+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched CD4+ T cell composition incubated with recombinant IL-15 may also be incubated with recombinant IL-7 and / or recombinant IL-2, such as in the amounts described. In some embodiments, the co-incubated recombinant IL-15t-enriched CD8+ T cell composition may also be incubated with recombinant IL-2, such as in the amounts described.

[0287] In certain embodiments, cells, e.g., enriched CD4+ T cells and / or enriched CD8+ T cells, are incubated with a stimulatory reagent in the presence of one or more antioxidants, such as tocopherol, tocotrienol, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, alpha-tocopherol quinone, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), flavonoids, isoflavones, and lycopene. In some embodiments, the incubation of the enriched T cell composition, e.g., enriched CD4+ T cells and / or enriched CD8+ T cells, with the antioxidant also includes the presence of a stimulatory reagent, e.g., anti-CD3 / anti-CD28 magnetic beads and one or more recombinant cytokines as described.

[0288] In some embodiments, one or more antioxidants are or include sulfur-containing oxidants. In certain embodiments, sulfur antioxidants can include thiol-containing antioxidants and / or antioxidants that, for example, have one or more sulfur moieties in their ring structures. In some embodiments, sulfur-containing antioxidants can include, for example, N-acetylcysteine ​​(NAC) and 2,3-dimercaptopropanol (DMP), L-2-oxo-4-thiozolidinecarboxylate (OTC) and lipoic acid. In certain embodiments, the sulfur-containing antioxidant is a glutathione precursor. In some embodiments, glutathione precursors are molecules that can be modified in one or more steps within cells to derive glutathione. In certain embodiments, glutathione precursors can include, but are not limited to, N-acetylcysteine ​​(NAC), L-2-oxothiozolidine-4-carboxylic acid (procysteine), lipoic acid, S-allyl cysteine, or methylmethionine sulfonium chloride.

[0289] In some embodiments, incubating cells, e.g., enriched CD4+ T cells and / or enriched CD8+ T cells, under stimulatory conditions includes incubating the cells in the presence of one or more antioxidants. In certain embodiments, the cells are stimulated with a stimulatory reagent in the presence of one or more antioxidants. In some embodiments, the cells are incubated in the presence of one or more antioxidants at between 1 ng / ml and 100 ng / ml, 10 ng / ml and 1 μg / ml, 100 ng / ml and 10 μg / ml, 1 μg / ml and 100 μg / ml, 10 μg / ml and 1 mg / ml, 100 μg / ml and 1 mg / ml, 1500 μg / ml and 2 mg / ml, 500 μg / ml and 5 mg / ml, 1 mg / ml and 10 mg / ml, or 1 mg / ml and 100 mg / ml. In some embodiments, the cells are at or near 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml or about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml, 0.2 mg The sample is incubated in the presence of one or more antioxidants at 0.5 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, or 500 mg / ml. In some embodiments, the one or more antioxidants are or comprise sulfur-containing antioxidants. In certain embodiments, the one or more antioxidants are or comprise glutathione precursors.

[0290] In some embodiments, the one or more antioxidants comprise or include N-acetylcysteine ​​(NAC). In some embodiments, incubating cells, e.g., enriched CD4+ T cells and / or enriched CD8+ T cells, under stimulatory conditions comprises incubating the cells in the presence of NAC. In certain embodiments, the cells are stimulated with a stimulatory reagent in the presence of NAC. In some embodiments, the cells are incubated in the presence of NAC at between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 μg / ml, between 100 ng / ml and 10 μg / ml, between 1 μg / ml and 100 μg / ml, between 10 μg / ml and 1 mg / ml, between 100 μg / ml and 1 mg / ml, between 1-500 μg / ml and 2 mg / ml, between 500 μg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml. In some embodiments, the cells are at a concentration of 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml or about 1 In some embodiments, the cells are incubated in the presence of NAC at or about 0.8 mg / ml.

[0291] In certain embodiments, incubating a composition of enriched T cells, e.g., enriched CD4+ T cells and / or enriched CD8+ T cells, in the presence of one or more antioxidants, e.g., NAC, reduces activation of the cells compared to cells incubated in an alternative and / or exemplary process in the absence of antioxidants. In certain embodiments, reduced activation is measured by the expression of one or more activation ma...

Claims

1. 1. A method for determining efficacy of a therapeutic cell composition, comprising: performing a plurality of incubations, each of the plurality of incubations comprising culturing cells of a therapeutic cell composition comprising cells engineered to express a recombinant receptor with a recombinant receptor stimulating agent; Binding of the recombinant receptor stimulating agent to the recombinant receptor stimulates recombinant receptor-dependent activity in the cell; performing a plurality of incubations, each of which comprises a different adjusted ratio of cells of the therapeutic cell composition to the recombinant receptor stimulating agent; measuring a recombinant receptor-dependent activity from each of the plurality of incubations, wherein the recombinant receptor-dependent activity includes or is cytokine expression or production; and determining an adjusted ratio that results in half-maximal recombinant receptor-dependent activity based on the recombinant receptor-dependent activity measured from each of the multiple incubations. A method comprising:

2. The method of claim 1, further comprising determining the relative potency of the therapeutic cell composition by comparing the adjusted ratio that results in half-maximal recombinant receptor-dependent activity of the therapeutic cell composition to the adjusted ratio that results in half-maximal recombinant receptor-dependent activity of the reference standard.

3. 1. A method for determining efficacy of a therapeutic cell composition, comprising: performing a plurality of incubations, each of the plurality of incubations comprising culturing cells of a therapeutic cell composition comprising cells engineered to express a recombinant receptor with a recombinant receptor stimulating agent; Binding of the recombinant receptor stimulating agent to the recombinant receptor stimulates recombinant receptor-dependent activity in the cell; performing a plurality of incubations, each of which comprises a different adjusted ratio of cells of the therapeutic cell composition to the recombinant receptor stimulating agent; measuring a recombinant receptor-dependent activity from each of the plurality of incubations, wherein the recombinant receptor-dependent activity includes or is cytokine expression or production; and determining the relative potency of the therapeutic cell composition by comparing the half-maximal recombinant receptor-dependent activity of the therapeutic cell composition to the half-maximal recombinant receptor-dependent activity of the reference standard; A method comprising:

4. The method of any of claims 1 to 3, wherein each of the multiple incubations comprises culturing a fixed number of cells of the therapeutic composition with a different amount of the recombinant receptor stimulating agent to generate a plurality of different adjusted ratios.

5. The method of any of claims 1 to 3, wherein each of the multiple incubations comprises culturing a fixed amount of the recombinant receptor stimulating agent with a different number of cells of the therapeutic composition to produce a plurality of different adjusted ratios.

6. A method according to any one of claims 1 to 5, wherein the multiple incubations are at least three incubations.

7. The method of claim 1, wherein the cytokine is TNF-alpha, IFN-gamma (IFNg) or IL-2.

8. The method of claim 1, wherein the cytokine is IFN gamma (IFNg).

9. 9. The method of any of claims 1 to 8, wherein the recombinant receptor-dependent activity measured in each of the multiple incubations is normalized to the maximum receptor-dependent activity measured for the therapeutic cell composition.

10. The method of any one of claims 1 to 9, wherein the recombinant receptor stimulating agent comprises a target antigen or an extracellular domain binding portion thereof.

11. The method of claim 10 , wherein the recombinant receptor stimulating agent comprises a recombinant antigen of a recombinant receptor.

12. 10. The method of any one of claims 1 to 9, wherein the recombinant receptor stimulating agent is an antibody specific for the extracellular binding domain of the recombinant receptor.

13. 13. The method of any one of claims 1 to 12, wherein the recombinant receptor stimulating agent is an anti-idiotypic antibody specific for the extracellular antigen-binding domain of the recombinant receptor.

14. 14. The method of any of claims 1 to 13, wherein the recombinant receptor stimulating agent is immobilized or attached to a solid support.

15. 15. The method of claim 14, wherein the solid support is the surface of a vessel in which the multiple incubations are performed.

16. The method of claim 14, wherein the solid support is a bead.

17. The method of any one of claims 1 to 9, wherein the recombinant receptor stimulating agent is an antigen-expressing cell.

18. 18. The method of any of claims 2 to 17, wherein the adjusted ratio achieves a linear dose-response range of recombinant receptor-dependent activity of the reference standard.

19. 20. The method of claim 18, wherein the adjusted ratio comprises the lower asymptote (minimum) recombinant receptor-dependent activity and the upper asymptote (maximum) recombinant receptor-dependent activity of the reference standard.

20. 20. The method of any of claims 1 to 19, wherein the therapeutic cell composition comprises primary cells.

21. 21. The method of any of claims 1 to 20, wherein the therapeutic cell composition comprises CD3+ T cells.

22. 22. The method of any of claims 1 to 21, wherein the therapeutic cell composition comprises CD4+ T cells and CD8+ T cells.

23. 23. The method of any one of claims 1 to 22, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

24. 24. The method of any of claims 1 to 23, wherein each of the multiple incubations is carried out individually in a well of a multi-well plate.

25. 25. The method of any of claims 1 to 24, further comprising determining a dose of cells of the therapeutic composition for administration to a subject in need thereof based on the adjusted ratio that results in half-maximal recombinant receptor-dependent activity.

26. 26. The method of any of claims 2 to 25, further comprising determining a dose of cells of the therapeutic composition for administration to a subject in need thereof based on the relative potency.

27. A method according to any one of claims 1 to 26, wherein the cytokine is measured by ELISA (enzyme-linked immunosorbent assay).

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