Methods for assessing cell proliferation of cell compositions

Abstract

Provided herein are an assay and methods for determining cell proliferation of a cell composition, such as a cell therapy composition. The cells of the cell composition can express recombinant receptors including chimeric receptors, e.g., chimeric antigen receptors (CARs), or recombinant receptors, e.g., T cell receptors (TCRs). The methods allow assessment cell proliferation (e.g., relative cell proliferation) (%), as a product quality attribute of a cell composition. The present disclosure also relates to methods of assessing quality of a cell composition, such as a cell therapy composition, based on the proliferation assay described herein.

Description

73504-2029640METHODS FOR ASSESSING CELL PROLIFERATION OF CELL COMPOSITIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 733,378, filed December 12, 2024, entitled “METHODS FOR ASSESSING CELL PROLIFERATION OF CELL COMPOSITIONS,” the contents of which are herein incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates to an assay and methods for assessing proliferation or relative proliferation of a cell composition, such as a cell therapy composition. The cells of the cell composition can express recombinant receptors such as chimeric receptors, e.g., chimeric antigen receptors (CARs), or T cell receptors (TCRs). The present disclosure also relates to methods for assessing quality of a cell composition, such as a cell therapy composition, based on the proliferation assay described herein.BACKGROUND

[0003] Various immunotherapy and / or cell therapy methods are available for treating diseases and conditions. For example, adoptive cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and / or other recombinant antigen receptors, as well as other adoptive immune cell therapies, e.g., T cell therapies) can be beneficial in the treatment of cancer or other diseases or disorders. Improved approaches are needed for characterizing cell therapy compositions that may be used for treating a subject. These approaches may be applied during ex vivo production of the compositions or after the compositions have been produced for treating a subject with a cell therapy. Provided herein are methods that address such needs.SUMMARY

[0004] In some aspects, provided herein is a method for assessing cell proliferation of a test engineered cell composition, wherein the test engineered cell composition comprises cells comprising a recombinant receptor, the method comprising: (a) culturing cells of the test engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated cell composition; (b) after the period of time adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition; (c) incubating the1MF-36448161473504-2029640 stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal; (d) detecting the luminescent signal from the stimulated cell composition; and, optionally, (e) determining cell proliferation of the test engineered cell composition using the results of the detecting.

[0005] In some of any of the provided embodiments, steps (a) to (d) are performed for two or more replicates, optionally wherein an average of the luminescent signal detected from the two or more replicates is used as the luminescent signal in the determining. In some embodiments, the replicates are separate samples of the test engineered cell composition. In some embodiments, the replicates are separate samples of cells from the test engineered cell composition. In some of any of the provided embodiments, steps (a) to (d) are performed for at least three replicates.

[0006] In some of any of the provided embodiments, determining cell proliferation comprises calculating the difference between (A) the luminescent signal detected from the stimulated cell composition and (B) a luminescent signal detected from unstimulated cultured cells of the test engineered cell composition, wherein the unstimulated cultured cells have been cultured for a second period of time in the absence of the recombinant receptor-stimulating agent.

[0007] In some of any of the provided embodiments, the second period of time has the same duration as the first period of time. In some of any of the provided embodiments, the method further comprises culturing unstimulated cells of the test engineered cell composition for the second period of time in the absence of the recombinant receptor-stimulating agent to generate the unstimulated cultured cells, and detecting the luminescent signal from the unstimulated cultured cells. In some of any of the provided embodiments, the method further comprises adding the luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the unstimulated cultured cells. In some of any of the provided embodiments, the method further comprises incubating the unstimulated cultured cells with the luciferase enzyme and the substrate or pro-substrate of the luciferase enzyme to generate a luminescent signal. In some of any of the provided embodiments, the method further comprises detecting the luminescent signal from the unstimulated cultured cells. In some of any of the provided embodiments, the steps of culturing the unstimulated cells of the test engineered cell composition for the second period of time and detecting the luminescent signal from the unstimulated cultured cells are performed for two or more replicates (e.g., at least three replicates), optionally wherein an average of the luminescent signal detected from said two or more replicates (e.g., at least three replicates) is used in the calculating as the luminescent signal detected from the unstimulated cultured cells. In some embodiments, the replicates are separate samples of the test engineered cell composition. In some embodiments, the replicates are separate samples of cells from the test engineered cell composition.2MF-36448161473504-2029640

[0008] In some of any of the provided embodiments, the method comprises calculating a relative cell proliferation of the test engineered cell composition by comparing the cell proliferation as determined in step (e) to the cell proliferation of a reference standard. In some of any of the provided embodiments, the comparing comprises calculating a ratio of the cell proliferation as determined in step (e) to the cell proliferation of the reference standard and optionally expressing the ratio as a percentage by multiplying it by 100.

[0009] In some aspects, provided herein is a method for assessing relative cell proliferation of a test engineered cell composition, wherein the test engineered cell composition comprises cells comprising a recombinant receptor, the method comprising: (a) culturing cells of the test engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated cell composition; (b) after the period of time, adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition and incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal; (c) detecting the luminescent signal from the stimulated cell composition; (d) determining cell proliferation of the test engineered cell composition by calculating the difference between (A) the luminescent signal detected from the stimulated cell composition and (B) a luminescent signal detected from unstimulated cultured cells of the cell composition, wherein the unstimulated cultured cells have been cultured for a second period of time in the absence of the recombinant receptor-stimulating agent, wherein the second period of time is equivalent in duration to the period of time; and (e) calculating a relative cell proliferation of the test engineered cell composition by calculating a ratio of the cell proliferation as determined in step (d) to the cell proliferation of a reference standard.

[0010] In some of any of the provided embodiments, steps (a) to (c) are performed for two or more replicates. In some such embodiments, calculating the difference in step (d) comprises calculating the difference between the average luminescent signal detected from the stimulated cell composition and the luminescent signal detected from unstimulated cultured cells of the cell composition. In some of any of the provided embodiments, the method further comprises culturing the unstimulated cells of the test engineered cell composition for the second period of time in the absence of the recombinant receptor stimulating agent to generate the unstimulated cultured cells, and detecting the luminescent signal from the unstimulated cultured cells. In some of any of the provided embodiments, the steps of culturing the unstimulated cells of the test engineered cell composition for the second period of time and detecting the luminescent signal from the unstimulated cultured cells are performed for two or more replicates. In some of any of the provided embodiments, calculating the difference in step (d) comprises calculating the difference between the luminescent signal (e.g. the average luminescent signal) detected from the stimulated cell composition and the luminescent signal3MF-36448161473504-2029640(e.g., the average luminescent signal) detected from unstimulated cultured cells of the cell composition.

[0011] In some of any of the provided embodiments, the period of time is at least about 46 hours. In some of any of the provided embodiments, the period of time is between about 46 hours and about 96 hours. In some of any of the provided embodiments, the period of time is between about 46 hours and about 72 hours. In some of any of the provided embodiments, the period of time is about 46 hours to about 50 hours.

[0012] In some of any of the provided embodiments, the culturing in step (a) is carried out at a temperature of 37°C ± 2°C, or at a temperature of about 37°C ± 2°C. In some of any of the provided embodiments, the culturing in step (a) comprises maintaining carbon dioxide (CO2) between 3% and 7%, or between about 3% and about 7%. In some of any of the provided embodiments, the culturing in step (a) comprises maintaining humidity level between 90% and 100% or between about 90% and about 100%.

[0013] In some of any of the provided embodiments, the incubating of the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate in step (c) is carried out at a temperature of 37°C ± 2°C or at a temperature of about 37°C ± 2°C. In some of any of the provided embodiments, the incubating of the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate in step (c) comprises maintaining carbon dioxide (CO2) between 3% and 7%, or between about 3% and about 7%. In some of any of the provided embodiments, the incubating of the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate in step (c) comprises maintaining humidity level between 90% and 100% or between about 90% and about 100%.

[0014] In some of any of the provided embodiments, culturing the unstimulated cells is carried out at a temperature of 37°C ± 2°C, or at a temperature of about 37°C ± 2°C. In some of any of the provided embodiments, culturing the unstimulated cells comprises maintaining carbon dioxide (CO2) between 3% and 7%, or between about 3% and about 7%. In some of any of the provided embodiments, culturing the unstimulated cells comprises maintaining humidity level between 90% and 100% or between about 90% and about 100%.

[0015] In some of any of the provided embodiments, the incubating of the unstimulated cultured cells with the luciferase enzyme and the substrate or pro-substrate in step (c) is carried out at a temperature of 37°C ± 2°C or at a temperature of about 37°C ± 2°C. In some of any of the provided embodiments, the incubating of the unstimulated cultured cells with the luciferase enzyme and the substrate or pro-substrate in step (c) comprises maintaining carbon dioxide (CO2) between 3% and 7%, or between about 3% and about 7%. In some of any of the provided embodiments, the incubating of the unstimulated cultured cells with the luciferase enzyme and the substrate or pro-substrate in step4MF-36448161473504-2029640(c) comprises maintaining humidity level between 90% and 100% or between about 90% and about 100%. In some of any of the provided embodiments, the incubating in step (c) is for about 10 minutes to about 50 minutes.

[0016] In some of any of the provided embodiments, after the incubating in step (c) and before the detecting in step (d), the method comprises equilibrating the stimulated cell composition and the unstimulated cultured cells to room temperature. In some of any of the provided embodiments, the equilibrating is for about 2 hours to about 4 hours. In some of any of the provided embodiments, the stimulated cell composition and the unstimulated cultured cells are protected from light during the equilibrating. In some of any of the provided embodiments, the method further comprises contacting the recombinant receptor-stimulating agent with cells of the test engineered cell composition prior to the culturing in step (a), optionally wherein the contacting comprises adding a volume of the test engineered cell composition to a vessel containing the recombinant receptor-stimulating agent. In some of any of the provided embodiments, one or more steps of the method including at least step (d) of detecting the luminescent signal from the stimulated cell composition is performed iteratively to allow assessment of cell proliferation at different timepoints.

[0017] In some of any of the provided embodiments, a step of incubating the stimulated cell composition is performed before each iteration of detecting the luminescent signal from the stimulated cell composition (e.g., step (d)). In some of any of the provided embodiments, the step of adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated composition (e.g., step (b)) is not repeated after the first time it is performed. In some of any of the provided embodiments, each of the steps of adding a luciferase enzyme and a substrate or prosubstrate of the luciferase enzyme to the stimulated cell composition, incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal and detecting the luminescent signal from the stimulated cell composition (e.g., each of steps (b), (c) and (d)) are performed iteratively. In some of any of the provided embodiments, after each iteration of the incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate and before each iteration of the detecting of the luminescent signal from the stimulated cell composition (e.g., in step (c)) and before each iteration of detecting the luminescent signal from the stimulated cell composition (e.g, in step (d)), the method comprises equilibrating the stimulated cell composition to room temperature. In some of any of the provided embodiments, any iteration of the step of detecting the luminescent signal from the stimulated cell composition (e.g., step (d) of the method) is carried out within 96 hours of first initiating the culturing of the cells of the test engineered cell composition with the recombinant receptor-stimulating agent according to step (a). In some of any of the provided embodiments, any iteration of the step of detecting the luminescent signal from the stimulated cell composition (e.g, step (d) of the method) is carried out5MF-36448161473504-2029640 within 72 hours of first initiating the culturing of the cells of the test engineered cell composition with the recombinant receptor-stimulating agent according to step (a).

[0018] In some aspects, provided herein is a method for assessing cell proliferation of a test cell composition, the method comprising: (a) culturing cells of a test cell composition for a period of time under conditions that support cell proliferation to make a cultured cell composition, optionally wherein the period of time is about 46 hours; (b) after the culturing in (a), adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the cultured cell composition to generate a luminescent signal; (c) detecting the luminescent signal from the cultured cell composition; and (d) calculating a relative cell proliferation of the test cell composition by comparing the luminescent signal that is detected in step (c) to the luminescent signal that is detected from a reference standard.

[0019] In some of any of the provided embodiments, the culturing in (a) is carried out in the presence of a stimulating agent to stimulate cells of the test cell composition and generate a stimulated test cell composition.

[0020] In some of any of the provided embodiments, the test cell composition is a test engineered cell composition that comprises cells comprising a recombinant receptor.

[0021] In some of any of the provided embodiments, the stimulating agent is a recombinant receptor-stimulating agent.

[0022] In some of any of the provided embodiments, calculating the relative cell proliferation comprises: (i) determining cell proliferation of the test cell composition by calculating the difference between (A) the luminescent signal detected from the stimulated test cell composition and (B) the luminescent signal detected from unstimulated cells of the test cell composition that have been cultured in the absence of the stimulating agent; and (ii) comparing the cell proliferation as calculated in step (i) with the cell proliferation of a reference standard.

[0023] In some of any of the provided embodiments, the comparing comprises calculating a ratio of the cell proliferation (as determined in step (i)) to the cell proliferation of the reference standard and optionally expressing the ratio as a percentage by multiplying it by 100.

[0024] In some of any of the provided embodiments, the period of time is at least about 46 hours. In some of any of the provided embodiments, the period of time is between about 46 hours and about 96 hours. In some of any of the provided embodiments, the period of time is between about 46 hours and about 72 hours. In some of any of the provided embodiments, the period of time is about 46 hours to about 50 hours.

[0025] In some of any of the provided embodiments, the test engineered cell composition has been produced ex vivo from primary cells from a subject by a cell engineering manufacturing process to comprise the recombinant receptor.6MF-36448161473504-2029640

[0026] In some of any of the provided embodiments, the test engineered cell composition is a cell composition produced for use as a cell therapy for treating a subject with a disease or condition. In some of any of the provided embodiments, the disease or condition is a cancer or is an autoimmune or inflammatory disease or condition.

[0027] In some of any of the provided embodiments, the cells of the cell therapy are primary cells that are autologous to the subject to be treated. In some of any of the provided embodiments, the cells of the cell therapy are primary cells that are allogeneic to the subject to be treated.

[0028] In some of any of the provided embodiments, the reference standard is a reference engineered cell composition comprising a reference recombinant receptor. In some of any of the provided embodiments, the reference recombinant receptor expressed by the reference engineered cell composition differs from the recombinant receptor expressed by the test engineered cell composition. In some of any of the provided embodiments, the reference recombinant receptor expressed by the reference engineered cell composition is the same as the recombinant receptor expressed by the test engineered cell composition. In some of any of the provided embodiments, the reference standard is a reference engineered cell composition having a validated cell proliferation.

[0029] In some of any of the provided embodiments, the validated proliferation index of the reference standard is determined by: (a) culturing cells of the reference engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated reference engineered cell composition; (b) after the period of time, adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated reference engineered cell composition and incubating the stimulated cell composition with the luciferase enzyme and the substrate or the prosubstrate to generate a luminescent signal; (c) equilibrating the stimulated reference engineered cell composition to room temperature; (d) detecting the luminescent signal from the stimulated reference engineered cell composition; and (e) determining cell proliferation of the reference standard by calculati ng the difference between (A) the luminescent signal detected from the stimulated reference engineered cell composition and (B) the luminescent signal detected from unstimulated cells of the reference engineered cell composition, wherein the unstimulated cells of the reference engineered cell composition have been cultured in the absence of the recombinant receptor-stimulating agent. In some of any of the provided embodiments, the reference engineered cell composition is produced ex vivo from primary cells from a subject by a cell engineering manufacturing process to comprise the recombinant receptor, wherein the subject is different from the subject used to produce the test engineered cell composition.

[0030] In some of any of the provided embodiments, the subject is a healthy subject not known or suspected of having a disease or condition.7MF-36448161473504-2029640

[0031] In some of any of the provided embodiments, the manufacturing process used to manufacture the test engineered cell composition differs from the manufacturing process used to manufacture the reference cell composition. In some of any of the provided embodiments, the manufacturing process used to manufacture the test engineered cell composition is the same as the manufacturing process used to manufacture the reference cell composition.

[0032] In some of any of the provided embodiments, the recombinant receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some of any of the provided embodiments, the recombinant receptor is a CAR and the CAR comprises an extracellular antigen binding domain specific for a target antigen, a transmembrane domain, and an intracellular signaling domain comprising a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta, and a signaling domain from a T cell costimulatory molecule, optionally wherein the T cell costimulatory molecule is 4-1BB and / or CD28. In some of any of the provided embodiments, the extracellular antigen binding domain comprises a antibody variable heavy chain domain and variable light chain domain specific for the target antigen. In some of any of the provided embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv) specific for the target antigen. In some of any of the provided embodiments, the extracellular antigen binding domain comprises a VHH domain. In some of any of the provided embodiments, the CAR is monospecific to the target antigen. In some of any of the provided embodiments, the CAR is bispecific and the target antigen is a first target antigen and the extracellular binding domain is further specific for a second target antigen. In some of any of the provided embodiments, the extracellular antigen binding domain comprises a first antigen binding domain comprising a first variable heavy (VH) chain domain and a first variable light (VL) chain domain specific for the first target antigen and a second antigen binding domain comprising a second VH chain domain and a second VL chain domain specific for the second target antigen. In some of any of the provided embodiments, the extracellular antigen binding domain has a tandem structure comprising a sequence comprising in order the first antigen binding domain and the second antigen binding domain.

[0033] In some of any of the provided embodiments, the extracellular antigen binding domain has a loop structure comprising a sequence comprising in order, N- to C-terminal: the first VH chain or first VL chain of the first antigen binding domain, the second VH chain or second VL chain of the second antigen binding domain, the other of the second VH chain and second VL chain of the second antigen binding domain, and the other of the first VH chain and second VL chain of the second antigen binding domain. In some of any of the provided embodiments, the extracellular antigen binding domain comprises a first VHH specific to the first target antigen and a second VHH specific8MF-36448161473504-2029640 to the second target antigen. In some of any of the provided embodiments, further comprising a hinge spacer sequence between the extracellular antigen binding domain and the transmembrane domain.

[0034] In some of any of the provided embodiments, the recombinant receptor-stimulating agent is or comprises a binding moiety recognized by or specific to the recombinant receptor, optionally to the extracellular antigen binding domain of the CAR.

[0035] In some of any of the provided embodiments, the binding moiety is a target antigen or an extracellular domain binding portion thereof of the recombinant receptor, optionally wherein the extracellular domain binding portion of the target antigen comprises an epitope recognized by the recombinant receptor. In some of any of the provided embodiments, the binding moiety is an antibody specific to an extracellular binding domain of the recombinant receptor. In some of any of the provided embodiments, the binding moiety is an anti-idiotypic antibody specific to an extracellular antigen binding domain of the recombinant receptor. In some of any of the provided embodiments, the binding moiety is immobilized or attached to a solid support during the culturing, optionally wherein the culturing is initiated when cells of the test engineered cell composition or test cell composition are added to the solid support.

[0036] In some of any of the provided embodiments, the culturing is initiated when cells of the test engineered cell composition or test cell composition are added to a solid support. In some of any of the provided embodiments, the solid support is a surface of a culture vessel. In some of any of the provided embodiments, the solid support is a bead.

[0037] In some of any of the provided embodiments, the culturing is initiated when cells of the test engineered cell composition or test cell composition and the binding moiety-immobilized beads are contacted in a culture vessel. In some of any of the provided embodiments, the culture vessel is a multi-well plate, optionally a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96 wellplate or a 384-well plate. In some of any of the provided embodiments, the culture vessel is a 96-well plate.

[0038] In some of any of the provided embodiments, the cells are added to the culture vessel at a cell density of between about 6.7 x 104 viable cells / mL and about 3 x 106 viable cells / mL. In some of any of the provided embodiments, the number of cells present in the culture vessel at the time the detecting is performed is not more than 6 x 106 viable cells / mL. In some of any of the provided embodiments, the number of cells present in the culture vessel at the time the detecting is performed is not less than 3.3 x 104 viable cells / mL.

[0039] In some of any of the provided embodiments, the cells of the test engineered cell composition or the test cell composition comprise a T cell. In some of any of the provided embodiments, the T cells are primary cells. In some of any of the provided embodiments, the T cells are autologous cells. In some of any of the provided embodiments, the T cells are allogeneic cells. In9MF-36448161473504-2029640 some of any of the provided embodiments, the T cells are CD3+. In some of any of the provided embodiments, the T cells are CD4+ and / or CD8+ T cells. In some of any of the provided embodiments, the T cells are CD4+ and CD8+ T cells.

[0040] In some of any of the provided embodiments, the substrate or pro-substrate is cell permeable and is able to enter cells. In some of any of the provided embodiments, the substrate or pro-substrate added to the cells is a luciferin-D substrate or analog or derivative thereof, or is a prosubstrate of any of the foregoing. In some of any of the provided embodiments, the substrate or prosubstrate added to the cells is a coelenterazine substrate or analog or derivative thereof, or is a prosubstrate of any of the foregoing. In some of any of the provided embodiments, the substrate or prosubstrate added to the cells is a furimazine substrate or analog or derivative thereof, or is a prosubstrate of any of the foregoing. In some of any of the provided embodiments, the substrate or prosubstrate added to the cells is a pro-substrate, wherein the pro-substrate is able to be modified by the actions of a cellular enzyme present in the cell to release the substrate inside the cell.

[0041] In some of any of the provided embodiments, the cellular enzyme is an esterase. In some of any of the provided embodiments, when present in the cells, the substrate is able to be reduced by molecular oxygen and released from the cells as a substrate for the luciferase.

[0042] In some of any of the provided embodiments, the luciferase enzyme is cell- impermeable and does not enter the cell, wherein the luciferase binds the reduced substrate after it exits the cell. In some of any of the provided embodiments, the luciferase is from Oplophorus (OLuc), Gaussia (GLuc), Renilla (RLuc), Pyrophorus, or Photin us or is a variant thereof that is able to produce luminescence when bound to substrate. In some of any of the provided embodiments, the luciferase is from Oplophorus (OLuc) or is a variant thereof that is able to produce luminescence when bound to substrate. In some of any of the provided embodiments, the luciferase is a OLuc variant that is the variant Cl A4E comprising at least 8 substitutions selected from the group consisting of A4E, QI 1R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, P115E, Q124K, and Y138IP, optionally 8, 9, 10, 11, 12, 13, 14 or 15 substitutions. In some of any of the provided embodiments, the luciferase is NanoLuc™.

[0043] In some of any of the provided embodiments, the luminescent signal is generated after the luciferase binds to the reduced substrate. In some of any of the provided embodiments, the luminescent signal is detected by a luminometer.

[0044] In some of any embodiments, the method is used as a release assay. In some of any embodiments, the method is used to assess stability, such as of a cell composition .

[0045] In some of any embodiments, the method has a recovery rate of between about 80% and about 120%. In some of any embodiments, the method has a coefficient of determination of at least 0.97. In some of any embodiments, the method has an intermediate precision of less than 20%.10MF-36448161473504-2029640BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 shows a general overview of a cellular redox proliferation assay method provided herein. More details are provided in Example 1. In summary, an engineered cell composition (e.g., a CAR-T cell composition or other engineered cell therapy composition) is seeded onto platebound anti-idiotypic (alD) antibodies (which are specific to an engineered construct expressed on the cells, e.g., a CAR) and a first incubation step (typically 48 hours or more) is performed, during which the cells proliferate as a result of alD induced stimulation / activation. Following this proliferation period, furimazine substrate and enzyme mixed in cell culture medium are added to the cells, a short incubation is performed to allow the luminescence to develop, followed by room temperature equilibration during which the cells are protected from light, and subsequently, a luminometer is used to read the luminescent signal. ART: ambient room temperature.

[0047] FIG. 2 shows proliferation of chimeric antigen receptor (CAR) expressing cells in CAR-T cell products stimulated with increasing concentrations (pg / mL) of anti-idiotype antibody (alD). Proliferation of CAR-T cells was assessed by measuring luminescence with a luminometer after 48 or 72 hours of alD stimulation. The measured luminescence is in Relative Luminescence Units (RLU).

[0048] FIG. 3 shows % confluence of chimeric antigen receptor (CAR) expressing cells in CAR-T cell products across 240 hours of culture in response to stimulation (stimulated or stim) with anti-idiotype antibody (alD) or without alD (unstimulated or unstim). Confluence was assessed using the IncuCyte® proliferation method.

[0049] FIG. 4 shows assay results obtained following the indicated proliferation time periods using either the IncuCyte® proliferation assay (top) or the cellular redox proliferation assay (bottom). Proliferation based on the IncuCyte® proliferation assay was calculated using confluence readouts and depicted as area under the curve (AUC) for the average stimulated condition minus the AUC for a single control unstimulated condition. Proliferation based on the cellular redox proliferation assay described herein was calculated as the average Relative Luminescence Units (RLU) of triplicate stimulated conditions minus the average RLU of triplicate unstimulated conditions.

[0050] FIGS. 5A-5B show correlation between readout (Relative Luminescence Units (RLU)) from the cellular redox proliferation assay described herein and readout (cells / mL) from a viable cell count (VCC) assay. FIG. 5A shows RLU at 48 hours correlated with VCC at 72 hours. FIG. 5B shows RLU at 72 hours correlated with VCC at 72 hours.

[0051] FIGS. 6A-6B show results from the cellular redox proliferation assay described herein (FIG. 6A) and the IncuCyte® proliferation assay (FIG. 6B). FIG. 6A shows relative proliferation (relative to a reference standard). FIG. 6B shows proliferation results for individual donors (Sample IDs 1-4, with the donor for the 4thsample being the patient) expressed as area under the curve (AUC).11MF-36448161473504-2029640

[0052] FIG. 7 shows the dynamic range of the cellular redox proliferation assay described herein in Jurkat T cells that were cultured and passaged for 15 days or freshly thawed. Proliferation is depicted as Relative Luminescence Units (RLU).

[0053] FIG. 8 shows that proliferation of chimeric antigen receptor (CAR) expressing cells from CAR-T cell products (CPI, CP2, CP3) occurred selectively, that is, only in response to stimulation with the anti-idiotype antibody (alD) that is specific to the CAR (depicted as “+” in FIG. 8). Non-specific alD stimulation (depicted as in FIG. 8) yielded Relative Luminescence Units (RLU) results that are similar to that of an unstimulated control.

[0054] FIGS. 9A-9B show results of stability or stress testing experiments. FIG. 9A shows viability (top panel) and relative proliferation (bottom panel) of cells from CAR-T cell products that were stored on dry ice for 1.5 hours, or exposed to ambient room temperature (ART) for 1.5 hours, in 37°C water bath for 10 minutes, stored at 4°C for 30 minutes for one or two cycles (IX, 2X), or stored at -20°C for 2 hours for one or two cycles (IX or 2X).FIG. 9B shows relative % recovery (relative to a control, which did not undergo forced degradation) for each indicated method for samples subjected to the indicated forced degradation conditions.

[0055] FIG. 10 shows the percent average relative proliferation (relative to a reference standard) of two independent CAR-engineered cell compositions. Samples of the compositions were diluted to eight different concentrations, ranging from 20% up to 270% of 20,000 cells per well. Linearity was assessed by analyzing the linear relationship of all relative proliferation results at each concentration level, which yielded a strong correlation with an R2value of 0.99.DETAILED DESCRIPTION

[0056] Provided herein are methods and assays for assessing proliferation of a cell composition, e.g., an engineered cell composition, such as a cell therapy composition. In particular embodiments, the engineered cell composition is a cell therapy composition that is intended for or manufactured for the purpose of administration to a subject, including an engineered T cell therapy. In some embodiments, the methods are for use in connection with monitoring ex vivo processes for producing the engineered cell composition, selecting engineered cell compositions for administration (for instance as part of a release assay following manufacture), assessing the stability of the engineered cell composition , and / or for determining doses for the treatment of diseases or conditions, including various cancers. The provided embodiments relate to engineered cell compositions (e.g., cell therapy compositions) containing engineered cells such as those engineered to express recombinant proteins such as recombinant receptors designed to recognize and / or specifically bind to molecules associated with the disease or condition and result in a response, such as an immune response against such molecules upon binding to such molecules. The receptors may include chimeric12MF-36448161473504-2029640 receptors, e.g., chimeric antigen receptors (CARs), and other transgenic antigen receptors including transgenic T cell receptors (TCRs).

[0057] Proliferation or proliferative capacity is considered an important functional attribute of engineered cell compositions, such as cell therapy compositions (Gatti noni et al., JCI (2005) 115.6:1616-1626; Hinrichs et al., PNAS (2009) 106.41:17469-17474). In clinical trials, proliferative capacity of T cells in response to a stimulus (e.g., anti-CD3 and anti-CD28 beads) has been used to predict the success of manufacturing cell therapy compositions (e.g., CD19 CAR-T cell compositions) and determine patient eligibility for enrollment (Turtle et al., Sci. Transl. Med. (2016) 8(355):ral 16). Further, proliferation is an important predictor for durable antitumor responses in patients (Tian et al., JHO (2020) 13(1)) and a recent study identified a positive correlation between proliferation and cell health, indicati ng that proliferation can provide a more accurate measure of cell health than traditional methods of measuring cell viability (Pierce et al., Regen. Med. (2024) 19.1:27-45).

[0058] Traditional proliferation assays use various markers (e.g., active metabolism markers) to determine cell proliferation including cellular conversion of indicator dyes (e.g., MTT, MTS, Resazurin), detection of protease markers or ATP content. However, there are many disadvantages associated with conventional proliferation assays. One disadvantage of conventional proliferation assays that rely on measuring cell confluency is that cells are required to proliferate over an extended period of time (e.g., over 144 hours) to achieve a sufficient cell number to precisely measure increases in cell number in response to a stimulus. This is largely due to tightly controlled, multistep processes involved in cell proliferation such as signaling pathways and protein synthesis that precede cellular division. This poses a problem for cells that reach 100% confluence before 144 hours. The timing aspect of traditional proliferation assays is further complicated in the context that cell cultures may grow in overlapping three-dimensional clusters (e.g., suspension cell cultures). As one example, T cells can be loosely adherent when stimulated by plate bound stimuli, such as anti-ID, and can form three dimensional clusters on the bottom of the plate, which can result in inaccurate measurements of proliferation. Other proliferation assays that assess surrogate mechanisms for proliferation may have several disadvantages, including high variability, poor correlation with actual cell numbers or proliferation outcomes, and the need for over 96 hours of culturing to allow for proliferation to achieve a sufficient signal -to-noise (S:N) ratio. Additionally, these assays may be unable to assess proliferation in real time across multiple time points. From a Chemistry, Manufacturing, and Control (CMC) perspective, there is a need for a fit-for-purpose and quality control (QC)-friendly proliferation assay to ensure cell therapy composition quality and consistency. The methods described herein address many existing issues with traditional proliferation assays.

[0059] Provided herein is are novel methods for assessing proliferation of cells in an engineered cell composition (e.g., a cell therapy composition). In some embodiments, the methods13MF-36448161473504-2029640 described herein relate to assessing proliferation of an engineered cell composition based on the cellular redox status of the cells in the composition. In some embodiments, cellular redox is used to predict proliferation and provides a measure of proliferative capacity. In some embodiments, the methods disclosed herein are stability indicating.

[0060] Multiple benefits are associated with the provided methods, including accuracy, range, robustness and precision. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) released guidelines titled “Q2(R2) Validation of Analytical Procedures” that provide a general framework for the principles of analytical procedure validation. The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samplings of the same homogeneous sample under the prescribed conditions. Precision can be considered at three levels: repeatability, intermediate precision and reproducibility. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. In some instances, repeatability is measured as relative standard deviation of a series of measurements. The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or as an accepted reference value and the value measured. For example, the accuracy can be measured as a recovery rate of the conventional true value or as an accepted reference value. The robustness of an analytical procedure is a measure of its capacity to meet the expected performance requirements during normal use. Robustness is tested by deliberate variations of analytical procedure parameters. The methods described herein provide accurate, robust and precise predictions and assessments of proliferation of engineered cell compositions. The methods are also stability-indicating; the methods provided herein were found to be sensitive to insults known to affect stability as exemplified herein. A further benefit of the provided methods is that the methods result in a robust signal-to-noise ratio in as short a time as 48 hours.

[0061] In some aspects, cell proliferation of an engineered cell composition can be assessed by measuring metabolic activity (e.g., cellular redox) of the engineered cell composition. Cellular redox can be measured by a number of cellular redox based assays, including luminescent-based cellular redox assays. In some embodiments, the methods and assays provided herein allow determination of the proliferation or proliferative capacity of an engineered cell composition (e.g., such as a composition produced for use as a cell therapy). In some embodiments, the proliferation or proliferative capacity is determined using quantitation of stimulated and unstimulated cells’ metabolic activity (e.g., cellular redox). In some embodiments, the method uses anti-idiotype antibodies (alD) specific to the recombinant receptor(s) expressed by cells of the engineered cell composition to stimulate the cells.14MF-36448161473504-2029640

[0062] In some embodiments, the methods provided herein can be a method as depicted in FIG. 1. FIG. 1 illustrates a method for assessing proliferation of a cell composition. As shown in FIG. 1, the method begins with coating a cell culture plate, such as an individual well of a multi-well cell culture plate, with alD according to any of the methods described herein (see, e.g., Section I.A.3.). The engineered cells are then seeded in the alD-coated well and cultured for a period of time (e.g., 46 hours or more) to allow the cells time to proliferate (see, “long incubation” in FIG. 1). After the period of time, which in some embodiments is at least 46 hours, an enzyme (e.g., a luciferase, e.g., a NanoLuc luciferase) and its substrate or pro-substrate are added to the cells. The cells are incubated in the presence of the enzyme and its substrate or pro-substrate at 37°C (see “short incubation” in FIG. 1) to generate a luminescent signal; this incubation is followed by an equilibration period at ambient room temperature (ART) (see “ART equilibration” in FIG. 1). The equilibration period at ART prevents the occurrence of hotspots that may impact uniformity of the luminescence read out. The luminescent signal is then detected using a luminometer.

[0063] As described herein, the luminescent signal detected from the method can be used to calculate cell proliferation and / or relati ve cell proliferation. Relati ve cell proliferation can be calculated, for instance, by calculating the difference between alD stimulated engineered cells and unstimulated engineered cells relative to such difference for a reference standard (see Example 1). Cell proliferation and / or relative cell proliferation can be used to determine whether the test engineered cell composition is stable.

[0064] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0065] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. CELL PROLIFERATION ASSAY

[0066] In some aspects, provided herein are methods for assessing proliferation of a cell composition, e.g., an engineered cell composition such as a cell therapy composition. In some of any of the provided embodiments, the methods of assessing cell proliferation (e.g., relative cell proliferation), provide a measure of proliferation capacity, e.g., relative proliferation capacity, of a cell composition, e.g., an engineered cell composition such as a cell therapy composition. In some embodiments, assessing cell proliferation can provide information about the potency of the cell composition by providing a measure of the biological activity of the cell composition. In some15MF-36448161473504-2029640 embodiments, assessing proliferation provides information about the quality of the cell composition for its use as a cell therapy. In some embodiments, the provided methods of assessing cell proliferation can be used as a potency assay to ensure that a manufacturing process produces a final cell therapy composition that meets quality standards. In some embodiments, a method of assessing cell proliferation provided herein can be used as a release assay (e.g., as one of a set of release assays) for a cell therapy composition. A release assay may be used for characterizing the cell composition to ensure it meets predetermined quality standards (or specifications) before being released for administration to a subject in need thereof (e.g., a patient).

[0067] In some embodiments, a method of assessing cell proliferation provided herein can be used as a stability assay (e.g., as one of a set of stability assays) for a cell therapy composition. A stability assay may be used for characterizing the cell composition, e.g., after the composition has been subjected to a condition that may impact its stability or before the cell composition is released for administration to a subject in need thereof (e.g., a patient). In some embodiments, a method of assessing cell proliferation provided herein is used for determining the shelf life of a cell therapy composition.

[0068] In some embodiments, the cell composition is an engineered cell composition. In some embodiments, the cells of the engineered cell composition are immune cells. In some embodiments, the engineered cell composition comprises an effector cell or a population of effector cells. In some embodiments, the effector cell or population of effector cells comprises a T cell, a B cell and / or a natural killer (NK) cell. In some embodiments, the T cell is a CD3+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is CD3+ and CD4+. In some embodiments, the T cell is CD3+ and CD8+. In some embodiments, the engineered cell compositions comprise any of the cell compositions described in Section I. A. In some embodiments, cells of the engineered cell composition comprise (e.g., express) a recombinant receptor. In some embodiments, the recombinant receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the engineered cell composition comprises any of the recombinant receptors described in Section II. A and II.B.

[0069] In some embodiments, the engineered cell composition is a test cell composition, e.g., a cell composition for which a specific characteristic (e.g., proliferation) is being assessed. In some embodiments, the characteristic being assessed is proliferation. In some embodiments, the engineered cell composition is a reference cell composition, e.g., a cell composition used for comparison to the test cell composition.

[0070] In some embodiments, the method for assessing cell proliferation of the engineered cell composition comprises culturing the cells of the engineered cell composition with a stimulating agent. In some embodiments, the stimulating agent binds to a recombinant receptor expressed on the surface16MF-36448161473504-2029640 of the cells of the engineered cell composition to lead to the activation of the engineered cell. In some embodiments, activation comprises proliferation (e.g., increased prolif eration). In some embodiments, the receptor is an engineered receptor, e.g., a recombinant antigen receptor such as a chimeric antigen receptor (CAR) or T cell receptor (TCR). Thus, in some embodiments, the stimulating agent is a recombinant receptor-stimulating agent. In some embodiments, culturing is initiated when cells of the engineered cell composition are contacted with the recombinant receptor-stimulating agent.

[0071] In some embodiments, the recombinant receptor-stimulating agent is or comprises a binding moiety recognized by or specific to the recombinant receptor expressed on the surface of the cells of the engineered cell composition. In some embodiments, the binding moiety is recognized by or specific to the extracellular antigen binding domain of the recombinant receptor. In some embodiments, the binding moiety recognized by the recombinant receptor is a target antigen of the recombinant receptor. In some embodiments, the target antigen comprises an epitope recognized by the recombinant receptor. In some embodiments, the binding moiety specific to the recombinant receptor is an extracellular domain binding portion of the recombinant receptor. In some embodiments, the binding moiety specific to the recombinant receptor is an antibody. In some embodiments, the antibody is an anti-idiotypic antibody (alD). In some embodiments, the alD is specific to the extracellular antigen binding domain of the recombinant receptor. In some embodiments, a recombinant receptor-stimulating agent comprises any agent described in Section I.A.3.

[0072] In some embodiments, the method for assessing cell proliferation of the engineered cell composition comprises culturing the cells of the engineered cell composition with a stimulating agent for a period of time and culturing cells of the engineered cell composition with the recombinantreceptor stimulating agent generates a stimulated engineered cell composition.

[0073] In some embodiments, the method for assessing cell proliferation of the engineered cell composition comprises adding a luciferase enzyme and a substrate or a pro-substrate of the luciferase enzyme to the stimulated engineered cell composition and incubating the stimulated cell composition with the luciferase enzyme and substrate or pro-substrate to generate a luminescent signal.

[0074] Any known luciferase enzyme and corresponding substrate or pro-substrate can be used in the provided methods, including the any of the luciferase enzymes and substrates or pro-substrates disclosed in Section I.B.l and I.B.2.

[0075] In some embodiments, the luciferase enzyme and substrate or pro-substrate of the luciferase enzyme are added after the period of time for culturing the cells of the engineered cell composition with a stimulating agent. In some embodiments, the period of time allows the cells of the engineered cell composition to proliferate. In some embodiments, the period of time depends on the cell type of the engineered cell composition. In some embodiments, the cell type may be a fast17MF-36448161473504-2029640 growing cell type. In some embodiments, the cell type may be a slow growing cell type. In some embodiments, the period of time is greater than 0 hours. In some embodiments, the period of time is between or between about 24 hours and 96 hours, between or between about 36 hours and 84 hours, between or between about 48 hours and 72 hours, between or between about 46 hours and 50 hours, between or between about 46 hours and 72 hours, between or between about 46 hours and 84 hours, between or between about 46 hours and 96 hours, between or between about 46 hours and 120 hours, between or between about 46 hours and 144 hours, between or between about 46 hours and 168 hours. In some embodiments, the period of time is between or between about 36 hours and 72 hours. In some embodiments, the period of time is between or between about 68 hours and 72 hours. In some embodiments, the period of time is at least about 69 hours, 71 hours, or 72 hours. In some embodiments, the period of time is between or between about 45 hours and 48 hours. In some embodiments, the period of time is at least about 45 hours, 46 hours, 47 hours or 48 hours. In some embodiments, the period of time is about 46 hours. In some embodiments, the period of time is about 48 hours. In some embodiments, the period of time is about 46 hours. In some embodiments, the period of time is about 50 hours.

[0076] In some embodiments, the stimulated cell composition is incubated under conditions that promote luciferase binding to substrate or pro-substrate to boost luminescent signal. In some embodiments, these conditions include a specific temperature. In some embodiments, these conditions include a specific percentage of carbon dioxide (CO2). In some embodiments, these conditions include a specific percentage of humidity. In some embodiments, the culturing is carried out in a cell culture incubator to regulate or maintain both temperature and CO2. In some embodiments, the culturing step is carried out at a temperature of at or about 30°C to 40°C. In some embodiments, the culturing step is carried out at a temperature of at or about 30°C to 33°C, 31°C to 34°C, 32°C to 35°C, 33°C to 36°C, 34°C to 37°C, 35°C to 38°C, or 36°C to 39°C. In some embodiments, the culturing step is carried out at 35°C to 39°C or 37°C ± 2°C. In some embodiments, the temperature is at or about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C. In some embodiments, the culturing step is carried out at a CO2 level between 1% and 10%. In some embodiments, the culturing step is carried out at a CO2 level between 3% and 7% or between about 3% and about 7%. In some embodiments, the culturing step is carried out at a humidity level between 90% and 100% or between about 90% and about 100%.

[0077] In some embodiments, the method for assessing cell proliferation further comprises equilibrating the stimulated cell composition under conditions that promote uniform reading of the luminescent signal. In some embodiments, these conditions include a specific temperature. In some embodiments, the stimulated cell composition is equilibrated at room temperature. In some embodiments, the equilibrating is carried out under light protected conditions.18MF-36448161473504-2029640

[0078] In some embodiments, the method for assessing cell proliferation of the engineered cell composition comprises detecting the luminescent signal from the stimulated cell composition. In some embodiments, the luminescent signal is detected by any means known in the art, including by using a device or apparatus, such as a luminometer.

[0079] In some embodiments, the method for assessing cell proliferation of the engineered cell composition comprises determining cell proliferation of the engineered cell composition according to the methods described herein, such as in Section I.C. In some embodiments, the methods provided herein further comprise calculating a relative cell proliferation of the engineered cell composition using the cell proliferation of the engineered cell composition and comparing the cell prol iteration of the engineered cell composition to the cell proliferation of a reference standard. Exemplary methods for calculating relative cell proliferation are provided in Section I.C.l.

[0080] In some aspects, provided herein are methods for assessing proliferation of a cell composition, e.g., an non-engineered cell composition. In some embodiments, the cell composition is a non-engineered cell composition. In some embodiments, the non-engineered cell composition comprises cells that are not engineered to express a recombinant receptor. In some embodiments, the non-engineered cell compositions comprise any of the compositions described in Section I. A. In some embodiments, the method for assessing proliferation of a cell composition comprises any of the methods provided herein for an engineered cell composition, with the exception that the nonengineered cells are not stimulated with a recombinant receptor-stimulating agent.

[0081] In some embodiments, the method for assessing proliferation of a cell composition comprises culturing cells of the cell composition under conditions that support cell proliferation. In some embodiments, the method for assessing proliferation of a cell composition comprises adding a luciferase enzyme and a substrate or a pro-substrate of the luciferase enzyme to the cell composition. In some embodiments, the method for assessing cell proliferation of a cell composition comprises detecting the luminescent signal generated from the cell composition. In some embodiments, the method for assessing proliferation of a cell composition comprises determining cell proliferation of the cell composition as disclosed herein.

[0082] In some embodiments, the non-engineered cell composition is a test cell composition, e.g., a cell composition for which a specific characteristic (e.g., proliferation) is being assessed. In some embodiments, the characteristic being assessed is proliferation. In some embodiments, the nonengineered cell composition is a reference cell composition, e.g., a cell composition used for comparison to the test cell composition.

[0083] In some embodiments, the receptor is a naturally occurring receptor. In some embodiments, when the receptor is a naturally occurring receptor, a naturally occurring ligand specific19MF-36448161473504-2029640 to or capable of being bound by the naturally occurring receptor is contemplated. In some embodiments, the ligand comprises a cytokine.

[0084] In particular embodiments, provided herein is a method for assessing cell proliferation of a test engineered cell composition, wherein the test engineered cell composition comprises cells comprising a recombinant receptor, the method comprising: (a) culturing cells of the test engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated cell composition; (b) after the period of time adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition; (c) incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal; and (d) detecting the luminescent signal from the stimulated cell composition.

[0085] In particular embodiments, provided herein is a method for assessing cell proliferation of a test engineered cell composition, wherein the test engineered cell composition comprises cells comprising a recombinant receptor, the method comprising: (a) culturing cells of the test engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated cell composition; (b) after the period of time, adding a luciferase enzyme and a substrate or a pro-substrate of the luciferase enzyme to the stimulated cell composition, (c) incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal; (d) detecting the luminescent signal generated from the stimulated cell composition. In particular embodiments, the method comprises a step (e) determining cell proliferation of the test engineered cell composition using the results of the detecting.

[0086] In particular embodiments, provided herein is a method for assessing relative cell proliferation of a test engineered cell composition, wherein the test engineered cell composition comprises cells comprising a recombinant receptor, the method comprising: (a) culturing cells of the test engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated cell composition; (b) after the period of time, adding a luciferase enzyme and a substrate or a pro-substrate of the luciferase enzyme to the stimulated cell composition and incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal; (c) detecting the luminescent signal detected from the stimulated cell composition; (d) determining cell proliferation of the test engineered cell composition by calculating the difference between (A) the luminescent signal detected from the stimulated cell composition and (B) the luminescent signal detected from unstimulated cultured cells of the cell composition, wherein the unstimulated cultured cells have been cultured for a second period of time in the absence of the recombinant receptor-stimulating agent, wherein the second period of time is equivalent in duration to the period of time; and (e) calculating a relative cell proliferation of the test engineered cell20MF-36448161473504-2029640 composition by calculating a ratio of the cell proliferation as determined in step (d) to the cell proliferation of a reference standard.

[0087] In particular embodiments, provided herein is a method for assessing relative cell proliferation of a test engineered cell composition, wherein the test engineered cell composition comprises cells comprising a recombinant receptor, the method comprising: (a) culturing cells of the test engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated cell composition, wherein the period of time is 46 hours or more; (b) after the period of time, adding a luciferase enzyme and a substrate or a pro-substrate of the luciferase enzyme to the stimulated cell composition and i ncubati ng the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate for about 10 to 50 minutes to generate a luminescent signal; (c) detecting the luminescent signal detected from the stimulated cell composition; (d) determining cell proliferation of the test engineered cell composition by calculating the difference between (A) the luminescent signal detected from the stimulated cell composition and (B) the luminescent signal detected from unstimulated cultured cells of the cell composition, wherein the unsti mulated cultured cells have been cultured for 46 hours or more in the absence of the recombinant receptor-stimulating agent; and (e) calculating a relative cell proliferation of the test engineered cell composition by calculating a ratio of the cell proliferation as determined in step (d) to the cell proliferation of a reference standard; and wherein steps (a) to (c) are performed for two or more replicates; and wherein the steps of culturing the unstimulated cells of the test engineered cell composition for the second period of time and detecting the luminescent signal from the unstimulated cultured cells are performed for two or more replicates.

[0088] In particular embodiments, provided herein is a method for assessing stability of a test engineered cell composition, the method comprising any of the methods for assessing cell proliferation disclosed herein. In some embodiments, the method for assessing stability of a test engineered cell composition further comprises comparing the cell proliferation (e.g., relative cell proliferation) of the test engineered cell composition to the relative cell proliferation (e.g., relative cell proliferation) of a control cell composition.

[0089] In particular embodiments, provided herein is a method for assessing stability of a test engineered cell composition, the method comprising: (a) culturing cells of the test engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated cell composition; (b) after the period of time, adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition; (c) incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal; (d) detecting the luminescent signal from the stimulated cell composition; (e) determining cell proliferation of the test engineered cell composition using the results of the detecting;21MF-36448161473504-2029640 and (f) comparing the proliferation of the test engineered cell composition to a reference standard to obtain a relative cell proliferation; and (g) determining whether the test engineered cell composition is stable based on the relative cell proliferation.

[0090] In particular embodiments, provided herein is a method for assessing stability of a test engineered cell composition, the method comprising: (a) culturing cells of the test engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated cell composition; (b) after the period of time, adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition; (c) incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal; (d) detecting the luminescent signal from the stimulated cell composition; (e) determining cell proliferation of the test engineered cell composition using the results of the detecting; and (f) comparing the proliferation of the test engineered cell composition with proliferation of a control cell composition. In some embodiments, cells of the test engineered cell composition have been subjected to a condition (e.g., a storage condition) that may impact stability. In some embodiments, cells of the control cell composition have not been subjected to said condition.

[0091] In particular embodiments, provided herein is a method for assessing stability of a test engineered cell composition, the method comprising: (a) culturing cells of the test engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated cell composition; (b) after the period of time, adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition; (c) incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal; (d) detecting the luminescent signal from the stimulated cell composition; (e) determining cell proliferation of the test engineered cell composition using the results of the detecting; and (f) comparing the proliferation of the test engineered cell composition to a reference standard to obtain a relative cell proliferation; and (g) determining whether the test engineered cell composition is stable based on comparison of the relative cell proliferation of the test engineered cell composition with a relative cell proliferation of a control cell composition. In some embodiments, cells of the test engineered cell composition have been subjected to a condition (e.g., a storage condition) that may impact stability. In some embodiments, cells of the control cell composition have not been subjected to said condition.

[0092] In particular embodiments, provided herein is a method for assessing cell proliferation of a test cell composition, the method comprising: (a) culturing cells of a test cell composition for a period of time under conditions that support cell proliferation to make a cultured cell composition, wherein the period of time is about 46 hours; (b) after initiating the culturing in (a), adding a luciferase enzyme and a substrate or a pro-substrate of the luciferase enzyme to the cultured cell22MF-36448161473504-2029640 composition to generate a luminescent signal; (c) detecting the luminescent signal from the cultured cell composition; and (d) calculating a relative cell proliferation of the test cell composition by comparing the luminescent signal that is detected in step (c) to the luminescent signal that is detected from a reference standard. In some embodiments, the culturing in (a) is carried out in the presence of stimulating agent to stimulate the cells and generate a stimulated test cell composition. In some embodiments, a test cell of the test cell composition only comprises (e.g., expresses) a naturally occurring receptor. In some embodiments, the stimulating agent is a binding agent (e.g., an antibody). In some embodiments, the stimulating agent is an anti-CD3 antibody and / or an anti-CD28 antibody. In some embodiments, the stimulating agent is a recombinant protein (e.g., a cytokine). In some embodiments, the stimulating agent is a selected from IL-2, IL-7, IL-15, and IL-21. In some embodiments, a test cell of the test cell composition comprises (e.g., expresses) a recombinant receptor. In some embodiments, the stimulating agent is a recombinant receptor-stimulating agent. In some embodiments, calculating the relative cell proliferation of the test cell composition comprises any of the calculations provided herein, such as in Section I.C.l.

[0093] In some embodiments, any of the foregoing steps (e.g., steps (a) to (d) or steps (a) to (e)) of the described methods are performed with a replicate of the cell composition (e.g., stimulated cell composition). In some embodiments, two or more replicate stimulated cell compositions are used in the methods. In some embodiments, two replicate stimulated cell compositions are used in the methods. In some embodiments, at least three replicate stimulated cell compositions are used in the methods. In some embodiments, three replicate stimulated cell compositions are used in the methods. In some embodiments, an average of the luminescent signal detected from the two or more replicate stimulated cell compositions is used to determine cell proliferation.

[0094] In some embodiments, any of the methods described herein, such as immediately above, are performed once. In some embodiments, any of the methods described herein, such as immediately above, are performed iteratively. In some embodiments, iterative performance of the method allows for the assessment of cell proliferation at different timepoints.

[0095] In some embodiments, at least one step of the methods described herein is performed iteratively with the same test cell composition (e.g., stimulated cell composition) to allow assessment of cell proliferation at different timepoints. In some embodiments, the entire method is performed iteratively. In some embodiments, only less than all of the steps are performed iteratively. In some embodiments, only one step is performed iteratively.

[0096] In some embodiments, the culturing step is not repeated with each iteration. In some embodiments, the incubating step is not repeated with each iteration.

[0097] In some embodiments, at least the detecting step is repeated. In some embodiments, equilibrating and detecting steps are repeated. In some embodiments, when the detecting step is23MF-36448161473504-2029640 repeated, the incubating step is performed before each iteration of the detecting step. In some such embodiments, the equilibrating step is performed before each iteration of the detecting step.

[0098] In some embodiments, the incubating, equilibrating, detecting and determining steps are repeated.

[0099] In some embodiments, iteration of the detecting step is carried out within a certain time period from the first performance of the culturing step (e.g., culturing with the recombinant receptorstimulating agent). In some embodiments, such period of time is 96 hours. In some embodiments, such period of time is 72 hours.

[0100] In some embodiments, a method disclosed herein is used as a release assay. In some embodiments, a method disclosed herein is used as a stability assay.A. Culturing a Cell Composition

[0101] In some aspects, the provided methods include culturing cells of an engineered cell composition with a recombinant receptor-stimulating agent to generate a stimulated cell composition. In some embodiments, the recombinant receptor-stimulating agent binds to recombinant receptors expressed by the cells of the engineered cell composition to activate the cell. In some embodiments, the engineered cell composition includes any of the cell compositions described herein, particularly in Section I. A. In some embodiments, the recombinant receptor includes any of the recombinant receptors described herein, particularly in Section ILA and II.B.

[0102] In some embodiments, the culturing step is initiated when cells of the engineered cell composition come into contact with the recombinant receptor-stimulating agent.

[0103] In some embodiments, the culturing step lasts for a certain period of time. In some embodiments, the cells (e.g., stimulated cells or unstimulated cells) are cultured for any period of time without limit. In some embodiments, the cells are cultured for at least about 40 hours. In some embodiments, the cells are cultured for at least 45 hours, at least 46 hours, at least 47 hours, or at least 48 hours. In some embodiments, the cells are cultured for at least about 46 hours. In some embodiments, the cells are cultured between about 40 hours and about 240 hours. In some embodiments, the cells are cultured between about 46 hours and about 96 hours, in some embodiments, the cells are cultured between about 46 hours and about 72 hours. In some embodiments, the cells are cultured between or between about 40 hours and about 50 hours, about 45 hours and about 55 hours, about 50 hours and about 60 hours, about 55 hours and about 65 hours, about 60 hours and about 70 hours, about 65 hours and about 75 hours, about 70 hours and about 80 hours, about 75 hours and about 85 hours, about 80 hours and about 90 hours, about 85 hours and about 95 hours, and about 90 hours and about 100 hours. In some embodiments, the cells are cultured between or between about 46 hours and about 50 hours. In some embodiments, the cells are cultured for about 46 hours. In some embodiments, the cells are cultured for about 48 hours. In some24MF-36448161473504-2029640 embodiments, the cells are cultured for about 50 hours. In some embodiments, the cells are cultured for about 72 hours. In some embodiments, the cells are cultured for about 96 hours.

[0104] In some embodiments, the culturing step is carried out under conditions that promote cell health and growth. In some embodiments, these conditions include a specific temperature. In some embodiments, these conditions include a specific percentage of carbon dioxide (CO2). In some embodiments, the culturing is carried out in a cell culture incubator to regulate or maintain both temperature and CO2. In some embodiments, these conditions include a specific percentage of humidity. In some embodiments, the culturing is carried out in a cell culture incubator to regulate or maintain temperature, CO2, and humidity.

[0105] In some embodiments, the culturing step is carried out at a temperature of at or about 30°C to 40°C. In some embodiments, the culturing step is carried out at a temperature of at or about 30°C to 33°C, 31°C to 34°C, 32°C to 35°C, 33°C to 36°C, 34°C to 37°C, 35°C to 38°C, or 36°C to 39°C. In some embodiments, the culturing step is carried out at 35°C to 39°C or 37°C ± 2°C. In some embodiments, the temperature is at or about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0106] In some embodiments, the culturing step is carried out while maintaining CO2 at a percentage between 1% and 10%. In some embodiments, the culturing step is carried out while maintaining CO2 at a percentage between 3% and 7%. In some embodiments, CO2 is maintained at a percentage of or of about 3%, 4%, 5%, 6%, or 7%.

[0107] In some embodiments, the culturing step is carried out while maintaining humidity at a percentage between 90% and 100%. In some embodiments, humidity is maintained at a percentage of or of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0108] In some embodiments, the method provided herein includes culturing cells in a cell culture vessel. In some embodiments, the cell culture vessel is a treated cell culture vessel. In some embodiments, the cell culture vessel is an untreated cell culture vessel. In some embodiments the cell culture vessel is a flask. In some embodiments, the vessel is a plate. In some embodiments, the cell culture vessel comprises a multi-well plate. In some embodiments, any multi-well plate known in the art can be used. In some embodiments, the multi-well plate is a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96 well plate or a 384-well plate. In some embodiments, the multi-well plate is a 96-well plate.

[0109] In some embodiments, the cells are added to the cell culture vessel at a density of between or between about 3.3 x 104viable cells / mL and about 6 x 106viable cells / mL. In some embodiments, the cells are added to the cell culture vessel at a density of between or between about 8 x 104viable cells / mL and about 6 x 106viable cells / mL. In some embodiments, the cells are added to the culture vessel at a cell density of between about 6.7 x 104viable cells / mL and about 3 x 106viable25MF-36448161473504-2029640 cells / mL. In some embodiments, the cells are added to the cell culture vessel at a density of between or between about 8 x 104viable cells / mL and 8 x 105viable cells / mL. In some embodiments, the cells are added to the cell culture vessel at a density of between or between about 6 x 105viable cells / mL and 6 x 106viable cells / mL.

[0110] In some embodiments, the cells are added to the cell culture vessel at a density of between or between about 8 x 104viable cells / mL and about 9 x 104viable cells / mL, about 8.5 x 104viable cells / mL and about 9.5 x 104viable cells / mL, about 9 x 104viable cells / mL and about 10 x 104viable cells / mL, about 9.5 x 104viable cells / mL and about 10.5 x 104viable cells / mL, about 1 x 105viable cells / mL and about 2 x 105viable cells / mL, about 1.5 x 105viable cells / mL and about 2.5 x 105viable cells / mL, about 2 x 105viable cells / mL and about 3 x 105viable cells / mL, about 2.5 x 105viable cells / mL and about 3.5 x 105viable cells / mL, about 3 x 105viable cells / mL and about 4 x 105x 105viable cells / mL, about 3.5 x 105x 105viable cells / mL and about 4.5 x 105x 105viable cells / mL, about 4 x 105viable cells / mL and about 5 x 105viable cells / mL, about 4.5 x 105viable cells / mL and about 5.5 x 105viable cells / mL, about 5 x 105viable cells / mL and about 6 x 105viable cells / mL, about 5.5 x 105viable cells / mL and about 6.5 x 105viable cells / mL, about 6 x 105viable cells / mL and about 7 x 105viable cells / mL, about 6.5 x 105viable cells / mL and about 7.5 x 105viable cells / mL, about 7 x 105viable cells / mL and about 8 x 105viable cells / mL, about 7.5 x 105viable cells / mL and about 8.5 x 105viable cells / mL, about 8 x 105viable cells / mL and about 9 x 105viable cells / mL, about 8.5 x 105viable cells / mL and about 9.5 x 105viable cells / mL, about 9 x 105viable cells / mL and about 10 x 105viable cells / mL, about 9.5 x 105viable cells / mL and about 10.5 x 105viable cells / mL, about 1 x 106viable cells / mL and about 2 x 106viable cells / mL, about 1.5 x 106viable cells / mL and about 2.5 x 106viable cells / mL, about 2 x 106viable cells / mL and about 3 x 106viable cells / mL, about 2.5 x 106viable cells / mL and about 3.5 x 106viable cells / mL, about 3 x 106viable cells / mL and about 4 x 106viable cells / mL, about 3.5 x 106viable cells / mL and about 4.5 x 106viable cells / mL, about 4 x 106viable cells / mL and about 5 x 106viable cells / mL, about 4.5 x 106viable cells / mL and about 5.5 x 106viable cells / mL, about 5 x 106viable cells / mL and about 6 x 106viable cells / mL.

[0111] In some embodiments, the cells are added to the cell culture vessel at a density of between or between about 8.3 x 104cells / mL and about 5.3 x 106cells / mL. In some embodiments, the cells are added to the cell culture vessel at a density of between or between about 1 x 105viable cells / mL and about 1 x 106viable cells / mL. In some embodiments, the cells are added to the cell culture vessel at a density of between or between about 1 x 105viable cells / mL and about 7 x 105viable cells / mL. In some embodiments, the cells are added to the cell culture vessel at a density of between or between about 1 x 105viable cells / mL and about 2 x 105viable cells / mL. In some embodiments, the cells are added to the cell culture vessel at a density of between or between about 1 x 105viable cells / mL and about 1.5 x 105viable cells / mL.26MF-36448161473504-2029640

[0112] In some embodiments, the cells are added to the cell culture vessel at a density of about 1 x 105viable cells / mL, about 1.1 x 105viable cells / mL, about 1.2 x 105viable cells / mL, about 1.3 x 105viable cells / mL, about 1.4 x 105viable cells / mL, or about 1.5 x 105viable cells / mL.

[0113] In some embodiments, the number of cells present in the culture vessel at the time the detecting is performed is not more than about 6 x 106viable cells / mL. In some embodiments, the number of cells present in the culture vessel at the time the detecting is performed is not less than about 3.3 x 104viable cells / mL. In some embodiments, the number of cells present in the culture vessel at the time the detecting is performed is between about 3.3 x 104viable cells / mL and about 6 x 106viable cells / mL. In some embodiments, the number of cells present in the culture vessel at the time the detecting is performed is between 3.3 x 104viable cells / mL and 6 x 106viable cells / mL.

[0114] In some embodiments, the provided methods include culturing cells of a cell composition in the absence of a recombinant receptor-stimulating agent. In some embodiments, cells of a cell composition that are cultured in the absence of a recombinant receptor-stimulating agent are referred to as unstimulated cells or an unstimulated cell composition (e.g., unstimulated cells of the test engineered cell composition). In some embodiments, the unstimulated cells comprise unstimulated engineered cells. In some embodiments, the unstimulated cells comprise unstimulated non-engineered cells. In some embodiments, the unstimulated cells comprise unstimulated reference cells, which can be used as the reference standard.

[0115] In some embodiments, culturing the unstimulated cells or unstimulated cell composition is typically performed under conditions that are the same as the conditions for culturing stimulated cells, except that the recombinant receptor-stimulating agent is not present. In some embodiments, the unstimulated cells are cultured under the same conditions of the stimulated cells, but without the recombinant receptor-stimulating agent, for a second period of time.

[0116] In some embodiments, culturing the unstimulated cells or unstimulated cell composition is performed with a replicate of the unstimulated cell composition. In some embodiments, two or more replicate unstimulated cell compositions are used in the methods. In some embodiments, an average of the luminescent signal detected from the two or more replicate unstimulated cell compositions is used to measure cell proliferation. In some embodiments, two replicate unstimulated cell compositions are used in the methods. In some embodiments, at least three replicate unstimulated cell compositions are used in the methods. In some embodiments, three replicate unstimulated cell compositions are used in the methods.

[0117] In some embodiments, incubating the unstimulated cells or unstimulated cell composition in the presence of a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme, is performed under conditions that are the same as the conditions for incubating stimulated cells, as described in Section LB. In some embodiments, luminescent signal from the unstimulated27MF-36448161473504-2029640 cells or unstimulated cell compositions is detected under conditions that are the same as the conditions for detecting luminescent signal from stimulated cells, as described in Section LB.1. Test Cell Compositions

[0118] In some embodiments, the provided methods comprise assessing cell proliferation of a test cell composition. In some embodiments, a test cell composition comprises any cell composition for which a specific characteristic (e.g., proliferation) is being assessed.

[0119] In some embodiments, cells of the test cell composition comprise (e.g., express) a recombinant receptor, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR) as described in Section II. A. and II.B. Thus, in some embodiments, the test cell composition is an engineered test cell composition. In some embodiments, the engineered test cell composition is generated according to any of the production methods described herein, e.g., any described in Section I. A. In some embodiments, the test engineered cell composition has been produced ex vivo from primary cells. In some embodiments, the primary cells are subjected to a manufacturing process such that at the end of the manufacturing process, the cells comprise (e.g. , express) a recombinant receptor (e.g., a CAR or TCR).

[0120] In some embodiments, the test engineered cell composition is for use as a cell therapy for treating any disease or condition. In some embodiments, cells used to make the test engineered cell composition are primary cells that are sourced from (or autologous to) the subject to be treated. In some embodiments, the cells used to make the test engineered cell composition are primary cells that are sourced from a different subject than (or are allogeneic to) the subject to be treated. In some embodiments, the disease or condition comprises a cancer. In some embodiments, the disease or condition comprises an autoimmune or inflammatory disease or disorder.

[0121] In some embodiments, the cells of the test cell composition do not comprise (e.g., do not express) any recombinant receptor. Thus, in some embodiments, the test cell composition is a non-engineered cell composition. In some embodiments, the non-engineered cells are generated according to any of the production methods described herein, e.g., any described in Section LA.

[0122] In some embodiments, the cells of the test cell composition are stimulated with a stimulating agent. In some embodiments, the stimulating agent comprises any of the stimulating agents described in Section I.A.3. In some embodiments, the stimulating agent is a recombinant receptor-stimulating agent, such as any of those described in in Section I.A.3. In some embodiments, the test engineered cell composition is stimulated by culturing the cells with the recombinant receptor-stimulating agent as described in Section I.A.3.28MF-36448161473504-2029640 a. Samples

[0123] In particular embodiments, the test cell composition is generated by isolating, selecting, and / or enriching cells from a biological sample. In some embodiments, the described methods include isolation of cells, or compositions, thereof from biological samples, such as those obtained from or derived from a subject, such as one having or suspected of having a particular disease or condition or in need of a cell therapy or to which cell therapy will be administered. In some embodiments, the subject is a human, such as a subject who is a patient in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. In some embodiments, the subject is a healthy donor not having or suspected of having a disease or condition. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples can be, e.g., tissue, fluid, and / or other samples taken directly from the subject. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0124] In some embodiments, the sample is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0125] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some embodiments, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some embodiments contains cells other than red blood cells and platelets.

[0126] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media 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 many or all divalent cations. In some embodiments, a washing step is accomplished a semi-automated “flow- through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions. In some embodiments, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer’s instructions. In some embodiments, the cells are29MF-36448161473504-2029640 resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++ / Mg++free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[0127] In some embodiments, the preparation methods include steps for freezing, e.g., cry opreserving, the cells, either before or after isolation, selection and / or enrichment of cells and / or incubation of cells for transduction and engineering, and / or after cultivation and / or harvesting of the engineered cells. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters, in some embodiments, may be used.

[0128] In some embodiments, isolation of the cells or populations includes one or more preparation and / or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0129] In some embodiments, at least a portion of the selection step includes incubation of cells with a selection reagent. The incubation with a selection reagent or reagents, e.g., as part of selection methods which may be performed using one or more selection reagents for selection of one or more different cell types based on the expression or presence in or on the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method using a selection reagent or reagents for separation based on such markers may be used. In some embodiments, the selection reagent or reagents result in a separation that is affinity- or immunoaffinity-based separation. For example, the selection in some embodiments includes incubation with a reagent or reagents for separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0130] In some embodiments of such processes, a volume of cells is mixed with an amount of a desired affinity-based selection reagent. The immunoaffinity-based selection can be carried out using any system or method that results in a favorable energetic interaction between the cells being30MF-36448161473504-2029640 separated and the molecule specifically binding to the marker on the cell, e.g., the antibody or other binding partner on the solid surface, e.g., particle. In some embodiments, methods are carried out using particles such as beads, e.g., magnetic beads, that are coated with a selection agent (e.g., antibody) specific to the marker of the cells. The particles (e.g., beads) can be incubated or mixed with cells in a container, such as a tube or bag, while shaking or mixing, with a constant cell density- to-particle (e.g., bead) ratio to aid in promoting energetically favored interactions. In other cases, the methods include selection of cells in which all or a portion of the selection is carried out in the internal cavity of a centrifugal chamber, for example, under centrifugal rotation. In some embodiments, incubation of cells with selection reagents, such as immunoaffinity-based selection reagents, is performed in a centrifugal chamber. In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus described in W02009 / 072003 or US 20110003380 Al. In one example, the system is a system as described in W02016 / 073602.

[0131] In some embodiments, by conducting such selection steps or portions thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads) in the cavity of a centrifugal chamber, the user is able to control certain parameters, such as volume of various solutions, addition of solution during processing and timing thereof, which can provide advantages compared to other available methods. For example, the ability to decrease the liquid volume in the cavity during the incubation can increase the concentration of the particles (e.g., bead reagent) used in the selection, and thus the chemical potential of the solution, without affecting the total number of cells in the cavity. This in turn can enhance the pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, carrying out the incubation step in the chamber, e.g., when associated with the systems, circuitry, and control as described herein, permits the user to effect agitation of the solution at desired time(s) during the incubation, which also can improve the interaction.

[0132] In some embodiments, at least a portion of the selection step is performed in a centrifugal chamber, which includes incubation of cells with a selection reagent. In some embodiments of such processes, a volume of cells is mixed with an amount of a desired affinitybased selection reagent that is far less than is normally employed when performing similar selections in a tube or container for selection of the same number of cells and / or volume of cells according to manufacturer’s instructions

[0133] In some embodiments, for selection, e.g., immunoaffinity-based selection of the cells, the cells are incubated in the cavity of the chamber in a composition that also contains the selection buffer with a selection reagent, such as a molecule that specifically binds to a surface marker on a cell that it desired to enrich and / or deplete, but not on other cells in the composition, such as an31MF-36448161473504-2029640 antibody, which optional I y is coupled to a scaffold such as a polymer or surface, e.g. , bead, e.g. , magnetic bead, such as magnetic beads coupled to monoclonal antibodies specific for CD4 and CD8.

[0134] In some embodiments, such process is carried out within the entirely closed system to which the chamber is integral. In some embodiments, this process (and in some embodiments also one or more additional step, such as a previous wash step washing a sample containing the cells, such as an apheresis sample) is carried out in an automated fashion, such that the cells, reagent, and other components are drawn into and pushed out of the chamber at appropriate ti mes and centrifugation effected, so as to complete the wash and binding step in a single closed system using an automated program.

[0135] In some embodiments, after the incubation and / or mixing of the cells and selection reagent and / or reagents, the incubated cells are subjected to a separation to select for cells based on the presence or absence of the particular reagent or reagents. In some embodiments, the separation is performed in the same closed system in which the incubation of cells with the selection reagent was performed. In some embodiments, after incubation with the selection reagents, incubated cells, including cells in which the selection reagent has bound are transferred into a system for immunoaffinity-based separation of the cells. In some embodiments, the system for immunoaffinitybased separation is or contains a magnetic separation column.

[0136] Such separation steps can be based on positive selection, in which the cells having bound the reagents, e.g., antibody or binding partner, are retained for further use, and / or negative selection, in which the cells having not bound to the reagent, e.g. , antibody or binding partner, are retained. In some examples, both fractions are retained for further use. In some embodiments, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0137] In some embodiments, the process steps further include negative and / or positive selection of the incubated and cells, such as using a system or apparatus that can perform an affinitybased selection. In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negati ve selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (marker111®11) on the positively or negatively selected cells, respectively. 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 subjected to the process for selection, such as by repeating a positive or negative selection step. In some embodiments, selection is repeated twice, three times, four times, five times,32MF-36448161473504-2029640 six times, seven times, eight times, nine times 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.

[0138] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0139] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types. In certain embodiments, one or more separation steps are repeated and / or performed more than once. In some embodiments, the positively or negatively selected fraction resulting from a separation step is subjected to the same separation step, such as by repeating the positive or negative selection step. In some embodiments, a single separation step is repeated and / or performed more than once, for example, to increase the yield of positively selected cells, to increase the purity of negatively selected cells, and / or to further remove the positively selected cells from the negatively selected fraction. In certain embodiments, one or more separation steps are performed and / or repeated two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more than ten times. In certain embodiments, the one or more selection steps are performed and / or repeated between one and ten times, between one and five times, or between three and five times. In certain embodiments, one or more selection steps are repeated three times.

[0140] For example, in some embodiments, specific subpopulations of T cells, such as cells positive or expressing 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 antibody or binding partner that specifically binds to such markers. In some embodiments, the antibody or binding partner can be conjugated, such as directly or indirectly, to a solid support or matrix to effect selection, such as a magnetic bead or paramagnetic bead. For example, CD3+,33MF-36448161473504-2029640CD28+ 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).

[0141] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some embodiments, 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 or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0142] In some embodiments, CD8+ T cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some embodiments is particularly robust in such sub-populations. See Terakura et al. , (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0143] In embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0144] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in some embodiments, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some embodiments, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L.

[0145] Such selections in some embodiments are carried out simultaneously and in other embodiments are carried out sequentially, in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ T cell population or subpopulation, also is used to generate the CD4+ T cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps. In some embodiments, the selection for the CD4+ T cell population and the selection for the CD8+ T cell34MF-36448161473504-2029640 population are carried out simultaneously. In some embodiments, the CD4+ T cell population and the selection for the CD8+ T cell population are carried out sequentially, in either order. In some embodiments, methods for selecting cells can include those as described in US20170037369. In some embodiments, the selected CD4+ T cell population and the selected CD8+ T cell population may be combined subsequent to the selecting. In some embodiments, 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 separately processed, whereby the selected CD4+ T cell population is enriched in CD4+ T cells and incubated with a stimulatory reagent (e.g., anti-CD3 / anti-CD28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g., CAR) and cultivated under conditions to expand T cells and the selected CD8+ T cell population is enriched in CD8+ T cell and incubated with a stimulatory reagent (e.g., anti-CD3 / anti-CD28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g., CAR), such as the same recombinant protein as for engineering of the CD4+ T cells from the same donor, and cultivated under conditions to expand T cells, such as in accord with the provided methods.

[0146] In particular embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD4+ T cells, where both the 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, where both the negative and positive fractions are retained. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0147] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ T cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD 14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

[0148] CD4+ T helper cells may be sorted into naive, central memory, and effector cells by identifying cell populations that have 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-.

[0149] In one example, to enrich for CD4+ T cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as35MF-36448161473504-2029640 a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or 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: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[0150] In some embodiments, the incubated sample or composition of cells to be separated is incubated with a selection reagent containing small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS® beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

[0151] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. Many well-known magnetically responsive materials for use in magnetic separation methods are known, e.g., those described in Molday, U.S. 4,452,773, and in EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 also may be used.

[0152] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[0153] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0154] In some embodiments, separation is achieved in a procedure in which the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and36MF-36448161473504-2029640 negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[0155] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic Activated Cell Sorting (MACS), e.g., CliniMACS systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0156] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and / or engineered; in some embodiments, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.2. Reference Cell Compositions

[0157] In some embodiments, the provided methods comprise assessing cell proliferation of a reference cell composition. In some embodiments, the provided methods comprise assessing cell proliferation of a reference cell composition, which is used as a reference to which proliferation of a test cell composition (e.g., an engineered cell composition) is compared. In some embodiments, the provided methods comprise calculating a relative proliferation by comparing the cell proliferation of a test cell composition (e.g., an engineered cell composition) to the cell proliferation of a reference cell composition. In some embodiments, the reference cell composition comprises any cell composition used for comparison to any of the test cell compositions described herein.

[0158] In some embodiments, the cells of the reference cell composition comprise (e.g., express) a reference recombinant receptor, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR) as described in Section ILA. and II.B. Thus, in some embodiments, the reference cell composition is a reference engineered cell composition. In some embodiments, the reference engineered cell composition is generated according to any of the production methods described herein, e.g., any described in Section I. A. In some embodiments, the reference cell composition is a reference engineered cell composition that has been engineered to comprise (e.g., express) the same37MF-36448161473504-2029640 recombinant receptor (such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR)) as the test cell composition. In some embodiments, the reference cell composition is a reference engineered cell composition that has been engineered to comprise (e.g., express) a different recombinant receptor than the test cell composition.

[0159] In some embodiments, the cells of the reference cell composition do not comprise (e.g., do not express) any recombinant receptor. Thus, in some embodiments, the reference cell composition is a non-engineered cell composition. In some embodiments, the non-engineered cells are generated according to any of the production methods described herein, e.g., any described in Section I.A.l.

[0160] In some embodiments, the cells of the reference cell composition are derived from a healthy donor.

[0161] In some embodiments, the cells of the reference cell composition are stimulated with a stimulating agent. In some embodiments, the stimulating agent comprises any of the stimulating agents described in Section I.A.3. In some embodiments, the stimulating agent is a recombinant receptor-stimulating agent, such as any of those described in in Section I.A.3. In some embodiments, the reference engineered cell composition is stimulated by culturing the cells with the recombinant receptor-stimulating agent as described in Section I.A.3.

[0162] In some embodiments, the reference standards described herein, such as in Section I.C.l.a, comprise a reference cell composition. In some embodiments, the control cell compositions described herein, such as in Section I.D, comprise a reference cell composition.3. Stimulating a Cell Composition

[0163] In some aspects, the provided methods include stimulating cells of a cell composition to generate a stimulated cell composition.

[0164] In some embodiments, the cells of the cell composition can comprise (e.g., express) a naturally occurring receptor. In some embodiments, stimulating a cells of a cell composition comprising a naturally occurring receptor can be accomplished by means known in the art. In some embodiments, the cells of the cell composition are T cells. In some embodiments, a stimulating agent for a T cell is an anti-CD3 antibody and / or an anti-CD28 antibody. In some embodiments, a stimulating agent for a T cell is a recombinant cytokine. In some embodiments, the stimulating agent is selected from IL-2, IL- 15, IL-7 and IL-21.

[0165] In some embodiments, the cells of the cell composition can comprise (e.g., express) a recombinant receptor. In some embodiments, the cells of the cell composition are engineered to express a recombinant receptor. In some embodiments, the recombinant receptor comprises any of the recombinant receptors described in Section II. In some embodiments, the recombinant receptor is a CAR or a TCR.38MF-36448161473504-2029640

[0166] In some embodiments, a protein-based recombinant receptor-stimulating agent, such as a surface immobilized binding moiety (e.g., target antigen or alD antibody), is contacted with a sample of cells of the engineered cell composition. In some embodiments, the recombinant receptorstimulating agent is in an amount of from 0.1 pg / mL to 100 pg / mL, such as from 0.5 pg / mL to 50 pg / mL, more generally 1 pg / mL to 10 pg / mL. In some embodiments, the recombinant receptorstimulating agent is in an amount of from 1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, 6 pg / mL, 7 pg / mL, 8 pg / mL, 9 pg / mL, 10 pg / mL, or any value between any of the foregoing. In some embodiments, for the contacting with the recombinant receptor-stimulating agent, the cells of the engineered cell composition are present at a concentration of from 0.1 x 106cells / mL to 100 x 106cells / mL, such as from 0.5 x 106cells / mL to 50 x 106cells / mL, more generally 0.5 x 106cells / mL to 10 x 106cells / mL. In some embodiments, for the contacting with the recombinant receptorstimulating agent, the cells of the engineered cell composition are present at a concentration of from 0.5 x 106cells / m, 1 x 106cells / mL, 2 x 106cells / mL, 3 x 106cells / mL, 4 x 106cells / mL, 0.5 x 106cells / mL, 6 x 106cells / mL, 7 x 106cells / mL, 8 x 106cells / mL, 9 x 106cells / mL, 10 x 106cells / mL, or any value between any of the foregoing.

[0167] In some embodiments, the method of stimulating cells of an engineered cell composition to generate a stimulated cell composition includes means of stimulating the recombinant receptor (e.g., CARs or TCRs) expressed by the cells of the cell composition. It is contemplated that any means suitable for stimulating the recombinant receptor that is also capable of being quantified may be used. In some embodiments, the means of stimulation of the recombinant receptor is achieved by a recombinant receptor-stimulating agent able to bind to and stimulate an intracellular signal by the recombinant receptor. Exemplary recombinant receptor-stimulating agents include antigens (e.g., purified or recombinant antigens) of the recombinant receptor, antibodies such as antiidiotype (alD) antibodies, and antigen-expressing cells.

[0168] In particular embodiments, the recombinant receptor-stimulating agent is composed of a binding moiety that is able to be bound by the recombinant receptor that is immobilized on a surface support. In provided embodiments, the binding moiety may be an antigen or a portion of an antigen of the recombinant receptor (e.g. extracellular portion of an antigen) or an antibody (e.g., an alD antibody) specific to the recombinant receptor. In some embodiments, the recombinant receptorstimulating agent is immobilized or bound to a surface support, such as a microwell plate or a solid particle (e.g. bead).

[0169] In some aspects, the binding moiety is an antibody (e.g., an anti-idiotype antibody (alD)) or antigen-binding fragment thereof that specifically recognizes a recombinant receptor(e.g. , CAR). In particular, an alD targets via binding an idiotype of an antibody, such as the antigen binding site of another antibody, such as the scFv of the extracellular antigen binding domain of a39MF-36448161473504-2029640CAR. In some embodiments, the idiotype is any single antigenic determinant or epitope within the variable portion of an antibody. In some cases it can overlap the actual antigen-binding site of the antibody, and in some cases it may comprise variable region sequences outside of the antigenbinding site of the antibody. The set of individual idiotypes of an antibody is in some embodiments referred to as the “idiotype” of such antibody. In some embodiments, the alD is able to bind to the recombinant receptor to stimulate a recombinant receptor-dependent activity.

[0170] The choice of alD can be made depending on the particular recombinant receptor, such as CAR. Exemplary alD against antigen-specific CARs are known or can be generated by standard antibody technologies. These include, but are not limited to, alD directed against a CD22-directed CAR, see e.g. PCT Publication No. WO2013188864; CD19-directed CAR, such as directed against FMC63 scFv, see e.g. PCT Publication No. WO 2018 / 023100; Cat. No. REA1297 (Miltenyi Biotech); clone Y45 (Fisher Scientific, e.g., Cat. No. 16871936); a GPRC5D-directed CAR, see e.g. PCT Application No. PCT / US2020 / 063497; and a BCMA-directed CAR, see e.g. PCT Application No. PCT / US2020 / 063492. The alD can be immobilized or attached to a surface support (e.g., bead) as described above for use as a recombinant receptor-stimulating agent against cells expressing the recombinant receptor (e.g., CAR) targeted by the alD.

[0171] In some embodiments, the recombinant receptor-stimulating agent is contacted with a sample of cells of the engineered cell composition and incubated under conditions suitable for activation of cells expressing the recombinant receptor. In some embodiments, a recombinant receptor-stimulating agent (e.g., surface immobilized antigen of the recombinant receptor, e.g., CAR, for example, plate-bound antigen) is incubated with the cells for 2 hours to 96 hours. In particular embodiments, the incubation is for 12 hours to 72 hours, such as 12 hours to 48 hours, for example at or about 24 hours. In some embodiments, the incubation is carried out at a temperature suitable for culture of the cells, such as a temperate of at or about 37°C ± 4°C, for example, at or about 37°C. In some embodiments, a stable carbon dioxide is maintained, such as at or about 5% CO2.

[0172] In some embodiments, the receptor stimulating agent binds to and activates a recombinant receptor. Thus, in some embodiments, the receptor stimulating agent is a recombinant receptor-stimulating agent. In other embodiments, the receptor stimulating agents binds to and activates a naturally occurring receptor.

[0173] In some embodiments, the recombinant receptor-stimulating agent is or comprises a binding moiety that is able to bind a recombinant receptor or is able to be recognized by (e.g., bound by) the recombinant receptor. In some embodiments, the recombinant receptor is a CAR, such as any of the CARs disclosed in Section II. A. In some embodiments, the recombinant receptor is a TCR, such as any of the TCRs disclosed in Section II.B. In some embodiments, the recombinant receptorstimulating agent binds to a CAR.40MF-36448161473504-2029640

[0174] In some embodiments, binding of the recombinant receptor-stimulating agent to a recombinant receptor (e.g., a CAR) is facilitated by a binding moiety on the recombinant receptorstimulating agent. In some embodiments, the recombinant receptor-stimulating agent is or comprises a binding moiety specific to the recombinant receptor. In some embodiments, the binding moiety recognizes an extracellular domain of the recombinant receptor (e.g., a CAR). In some embodiments, the recombinant receptor-stimulating agent binds to an extracellular antigen binding domain of a CAR.

[0175] In some embodiments, the binding moiety is a target antigen or an extracellular domain binding portion thereof of the recombinant receptor. In some embodiments, the extracellular domain binding portion of the target antigen comprises an epitope recognized by the recombinant receptor. In some embodiments, the binding moiety is an antibody. In some embodiments, the binding moiety is an alD, such as any of the alDs described above.

[0176] In some embodiments, the binding moiety is immobilized or attached to a solid support prior to introduction to the cell compositions described herein. The binding moiety can be immobilized to any solids support known in the art. In some embodiments, a solid support comprises any solid material suitable for use with cells or cell culture. In some embodiments, the solid support is a culture vessel. In some embodiments, the culture vessel is a microwell plate. In some embodiments, the solid support is a bead.

[0177] In some embodiments, the microwell plate comprises any microwell plate known in the art. In some embodiments, the microwell plate is a multi-well plate (e.g., comprising multiple wells). In some embodiments, the multi-well plate comprises a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate. In some embodiments, the microwell plate is a 96-well plate.B. Generating and Detecting a Luminescent Signal

[0178] In some embodiments, provided herein are methods for generating and detecting a luminescent signal.

[0179] In some embodiments, the luminescent signal is generated and detected in an engineered cell composition, e.g., a cell therapy composition such as a CAR-T cell therapy. In some embodiments, the engineered cell composition comprises any of the engineered cell compositions described herein (e.g., test engineered cell composition). In some embodiments, the engineered cell composition is a test engineered cell composition. In some embodiments, the test engineered cell composition is a stimulated cell composition. In some embodiments, the luminescent signal is generated and detected in a reference cell composition (e.g., a composition as described in Section I.A.2).41MF-36448161473504-2029640

[0180] In some embodiments, the method of generating the luminescent signal comprises adding a luciferase enzyme and a substrate or a pro-substrate of the luciferase enzyme to the stimulated cell composition.

[0181] In some embodiments, the luciferase enzyme binds to a specific substrate or prosubstrate. In some embodiments, any luciferase enzyme and its substrate or pro-substrate can be used in the method of generating the luminescent signal. In some embodiments, the luciferase enzyme comprises any of the luciferase enzymes described herein, such as below. In some embodiments, the substrate or pro-substrate comprises any of the substrates or pro-substrates described herein, such as below.

[0182] In some embodiments, the luciferase enzyme and substrate or pro-substrate are added to the stimulated cell composition within a period of time after initiating the culturing described in Section I above. In some embodiments, the period of time is an amount of time sufficient for cells to proliferate and / or divide.

[0183] In some embodiments, the period of time can be any period of time after initiation of the culturing.

[0184] In some embodiments, the period of time is at least 24 hours. In some embodiments, the period of time is between or between about 24 hours and 96 hours. In some embodiments, the period of time is between or between about 36 hours and 84 hours. In some embodiments, the period of time is between or between about 48 hours and 72 hours.

[0185] In some embodiments, the period of time is at least about 40 hours. In some embodiments, the period of time is at least about 45 hours, at least about 46 hours, at least about 47 hours or at least about 48 hours. In some embodiments, the period of time is about 48 hours.

[0186] In some embodiments, the period of time is at least about 60 hours. In some embodiments, the period of time is at least about 68 hours, at least about 69 hours, at least about 70 hours, at least about 71 hours or at least about 72 hours. In some embodiments, the period of time is about 72 hours.

[0187] In some embodiments, the method of generating the luminescent signal comprises incubating the stimulated cell composition with the luciferase enzyme and the substrate or prosubstrate of the luciferase enzyme. In some embodiments, the incubating step is carried out under conditions that promote luciferase binding to substrate or pro-substrate to boost the luminescent signal generated.

[0188] In some embodiments, these conditions include a specific temperature. In some embodiments, these conditions include a specific percentage of carbon dioxide (CO2). In some embodiments, the incubating is carried out in a cell culture incubator to regulate or maintain both temperature and CO2. In some embodiments, the incubating step is carried out at a temperature of at42MF-36448161473504-2029640 or about 30°C to 40°C. In some embodiments, the incubating step is carried out at a temperature of at or about 30°C to 33°C, 31°C to 34°C, 32°C to 35°C, 33°C to 36°C, 34°C to 37°C, 35°C to 38°C, or 36°C to 39°C. In some embodiments, the incubating step is carried out at 35°C to 39°C or 37°C ± 2°C. In some embodiments, the temperature is at or about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C. In some embodiments, the incubating step is carried out while maintaining CO2 at a percentage between 1% and 10%. In some embodiments, the incubating step is carried out while maintaining CO2 at a percentage between 3% and 7%. In some embodiments, CO2 is maintained at a percentage of or of about 3%, 4%, 5%, 6%, or 7%.

[0189] In some embodiments, the culturing step is carried out while maintaining humidity at a percentage between 90% and 100%. In some embodiments, humidity is maintained at a percentage of or of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0190] In some embodiments, these conditions include a specific period of time. In some embodiments, the incubating step lasts for a specific period of time. In some embodiments, the incubating step is for any period of time. In some embodiments, the incubating step is for about 5 minutes to 2 hours. In some embodiments, the incubating step is for about 10 minutes to 30 minutes, 20 minutes to 40 minutes, 30 minutes to 50 minutes, 40 minutes to 60 minutes, 50 minutes to 70 minutes, 60 minutes to 80 minutes, 70 minutes to 90 minutes, 80 minutes to 100 minutes, 90 minutes to 110 minutes, or 100 minutes to 120. In some embodiments, the incubating step is for about 5 minutes to about 55 minutes. In some embodiments, the incubating step is for about 10 minutes to 50 minutes.

[0191] In some embodiments, the method of generating the luminescent signal further comprises equilibrating the stimulated cell composition under conditions that promote uniform reading of the luminescent signal detected from the stimulated cell composition. In some embodiments, the equilibrating is performed after the incubating step and before the detecting step described herein, such as below.

[0192] In some embodiments, the equilibrating conditions comprise a specific temperature or a temperature within a temperature range. In some embodiments, the temperature comprises ambient room temperature (ART). In some embodiments, the equilibrating step is carried out at a temperature at or about 68°C to 77°C.

[0193] In some embodiments, the equilibrating conditions comprise a specific period of time. In some embodiments, the period of time is for about 1 hour to about 6 hours, about 2 hours to about 5 hours, or about 3 hours to about 4 hours. In some embodiments, the equilibrating is for about 1 hour to about 2 hours, about 1.5 hours to about 2.5 hours, about 2 hours to about 3 hours, about 2.5 hours to about 3.5 hours, about 3 hours to about 4 hours, about 3.5 hours to about 4.5 hours, about 4 hours to about 5 hours, about 4.5 hours to about 6 hours. In some embodiments, the equilibrating is43MF-36448161473504-2029640 for about 1 hour to about 6 hours. In some embodiments, the equilibrating is for about 2 hours to about 4 hours. In some embodiments, the equilibrating is for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours. In some embodiments, the equilibrating is for about 3 hours.

[0194] In some embodiments, the equilibrating conditions comprise protection from light. In some embodiments, the equilibrating step is performed under low light. In some embodiments, the equilibrating step is performed under no light or in the absence of light. In some embodiments, the equilibrating step is performed in the dark.

[0195] In some embodiments, the method of detecting the luminescent signal comprises visualizing or detecting the light emitted or luminescent signal generated in or by the stimulated cell composition. In some embodiments, the luminescent signal is detected by any means known in the art, including a device or apparatus, such as a luminometer. Any luminometer known in the art can be used to detect luminescent signal generated according to the methods provided herein. In some embodiments, the luminometer is a IncuCyte® S3 Live-Cell Imaging and Analysis System.

[0196] In some embodiments, the luminescent signal is generated and detected in a cell composition, e.g., a non-engineered cell. In some embodiments, the cell composition comprises any of the cell compositions described herein. In some embodiments, the cell composition is a test cell composition. In some embodiments, the cell composition is an unstimulated cell composition. In some embodiments, the cell composition is a stimulated cell composition.

[0197] In some embodiments, the method of generating the luminescent signal comprises adding a luciferase enzyme and a substrate or a pro-substrate of the luciferase enzyme to the test cell composition. In some embodiments, the luciferase enzyme and substrate or pro-substrate are added to the test cell composition within a period of time after initiating the culturing of the test cell composition as described in Section I above. In some embodiments, the period of time is an amount of time sufficient for cells to proliferate and / or divide.

[0198] In some embodiments, the period of time can be any period of time after initiation of the culturing. In some embodiments, the period of time is at least 24 hours. In some embodiments, the period of time is between or between about 24 hours and 96 hours. In some embodiments, the period of time is between or between about 36 hours and 84 hours. In some embodiments, the period of time is between or between about 48 hours and 72 hours.

[0199] In some embodiments, the period of time is at least about 40 hours. In some embodiments, the period of time is at least about 45 hours, at least about 46 hours, at least about 47 hours or at least about 48 hours. In some embodiments, the period of time is about 48 hours.

[0200] In some embodiments, the period of time is at least about 60 hours. In some embodiments, the period of time is at least about 68 hours, at least about 69 hours, at least about 7044MF-36448161473504-2029640 hours, at least about 71 hours or at least about 72 hours. In some embodiments, the period of time is about 72 hours.

[0201] In some embodiments, the method of detecting the luminescent signal comprises visualizing or detecting the light emitted or luminescent signal generated in or by the stimulated cell composition. In some embodiments, the luminescent signal is detected by any means known in the art, including a device or apparatus, such as a luminometer.

[0202] In some embodiments, the luminescent signal is generated when a luciferase binds to its corresponding substrate or pro-substrate. In some embodiments, the luciferase does not produce any detectable signal (e.g., a luminescent signal). In some embodiments, the luminescent signal is generated after the luciferase binds to its corresponding substrate or pro-substrate.

[0203] In some embodiments, luminescent signal is detected by any suitable device, detector, apparatus or system known in the art. In some embodiments, the luminescent signal is detected using a luminometer. Various luminometer s are known in the art and can be used. In some embodiments, luminometers provide luminescent signal readouts as Relative Luminescence Units (RLUs), which is a measure of the number of photons emitted.

[0204] In some embodiments, the luciferase cannot bind to the pro-substrate until the prosubstrate is modified to allow for the binding of the luciferase. In some embodiments, the prosubstrate comprises a functional moiety (e.g., a blocking moiety) and a substrate moiety. In some embodiments, the blocking moiety prevents the luciferase from binding the substrate moiety. In some embodiments, the blocking moiety is an entity or functional group (e.g., peptide, organic molecule, etc.) that can be severed from the substrate moiety by an event (e.g., enzymatic cleavage, chemical cleavage, etc.). When the functional moiety is removed from the substrate moiety, the resulting substrate is liberated and can bind to the luciferase. In some embodiments, the blocking moiety is removed from the substrate by an intracellular event. In some embodiments, the intracellular event comprises reduction of the pro-substrate. Since only metabolically active cells can reduce the pro-substrate, the methods provided herein determine or assess cell proliferation of the cell composition based on the redox potential of the cells. The light produced once the luciferase binds the substrate is proportional to the number of live or proliferating cells in the culture. In some embodiments, the cells of the cell composition continuously reduce the pro-substrate.

[0205] Once the blocking moiety is liberated from the pro-substrate, the substrate can bind to the luciferase to result in the generation of the bioluminescent. In some embodiments, the blocking moiety is removed from the pro-substrate intracellularly to result in the substrate. In some embodiments, the substrate is cell permeable and exits the cell. The chemical reaction responsible for generation of the bioluminescent signal varies based on the specific luciferase used. In some embodiments, the chemical reaction occurs extracellularly. In some embodiments, the chemical45MF-36448161473504-2029640 reaction occurs in the absence of a co-factor. In some embodiments, the chemical reaction occurs in the presence of a co-factor. In some embodiments, the co-factor comprises an ion or molecule. In some embodiments, the co-factor comprises adenosine triphosphate (ATP), oxygen (O2) and / or magnesium (Mg2). In some embodiments, the luciferase reacts with the substrate in the presence of ATP, O2 andMg2. In some embodiments, the luciferase reacts with the substrate in the presence of O2. In some embodiments, the bioluminescent signal is generated when the luciferase binds the substrate in the presence of O2. In some embodiments, the Chis part of the substrate. In some embodiments, the luminescent signal is generated when the oxygen molecule is reduced by the luciferase to carbon dioxide (CO2).

[0206] In some embodiments, the bioluminescent assay can be performed in two formats: a continuous -read measurement or an endpoint measurement. In the continuous-read measurement, the cell compositions described here are cultured in the presence of a recombinant receptor-stimulating agent concurrently in the presence of the luciferase and substrate or pro-substrate, and luminescent signal is detected and analyzed over an extended period of time. In the endpoint measurement format, the cell compositions described herein are cultured in the presence of a recombinant receptor-stimulating agent and at the end of culturing, the luciferase and substrate or pro-substrate are added to the cell culture and the luminescent signal is detected an analyzed. In some embodiments, luminescent signal is generated and detected using the endpoint measurement format. In some embodiments, the endpoint measurement format is performed iteratively or repeated.

[0207] Various luciferase enzymes and substrates or pro-substrates are known in the art and can be used in connection with the methods provided herein. In some embodiments, any luciferase and substrate or pro-substrate combination known in the art can be employed in the methods described herein. In some embodiments, the luciferase enzyme comprises any of the luciferase enzymes described herein, such as below. In some embodiments, the substrate or pro-substrate comprises any of the substrates or pro-substrates described herein, such as below and corresponding to the luciferase enzymes described herein.1. Luciferase Enzymes

[0208] In some embodiments, any luciferase enzyme known in the art can be used in connection with the provided methods. In some embodiments, any commercially available luciferase can be used in the provided methods. In some embodiments, the luciferase enzyme is cell impermeable or does not enter the cell. In some embodiments, the luciferase is derived from any species known to produce or express a luciferase.

[0209] In some embodiments, the species can be an insect (e.g., firefly or beetle), a crustacean (e.g., shrimp), or an aquatic animal (e.g., coral). In some embodiments, the luciferase is derived from Oplophorus (OLuc), Gaussia (GLuc), Renilla (RLuc), Pyrophorus, or Photinus. In some46MF-36448161473504-2029640 embodiments, the luciferase comprises Renilla luciferase (RLuc). In some embodiments, the luciferase is a Renilla reniformis luciferase. In some embodiments, the luciferase is a Photinus pyralis luciferase. In some embodiments, the luciferase is a Pyrophorus plagiophthalamus luciferase. In some embodiments, the luciferase is a Gaussia princeps luciferase. In some embodiments, the luciferase is an Oplophorus gracilirostris luciferase. In some embodiments, the luciferase is any one or more of GLuc, NanoLuc (NLuc), MLuc7, HtLuc, LoLuc, PaLucl, PaLuc2, MpLucl, McLucl, MaLucl, MoLucl, MoLuc2, MLuc39, PsLucl, LocLucl-3, HtLuc2 Renilla, TurboLucl6 (TLuc) or homologs or orthologs thereof or mutants or functional derivatives thereof.

[0210] In some embodiments, the luciferase is a variant luciferase.

[0211] In some embodiments, the variant luciferase has one or more amino acid modifications, e.g., substitution, insertion and / or deletion in a wild-type or unmodified luciferase sequence. In some embodiments, the one or more amino acid modifications comprise a substitution, insertion and / or a deletion. In some embodiments, the variant luciferase is modified to optimize luminescent signal. In some embodiments, the variant luciferase has increased thermostability, a smaller size, and produces increased luminescence signal compared to a wild-type or an unmodified luciferase. In some embodiments, luminescent signal detected from a modified luciferase is at least 2-fold brighter than luminescent signal detected from wild-type or unmodified luciferase. In some embodiments, luminescent signal detected from a modified luciferase is at least 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19- fold, or 20-fold brighter than luminescent signal detected from wild-type or unmodified luciferase. In some embodiments, luminescent signal detected from a modified luciferase is up to 100,000-fold brighter than luminescent signal detected from wild-type or unmodified luciferase.

[0212] In some embodiments, any variant luciferase known in the art can be used in connection with the provided methods. Many luciferase variants are known in the art. See, e.g., Cosby et al. Cell Notes 18:9-1 1 , 2007; Sharma et al. Biochemistry and biophysics reports 24:100861, 2002; US Publication No 2019 / 0010157 Al ; Hall et al. ACS Chem Biol 7: 1848-1857, 2012; Endo and Ozawa. International Journal of Molecular Sciences 21.18:6538, 2020; De et al. Engineering in translational medicine. London: Springer London, 257-300, 2013 the disclosures of which are incorporated by reference herein in their entireties.

[0213] In some embodiments, the variant luciferase is from Oplophorus (OLuc). In some embodiments, the OLuc variant comprises one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions comprise A4E, Q11R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, P115E, Q124K, and Y138IP. In some embodiments, the one or more amino acid substitutions are selected from tire group consisting of A4E, Q11R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or47MF-36448161473504-2029640F68D, L72Q, M75K, I90V, P115E, Q124K, and Y138IP. In some embodiments, the OLuc variant comprises at least 8 substitutions selected from the group consisting of A4E, QI 1R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, Pl 15E, Q124K, and Y138IP. In some embodiments, the OLuc variant comprises at least 9, 10, 11, 12, 13, 14 or 15 substitutions selected from the group consisting of A4E, QI 1R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, P115E, Q124K, and Y138IP. In some embodiments, the OLuc variant comprises at least 9 substitutions selected from the group consisting of A4E, Q11R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, P115E, Q124K, and Y138IP. In some embodiments, the OLuc variant comprises at least 10 substitutions selected from the group consisting of A4E, Q11R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, Pl 15E, Q124K, and Y138IP. In some embodiments, the OLuc variant comprises at least 11 substitutions selected from die group consisting of A4E, Q11R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, P115E, Q124K, and Y138IP. In some embodiments, the OLuc variant comprises at least 12 substitutions selected from the group consisting of A4E, QI 1R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, Pl 15E, Q124K, and Y138IP. In some embodiments, the OL uc variant comprises at least 13 substitutions selected from the group consisting of A4E, Q11R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, P115E, Q124K, and Y138IP. In some embodiments, the OLuc variant comprises at least 14 substitutions selected from the group consisting of A4E, QI 1R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, Pl 15E, Q124K, and Y138IP. In some embodiments, the OLuc variant comprises at least 15 substitutions selected from the group consisting of A4E, QI 1R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, P115E, Q124K, and Y138IP.

[0214] In some embodiments, the OLuc variant comprises or consists of A4E, Q11R, A33K, V44I, A54F, P115E, Q124K and Y138I. In some embodiments, the OLuc variant comprises or consists of A4E, Q11R, A33K, V44I, A54F, P115E, Q124K, Y138I and N166R (also known as C1A4E). In some embodiments, the OLuc variant comprises or consists of A4E, Q11R, Q18L, 1.27 V. A33N, K43R, V44I, A54I, F68D, L72Q, M75K, I90V, P115E, Q124K, Y138I, N166R (also known as NLuc). In some embodiments, the OLuc variant comprises or consists of A4E, QI 1R, Q18L, D19S, L27V, A33N, K43R, V44I, A54I, F68D, L72Q, M75K, D85N, I90V, P115E, QI24K, Y138I, C164H, N166R (also known as teLuc). In some embodiments, the OLuc variant comprises or consists of F1L, A4E, Q11R, A14D, L27V, D19A, V27L, S28T, A33N, K43R, V44I, A54I, F68D, Q69R, L72Q, M75K, I90V, RI 12Q, Pl 15E, QI 24K, Y138I, L142R, C164S, and N166R (also known as yeLuc). In some embodiments, the OLuc variant comprises or consists of A4G, QI 1R,48MF-36448161473504-2029640D19A, S28T, A33N, K43R, V44I, A54I, G67C, F68D, G71 A, L72Q, M75K, I90A, R112Q, Pl 15E, V119K, Q124K, K136T, Y138I, C164H, and N166R (also known as LumiLuc).

[0215] In some embodiments, the OLuc variant is the Cl A4E variant.

[0216] In some embodiments, the variant luciferase is mutated Oplophorus gracilirostris luciferase, known as NanoLuc® (NLuc). In some embodiments, the NLuc is a Promega luciferase (NanoLuc® enzyme; Part # E499A). In some embodiments, NanoLuc® is used in accordance with manufacturer instructions. In some embodiments, the NLuc can be further modified. In some embodiments, the modified NLuc further comprises D19S, D85N, and C164H. In some embodiments, the modified NLuc is teLuc. In some embodiments, the modified NLuc comprises F1L, A14D, L18Q, D19A, S28T, V27L, Q69R, R112Q, L142R, and C164S. In some embodiments, the modified NLuc is yeLuc.

[0217] In some embodiments, NanoLuc® is used at a concentration range between or between about 1 ng / mL and about 150 ng / mL. In some embodiments, NanoLuc® is used at a concentration range between or between about 20 ng / mL and about 120 ng / mL, about 30 ng / mL and about 110 ng / mL, about 40 ng / mL and about 100 ng / mL, about 50 ng / mL and about 90 ng / mL, or about 60 ng / mL and about 80 ng / mL. In some embodiments, NanoLuc® is used at a concentration of about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In some embodiments, NanoLuc® is used at a concentration of about 40 ng / mL. NanoLuc® is used at a concentration of about 100 ng / mL.2. Substrate

[0218] In some embodiments, any substrate of a luciferase enzyme known in the art can be used in connection with the provided methods. In some embodiments, any commercially available substrate of a luciferase enzyme can be used in the provided methods. In some embodiments, the substrate is used to generate a luminescent signal in the cell.

[0219] In some embodiments, the substrate is cell-permeable and is able to enter and exit a cell, such as cells of any of the cell compositions described herein (e.g., engineered and nonengineered cell compositions and reference cell compositions)

[0220] In some embodiments, the substrate is one that includes a luciferin or a luciferin derivative or analog.

[0221] In some embodiments, the substrate includes D-luciferin which is a luciferin substrate for the enzyme firefly luciferase.

[0222] In some embodiments, the substrate is a D-luciferin derivative. In some embodiments, the D-luciferin derivative is D,L-homoluciferin, 5-methyl and 5, 5 -dimethylluciferin, Deshydroxy,49MF-36448161473504-2029640 decarboxyluciferin, 6'-methoxyluciferin, 6'-amino and 6'-acetylaminoluciferin, 5 '-chloroluciferin, Aminoluciferin (AL).

[0223] In some embodiments, the luciferin substrate is coelenterazine. Coelenterazines are luciferase molecules known to luminesce when acted on by luciferase enzymes, and particularly marine luciferases. Examples of marine luciferases include Renilla luciferase, aequorin, Gaussia luciferase, Oplophorus luciferase, and Cypridina luciferase. Useful, but non-limiting, coelenterazines are disclosed in U.S. patent application no. 10 / 053,482 and U.S. patent application no. 10 / 665,314, the disclosure which is hereby incorporated by reference in its entirety. Coelenterazines are commercially available, such as available from Promega Corporation, Madison, WI and from Molecular Probes, Inc., Eugene, OR. Coelenterazines may also be synthesized as described for example in Shimomura et al, Biochem. J. 261: 913-20, 1989: Louye et al, Biochem: Biophys. Res. Comm. 233: 349-53, 1997; and Teranishi et al, Anal. Biochem. 249: 37-43, 1997.

[0224] In some embodiments, the substrate comprises coelenterazine or a derivative or analog thereof. In some embodiments, the substrate is coelenterazine. In some embodiments, the substrate is a coelenterazine derivative or analog. Non-limiting examples of a coelenterazine derivatives or analogs include coelenterazine 400a (DeepBlueC™), coelenterazine c, coelenterazine cp, coelenterazine-e, coelenterazine f, coelenterazine fcp, coelenterazine h, coelenterazine-h-h, coelenterazine hep, coelenterazine-1, coelenterazine-icp, coelenterazine-n, coelenterazine-v, furimazine, JRW-0238, JRW-743, JRW-1744, methyl coelenterazine, 2-metyyl coelenterazine, 2- benzyl-8-(4-chlorophenylthio)-6-(4-hydroxyphenyl)imidazo[l ,2-a]pyrazin-3(7H)-one. In some embodiments, the compound may be a coelenterazine analog described in WO 2003 / 040100; U.S. Pat. Pub. 2008 / 0248511 (e.g., paragraph

[0086] ); U.S. Pat. No. 8,669,103; WO 2012 / 061529; U.S. Pat. Pub. 2017 / 0233789; U.S. Pat. No. 9,924,073; U.S. Pat. Pub. 2018 / 0030059; U.S. Pat. No. 10,000,500; U.S. Pat. Pub. 2018 / 0155350; U.S. Provisional Pat. App. No. 62 / 665,346; U.S. application Ser. No. 16 / 399,410; U.S. Provisional Pat. App. No. 62 / 721,708; U.S. application Ser. No. 16 / 548,214; U.S. Pat. Pub. 2014 / 0227759; U.S. Pat. Nos. 9,840,730; 7,268,229; 7,537,912; 8,809,529; 9,139,836; 10,077,244; 9,487,520; 9,924,073; 9,938,564; 9,951,373; 10,280,447; 10,308,975; 10,428,075; the disclosures of which are incorporated by reference herein in their entireties.

[0225] In some embodiments, the luciferin substrate includes furimazine (2-furanylmethyl- deoxycoelenterazine). Furimazine contains a furan group at the C-2 position of coelenterazine, and is a substrate for Oplophorus luciferase, particularly the Oplophorus luciferase variant NanoLuc. Also among luciferin substrates that can be used in the provided methods are any that include a furimazine analog or derivative, such as but not limited to 3-methoxy-furimazine, hydrofurimazine (HFz),50MF-36448161473504-2029640 fluorofurimazine (FFz), cephalofurimazine (CFz), or a hikarazines or deacetylated form thereof (e.g., hikarazine-001, hikarazine-003, hikarazine-097, hikarzaine-103, hikarazine-108).

[0226] In some embodiments, the substrate is a pro-substrate (also referred to as “proluciferin”) when added to the cells, in which a luciferase substrate is released as a result of a cellular event or response (e.g. presence of enzymes) present in the cell that converts the pro-substrate to a substrate that can be acted upon by a luciferase. In provided embodiments, the pro-substrate itself does not produce any detectable signal (e.g., a luminescent signal) until it is converted to a substrate that can be recognized by a luciferase. In some embodiments, the luminescent signal is generated after intracellular reduction of the pro-substrate to a substrate, diffusion of the substrate to the extracellular space and binding of the substrate to a luciferase.

[0227] In some embodiments, the pro-substrate comprises an R group (e.g., organic moiety, peptide, nucleic acid, etc.) that prevents the substrate from being bound by the luciferase. In some embodiments, the R group comprises both a blocking functionality (e.g., preventing the pro-substrate from being utilized by the protein sensor) and functional utility (e.g., capable of being removed or altered by some intracellular agent, event, or response). In some embodiments, the R group is removed or modified by an enzyme or molecule that may be attributed to a cellular event or response. In some embodiments, the cellular event or response comprises reduction by molecular oxygen. Following conversion of the pro-substrate into the substrate by the cellular event, response, condition or agent, the substrate is utilized by the luciferase enzyme. In some embodiments, the substrate does not accumulate such that any signal detected from a luciferase is the result of substrate that was recently converted from the pro-substrate (e.g., within the preceding: 10 minutes, 5 minutes, 2 minutes, 1 minute, 30 seconds, 10 seconds, 5 seconds, 2 seconds, 1 second, or less). In other embodiments, the substrate accumulates such that a signal may be generated from the substrate e over the course of the assay.

[0228] In some embodiments, the pro-substrate can be a pro-luciferin-D or analogs or derivatives thereof. In some embodiments, the pro-substrate can e a pro-coelenterazine or analogs or derivatives thereof.

[0229] Examples of pro-luciferin substrates include, but are not limited to, Luciferin 6' 2- fluorobenzyl ether (Luciferin- 2FBE), Luciferin 6' 3 -fluorobenzyl ether (Luciferin-3FBE), Luciferin 6' 2-furfuryl ether (Luciferin- 2FE), Luciferin 6' 3-furfuryl ether (Luciferin-3FE), Luciferin 6' 2-(5- trifluoromethyl)furfuryl ether (Luciferin-TFM2FE), Luciferin 6' 3-thenyl ether (Luciferin-3TE) or Luciferin-3FBEME.

[0230] In some embodiments, the luciferin substrate is a pro-substrate of a coelenterazine or derivative or analog thereof such as, for example, those described in U.S. Pat. Pub. 2008 / 0248511; U.S. Pat. Pub. 2012 / 0707849; U.S. Pat. Pub. 2014 / 0099654; U.S. Pat. Nos. 9,927,430; 10,316,070;51MF-36448161473504-2029640 herein incorporated by reference in their entireties. In some embodiments, the pro-substrate is a prosubstrate of Formula I as described in U.S. Pat. Pub. 2014 / 0099654, in which the R of the Formula I comprises an organic moiety, peptide, amino acid, nucleotide, or nucleic acid that can be acted upon, e.g., removed or modified, by an enzyme of interest. In some embodiments, the R group is removed or modified by an enzyme, molecule or agent that may be attributed to an intracellular agent, event, or response, thereby creating a substrate for the luciferase.

[0231] In some embodiments, the pro-substrate is a pro-coelenterazine, such as pro- coelenterazine derivatives or analogs including pro-furimazine compounds, which have been structurally modified from coelenterazine and furimazine or derivatives thereof, such that they no longer interact with a luciferase to luminesce. In some embodiments the pro-substrate is a pro- furimazine or a derivative thereof. In some embodiments, the pro-substrate is a pro-furimazine. In some embodiments, the pro-substrate is a pro-furimazine derivative. In some embodiments, the substrate is a pro-substrate when added to the cells, which then is converted to a furimazine substrate or derivative inside the cell. In some embodiments, the pro-substrate contains an R group that may react with a deprotection enzyme (such as an esterase) to release the luciferase substrate. In some embodiments, the R group may be a structure modification that is an enzyme-removable group (such as formation of an ester group). Interaction of the pro-substrate, such as a pro-furimazine, with an appropriate enzyme (such as an esterase) may yield an active luminophore compound, including coelenterazine, furimazine, or derivatives thereof.

[0232] In some embodiments, the pro-coelenterazine substrate, such as pro-furimazine substrate, is a furimazine-O-methyl carboxyl ester. In some embodiments, the pro-coelenterazine substrate, such as pro-furimazine substrate, is a compound of formula (I), formula (II) or formula (III) as described in U.S. published patent application US20190010157, which is incorporated by reference in its entirety. In some embodiments, the pro-coelenterazine substrate, such as pro- furimazine substrate, is a compound of formula I as described in CN114394970, which is incorporated by reference in its entirety. In some embodiments, the presence of a cellular esterase in the cell releases the coelenterazine, such as furimazine. In any of such embodiments, when added to cells, a slow rate of ester hydrolysis catalyzed by cellular esterase leads to the steady release of the coelenterazine or analogs or derivatives, such as furimazine.

[0233] In some embodiments the pro-substrate is ((8-benzyl-2-(furan-2-ylmethyl)-6- phenylimidazo[ 1 ,2-a]pyrazin-3-yl)oxy)methyl 3-methoxy-2,2-dimethylpropanoate; ((8-benzyl-2- (furan-2-ylmethyl)-6-phenylimidazo[ 1 ,2-a]pyrazin-3-yl)oxy)methyl 3-(2-(2-methoxyethoxy)ethoxy)- 2,2-dimethylpropanoate; ((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[ 1 ,2-a]pyrazin-3- yl)oxy)methyl 3-(2-methoxyethoxy)-2,2-dimethylpropanoate; ((8-benzyl-2-(furan-2-ylmethyl)-6- phenylimidazo[ 1 ,2-a]pyrazin-3-yl)oxy)methyl 13 , 13-dimethyl-2,5 ,8,11 -tetraoxatetradecan- 14-oate ;52MF-36448161473504-2029640((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[l,2-a]pyrazin-3-yl)oxy)methyl 16,16-dimethyl- 2,5,8,ll,14-pentaoxaheptadecan-17-oate; ((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[l,2- a]pyrazin-3-yl)oxy)methyl 2-methoxy-2-methylpropanoate; or ((8-benzyl-2-(furan-2-ylmethyl)-6- phenylimidazo[l,2-a]pyrazin-3-yl)oxy)methyl 2-acetamido-2-methylpropanoate. In some embodiments, the pro-substrate is ((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[l,2-a]pyrazin-3- yl)oxy)methyl furan-2-carboxylate; ((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[l ,2-a]pyrazin- 3-yl)oxy)methyl furan-3 -carboxylate; or ((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[l,2- a]pyrazin-3-yl)oxy)methyl benzoate. In some embodiments, the pro-substrate is ((8-benzyl-2-(furan- 2-ylmethyl)-6-phenylimidazo[l,2-a]pyrazin-3-yl)oxy)methyl methyl carbonate. In some embodiments, the presence of a cellular esterase in the cell releases the coelenterazine, such as furimazine.

[0234] In some embodiments, the pro-substrate is pro-furimazine derivative substrate that is an ester furimazine derivative having the chemical structure of formula I below, wherein R is 1 Tertbutyl, phenyl, furyl, tert-butoxy methyl, 2-phenyl-2-methylethyl, 2- (benzoyloxy) -2-methylethyl, 1- methoxyethyl, propoxy.I

[0235] In some embodiments, the pro-substrate is ((2- (furan-2-ylmethyl) -6-phenyl-8- (phenylsulfanyl) imidazo [1,2-a ] pyrazin-3-yl) oxy) pivalic acid methyl ester, ((2- (furan-2- ylmethyl) -6-phenyl-8- (phenylsulfanyl) imidazo [1,2-a ] pyrazin-3-yl) oxy) benzoic acid methyl ester, ((2- (furan-2-ylmethyl) -6-phenyl-8- (phenylsulfanyl) imidazo [1,2-a ] pyrazin-3-yl) oxy) furan-2-carboxylic acid methyl ester, ((2- (Furan-2-ylmethyl) -6-phenyl-8- (phenylsulfanyl) imidazo [1,2-a ] pyrazin-3-yl) oxy) 2- (tert-butoxy) acetic acid methyl ester, ((2- (Furan-2-ylmethyl) -6- phenyl-8- (phenylsulfanyl) imidazo [1,2-a ] pyrazin-3-yl) oxy) 2-methyl-2-phenylpropionic acid methyl ester, 1- (((2- (furan-2-ylmethyl) -6-phenyl-8- (phenylsulfanyl) imidazo [1,2-a ] pyrazin-3-yl) oxy) methoxy) -2-methyl-l -oxypropane benzoic acid 2-yl ester, ((2- (Furan-2-ylmethyl) -6-phenyl-8-53MF-36448161473504-2029640(phenylsulfanyl) imidazo [1,2-a ] pyrazin-3-yl) oxy) 2-methoxypropionic acid methyl ester, or ((2- (furan-2-ylmethyl) -6-phenyl-8- (phenylsulfanyl) imidazo [1,2-a ] pyrazin-3-yl) oxy) methylpropyl carbonate. In some embodiments, the pro-substrate is ((2- (furan-2-ylmethyl) -6-phenyl-8- (phenylthio) imidazo [1,2-a ] pyrazin-3-yl) oxy) furan- 2-carboxylic acid methyl ester. In some embodiments, the pro-sbustrate is ((2- (furan-2-ylmethyl) -6-phenyl-8- (phenylthio) imidazo [1,2-a ] pyrazin-3-yl) oxy) methylpropyl carbonate.

[0236] In some embodiments, the pro-substrate is a methyl pivalate ester-protected furimazine Endurazine™. or Vivazine®. In some embodiments, the presence of a cellular esterase in the cell releases the coelenterazine, such as furimazine.

[0237] In some embodiments, the substrate used herein is the MT Cell Viability Substrate (Promega; Part # G971A). In some embodiments, the MT Cell Viability Substrate is used in accordance with manufacturer instructions.

[0238] In some embodiments, the substrate added to the cells, including a pro-substrate added to the cells for conversion to a substrate in the cell, is added to cells at a concentration range between or between about 1 pM and about 100 pM. In some embodiments, added to the cells, including a pro-substrate added to the cells for conversion to a substrate in the cell, is added at a concentration range between or between about 10 pM and about 90 pM, about 20 pM and about 80 pM, about 30 pM and about 70 pM, or about 40 pM and about 60 pM. In some embodiments, substrate added to the cells, including a pro-substrate added to the cells for conversion to a substrate in the cell, is added at a concentration of about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, or about 100 pM. In some embodiments, the substrate added to the cells, including a pro-substrate added to the cells for conversion to a substrate in the cell, is added at a concentration of about 40 pM. In some embodiments, the substrate is added to the cells, including a pro-substrate added to the cells for conversion to a substrate in the cell, is added at a concentration of about 50 pM.3. Luminescent Signal

[0239] In some embodiments, the luminescent signal is measured in Relative Luminescence Units (RLU). RLU is a unit of measurement used to quantify the amount of light (photons) that is emitted during a reaction and that the luminometer can quantify. In some embodiments, a measured luminescent signal is normalized, e.g., as described herein.C. Determining Cell Proliferation

[0240] In some embodiments, a method provided herein comprises determining cell proliferation of an engineered cell composition, e.g., a cell therapy composition such as a CAR-T cell therapy, using the results of the luminescent signal detection as described in Section I. B.3. In54MF-36448161473504-2029640 some embodiments, the engineered cell composition is stimulated according to any of the methods described herein, including as described in Section I.A.3. In some embodiments, the stimulated cell composition is a first stimulated cell composition having been cultured once with a recombinant receptor-stimulating agent. In some embodiments, the stimulated cell composition is a second stimulated cell composition having been cultured iteratively or repeatedly with the recombinant receptor-stimulating agent. In some embodiments, the engineered cell composition is unstimulated. In some embodiments, the unstimulated engineered cell composition has not been cultured with any of the recombinant receptor-stimulating agents described herein, including as described in Section I.A.3.

[0241] In some embodiments, determining cell proliferation of a stimulated cell composition (e.g., the test engineered cell composition) comprises calculating the difference between the luminescent signal detected from the stimulated cell composition and a luminescent signal detected from unstimulated cells (e.g., unstimulated cultured cells of the test engineered cell composition ).

[0242] In some embodiments, the unstimulated cells are generated by culturing cells of the test engineered cell composition in the absence of the recombinant-receptor stimulating agent. Thus, in some embodiments, the stimulated cells and the unstimulated cells are comparable (e.g., express the same receptors, are derived from the same source, etc.). In some embodiments, the stimulated and unstimulated cells have been subjected to the same experimental conditions except that the stimulated cells are cultured in the presence of the recombinant receptor-stimulating agent and the unstimulated cells are cultured in the absence of the recombinant receptor-stimulating agent.

[0243] In some embodiments, the test engineered cell composition has been cultured with the recombinant receptor-stimulating agent for a period of time and the unstimulated cultured cells of the test engineered cell composition have been cultured in the absence of the recombinant receptorstimulating agent for a second period of time. In some embodiments, the period of time and the second period of time are the same duration.

[0244] In some embodiments, detecting the luminescent signal from the unstimulated cultured cells comprises adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the unstimulated cultured cells. In some embodiments, any of the luciferase enzymes and substrates or pro-substrates of the luciferase enzymes described herein are used.

[0245] In some embodiments, the unstimulated cultured cells are incubated with the luciferase and the substrate or pro-substrate of the luciferase enzyme to generate a luminescent signal. In some embodiments, the unstimulated cells are incubated with the luciferase enzyme the substrate or prosubstrate of the luciferase enzyme under the same conditions described in Section LB. In some embodiments, generating the luminescent signal further comprises equilibrating the unstimulated55MF-36448161473504-2029640 cultured cells under conditions that promote uniform reading of the luminescent signal, such as those conditions described in Section LB.

[0246] In some embodiments, the luminescent signal is detected from the unstimulated cultured cells. In some embodiments, the luminescent signal is detected according to the methods described in Section I.B.

[0247] In some embodiments, detecting the luminescent signal from the unstimulated cultured cells is performed with a replicate of the unstimulated cell composition. In some embodiments, two or more replicate unstimulated cell compositions are used in the methods. In some embodiments, two replicate cell unstimulated cell compositions are used in the methods. In some embodiments, at least three replicate unstimulated cell compositions are used in the methods. In some embodiments, three replicate unstimulated cell compositions are used in the methods. In some embodiments, an average of the luminescent signal detected from the two or more replicate unstimulated cell compositions is used to measure cell proliferation.

[0248] In some embodiments, the methods provided herein include determining cell proliferation of a non-engineered cell composition. In some embodiments, the non-engineered cell composition comprises primary cells (e.g., primary immune cells or an immune cell line, such as primary T cells or a T cell line). In some embodiments, the non-engineered cell composition comprises Jurkat T cells. In some embodiments, the non-engineered cell composition is stimulated with a stimulating agent according to any of the methods described herein, including as described in Section I.A.3. In some embodiments, the non-engineered cell composition is unstimulated. In some embodiments, an unstimulated non-engineered cell composition has not been cultured with any of the stimulating agents described herein, including as described in Section I.A.3.

[0249] In some embodiments, the method of determining cell proliferation of a stimulated non-engineered cell composition comprises calculating the difference between a luminescent signal detected from a stimulated non-engineered cell composition and a luminescent signal detected from an unstimulated non-engineered cell composition. In some embodiments, the luminescent signal detected from the stimulated non-engineered cell composition is increased compared to the luminescent signal detected from the unstimulated non-engineered cell composition. In some embodiments, an increased luminescent signal indicates increased cell proliferation. In some embodiments, the luminescent signal detected from the stimulated non-engineered cell composition is unchanged or decreased compared to the luminescent signal detected from the unstimulated nonengineered cell composition. In some embodiments, a decreased or unchanged luminescent signal indicates decreased or unchanged cell proliferation.56MF-36448161473504-20296401. Relative Cell Proliferation

[0250] In some embodiments, a method disclosed herein includes calculating a relative cell proliferation for an engineered cell composition, e.g., a CAR-T cell therapy composition. In some embodiments, the engineered cell composition is a test engineered cell composition, e.g. , an engineered cell composition that has been stimulated by culturing the cells with any of the recombinant receptor-stimulating agents described herein, such as in Section I.A.3. In some embodiments, the method includes comparing the cell proliferation of the test engineered cell composition to the cell proliferation of a reference standard (e.g., a reference cell composition as described in Section I.A.2), e.g., as described below.

[0251] In some embodiments, a relative cell proliferation of a test engineered cell composition is calculated using a luminescent signal detected from the test engineered cell composition (e.g. , an average of a detected luminescent signal) according to the following formula, wherein the sample employed is or comprises the test engineered cell composition.Average Sample stimulated — Avera e Sample unstimulated - - - - - - - - x 100% Average Reference Standard (RS) stimulated — Average RS unstimulated

[0252] As a person of skill in the art would appreciate, in the above formula, an unstimulated cell composition is used for comparison. However, alternative approaches to calculating a relative cell proliferation could be used that do not require an unstimulated cell composition. For instance, a relative cell proliferation could be calculated by comparing the luminescent signal for a sample (e.g., a stimulated sample) to the luminescent signal for a reference standard (e.g., a stimulated reference standard). For instance, a ratio could be calculated as follows:Average Sample stimulated Average Ref er ence Standard (RS) stimulated'

[0253] Optionally, this ratio could be multiplied by 100 to express the relative proliferation in the form of a percentage. Also, as a person of skill in the art would appreciate, instead of an average, an alternative value for a measured luminescent signal (e.g., a set of measured luminescent signals) could be used (e.g., a predetermined percentile, or an alternative measure of central tendency, such as a median).

[0254] In some embodiments, the luminescent signal detected from the test engineered cell composition is increased compared to the luminescent signal detected from the reference standard (i.e., the relative cell proliferation is >100%). In some embodiments, an increased luminescent signal indicates increased relative cell proliferation. In some embodiments, the luminescent signal detected from the test engineered cell composition is unchanged or decreased compared to the luminescent signal detected from the reference standard (i.e., the relative cell proliferation is <100%). In some embodiments, a decreased or unchanged luminescent signal indicates a decreased or unchanged relative cell proliferation.57MF-36448161473504-2029640 a. Reference Standards

[0255] Particular embodiments contemplate that cell proliferation for a test engineered cell composition can be compared to cell proliferation of a reference standard to, for example, determine a relative cell proliferation. In some embodiments, cell proliferation of the reference standard is assessed according to the methods disclosed herein. The reference can be any of the reference cell compositions described in Section I.A.2.

[0256] In some embodiments, a reference standard is a highly characterized, uniform, and stable material that serves as the benchmark against which all test samples are compared. In some embodiments, the reference standard has been validated. As a person of skill in the art would appreciate, to be validated means to demonstrate, through documented laboratory studies, that a reference standard is suitable for its intended purpose and consistently produces accurate, precise, and reliable results.

[0257] In some embodiments, the reference standard is selected and validated through one of more of the criteria selected from below:

[0258] (i) Extensive Characterization Data: It is subjected to a wide set of analytical tests to confirm its identity, purity, and potency.

[0259] (ii) Internal (In-house) Primary Standard: For biologies and cell therapies, the primary reference standard is typically a representative batch from pivotal clinical or commercial manufacturing runs that has been extensively characterized and approved internally.

[0260] (iii) Official / External Standard: Where available, it might be calibrated against an officially recognized standard from a public agency like the USP (US Pharmacopeia), NIBSC, or NIST.

[0261] (iv) Traceability and Certified Values: The reference standard is traceable to a certified source or an international standard (where one exists), establishing a documented, unbroken chain of calibrations back to a fundamental unit of measurement.

[0262] (v) Intended Use and Suitability: Its quality and characteristics are judged against its specific intended application. For a stability-indicating test, it is demonstrably stable over a long period to ensure the test can be used for comparing current production batches to a consistent historical benchmark.

[0263] In some embodiments, the reference standard is a commercially available cell therapy composition. In some embodiments, the reference standard is a reference engineered cell composition.

[0264] In some embodiments, the reference engineered cell composition is made using primary cells from a subject. In some embodiments, the cells that are used to make the reference engineered cell composition and the cells that are used to make the test engineered cell composition58MF-36448161473504-2029640 are from the same subject. In some embodiments, the reference engineered cell composition and the test engineered cell composition are made using cells from different subjects. In some embodiments, the reference engineered cell composition is made from cells from a healthy subject or donor. In some embodiments, the reference engineered cell composition is made from cells from a subject having a disease or condition. In some embodiments, the reference engineered cell composition is made from cells from a subject having cancer.

[0265] In some embodiments, the reference engineered cell composition is produced ex vivo by a cell engineering manufacturing process to comprise the recombinant receptor. In some embodiments, the reference standard is an engineered cell composition manufactured using a manufacturing process that is identical to a manufacturing process used to manufacture the test engineered cell composition to which it is compared. In some embodiments, the reference standard is an engineered cell composition manufactured using a manufacturing process that is different from the manufacturing process used to manufacture the test engineered cell composition to which it is compared.

[0266] In some embodiments, the reference engineered cell composition comprises cells engineered to comprise (e.g., express) a recombinant receptor. In some embodiments, the reference recombinant receptor expressed by the cells of the reference engineered cell composition differs from the recombinant receptor expressed by the cells of the test engineered cell composition. In some embodiments, the reference recombinant receptor expressed by the cells of the reference engineered cell composition is the same as the recombinant receptor expressed by the cells of the test engineered cell composition. In some embodiments, the cells of the reference engineered cell composition comprises any of the recombinant receptors described herein, such as in Sections II. A and II.B.

[0267] In some embodiments, the reference standard may be a combination of one or more of those reference engineered cell compositions described above.

[0268] In some embodiments, the reference standard has a validated proliferation index. In some embodiments, the validated proliferation index is determined by culturing cells of the reference engineered cell composition with a recombinant receptor-stimulating agent for a period of time to generate a stimulated reference engineered cell composition. In some embodiments, the recombinant receptor-stimulating agent includes any of the recombinant receptor-stimulating agents described herein, including those described in Section I.A.3. In some embodiments, the reference engineered cell composition is cultured under the same conditions described above in Section I. A.

[0269] In some embodiments, after the period of time, a luciferase enzyme and a substrate or a pro-substrate of the luciferase enzyme is added to the stimulated reference engineered cell composition and incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal. In some embodiments, the incubating step59MF-36448161473504-2029640 is carried out under certain conditions that promote binding of the substrate or pro-substrate by the luciferase enzyme. In some embodiments, these conditions include a specific temperature. In some embodiments, these conditions include a specific percentage of carbon dioxide (CO2). In some embodiments, these conditions include a specific percentage of humidity. In some embodiments, the reference engineered cell composition is incubated under the same conditions described in Section LB. In some embodiments, after the incubating step, the method comprises equilibrating the reference engineered cell composition to room temperature. In some embodiments, the reference engineered cell composition is equilibrated under the same conditions described in Section I.B.

[0270] In some embodiments, the validated proliferation index is determined by detecting the luminescent signal from the stimulated reference engineered cell composition.

[0271] In some embodiments, the validated proliferation index is determined by determining cell proliferation of the reference standard by calculating the difference between the luminescent signal detected from the stimulated reference engineered cell composition and the luminescent signal detected from unstimulated cells of the reference engineered cell composition. In some embodiments, the unstimulated cells of the reference engineered cell composition have been cultured in the absence of the recombinant receptor-stimulating agent.

[0272] In some embodiments, the reference standard is a reference non-engineered cell composition. In some embodiments, the non-engineered cell composition comprises primary cells or a cell line, e.g., primary T cells or a T cell line. In some embodiments, the reference standard that is a reference non-engineered cell composition comprises any of the features or characteristics of the reference engineered cell compositions described above.D. Determining Cell Stability

[0273] In some embodiments, the methods disclosed herein are highly stability-indicating. In some embodiments, a method described herein is stability-indicating and measures the ability of engineered cells to proliferate in response to recombinant receptor stimulation. In some embodiments, the method can rapidly and accurately detect when a sample of the engineered cell therapy composition no longer meets a desired specification indicative of potency and / or stability.

[0274] For instance, in some embodiments, the methods disclosed herein can be used as part of a stability assay to assess stability of a test engineered cell composition that has been subjected to a stability-impacting condition. Non-limiting examples of stability-impacting conditions include any of the following: storage for a defined period of time, such as storage for about 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 2 years, about 3 years, or longer (e.g., under recommended storage conditions); changes in storage temperature or temperature excursions, such as storage at a different temperature than the specified storage temperature, transient warming or cooling outside of a specified temperature range, or exposure to room60MF-36448161473504-2029640 temperature during handling or transport; freeze-thaw related conditions, such as subjecting the composition to one or more additional freeze-thaw cycles beyond those used during validated manufacturing, altering the thawing procedure (e.g., different thaw rate or method), or holding the thawed product for an extended period prior to administration; changes in container or closure system, such as switching between cryovials and cryobags, modifying closure or overwrap components, altering fill volume or headspace, or changing the orientation of containers during storage (e.g., verdeal versus horizontal storage); changes in formulation or excipients, such as changes in cryoprotectant composition or concentration (e.g., DMSO percentage, presence or absence of serum, albumin, sugars, or other excipients), changes in diluent or buffer composition, or introduction of alternative stabilizing agents; mechanical or physical stress, such as exposure to agitation or vibration during transportation, pumping through tubing sets or filters, mixing, or other operations that can impose shear stress on the cells; changes in storage atmosphere, such as variations in oxygen or carbon dioxide content in the container headspace or storage in an atmosphere differing from a qualified gas composition; light exposure, such as extended exposure to ambient or ultraviolet light during storage or handling, where such exposure may impact the stability of the composition; transportation conditions, including shipment between manufacturing, storage, and clinical sites under controlled or uncontrolled conditions, as well as changes in shipping container, route, or duration; manufacturing process changes, such as changes to culture medium components, cell expansion duration, activation conditions, washing steps, cry opreservation protocol, or any other process parameter that could affect the quality attributes of the engineered cell therapy composition.

[0275] In various embodiments, the method of assessing cell proliferation disclosed herein is performed on test samples (e.g., an engineered cell composition as described in Section I.A.l) collected after one or more of the above stability-impacting conditions have occurred. For example, test samples may be collected from a manufactured lot and stored for predetermined periods (e.g., 3, 6, and 12 months) prior to testing, or may be collected before and after a manufacturing process change, a change in shipping conditions, or an intended change to storage configuration.

[0276] In some embodiments, the method of assessing cell proliferation disclosed herein is employed as part of a stability program for an engineered cell therapy drug product. Test samples from one or more lots can be stored under proposed shelf-life conditions and evaluated at multiple time points to generate recovery rate profiles over time. These data can be used to assign, confirm, or extend the product shelf-life, demonstrate comparability following manufacturing or process changes, and / or support regulatory submissions. Acceptance criteria can be defined for a single storage time point and / or for multiple time points, and can be established based on developmental and validation data, regulatory guidance, and clinical considerations.61MF-36448161473504-2029640

[0277] In some embodiments, the stability of the engineered cell composition is determined by the proliferation (e.g., relative proliferation as calculated in Section I.C.l) of the engineered cell composition. In some embodiments, the engineered cell composition is deemed to be stable if the relative proliferation is within a predefined potency range (e.g. 70%-120%). In some embodiments, the engineered cell composition is deemed to be stable if the relative proliferation is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0278] In some embodiments, the methods disclosed herein further comprise comparing the proliferation (e.g., relative proliferation) of the engineered cell composition to the proliferation (e.g., relative proliferation) of a control cell composition. In some embodiments, the methods disclosed herein further comprise calculating a ratio between the relative proliferation of the engineered cell composition and the relative proliferation of the control cell composition (proliferation of test engineered cell composition ^-proliferation of the control cell composition). In some embodiments, the engineered cell composition is deemed to be stable if the ratio is within a predefined potency range (e.g. 0.7-1.2). In some embodiments, the engineered cell composition is deemed to be stable if the ratio between the relati ve proliferation of the engineered cell composition and the relative proliferation of the control cell composition is at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, or at least 0.95.

[0279] The use of a control cell composition can aid in verifying that the method is performing correctly during each run. The control cell composition can be a cell composition comprising a recombinant receptor as is included in the cells of the test engineered cell composition. In some embodiments, the control cell composition has not been subjected to condition changes (e.g., changes in storage conditions) that engineered cell composition have been. In other embodiments, the control cell composition is a pooled composition, a previously qualified lot, or an internal assay control that has been characterized to represent acceptable potency. In some embodiments, the control cell composition is any of the reference cell compositions as described in Section I. A.2.

[0280] In certain embodiments, the method of assessing proliferation and stability is used in conjunction with other quality attributes, such as viable cell count, phenotype markers, or other potency assays. Because the proliferation-based assay directly measures the capacity of the engineered cells to respond to recombinant receptor stimulation and expand, it can serve as a highly stability-indicating assay that sensitively detects loss of functional potency even in cases where viable cell counts remain within specification. Thus, the assay provides an early and accurate indication that a sample is “no longer good,” i.e., that it no longer meets the established potency or stability criteria for clinical use.62MF-36448161473504-2029640IL RECOMBINANT RECEPTORS AND ENGINEERED CELLS

[0281] In some embodiments, the provided methods and uses relate to methods of assessing cell proliferation of cells of an engineered cell composition (e.g., a cell therapy composition ), wherein the cells of the engineered cell composition comprise a recombinant receptor. In some embodiments, a cell that comprises a recombinant receptor expressed the recombinant receptor.

[0282] In some embodiments, the cells of the engineered cell composition include one or more nucleic acids introduced via genetic engineering to express the recombinant receptor or other genetically engineered products of the nucleic acids. In some embodiments, gene transfer is accomplished by first stimulating non-engineered cells, such as by combining the cells with a stimulus that induces a response such as proliferation, survival, and / or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.

[0283] In some embodiments, the recombinant receptor can be any known recombinant receptor, including any of the recombinant receptors described below. In some embodiments, the recombinant receptor comprises a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor comprises a T cell receptor (TCR).

[0284] In some embodiments, the engineered cells can comprise any nucleic acid encoding any recombinant receptor, to thereby express said recombinant receptor, including any of the nucleic acids described below.A. Chimeric Antigen Receptors (CARs)

[0285] In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen-binding domain specific for a target antigen, a hinge spacer, a transmembrane domain, and an intracellular signaling domain containing a costimulatory signaling region and an activating domain that provides a primary activation signal. In some aspects, the hinge spacer is between the antigen-binding domain and the transmembrane domain. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0286] In some embodiments, the antigen-binding domain is specific for a single target antigen (i.e., monospecific). In some embodiments, the antigen-binding domain is specific for more than one target antigen (i.e., bispecific). In some embodiments, the bispecific antigen-binding domain is specific for a first target antigen and a second target antigen. In some embodiments, the antigen-binding domain is an antibody or antigen-binding fragment or portion that targets any target63MF-36448161473504-2029640 antigen. In some embodiments, the antigen-binding domain is a portion of an antibody molecule, generally a variable heavy (VH) chain region and / or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb). In some embodiments, an antigen-binding fragment comprises antibody variable regions, VH and VL, joined by a flexible linker. In such embodiments, the antibody or an antigen-binding fragment (e.g., scFv) contains a variable heavy chain and a variable light chain with six CDRs, CDRH1-3 and CDRL1-3, that confer binding to the target antigen. In some embodiments, the antigen-binding domain is a single domain antibody (sdAb), such as sdFv, nanobody, VHH and VNAR. In some embodiments, a single domain antibody is a VHH that contains three CDRs, CDRH1-3. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to an antigen associated with the disease or disorder. In some embodiments, the antigen associated with the disease or disorder is CD 19, CD20, CD22, R0R1, BCMA, CD45, CD21, CD5, CD33, Igkappa (IgK), Iglambda (IgX), CD79a, CD79b, CD30, GPRC5D.

[0287] In some embodiments, the extracellular antigen binding domain comprises a first antigen binding domain and a second antigen binding domain. In some embodiments, the first antigen binding domain comprises a VH and VL that binds a first target antigen and the second antigen binding domain comprises a VH and VL that binds a second target antigen. In some embodiments, the first antigen binding domain comprises a first VHH specific to a first target antigen and a second VHH specific to a second target antigen.

[0288] In some embodiments, the extracellular antigen binding domain comprises any structure known in the art. In some embodiments, the extracellular antigen binding domain comprises a tandem structure. In some embodiments, the tandem structure comprises a sequence comprising in order (e.g., N- to C-terminal) the first antigen binding domain and the second antigen binding domain. In some embodiments, the extracellular antigen binding domain comprises a loop structure. In some embodiments, the loop structure comprises a sequence comprising in order (e.g., N- to C-terminal), the first VH chain or first VL chain of the first antigen binding domain, the second VH chain or second VL chain of the second antigen binding domain, the other of the second VH chain and second VL chain of the second antigen binding domain, and the other of the first VH chain and second VL chain of the second antigen binding domain.

[0289] In some embodiments, the CAR includes a spacer containing a hinge domain. In some embodiments, the hinge domain comprises or consists of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine. The spacer may be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the64MF-36448161473504-2029640 spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol Res. 3(2): 125-135 or international patent application publication number WO2014031687. In some embodiments, the spacer includes an immunoglobulin hinge domain. In some embodiments, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl. In some embodiments, the spacer is an Ig hinge, e.g., and IgG4 hinge, linked to a CH2 and / or CH3 domains. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers. In some embodiments, the constant region or portion is of IgD.

[0290] In some embodiments, the transmembrane domain of the CAR is linked or fused between the antigen binding domain (e.g., scFv) or optionally the hinge domain and the intracellular signaling domain. The antigen binding domain and transmembrane may be linked directly or indirectly. In some embodiments, the antigen binding domain and transmembrane are between a hinge domain spacer, such as any described herein. In some embodiments, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (4-1BB), or CD154. Alternatively, the transmembrane domain in some embodiments is synthetic. In some embodiments, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some65MF-36448161473504-2029640 embodiments, the linkage is by linkers, spacers, and / or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28 or a variant thereof.

[0291] In some embodiments, the intracellular signaling domain of the CAR provides for activation of the cell (e.g. , T cell) into which it is engineered when engaged or ligated with the antigen recognized by the antigen binding domain. T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigendependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components. In particular embodiments, the intracellular signaling region mimics or approximate a signal of a natural antigen receptor through the primary activation domain and a signal through such a receptor in combination with a costimulatory receptor through the costimulatory domain. In some embodiments, upon ligation of the CAR by antigen, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling region of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the primary activation domain of the intracellular signaling domain comprises an IT AM. In some embodiments, the primary activation domain regulates primary activation of the TCR complex. Examples of IT AM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta. In some embodiments, the CD3-zeta chain is a human CD3-zeta chain. In some embodiments, the intracellular domain also includes a costimulatory domain. In some embodiments, the costimulatory domain is an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR includes a signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40 (CD134), CD27, DAP10, DAP12, ICOS and / or other costimulatory receptors. In some embodiments, the CAR includes a costimulatory region or domain of CD28 or 4- IBB, such as of human CD28 or human 4- 1BB. In some embodiments, the intracellular signaling region further comprises a CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain. In some embodiments, the CD28 is a human CD28. In some embodiments, the 4-1BB is a human 4-1BB.66MF-36448161473504-2029640

[0292] Exemplary antigen receptors, e.g. , CARs, also include the CARs of FDA-approved products BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), and YESCARTA™ (axicabtagene ciloleucel). In some of any of the provided embodiments, the CAR is the CAR of BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), YESCARTA™ (axicabtagene ciloleucel). In some of any of the provided embodiments, the CAR is the CAR of BREYANZI® (lisocabtagene maraleucel, see Sehgal et al., 2020, Journal of Clinical Oncology 38:15_suppl, 8040; Teoh et al., 2019, Blood 134(Supplement_l):593; and Abramson et al., 2020, The Lancet 396(10254): 839-852). In some of any of the provided embodiments, the CAR is the CAR of TECARTUS™ (brexucabtagene autoleucel, see Mian and Hill, 2021, Expert Opin Biol Ther; 21(4):435-441; and Wang et al., 2021, Blood 138(Supplement 1):744). In some of any of the provided embodiments, the CAR is the CAR of KYMRIAH™ (tisagenlecleucel, see Bishop et al., 2022, N Engl J Med 386:629:639; Schuster et al., 2019, N Engl J Med 380:45-56; Halford et al., 2021, Ann Pharmacother 55(4):466-479; Mueller et al., 2021, Blood Adv. 5(23):4980-4991; and Fowler et al., 2022, Nature Medicine 28:325-332). In some of any of the provided embodiments, the CAR is the CAR of YESCARTA™ (axicabtagene ciloleucel, see Neelapu et al. , 2017, N Engl J Med 377(26):2531-2544; Jacobson et al., 2021, The Lancet 23(l):P91-103; and Locke et al., 2022, N Engl J Med 386:640-654). Additional exemplary antigen receptors, e.g., CARs, also include the CARs of YTB323 (rapcabtagene autoleucel), MB-CART19.1 (Mougiakakos et al., CD19-targeted CAR-T cells in refractory systemic lupus erythematosus. N Engl J Med 2021;385:567-9), MB- CART2019.1 (zamtocabtagene autoleucel, see W02019 / 028051), prizloncabtagene autoleucel (C- CAR038, JNJ-90014496, see WO2021 / 188681), CABA-101 and CABA-201 (Peng et al., Molecular Therapy Methods & Clinical Development, Volume 32, Issue 2, 101267), KYV-101 (US10,287,350B2, WO2023 / 133092), IMPT-514 (see the CAR described in Larson et al., Cancer Discovery. 2023; 13(3):580-597).B. T Cell Receptors (TCRs)

[0293] In some embodiments, the recombinant receptor is a T cell receptor (TCR) or antigenbinding portion thereof that recognizes an peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, viral, or autoimmune protein.

[0294] In some embodiments, a “T cell receptor” or “TCR” is a molecule that contains a variable a and [3 chains (also known as TCRa and TCR , respectively) or a variable y and 5 chains (also known as TCRa and TCR , respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the af> form. Typically, TCRs that exist in a[3 and y5 forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can67MF-36448161473504-2029640 be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0295] Unless otherwise stated, the term “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, including TCRs in the a[3 form or y5 form. In some embodiments, the TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of a TCR contain complementarity determining regions involved in recognition of the peptide, MHC and / or MHC-peptide complex.

[0296] In some embodiments, the variable domains of the TCR contain hypervariable loops, or complementarity determining regions (CDRs), which generally are the primary contributors to antigen recognition and binding capabilities and specificity. In some embodiments, a CDR of a TCR or combination thereof forms all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs within a variable region of a TCR chain generally are separated by framework regions (FRs), which generally display less variability among TCR molecules as compared to the CDRs (see, e.g., lores et al., Proc. Nat’l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDR responsible for antigen binding or specificity, or is the most important among the three CDRs on a given TCR variable region for antigen recognition, and / or for interaction with the processed peptide portion of the peptide-MHC complex. In some contexts, the CDR1 of the alpha chain can interact with the N-terminal part of certain antigenic peptides. In some contexts, CDR1 of the beta chain can interact with the C-terminal part of the peptide. In some contexts, CDR2 contributes most strongly to or is the primary CDR responsible for the interaction with or recognition of the MHC portion of the MHC-peptide complex. In some embodiments, the variable region of the [3-chain can contain a further hypervariable region (CDR4 or HVR4), which generally is involved in superantigen binding and not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0297] In some embodiments, a TCR also can contain a constant domain, a transmembrane domain and / or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune68MF-36448161473504-2029640System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C- terminal end. In some embodiments, a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction.

[0298] In some embodiments, a TCR chain contains one or more constant domain. For example, the extracellular portion of a given TCR chain (e.g., a-chain or b-chain) can contain two immunoglobulin-like domains, such as a variable domain (e.g., Va or Vb; typically amino acids 1 to 116 based on Kabat numbering Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and a constant domain (e.g., a-chain constant domain or Ca, typically positions 117 to 259 of the chain based on Kabat numbering or b chain constant domain or Cb, typically positions 117 to 295 of the chain based on Kabat) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains, which variable domains each contain CDRs. The constant domain of the TCR may contain short connecting sequences in which a cysteine residue forms a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, a TCR may have an additional cysteine residue in each of the a and 0 chains, such that the TCR contains two disulfide bonds in the constant domains.

[0299] In some embodiments, the TCR chains contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules like CD3 and subunits thereof. For example, a TCR containing constant domains with a transmembrane region may anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (e.g., CD3y, CD35, CD3s and CD3^ chains) contain one or more immunoreceptor tyrosine-based activation motif or IT AM that are involved in the signaling capacity of the TCR complex.C. Nucleic Acids

[0300] In some embodiments, the cells of the engineered cell composition, e.g., T cells, are genetically engineered to express a recombinant receptor. In some embodiments, the engineering is carried out by introducing polynucleotides that encode the CAR. In some embodiments, the nucleic acid sequence encoding the CAR contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide. Also provided69MF-36448161473504-2029640 are polynucleotides encoding the CAR, and vectors or constructs containing such nucleic acids and / or polynucleotides.

[0301] In some embodiments, the nucleic acid sequence encoding the CAR contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. Non-limiting exemplary examples of signal peptides include, for example, the GMCSFR alpha chain signal peptide , or the CD8 alpha signal peptide. Cells and Methods of Engineering Cells

[0302] In some embodiments, engineered cells, such as those that express a CAR as described herein, used in accord with the provided methods and uses are produced or generated by exemplary processes as described in, for example, PCT / US2019 / 046062, WO 2019 / 089855 and WO 2015 / 164675.

[0303] The cells for introduction of the nucleic acid encoding the transgenic receptor such as the CAR, may be isolated from a sample, such as a biological sample, e.g. , one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered.

[0304] Accordingly, the cells in some embodiments are primary cells, e.g. , primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and / or incubation. The biological sample may be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0305] In some aspects, the sample is blood or a blood-derived sample, or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0306] In some embodiments, immune cells to be engineered, such as T cells, are selected, isolated or enriched from the sample. In some embodiments, the method includes immunoaffinity-70MF-36448161473504-2029640 based selection of T cells. For example, the selection in some aspects includes incubation with a reagent or reagents for separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0307] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0308] In some aspects of such processes, a volume of cells is mixed with an amount of a desired affinity-based selection reagent. The immunoaffinity-based selection may be carried out using any system or method that results in a favorable energetic interaction between the cells being separated and the molecule specifically binding to the marker on the cell, e.g., the antibody or other binding partner on the solid surface, e.g., particle. Non-limiting methods for cell selection include, for example, magnetic bead-based separation methods, chromatography-based methods and flow cytometry by fluorescence-activated cell sorting (FACs). In some embodiments, methods are carried out using particles such as beads, e.g., magnetic beads, that are coated with a selection agent (e.g., antibody) specific to the marker of the cells. The particles (e.g., beads) may be incubated or mixed with cells in a container, such as a tube or bag, while shaking or mixing, with a constant cell density- to-particle (e.g., bead) ratio to aid in promoting energetically favored interactions.

[0309] In some aspects, the separation and / or other steps are carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components may include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.

[0310] In certain embodiments, separation and / or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy® system in some aspects is71MF-36448161473504-2029640 equipped with a cell processing unit that permits automated washing and fracdonation of cells by centrifugation. The CliniMACS Prodigy® system may also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy® system may also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports may allow for the sterile removal and replenishment of media and cells may be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0311] In some embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, is subjected to selection of T cells. In some embodiments, the selection results in an enriched composition of input cells in which at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in the composition are T cells. In some embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD3+ T cells. In some embodiments, the selection results in an enriched composition of input cells in which at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in the composition are CD3+ T cells. In some embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD4+ T cells and CD8+ T cells. In some embodiments, the selection results in an enriched composition of input cells in which at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in the composition are CD4+ and CD8+ T cells.

[0312] In some embodiments, gene transfer is accomplished by first stimulating the cells, such as by combining it with a stimulus that induces a response such as proliferation, survival, and / or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications. In particular embodiments, the T cells are activated or stimulated by contacting the cells with T cell stimulatory reagent. T cell stimulating reagents include one or more agents, such as antibodies, that are able to engage the TCR to initiate a primary signal in the cells and engage a costimulatory receptor signal. In some embodiments, the agents include an agent specific for a TCR, e.g., anti-CD3, and also an agent for stimulating a costimulatory receptor, e.g., anti-CD28. In some embodiments, the T cell stimulatory reagents include an anti-CD3 antibody or antigen binding fragment (e.g., Fab) and an anti-CD28 antibody or antigen binding fragment (e.g., Fab). In some embodiments, such agents may be, bound to solid support such as a bead. In particular embodiments, the one or more agents comprise a streptavidin-based oligomer, such as a streptavidin mutein oligomer, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fabs. In some72MF-36448161473504-2029640 embodiments, the oligomeric particle reagent is any as described in WO2015 / 158868 or WO2018 / 197949. Among the stimulatory reagents are anti-CD3 / anti-CD28 beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander, Detachable Dynabeads™, ExpACT® beads, or EXP AMER™). In some embodiments, the stimulation further includes culture of the cells with one or more cytokines. In some embodiments, the cytokine includes one or more of IL-2, IL-15 and / or IL-7. In particular embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25°C, generally at least about 30°C, and generally at or about 37°C. In some embodiments, the incubation is performed in serum free media.

[0313] In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427- 437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[0314] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lenti viral vectors or retroviral vectors, such as gamma- retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy, 2014 Apr 3. doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp. Hematol., 28(10): 1137-46; Alonso-Camino et al. (2013) Mol. Ther. Nucl. Acids., 2, e93; Park er al., Trends Biotechnol. , 2011 November 29(11): 550-557). Methods of lenti viral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0315] In particular embodiments, genetic engineering, such as by transforming (e.g., transducing) the cells with a viral vector, further includes one or more steps of incubating the cells after the introducing or contacting of the cells with the viral vector.

[0316] In some such embodiments, the further incubation is effected under conditions to result in integration of the viral vector into a host genome of one or more of the cells. For example, the73MF-36448161473504-2029640 further incubation provides time for the viral vector that may be bound to the T cells following transduction, e.g., via spinoculation, to integrate within the genome of the cell to delivery the gene of interest. In some aspects, the further incubation is carried out under conditions to allow the cells, e.g., transformed cells, to rest or recover in which the culture of the cells during the incubation supports or maintains the health of the cells. In particular embodiments, the cells are incubated under static conditions, such as conditions that do not involve centrifugation, shaking, rotating, rocking, or perfusion, e.g., continuous or semi-continuous perfusion of the media. In some embodiments, the incubation includes culture of the cells with one or more cytokines. In some embodiments, the cytokine includes one or more of IL-2, IL-15 and / or IL-7. In particular embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In some embodiments, the incubation is for between about 1 day and about 6 days, such as between about 2 days and about 4 days.

[0317] In some embodiments, the methods of engineering the cells produce a cell therapy for treating the subject. Thus, in some embodiments, the methods provide for a therapeutically effective dose for treating the subject. In some embodiments, the engineered cell therapy is autologous to the subject. In some embodiments, cells of the cell therapy are harvested after the incubation and are formulated for administration to a subject. In some embodiments, the cells are formulated in a pharmaceutically acceptable buffer, which may, in some aspects, include a pharmaceutically acceptable carrier or excipient. In some embodiments, the cells are formulated with a cryoprotectant (e.g., DMSO), such as 5% to 10% DMSO soludon, e.g., about 7.5% DMSO. In some embodiments, the cells are further formulated with human serum albumin (HSA) at a concentration between about 0.1% w / v and about 4% w / v, such as about 0.1% and about 1% w / v.III. DOWNSTREAM APPLICATIONS

[0318] In some aspects, the methods provided herein have various downstream applications that extend beyond prediction of cell proliferation and relative cell proliferation. In some embodiments, the provided methods offer an easy and fast way to predict cell proliferation and quality, including stability, that is accurate, robust and precise. As described herein, proliferative capacity of engineered cells in response to a stimulus (e.g., anti-CD3 and anti-CD28 beads or recombinant-receptor stimulating agents) has been used to predict the success of manufacturing cell therapy compositions (e.g., CD 19 CAR-T cell compositions) and determine patient eligibility for enrollment (Turtle etal., Sci. Transl. Med. (2016) 8(355):ral 16). Further, proliferation is an important predictor for durable antitumor responses in patients (Tian et al., JHO (2020) 13(1)). Thus, it is contemplated that in some embodiments, the methods provided herein can be used to select a subject for treatment with an engineered cell composition (e.g., a cell therapy composition), predict whether a subject will respond to the cell therapy composition, and / or predict efficacy of the cell74MF-36448161473504-2029640 therapy composition based on results obtained from the provided methods before treatment and after manufacture.

[0319] Additionally, existing methods for generating cell therapy compositions include steps, stages or phases of proliferating or expanding cells, such as to produce a dose of a cell therapy composition having a sufficient number of cells. However, some cell therapy compositions may not display any proliferation or expansion potential, or may expand slowly, thereby requiring extra days to achieve a sufficient number of cells for a dose of a cell therapy. In this way, generation of a cell therapy composition by genetic engineering of a subject’s cells can result in a non-conforming cell therapy composition because the cells fail to sufficiently proliferate or expand during manufacture. Such manufacturing failures not only deplete resources, but also fail to provide a cell therapy composition for patients in need. For example, in some cases, engineered populations of cells (e.g., T cells) obtained from a subject (e.g., from a biological sample from a subject) fail to achieve at least about five population doublings within about 10 days from the initiation of incubation (i.e., activation). Therefore, in some cases, existing manufacturing methods are not satisfactory for producing a sufficient number of engineered cells from a biological sample obtained from a subject, thereby leading to manufacturing failures. Thus, in some embodiments, improved methods are needed to predict and select biological samples that will sufficiently expand or proliferate to produce a dose of a cell therapy composition having a sufficient number of cells. The provided methods address this need by first allowing a manufacturer, or quality control engineer, to predict cells that do not have a desired proliferation capacity and then adjust manufacturing conditions (e.g., stimulation conditions and / or reagents, transduction reagents, temperature, etc.) prior to completion of the manufacturing process to improve or promote engineering and / or cell proliferation.

[0320] In view of the above, the provided methods are suited for various alternative methods including a method of selecting a subject for treatment and / or predicting response to and / or efficacy of an engineered cell composition, e.g., a cell therapy composition such as a CAR-T cell therapy composition. In other embodiments, the provided methods are suited for use in a method of adaptive manufacturing of a cell therapy composition and / or adaptive treatment of a subject with a cell therapy composition.

[0321] In some embodiments, the methods provided herein can be used to select a subject for treatment with an engineered cell composition, e.g., a cell therapy composition such as a CAR-T cell therapy composition. In some embodiments, a method of selecting a subject for treatment comprises subjecting the cells of the cell therapy composition, of which the cells are obtained from the subject (i.e., autologous) or from a different subject (i.e., allogeneic), to the methods provided herein and calculating a relative cell proliferation of the cell therapy composition using a reference standard (e.g., any reference standard disclosed herein). If the relative cell proliferation of the cell therapy75MF-36448161473504-2029640 composition is increased, then the subject is selected for treatment. If the relative cell proliferation of the cell therapy composition is unchanged or decreased, then the subject is not selected for treatment with the cell therapy composition and / or the subject is selected for an alternative treatment (e.g., adaptive treatment). In some embodiments, an adaptive treatment is a treatment that changes depending on the presence or absence of a characteristic (e.g., cell activity) in a subject that is predictive of response to a treatment.

[0322] In some embodiments, the methods provided herein can be used to predict response to and / or efficacy of an engineered cell composition, e.g., a cell therapy composition such as a CAR-T cell therapy composition. In some embodiments, a method of predicting response to and / or efficacy of a cell therapy composition, comprises using the methods provided herein and calculating a relative cell proliferation of the cell therapy composition using a reference standard (e.g., any reference standard disclosed herein). If the relative cell proliferation of the cell therapy composition is increased, then the subject is selected for treatment. If the relative cell proliferation of the cell therapy composition is unchanged or decreased, then the subject is not selected for treatment or the subject is selected for an adaptive treatment. In some embodiments, the provided method can be used throughout the treatment course or cycle to predict response to and / or efficacy of the cell therapy composition.

[0323] In some embodiments, the methods provided herein can be used for adaptive manufacturing. In some embodiments, a method of adaptive manufacturing of cells of a cell therapy composition, of which the cells are obtained from the subject (i.e., autologous) or from a different subject (i.e., allogeneic), comprises using the cell proliferation methods provided herein and calculating a relative cell proliferation of the cell therapy composition using a reference standard (e.g., any reference standard disclosed herein). If the relative cell proliferation of the cell therapy composition is increased, then manufacturing continues as scheduled without adjustment. If the relative cell proliferation of the cell therapy composition is unchanged or decreased, then manufacturing conditions are adjusted. In some embodiments, the provided method can be used throughout the manufacturing process. In some embodiments, the manufacturing conditions include transduction titer, concentration of stimulating reagents (e.g., recombinant receptor stimulating agents or anti-CD3 / anti-CD28 beads), culturing conditions (e.g., temperature and % CO2), etc.

[0324] In some embodiments, the provided methods can be used in methods for adaptive treatment. In some embodiments, a method of adaptive treatment comprises subjecting the cells of the cell therapy composition, of which the cells are obtained from the subject (i.e., autologous) or from a different subject (i.e., allogeneic), to the cell proliferation methods provided herein and calculating a relative cell prol iteration of the cell therapy composition relati ve to a reference standard (e.g., any reference standard disclosed herein). If the relative cell proliferation of the cell therapy76MF-36448161473504-2029640 composition is increased, then the subject is treated with the cell therapy composition. If the relative cell proliferation is unchanged or decreased, then the subject treated with the cell therapy composition in combination with another agent. In some embodiments, the provided method can be used throughout the treatment course or cycle to maintain response to the cell therapy composition.

[0325] In some embodiments, the disease or condition is an autoimmune or inflammatory disease or disorder. In some embodiments, the subject has an autoimmune or inflammatory disease or disorder and is a candidate for treatment with a cell therapy composition (e.g. , T cell therapy composition). In some embodiments, the autoimmune or inflammatory disease or condition is arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave’s disease, Crohn’s disease multiple sclerosis, asthma, and / or a disease or condition associated with transplant.

[0326] In some embodiments, the disease or condition is a cancer or tumor. In some embodiments, the cancer or tumor is a hematological malignancy or a blood cancer. In some embodiments, the cancer is a B cell malignancy. In some embodiments, the cancer is a lymphoma or leukemia. In some embodiments, the cancer is acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), Anaplastic large cell lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM), a B cell malignancy is selected from among acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), and Diffuse Large B-Cell Lymphoma (DLBCL).

[0327] In some embodiments, the cancer is lymphoma. In some embodiments, the cancer is selected from small cell lymphoma, lymphoplasmacytic lymphoma (e.g., Waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell neoplasms (e.g., plasma cell myeloma such as multiple myeloma, or plasmacytoma), extranodal marginal zone B cell lymphoma (e.g., MALT lymphoma), nodal marginal zone B cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneous follicle center lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), Epstein-Barr virus- positive DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B- cell lymphoma (PMBCL), Intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, Burkitt lymphoma, adult T-cell lymphoma, extranodal K / T-cell lymphoma, enteropathy-associated T-cell lymphoma, Hepatosplenic T-cell lymphoma, blastic NK cell77MF-36448161473504-2029640 lymphoma, Mycosis fungoides / Sezary syndrome, Primary cutaneous anaplastic large cell lymphoma, Lymphomatoid papulosis, Peripheral T-cell lymphoma, Angioimmunoblastic T cell lymphoma, Anaplastic large cell lymphoma, B- lymphoblastic lymphoma, B-lymphoblastic lymphoma with recurrent genetic abnormalities, T-lymphoblastic lymphoma, and Hodgkin lymphoma. In some embodiments, the cancer is refractory to one or more prior treatments, and / or the cancer has relapsed after one more prior treatments. In some embodiments, the lymphoma is large B cell lymphoma (LBCL). In some embodiments, the lymphoma is diffuse large B cell lymphoma (DLBCL).

[0328] In some embodiments, the cancer is a pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancers, CNS cancers, brain tumors, bone cancer, or soft tissue sarcoma.

[0329] In some embodiments, the cancer is refractory to or the cancer has relapsed following one or more of chemotherapy, radiotherapy, immunotherapy (including a T cell therapy and / or treatment with an antibody or antibody-drug conjugate), an autologous stem cell transplant, or any combination thereof.IV. DEFINITIONS

[0330] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0331] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and / or “consisting essentially of’ aspects and variations.

[0332] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0333] As used herein, the term “area under the curve” or “AUC” is a method that can be used to measure cell proliferation kinetics, calculating the total area beneath a cell growth curve or a specific time period. The AUC integrates the initial cell density and the cell density over time78MF-36448161473504-2029640 throughout the experiment (i.e., proliferation rate) to provide a single value that represents cell proliferation. In some embodiments, the cell growth curve is based on a cell confluence curve.

[0334] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers, may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0335] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. The substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. Amino acid substitutions may be introduced into a binding molecule, e.g., antibody, of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0336] Amino acids generally can be grouped according to the following common side-chain properties:(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;(3) acidic: Asp, Glu;(4) basic: His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro;(6) aromatic: Trp, Tyr, Phe.

[0337] In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, nonconservative amino acid substitutions can involve exchanging a member of one of these classes for another class.

[0338] As used herein, the term “proliferative capacity” refers to the capacity or ability of a cell to proliferate or divide. As used herein, the term “relative cell proliferation” refers to the proliferation observed in the test sample relative or compared to the proliferation observed in a reference sample. In some embodiments, the test sample comprises a test sample described herein, e.g., a test engineered cell composition or test cell composition. In some embodiments, the reference sample comprises a reference sample described herein, e.g., a reference engineered cell composition.79MF-36448161473504-2029640

[0339] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.

[0340] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0341] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.V. EXEMPLARY EMBODIMENTS

[0342] Among the provided embodiments are:1. A method for assessing cell proliferation of a test engineered cell composition, wherein the test engineered cell composition comprises cells comprising a recombinant receptor, the method comprising:(a) culturing cells of the test engineered cell composition with a recombinant receptorstimulating agent for a period of time to generate a stimulated cell composition;(b) after the period of time adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition;(c) incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal;(d) detecting the luminescent signal from the stimulated cell composition; and, optionally,(e) determining cell proliferation of the test engineered cell composition using the results of the detecting.80MF-36448161473504-20296402. The method of embodiment 1, wherein steps (a) to (d) are performed for two or more replicates, optionally wherein an average of the luminescent signal detected from the two or more replicates is used as the luminescent signal in the determining.3. The method of embodiment 2, wherein steps (a) to (d) are performed for at least three replicates.4. The method of embodiment 1 , wherein determining cell proliferation comprises calculati ng the difference between (A) the luminescent signal detected from the stimulated cell composition and (B) a luminescent signal detected from unstimulated cultured cells of the test engineered cell composition, wherein the unstimulated cultured cells have been cultured for a second period of time in the absence of the recombinant receptor-stimulating agent.5. The method of embodiment 4, wherein the second period of time has the same duration as the first period of time.6. The method of embodiment 4 or 5, wherein the method further comprises culturing unstimulated cells of the test engineered cell composition for the second period of time in the absence of the recombinant receptor-stimulating agent to generate the unstimulated cultured cells, and detecting the luminescent signal from the unstimulated cultured cells.7. The method of any one of embodiments 4 to 6, wherein the method further comprises adding the luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the unstimulated cultured cells.8. The method of any one of embodiments 4 to 7, wherein the method further comprises incubating the unstimulated cultured cells with the luciferase enzyme and the substrate or prosubstrate of the luciferase enzyme to generate a luminescent signal.9. The method of any one of embodiments 4 to 8, wherein the method further comprises detecting the luminescent signal from the unstimulated cultured cells.10. The method of any one of embodiments 4 to 9, wherein the steps of culturing the unstimulated cells of the test engineered cell composition for the second period of time and detecting the luminescent signal from the unstimulated cultured cells are performed for two or more replicates, optionally wherein an average of the luminescent signal detected from said two or more replicates is used in the calculating as the luminescent signal detected from the unstimulated cultured cells.11. The method of any one of embodiments 1 to 10, comprising calculating a relative cell proliferation of the test engineered cell composition by comparing the cell proliferation as determined in step (e) to the cell proliferation of a reference standard.12. The method of embodiment 11, wherein the comparing comprises calculating a ratio of the cell proliferation as determined in step (e) to the cell proliferation of the reference standard and optionally expressing the ratio as a percentage by multiplying it by 100.81MF-36448161473504-202964013. A method for assessing relative cell proliferation of a test engineered cell composition, wherein the test engineered cell composition comprises cells comprising a recombinant receptor, the method comprising:(a) culturing cells of the test engineered cell composition with a recombinant receptorstimulating agent for a period of time to generate a stimulated cell composition;(b) after the period of time, adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition and incubating the sti undated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal;(c) detecting the luminescent signal from the stimulated cell composition;(d) determining cell proliferation of the test engineered cell composition by calculating the difference between (A) the luminescent signal detected from the stimulated cell composition and (B) the luminescent signal detected from unstimulated cultured cells of the cell composition, wherein the unstimulated cultured cells have been cultured for a second period of time in the absence of the recombinant receptor-stimulating agent, wherein the second period of time is equivalent in duration to the period of time; and(e) calculating a relative cell proliferation of the test engineered cell composition by calculating a ratio of the cell proliferation as determined in step (d) to the cell prol iteration of a reference standard.14. The method of embodiment 13, wherein steps (a) to (c) are performed for two or more replicates.15. The method of embodiment 13 or embodiment 14, wherein calculating the difference in step (d) comprises calculating the difference between the average luminescent signal detected from the stimulated cell composition and the luminescent signal detected from unstimulated cultured cells of the cell composition.16. The method of any one of embodiments 13 to 15, further comprising culturing the unstimulated cells of the test engineered cell composition for the second period of time in the absence of the recombinant receptor stimulating agent to generate the unstimulated cultured cells, and detecting the luminescent signal from the unstimulated cultured cells.17. The method of embodiment 15, wherein the steps of culturing the unstimulated cells of the test engineered cell composition for the second period of time and detecting the luminescent signal from the unstimulated cultured cells are performed for two or more replicates.18. The method of embodiment 17, wherein calculating the difference in step (d) comprises calculating the difference between the luminescent signal (e.g. the average luminescent signal) detected from the stimulated cell composition and average the luminescent signal detected from unstimulated cultured cells of the cell composition.82MF-36448161473504-202964019. The method of any one of embodiments 1 tol8, wherein the period of time is at least about 46 hours.20. The method of any one of embodiments 1 to 19, wherein the period of time is between about 46 hours and about 96 hours.21. The method of any one of embodiments 1 to 20, wherein the period of time is between about 46 hours and about 72 hours.22. The method of any one of embodiments 1 to 21, wherein the period of time is about 46 hours to about 50 hours.23. The method of any one of embodiments 1 to 22, wherein the culturing in step (a) is carried out at a temperature of 37°C ± 2°C, or at a temperature of about 37°C ± 2°C.24. The method of any one of embodiments 1 to 23, wherein the culturing in step (a) comprises maintaining carbon dioxide (CO2) between 3% and 7%, or between about 3% and about 7%.25. The method of any one of embodiments 1 to 24, wherein the culturing in step (a) comprises maintaining humidity level between 90% and 100% or between about 90% and about 100%.26. The method of any one of embodiments 1-25, wherein the incubating of the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate in step (c) is carried out at a temperature of 37°C ± 2°C or at a temperature of about 37°C ± 2°C.27. The method of any one of embodiments 1 to 26, wherein the incubating of the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate in step (c) comprises maintaining carbon dioxide (CO2) between 3% and 7%, or between about 3% and about 7%.28. The method of any one of embodiments 1 to 27, wherein the incubating of the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate in step (c) comprises maintaining humidity level between 90% and 100% or between about 90% and about 100%.29. The method of any one of embodiments 4 to 28, wherein culturing the unstimulated cells is carried out at a temperature of 37°C ± 2°C, or at a temperature of about 37°C ± 2°C.30. The method of any one of embodiments 4 to 29, wherein culturing the unstimulated cells comprises maintaining carbon dioxide (CO2) between 3% and 7%, or between about 3% and about 7%.31. The method of any one of embodiments 4 to 30, wherein culturing the unstimulated cells comprises maintaining humidity level between 90% and 100% or between about 90% and about 100%.83MF-36448161473504-202964032. The method of any one of embodiments 9 to 31 , wherein the incubating of the unstimulated cultured cells with the luciferase enzyme and the substrate or pro-substrate in step (c) is carried out at a temperature of 37°C ± 2°C or at a temperature of about 37°C ± 2°C.33. The method of any one of embodiments 9 to 32, wherein the incubating of the unstimulated cultured cells with the luciferase enzyme and the substrate or pro-substrate in step (c) comprises maintaining carbon dioxide (CO2) between 3% and 7%, or between about 3% and about 7%.34. The method of any one of embodiments 9 to 33, wherein the incubating of the unstimulated cultured cells with the luciferase enzyme and the substrate or pro-substrate in step (c) comprises maintaining humidity level between 90% and 100% or between about 90% and about 100%.35. The method of any one of embodiments 1 to 34, wherein the incubating in step (c) is for about 10 minutes to about 50 minutes.36. The method of any one of embodiments 1 to 35, wherein after the incubating in step (c) and before the detecting in step (d), the method comprises equilibrating the stimulated cell composition and the unstimulated cultured cells to room temperature.37. The method of embodiment 36, wherein the equilibrating is for about 2 hours to about 4 hours.38. The method of embodiment 37, wherein the stimulated cell composition and the unstimulated cultured cells are protected from light during the equilibrating.39. The method of any one of embodiments 1 to 38, further comprising contacting the recombinant receptor-stimulating agent with cells of the test engineered cell composition prior to the culturing in step (a), optionally wherein the contacting comprises adding a volume of the test engineered cell composition to a vessel containing the recombinant receptor-stimulating agent.40. The method of any one of embodiments 1 to 39, wherein one or more steps of the method including at least step (d) of detecting the luminescent signal from the stimulated cell composition is performed iteratively to allow assessment of cell proliferation at different timepoints.41. The method of embodiment 40, wherein a step of incubating the stimulated cell composition is performed before each iteration of step (d).42. The method of embodiment 41 , wherein step (b) is not repeated after the first time it is performed.43. The method of embodiment 40, wherein each of steps (b), (c) and (d) are performed iteratively.44. The method of any one of embodiments 41 to 43, wherein after each iteration of the incubating in step (c) and before each iteration of the detecting in step (d), the method comprises equilibrating the stimulated cell composition to room temperature.84MF-36448161473504-202964045. The method of any one of embodiments 39 to 44, wherein any iteration of step (d) of the method is carried out within 96 hours of first initiating the culturing of the cells of the test engineered cell composition with the recombinant receptor-stimulating agent according to step (a).46. The method of embodiment 25, wherein any iteration of step (d) of the method is carried out within 72 hours of first initiating the culturing of the cells of the test engineered cell composition with the recombinant receptor-stimulating agent according to step (a).47. A method for assessing cell proliferation of a test cell composition, the method comprising:(a) culturing cells of a test cell composition for a period of time under conditions that support cell proliferation to make a cultured cell composition, optionally wherein the period of time is about 46 hours;(b) after the culturing in (a), adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the cultured cell composition to generate a luminescent signal;(c) detecting the luminescent signal from the cultured cell composition; and(d) calculating a relative cell proliferation of the test cell composition by comparing the luminescent signal that is detected in step (c) to the luminescent signal that is detected from a reference standard.48. The method of embodiment 47, wherein the culturing in (a) is carried out in the presence of a stimulating agent to stimulate cells of the test cell composition and generate a stimulated test cell composition.49. The method of embodiment 47 or embodiment 48, wherein the test cell composition is a test engineered cell composition that comprises cells comprising a recombinant receptor.50. The method of embodiment 49, wherein the stimulating agent is a recombinant receptor-stimulating agent.51. The method of any one of embodiments 47-50, wherein calculating the relative cell proliferation comprises:(i) determining cell proliferation of the test cell composition by calculating the difference between (A) the luminescent signal detected from the stimulated test cell composition and (B) the luminescent signal detected from unstimulated cells of the test cell composition that have been cultured in the absence of the stimulating agent; and(ii) comparing the cell proliferation as calculated in step (i) with the cell proliferation of a reference standard.52. The method of embodiment 51 , wherein the comparing comprises calculating a ratio of the cell proliferation as determined in step (i) to the cell proliferation of the reference standard and optionally expressing the ratio as a percentage by multiplying it by 100.85MF-36448161473504-202964053. The method of any one of embodiments 47 to 52, wherein the period of time is at least about 46 hours.54. The method of any one of embodiments 47 to 53, wherein the period of time is between about 46 hours and about 96 hours.55. The method of any one of embodiments 47 to 54, wherein the period of time is between about 46 hours and about 72 hours.56. The method of any one of embodiments 47 to 53, wherein the period of time is about 46 hours to about 50 hours.57. The method of any one of embodiments 1 to 46 and 49 to 56, wherein the test engineered cell composition has been produced ex vivo from primary cells from a subject by a cell engineering manufacturing process to comprise the recombinant receptor.58. The method of any one of embodiments 1 to 46 and 49 to 57, wherein the test engineered cell composition is a cell composition produced for use as a cell therapy for treating a subject with a disease or condition.59. The method of embodiment 58, wherein the disease or condition is a cancer or is an autoimmune or inflammatory disease or condition.60. The method of embodiment 58 or embodiment 59, wherein the cells of the cell therapy are primary cells that are autologous to the subject to be treated.61. The method of embodiment 58 or embodiment 59, wherein the cells of the cell therapy are primary cells that are allogeneic to the subject to be treated.62. The method of any one of embodiments 11 to 61, wherein the reference standard is a reference engineered cell composition comprising a reference recombinant receptor.63. The method of embodiment 62, wherein the reference recombinant receptor expressed by the reference engineered cell composition differs from the recombinant receptor expressed by the test engineered cell composition.64. The method of embodiment 62, wherein the reference recombinant receptor expressed by the reference engineered cell composition is the same as the recombinant receptor expressed by the test engineered cell composition.65. The method of any one of embodiments 11 to 64, wherein the reference standard is a reference engineered cell composition having a validated cell proliferation.66. The method of embodiment 65, wherein the validated proliferation index of the reference standard is determined by:(a) culturing cells of the reference engineered cell composition with a recombinant receptorstimulating agent for a period of time to generate a stimulated reference engineered cell composition;(b) after the period of time, adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated reference engineered cell composition and incubati ng the86MF-36448161473504-2029640 stimulated cell composition with the luciferase enzyme and the substrate or the pro-substrate to generate a luminescent signal;(c) equilibrating the stimulated reference engineered cell composition to room temperature;(d) detecti ng the luminescent signal from the stimulated reference engineered cell composition; and(e) determining cell proliferation of the reference standard by calculati ng the difference between (A) the luminescent signal detected from the stimulated reference engineered cell composition and (B) the luminescent signal detected from unstimulated cells of the reference engineered cell composition, wherein the unstimulated cells of the reference engineered cell composition have been cultured in the absence of the recombinant receptor-stimulating agent.67. The method of any one of embodiments 62 to 66, wherein the reference engineered cell composition is produced ex vivo from primary cells from a subject by a cell engineering manufacturing process to comprise the recombinant receptor, wherein the subject is different from the subject used to produce the test engineered cell composition.68. The method of embodiment 67, wherein the subject is a healthy subject not known or suspected of having a disease or condition.69. The method of any one of embodiments 57 to 68, wherein the manufacturing process used to manufacture the test engineered cell composition differs from the manufacturing process used to manufacture the reference cell composition.70. The method of any one of embodiments 57 to 68, wherein the manufacturing process used to manufacture the test engineered cell composition is the same as the manufacturing process used to manufacture the reference cell composition.71. The method of any one of embodiments 1 to 46 and 49 to 70, wherein the recombinant receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).72. The method of embodiment 71 , wherein the recombinant receptor is a CAR and the CAR comprises an extracellular antigen binding domain specific for a target antigen, a transmembrane domain, and an intracellular signaling domain comprising a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optional I y is a CD3zeta, and a signaling domain from a T cell costimulatory molecule, optional I y wherein the T cell costimulatory molecule is 4-1BB and / or CD28.73. The method of embodiment 72, wherein the extracellular antigen binding domain comprises a antibody variable heavy chain domain and variable light chain domain specific for the target antigen.74. The method of embodiment 72 or embodiment 73, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv) specific for the target antigen.87MF-36448161473504-202964075. The method of embodiment 72, wherein the extracellular antigen binding domain comprises a VHH domain.76. The method of any one of embodiments 71 to 75, wherein the CAR is monospecific to the target antigen.77. The method of any one of embodiments 71 to 75, wherein the CAR is bispecific and the target antigen is a first target antigen and the extracellular binding domain is further specific for a second target antigen.78. The method of embodiment 77, wherein the extracellular antigen binding domain comprises a first antigen binding domain comprising a first variable heavy (VH) chain domain and a first variable light (VL) chain domain specific for the first target antigen and a second antigen binding domain comprising a second VH chain domain and a second VL chain domain specific for the second target antigen.79. The method of embodiment 78, wherein the extracellular antigen binding domain has a tandem structure comprising a sequence comprising in order the first antigen binding domain and the second antigen binding domain.80. The method of embodiment 78, wherein the extracellular antigen binding domain has a loop structure comprising a sequence comprising in order, N- to C-terminal: the first VH chain or first VL chain of the first antigen binding domain, the second VH chain or second VL chain of the second antigen binding domain, the other of the second VH chain and second VL chain of the second antigen binding domain, and the other of the first VH chain and second VL chain of the second antigen binding domain.81. The method of embodiment 77, wherein the extracellular antigen binding domain comprises a first VHH specific to the first target antigen and a second VHH specific to the second target antigen.82. The method of any one of embodiments 72 to 81, further comprising a hinge spacer sequence between the extracellular antigen binding domain and the transmembrane domain.83. The method of any one of embodiments 1 to 46 and 50 to 82, wherein the recombinant receptor-stimulating agent is or comprises a binding moiety recognized by or specific to the recombinant receptor, optionally to the extracellular antigen binding domain of the CAR.84. The method of embodiment 83, wherein the binding moiety is a target antigen or an extracellular domain binding portion thereof of the recombinant receptor, optionally wherein the extracellular domain binding portion of the target antigen comprises an epitope recognized by the recombinant receptor.85. The method of embodiment 83, wherein the binding moiety is an antibody specific to an extracellular binding domain of the recombinant receptor.88MF-36448161473504-202964086. The method of embodiment 83 or embodiment 85, wherein the binding moiety is an anti-idiotypic antibody specific to an extracellular antigen binding domain of the recombinant receptor.87. The method of any one of embodiments 83 to 86, wherein the binding moiety is immobilized or attached to a solid support during the culturing, optionally wherein the culturing is initiated when cells of the test engineered cell composition or test cell composition are added to the solid support.88. The method of any one of embodiments 1 to 87, wherein the culturing is initiated when cells of the test engineered cell composition or test cell composition are added to a solid support.89. The method of embodiment 87 or embodiment 88, wherein the solid support is a surface of a culture vessel.90. The method of embodiment 87 or embodiment 88, wherein the solid support is a bead.91. The method of embodiment 90, wherein the culturing is initiated when cells of the test engineered cell composition or test cell composition and the binding moiety-immobilized beads are contacted in a culture vessel.92. The method of embodiment 89 or embodiment 91 , wherein the culture vessel is a multi-well plate, optionally a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96 wellplate or a 384-well plate.93. The method of embodiment 92, wherein the culture vessel is a 96-well plate.94. The method of any one of embodiments 89 and 91 to 93, wherein the cells are added to the culture vessel at a cell density of between about 6.7 x 104viable cells / mL and about 3 x 106viable cells / mL.95. The method of any one of embodiments 89 and 91 to 94, wherein the cells number of cells present in the culture vessel at the time the detecting is performed is not more than 6 x 106viable cells / mL.96. The method of any one of embodiments 89 and 91 to 95, wherein the number of cells present in the culture vessel at the time the detecting is performed is not less than 3.3 x 104viable cells / mL.97. The method of any one of embodiments 1 to 96, wherein the cells of the test engineered cell composition or the test cell composition comprise a T cell.98. The method of embodiment 97, wherein the T cells are primary cells.99. The method of embodiment 97 or embodiment 98, wherein the T cells are autologous cells.100. The method of embodiment 97 or embodiment 98, wherein the T cells are allogeneic cells.101. The method of any one of embodiments 97 to 100, wherein the T cells are CD3+.89MF-36448161473504-2029640102. The method of any one of embodiments 97 to 101, wherein the T cells are CD4+ and / or CD8+ T cells.103. The method of any one of embodiments 97 to 102, wherein the T cells are CD4+ and CD8+ T cells.104. The method of any one of embodiments 1 to 103, wherein the substrate or pro-substrate is cell permeable and is able to enter cells105. The method of any one of embodiments 1 to 104, wherein the substrate or pro-substrate added to the cells is a luciferin-D substrate or analog or derivative thereof, or is a pro-substrate of any of the foregoing.106. The method of any one of embodiments 1 to 104, wherein the substrate or prosubstrate added to the cells is a coelenterazine substrate or analog or derivative thereof, or is a prosubstrate of any of the foregoing.107. The method of any one of embodiments 1 to 104, wherein the substrate or pro-substrate added to the cells is a furimazine substrate or analog or derivative thereof, or is a pro-substrate of any of the foregoing.108. The method of any one of embodiments 1 to 107, wherein the substrate or pro-substrate added to the cells is a pro-substrate, wherein the pro-substrate is able to be modified by the actions of a cellular enzyme present in the cell to release the substrate inside the cell.109.The method of embodiment 108, wherein the cellular enzyme is an esterase.110. The method of any one of embodiments 1 to 109, wherein, when present in the cells, the substrate is able to be reduced by molecular oxygen and released from the cells as a substrate for the luciferase.111. The method of any one of embodiments 1 to 110, wherein the luciferase enzyme is cell-impermeable and does not enter the cell, wherein the luciferase binds the reduced substrate after it exits the cell.112. The method of any one of embodiments 1 to 111, wherein the luciferase is from Oplophorus (OLuc), Gaussia (GLuc), Renilla (RLuc), Pyrophorus, or Photinus or is a variant thereof that is able to produce luminescence when bound to substrate.113. The method of any one of embodiments 1 to 112, wherein the luciferase is from Oplophorus (OLuc) or is a variant thereof that is able to produce luminescence when bound to substrate.114. The method of any one of embodiments 1 to 113, wherein the luciferase is a OLuc variant that is the variant C1A4E comprising at least 8 substitutions selected from the group consisting of A4E, QI 1R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, P115E, Q124K, and Y138IP, optionally 8, 9, 10, 11, 12, 13, 14 or 15 substitutions.90MF-36448161473504-2029640115. The method of any one of embodiments 1 to 114, wherein the luciferase is NanoLuc™.116. The method of any one of embodiments 1 to 115, wherein the luminescent signal is generated after the luciferase binds to the reduced substrate.117. The method of any one of embodiments 1 to 116, wherein the luminescent signal is detected by a luminometer.VI. EXAMPLES

[0343] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1: Development of a Non-Lytic Bioluminescent Assay For Assessing Proliferative Capacity of Engineered Cell Compositions

[0344] Proliferative capacity of T cells engineered to express a chimeric antigen receptor (CAR) was assessed using a non-lytic bioluminescent proliferation assay that uses a cell permeable substrate (furimazine or derivative or a pro-substrate thereof) and a bioluminescent enzyme (NanoLuc® enzyme). When released as a substrate in the cell, the substrate diffuses into the cells where it is reduced by molecular oxygen. Once reduced, the substrate exits the cell and reacts with a luciferase bioluminescent enzyme producing a light signal.

[0345] The CAR-T cell composition was generated by a process including immunoaffinitybased (e.g., immunomagnetic selection) enrichment of CD4+ and CD8+ cells from leukapheresis samples, activation with an anti-CD3 / anti-CD28 antibody reagent, transduction with a lentiviral vector encoding a CAR, and incubation with recombinant cytokines. The harvested CAR-T cell compositions were formulated with a cryoprotectant (e.g., DMSO), cryopreserved and then thawed before use. For the cellular redox proliferation assay, thawed cells of the engineered composition were stimulated by contacting the cells with a plate bound anti-idiotypic antibody (alD) specific to an extracellular antigen binding domain of the CAR and then measuring readout of the luminescent signal. Specifically, the proliferation assay was performed by coating each well of a 96-well plate with 8 pg / mL of alD suspended in sterile IX PBS. The 96-well plates were coated with alD soludon and incubated overnight for at least 16 hours at 4°C to allow alD to become plate bound. Any wells designated as unstimulated conditions were covered with IX PBS only for the same duration at 4°C. On the day of cell seeding, the alD solution or IX PBS was removed and all wells were blocked for a minimum of 30 minutes at ambient room temperature (ART) with cell culture medium. The blocking cell culture medium was removed and cells that had been washed and resuspended in culture medium were seeded in each well at 1.33x 105cells / mL; a total volume of 150 pL was added to each of the wells that contained alD or to wells that did not contain alD (unstimulated condition). The 96-well plate was covered with breathable plate sealer and cultured in an incubator at 37°C ± 2°C, 5% ± 2% CO2, 95 ±91MF-36448161473504-20296405% humidified. The culturing was conducted for a certain amount of time (e.g., for a time of 48 to 70 hours) to allow cells to proliferate (see “long incubation” in FIG. 1).

[0346] Following the culturing, the cells were incubated with substrate and enzyme at 37°C to allow luminescence to develop (see “short incubation” in FIG.l) followed by equilibration to ART (see “ART equilibration” in FIG. 1). Equal volumes of a furimazine pro-substrate (2X concentration; e.g., MT cell viability substrate from RealTime-Glo™; Promega) and enzyme (2X concentration; NanoLuc® Enzyme; Promega) were mixed into cell culture medium (IX), and 100 pL of the resulting mixture was added to each well of the 96-well plate. The plate was covered with a breathable plate sealer and incubated at 37°C ± 2°C, 5% ± 2% or 5% ± 1% CO2, 95 ± 5% humidified, for 30 ± 20 minutes. The plate was then equilibrated at ART in the dark for an additional 3 + 1 hours and read on a luminometer, which measures the emission of Relative Luminescence Units (RLU). The equilibration step at ART prevents the occurrence of hotspots that might affect the luminescence readout. An overview of the cellular redox proliferation assay is shown in FIG. 1.

[0347] For calculating relative cell proliferation, comparison of proliferation was made to a validated reference standard, which was an engineered T cell composition produced from material obtained from a healthy donor. Relative proliferation using this assay was calculated as follows. Sample engineered compositions along with one reference standard were each tested in triplicate format in both stimulated and unstimulated conditions. The average signal difference between stimulated and unstimulated conditions for the sample engineered compositions was divided by the average signal difference between sti mulated and unstimulated conditions for the reference standard, as shown in the formula below.Average Sample stim - - - - - Average Reference Standar

[0348] As shown in FIG. 2, a robust measure of CAR-T cell proliferation (high signal-to-noise ratio) was quantifiable even after only 48 hours of alD stimulation. Relative proliferation of CAR expressing cells in the engineered cell composition was dose-dependent with higher proliferation observed when cells were stimulated with increasing concentrations of alD.

[0349] Because most proliferation assays rely on cells proliferating over an extended period of time (e.g., 96 to 240 hours depending on the method) to achieve a sufficient cell number for the method to more precisely measure cell proliferation in response to antigen stimulation, studies were performed to determine if a single time-point luminescent early readout was correlated with and predictive of cell proliferation of engineered compositions post antigen sti mulation, as determined using conventional methods for assessing cell proliferation. Specifically, the cellular redox proliferation assay that is disclosed herein was compared to the IncuCyte® proliferation assay. For both methods, CAR-T cell compositions were generated from T cells from leukapheresis samples92MF-36448161473504-2029640 obtained from five different donor subjects with multiple myeloma by immunoaffinity-based selection of CD4+ and CD8+ T cells and transduction with a viral vector encoding the CAR.

[0350] The IncuCyte® S3 Live-Cell Imaging and Analysis System (Sartorius) was used to measure confluence over time. Each CAR-T cell composition was either stimulated with an alD against the CAR or was unstimulated and confluence over time for 240 hours was assessed every 2 hours. Readout for this assay is the area under the curve (AUC) at each 24-hour interval. Notably, with the IncuCyte® proliferation assay, three donor samples (1, 2 and 4) reached the confluence quantitation limit (100%) at around 96 hours (FIG. 3) after which point proliferation could not be accurately estimated.

[0351] The cellular redox proliferation assay as described above using the RTG reagent was carried out on the same CAR-T cell compositions generated from the subjects with multiple myeloma. FIG. 4 shows that readouts obtained using the cellular redox proliferation assay after 48 hours of proliferation were highly consistent with results obtained from the IncuCyte® method at later timepoints (e.g., after 144 hours of proliferation).

[0352] To confirm correlation between the cellular redox proliferation assay and actual cell growth, the relative luminescence unit (RLU) was compared to actual viable cell count (VCC). Briefly, the same engineered CAR-T cell composition was used for both correlation studies. The y- axis represents the output of VCC (cells / mL), and the X-axis represents the output of the cellular redox proliferation assay (RLU). Three seeding densities are tested in triplicate and are represented by individual data points on the plot. Results are shown in FIGS. 5A-5B. FIGS. 5A-5B show the cellular redox proliferation assay is an excellent prediction tool because RLU at 48 hours (FIG. 5A) and at 72 hours (FIG. 5B) correlates well with a cell enumeration method (VCC) at 72 hours. A linear trendline was fitted to the data, demonstrating a strong positive correlation between the two readout methods, with an R-squared value greater than 0.98. The high correlation indicates that the cellular redox proliferation assay (at both the 48 hour and 72 hour timepoints) provides results that are comparable to those obtained with the VCC method (at 72 hours). In the context of manufacturing cell products, these results indicate that the cellular redox proliferation assay can be used to characterize cell proliferation to ensure manufacturing consistency and efficacy.

[0353] Notably, the cellular redox proliferation assay and the standard IncuCyte® proliferation method provided consistent results for engineered cell compositions with different proliferative profiles. These results thus confirm the utility of readout of cellular redox after only 48 hours of proliferation time as a predictive readout of relative proliferation potential. The cellular redox proliferation assay described herein provides an early readout (after only 48 hours of proliferation) that accurately predicts proliferation outcomes at later times. This provides an improvement over traditional proliferation assays, for which 48 hours is too early to reach a sufficient number of cells to93MF-36448161473504-2029640 precisely measure proliferation or distinguish proliferative profiles and, in many instances, 96 hours is too late because cells (e.g., engineered cell compositions) with higher proliferative potential will have reached the upper limit of quantitation of the instrument (e.g., 100% confluence). Moreover, traditional proliferation assays such as the IncuCyte® method lack reference controls leading to variabilities between different assay runs and difficulty in establishing equivalency between runs, analysts, sites, etc. Because the cellular redox proliferation assay described herein provides an accurate and precise readout after a proliferation time of only 48 hours, it is an amenable assay to be used for routine characterization and testing of cell therapy drug products.Example 2: Comparing Cell Proliferation of Engineered Cell Compositions Produced Using Different Activation Reagents

[0354] Process variability can change engineered cell composition quality and attributes. Thus, the cell proliferation assay described in Example 1 was used to assess and compare different test engineered cell compositions produced using different manufacturing processes. Specifically, in this example, the impact of different activation reagents (Expamer, Dynabeads, detachable Dynabeads, and TransAct) in process on cell proliferation was determined.

[0355] During the CAR-T cell manufacturing process used in this example, T cells were activated prior to engineering the cells. Different activation reagents are available and known (see e.g., Noaks et al., Molecular Therapy Methods & Clinical Development (2021) 20:675-687). In this example, four different activation reagents were used to assess impacts on the proliferation of the produced T cell composition.

[0356] Engineered cell compositions were generated by a process substantially as described in Example 1 including immunoaffinity-based (e.g., immunomagnetic selection) enrichment of CD4+ and CD8+ cells from leukapheresis samples, activation with an anti-CD3 / anti-CD28 antibody reagent in a medium containing recombinant cytokines, transduction with a lentiviral vector encoding a CAR, and incubation in a medium containing recombinant cytokines. In this example, engineered cell compositions were generated from material obtained from 4 different donors (3 healthy donors and 1 donor with an autoimmune condition) and were cryopreserved before use. Enriched CD4+ and CD8+ T cells from each donor were used in 4 different processes for producing the engineered CAR-T cells. The processes employed differed in the T cell activation reagent that was used prior to transduction with the viral vector encoding the CAR including: (1) anti-CD3 / anti-CD28 antibody-conjugated Dynabeads™ (DBs; ThermoFisher Scientific, Cat. No. 11132D), (2) detachable anti-CD3 / anti-CD28 DBs (CTS™ Detachable Dynabeads™ CD3 / CD28; Cat. No. A56996, (3) anti-CD3 / antiCD28 polymeric nanomatrix (TransAct™; Miltenyi Biotec); and (4) anti-CD3 / anti-CD28 polymeric soluble cross-linked streptavidin muteins (Expamer).94MF-36448161473504-2029640

[0357] Cells were thawed and resuspended in culture media, then stimulated by contacting the cells with a plate bound anti-idiotypic antibody (alD) specific to an extracellular antigen binding domain of the CAR. Subsequent to stimulation, the readout of the luminescent signal was measured. Relative proliferation was determined as previously described herein. Proliferation was also assessed using the traditional IncuCyte® method.

[0358] The cellular redox proliferation assay results, as assessed following a 72-hour proliferation time, are shown in FIG. 6A. The results of the cellular redox proliferation assay showed that the relative proliferation of the cell compositions was comparable when produced using each of the Expamer, CTS Dynabeads™ and Detachable Dynabeads™ activation reagents, whereas the relative proliferation of the compositions was statistically significantly lower when produced using the TransAct™ activation reagent.

[0359] Using the traditional IncuCyte® method, the relative proliferation for each donor as determined by the AUC at 144 hours (relative to the mean of the Expamer condition) is shown in FIG. 6B and the summary results averaged across donors is shown in Table El.

[0360] Table El: Proliferation (mean AUC) in response to various activation reagents in engineered cell compositions produced from material obtained from several donors.

[0361] Like the cellular redox proliferation assay, the IncuCyte™ method results indicated that when using Expamer, CTS Dynabeads™ and Detachable Dynabeads™, the observed proliferation outcomes did not show statistically significant differences, whereas the use of TransAct reduced the observed proliferation. Statistical testing of the results obtained with both assays showed that the proliferation was significantly lower when TransAct was used than when Expamer was used (See Table El).

[0362] Together, these results establish the utility of the cellular redox proliferation assay to measure proliferation capacity of cells. The cellular redox proliferation assay provides an outcome measure that can be obtained at an earlier timepoint and is predictive of proliferation outcomes measured at later timepoints with a traditional method. Furthermore, the results show that the cellular redox proliferation assay can characterize differences in proliferation capacity of cell compositions that are produced with different manufacturing processes (e.g., with different activation reagents).95MF-36448161473504-2029640Example 3: Assessment of Dynamic Range of the Cellular Redox Proliferation Assay Using .Turkat T cells

[0363] Jurkat is a T lymphocyte cell line that can proliferate in the absence of antigen and / or cytokine stimulation. Furthermore, Jurkat cells are well characterized, robust and homogenous, making them the ideal candidate to be used for evaluation of the dynamic range and accuracy of the cellular redox proliferation assay described herein. In two independent experiments carried out on different days, either passaged Jurkat cells (15 days in culture) or freshly thawed Jurkat cells were used. Cells were plated in a 96-well plate at increasing seeding densities up to about 800,000 cells / mL. Then, equal volumes of furimazine pro-substrate (e.g., MT cell viability substrate from RealTime-Glo™; Promega), enzyme (NanoLuc® Enzyme; Promega) and cell culture medium were mixed and 100 pL of the resulting mixture was added to each well. The plate was covered with a breathable plate sealer and incubated for 30 minutes in an incubator at 37°C ± 2°C, 5% ± 2% CO2, 95 ± 5% humidity. The plate was then equilibrated in the dark for an additional 2 hours at ambient room temperature (ART) and read on a luminometer to determine the signal via Relative Luminescence Units (RLU). In this experiment, cells were not stimulated with alD because the aim was to directly assess whether the proliferation assay readout would correlate to the known cell seeding densities without subjecting cells to proliferation.

[0364] Results are shown in FIG. 7. While the cellular redox proliferation assay described herein can determine proliferation capacity at early time points with readouts as early as 48 hours, a broad method range can allow consistent readouts still at later time points if needed. The results demonstrate that the method has a wide dynamic range between 12,500 to 800,000 viable cells per well (about 8.3 x 104to 5.3 x 106cells / mL). The results also demonstrated low readout variability as evidenced by the similar results between the two independent experiments, with a %CV of less than 5%.Example 4: Assessment of Specificity of the Cellular Redox Proliferation Assay

[0365] The cellular redox proliferation assay described in Example 1 was used to assess CAR- dependent specificity of the cellular redox proliferation assay. In this experiment, 3 different engineered CAR-T cell compositions, each expressing a CAR directed to a different antigen, were monitored for cellular redox proliferation by contacting the cells with an alD (8 pg / mL) specific to the antigen binding domain of the CAR (shown as + in FIG. 8) or by contacting the cells with a nonspecific alD (shown as - in FIG. 8). For further comparison, as a negative control, the CAR-T cell compositions were incubated without any stimulation (unstimulated).

[0366] After 48 hours of incubation with specific or non-specific alD, the luminescent signal in relative luminescence units was determined using a luminometer, as described in Example 1. As96MF-36448161473504-2029640 shown in FIG. 8, CAR-T cells that were stimulated with alD specific to the CAR all resulted in substantial proliferation. In contrast, CAR-T cells that were contacted with non-specific alD yielded a luminescent signal similar to that of the unstimulated control (bottom panel). This result demonstrates that the cellular redox proliferation assay is antigen specific.Example 5 : Assessment of Stability of Different Engineered Cell Compositions Using the Cellular Redox Proliferation Assay

[0367] Stability testing may reveal whether or how the quality of a drug product (for instance, a cell therapy composition) changes under various environmental conditions (Blessy et al., Journal of Pharmaceutical Analysis, 2014). Stability testing through forced degradation is important to ensure safety, efficacy and quality of a drug product. Results from stability testing can be used to aid in the development of formulations and packaging that prevent degradation. In order to assess whether the cellular redox proliferation assay described herein can detect changes in stability of cell compositions, a forced degradation study was performed on five CAR-T cell compositions in which the cell compositions were subjected to different conditions expected to differentially and negatively impact their ability to proliferate.

[0368] The CAR-T cell compositions were generated by the same process that included immunoaffinity-based (e.g., immunomagnetic selection) enrichment of CD4+ and CD8+ cells from leukapheresis samples, activation with an anti-CD3 / anti-CD28 antibody reagent, transduction with a lenti viral vector encoding a chimeric antigen receptor (CAR), incubation and cry opreservation of the engineered cell composition. Then, each cryopreserved engineered cell composition was either (1) not subjected to a degrading condition (control treatment, which was keeping them cryopreserved with liquid nitrogen), or (2) subjected to one of the following degradation conditions: (a) storage on dry ice for 1.5 hours, partly exposed to ART, and (b) storage at ambient room temperature (ART) for 1.5 hours; and (c) left in 37°C water bath for 10 minutes; and (d) one cycle of storage at 4°C for 30 minutes followed by storage in liquid nitrogen; and (e) two cycles of storage at 4°C for 30 minutes followed by storage in liquid nitrogen (with return to liquid nitrogen storage between and after cycles); and (f) one cycle of storage at -20°C for 3 hours / cycle followed by liquid nitrogen storage; and (g) two cycles of storage at -20°C for 3 hours / cycle (with return to liquid nitrogen storage between and after cycles). Samples from all of these conditions were then thawed in 37°C water bath if frozen prior to resuspension in culture medium before being cultured at 37°C for 48 hours (the “long incubation” as described in Example 1 and FIG. 1) with or without anti-idiotypic antibody (alD) specific to an extracellular antigen binding domain of the relevant CAR.

[0369] Subsequently, the CAR-T cell composition was incubated with the substrate and enzyme mixture at 37C (the “short incubation” as described in Example 1 and FIG. 1) followed by97MF-36448161473504-2029640 incubation at ART (the “ART equilibration” as described in Example 1 and FIG. 1). Relative proliferation was determined as described in Example 1. As a comparison, cell viability of cells of the CAR-T cell compositions was measured by staining with acridine orange and propidium iodine (AO / PI) immediately after resuspension in culture media.

[0370] As shown in FIG. 9A, CAR-T cell compositions exposed to different temperatures (as shown) all resulted in reduced proliferation as measured by the cellular redox proliferation assay, with the exception of dry ice storage. Specifically, under milder degradation conditions in which cell compositions were exposed to ambient room temperature (ART) for 1.5 hours or 37°C water bath, there were substantial reductions in relative proliferation as assessed with the cellular redox proliferation assay (FIG. 9A, bottom panel). In contrast, viability measurements by AO / PI staining are less sensitive in detecting impacts of milder treatment-induced declines (FIG. 9A, top panel). CAR-T cell compositions cycled twice through -20°C each for 3 hours had the lowest post-thaw viability, which a...

Claims

73504-2029640CLAIMSWHAT IS CLAIMED IS:

1. A method for assessing cell proliferation of a test engineered cell composition, wherein the test engineered cell composition comprises cells comprising a recombinant receptor, the method comprising:(a) culturing cells of the test engineered cell composition with a recombinant receptorstimulating agent for a period of time to generate a stimulated cell composition;(b) after the period of time, adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition;(c) incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal; and(d) detecting the luminescent signal from the stimulated cell composition; and, optionally,(e) determining cell proliferation of the test engineered cell composition using the results of the detecting.

2. The method of claim 1, wherein steps (a) to (d) are performed for two or more replicates, optionally wherein an average of the luminescent signal detected from the two or more replicates is used as the luminescent signal in the determining.

3. The method of claim 2, wherein steps (a) to (d) are performed for at least three replicates.

4. The method of claim 1 , wherein the method comprises (e) determining cell proliferation of the test engineered cell composition using the results of the detecting, and determining cell proliferation comprises calculating the difference between (A) the luminescent signal detected from the stimulated cell composition and (B) a luminescent signal detected from unstimulated cultured cells of the test engineered cell composition, wherein the unstimulated cultured cells have been cultured for a second period of time in the absence of the recombinant receptor-stimulating agent.

5. The method of claim 4, wherein the second period of time has the same duration as the period of time.104MF-36448161473504-20296406. The method of any one of claims 4 to 5, wherein the method further comprises adding the luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the unstimulated cultured cells.

7. The method of any one of claims 4 to 6, wherein the method further comprises incubating the unstimulated cultured cells with the luciferase enzyme and the substrate or prosubstrate of the luciferase enzyme to generate a luminescent signal.

8. The method of any one of claims 4 to 7, wherein the method further comprises detecting the luminescent signal from the unstimulated cultured cells.

9. The method of any one of claims 4 to 8, wherein the steps of culturing the unstimulated cells of the test engineered cell composition for the second period of time and detecting the luminescent signal from the unstimulated cultured cells are performed for two or more replicates, optionally wherein an average of the luminescent signal detected from said two or more replicates is used in the calculating as the luminescent signal detected from the unstimulated cultured cells.

10. The method of any one of claims 1 to 9, comprising calculating a relative cell proliferation of the test engineered cell composition by comparing the cell proliferation as determined in step (e) to the cell proliferation of a reference standard.

11. The method of claim 10, wherein the comparing comprises calculating a ratio of the cell proliferation as determined in step (e) to the cell proliferation of the reference standard and optionally expressing the ratio as a percentage by multiplying it by 100.

12. A method for assessing relative cell proliferation of a test engineered cell composition, wherein the test engineered cell composition comprises cells comprising a recombinant receptor, the method comprising:(a) culturing cells of the test engineered cell composition with a recombinant receptorstimulating agent for a period of time to generate a stimulated cell composition;(b) after the period of time, adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition and incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal;(c) detecting the luminescent signal from the stimulated cell composition;(d) determining cell proliferation of the test engineered cell composition by calculating the difference between (A) the luminescent signal detected from the stimulated cell composition and (B)105MF-36448161473504-2029640 the luminescent signal detected from unstimulated cultured cells of the cell composition, wherein the unstimulated cultured cells have been cultured for a second period of time in the absence of the recombinant receptor-stimulating agent, wherein the second period of time is equivalent in duration to the period of time; and(e) calculating a relative cell proliferation of the test engineered cell composition by calculating a ratio of the cell proliferation as determined in step (d) to the cell prol iteration of a reference standard.

13. The method of claim 12, wherein steps (a) to (c) are performed for two or more replicates.

14. The method of claim 12 or claim 13, wherein calculating the difference in step (d) comprises calculating the difference between an average luminescent signal detected from the stimulated cell composition and the luminescent signal detected from unstimulated cultured cells of the cell composition.

15. The method of any one of claims 4, 5, and 12 to 14, further comprising: culturing the unstimulated cells of the test engineered cell composition for the second period of time in the absence of the recombinant receptor stimulating agent to generate the unstimulated cultured cells, and detecting the luminescent signal from the unstimulated cultured cells.

16. The method of claim 15, wherein the steps of culturing the unstimulated cells of the test engineered cell composition for the second period of time and detecting the luminescent signal from the unstimulated cultured cells are performed for two or more replicates.

17. The method of claim 16, wherein calculating the difference in step (d) comprises calculating the difference between an average luminescent signal detected from the sti mulated cell composition and an average luminescent signal detected from unstimulated cultured cells of the cell composition.

18. The method of any one of claims 1 to 17, wherein the period of time is at least about 46 hours.

19. The method of any one of claims 1 to 18, wherein the period of time is between about 46 hours and about 96 hours.106MF-36448161473504-202964020. The method of any one of claims 1 to 19, wherein the period of time is between about 46 hours and about 72 hours.

21. The method of any one of claims 1 to 20, wherein the period of time is between about 46 hours and about 50 hours.

22. The method of any one of claims 1 to 21, wherein the culturing in step (a) is carried out at a temperature of 37°C ± 2°C, or at a temperature of about 37°C ± 2°C.

23. The method of any one of claims 1 to 22, wherein the culturing in step (a) is carried out at a carbon dioxide (CO2) level between 3% and 7%, or between about 3% and about 7%.

24. The method of any one of claims 1 to 23, wherein the culturing in step (a) is carried out at a humidity level between 90% and 100%, or between about 90% and about 100%.

25. The method of any one of claims 1 to 24, wherein the incubating of the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate is carried out at a temperature of 37°C ± 2°C or at a temperature of about 37°C ± 2°C.

26. The method of any one of claims 1 to 25, wherein the incubating of the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate is carried out at a carbon dioxide (CO2) level between 3% and 7%, or between about 3% and about 7%.

27. The method of any one of claims 1 to 26, wherein the incubating of the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate is carried out at a humidity level between 90% and 100%, or between about 90% and about 100%.

28. The method of any one of claims 4 to 27, wherein culturing the unstimulated cells is carried out at a temperature of 37°C ± 2°C, or at a temperature of about 37°C ± 2°C.

29. The method of any one of claims 4 to 28, wherein culturing the unstimulated cells is carried out at a carbon dioxide (CO2) level between 3% and 7%, or between about 3% and about 7%.

30. The method of any one of claims 4 to 29, wherein culturing the unstimulated cells is carried out at a humidity level between 90% and 100%, or between about 90% and about 100%.107MF-36448161473504-202964031. The method of any one of claims 9 to 30, wherein the incubating of the unstimulated cultured cells with the luciferase enzyme and the substrate or pro-substrate is carried out at a temperature of 37°C ± 2°C, or at a temperature of about 37°C ± 2°C.

32. The method of any one of claims 9 to 31 , wherein the incubating of the unstimulated cultured cells with the luciferase enzyme and the substrate or pro-substrate is carried out at a carbon dioxide (CO2) level between 3% and 7%, or between about 3% and about 7%.

33. The method of any one of claims 9 to 32, wherein the incubating of the unstimulated cultured cells with the luciferase enzyme and the substrate or pro-substrate is carried out at a humidity level between 90% and 100% or between about 90% and about 100%.

34. The method of any one of claims 1 to 33, wherein the incubating is for about 10 minutes to about 50 minutes.

35. The method of any one of claims 1 to 34, wherein after the incubating and before the detecting in step (d), the method comprises equilibrating the stimulated cell composition and the unstimulated cultured cells to room temperature.

36. The method of claim 35, wherein the equilibrating is for about 2 hours to about 4 hours.

37. The method of claim 35 or 36, wherein the stimulated cell composition and the unstimulated cultured cells are protected from light during the equilibrating.

38. The method of any one of claims 1 to 37, further comprising contacting the recombinant receptor-stimulating agent with cells of the test engineered cell composition prior to the culturing in step (a), optionally wherein the contacting comprises adding a volume of the test engineered cell composition to a vessel containing the recombinant receptor-stimulating agent.

39. The method of any one of claims 1 to 38, wherein one or more steps of the method, including at least the step of detecting the luminescent signal from the stimulated cell composition, is performed iteratively to allow assessment of cell proliferation at different timepoints.108MF-36448161473504-202964040. The method of claim 39, wherein a step of incubating the stimulated cell composition is performed before each iteration of detecting the luminescent signal from the stimulated cell composition.

41. The method of claim 39 or 40, wherein the step of adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated composition is not repeated after the first ti me it is performed.

42. The method of claim 39 or 40, wherein the method comprises iteratively performing each of the steps of adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated cell composition, incubating the stimulated cell composition with the luciferase enzyme and the substrate or pro-substrate to generate a luminescent signal and detecting the luminescent signal from the stimulated cell composition.

43. The method of any one of claims 40 to 42, wherein after each iteration of the incubating the stimulated cell composition with the luciferase enzyme and the substrate or prosubstrate and before each iteration of the detecting of the luminescent signal from the stimulated cell composition, the method comprises equilibrating the stimulated cell composition to room temperature.

44. The method of any one of claims 39 to 43, wherein any iteration of the step of detecting the luminescent signal from the stimulated cell composition is carried out within 96 hours of first initiating the culturing of the cells of the test engineered cell composition with the recombinant receptor-stimulating agent according to step (a).

45. The method of any one of claims 39 to 43, wherein any iteration of the step of detecting the luminescent signal from the stimulated cell composition is carried out within 72 hours of first initiating the culturing of the cells of the test engineered cell composition with the recombinant receptor-stimulating agent according to step (a).

46. A method for assessing cell proliferation of a test cell composition, the method comprising:(a) culturing cells of a test cell composition for a period of time under conditions that support cell proliferation to make a cultured cell composition, optionally wherein the period of time is about 46 hours;(b) after the culturing in (a), adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the cultured cell composition to generate a luminescent signal;109MF-36448161473504-2029640(c) detecting the luminescent signal from the cultured cell composition; and(d) calculati ng a relative cell proliferation of the test cell composition by comparing the luminescent signal that is detected in step (c) to the luminescent signal that is detected from a reference standard.

47. The method of claim 46, wherein the culturing in (a) is carried out in the presence of a stimulating agent to stimulate cells of the test cell composition and generate a stimulated test cell composition.

48. The method of claim 46 or claim 47, wherein the test cell composition is a test engineered cell composition that comprises cells comprising a recombinant receptor.

49. The method of claim 48, wherein the stimulating agent is a recombinant receptorstimulating agent.

50. The method of any one of claims 48-49, wherein calculating the relative cell proliferation comprises:(i) determining cell proliferation of the test cell composition by calculating the difference between (A) the luminescent signal detected from the stimulated test cell composition and (B) the luminescent signal detected from unstimulated cells of the test cell composition that have been cultured in the absence of the stimulating agent; and(ii) comparing the cell proliferation as calculated in step (i) with the cell proliferation of a reference standard.

51. The method of claim 50, wherein the comparing comprises calculating a ratio of the cell proliferation as determined in step (i) to the cell proliferation of the reference standard and optionally expressing the ratio as a percentage by multiplying it by 100.

52. The method of any one of claims 46 to 51 , wherein the period of time is at least about 46 hours.

53. The method of any one of claims 46 to 52, wherein the period of time is between about 46 hours and about 96 hours.

54. The method of any one of claims 46 to 53, wherein the period of time is between about 46 hours and about 72 hours.110MF-36448161473504-202964055. The method of any one of claims 46 to 52, wherein the period of time is between about 46 hours and about 50 hours.

56. The method of any one of claims 1 to 45 and 48 to 55, wherein the test engineered cell composition has been produced ex vivo from primary cells from a subject by a cell engineering manufacturing process to comprise the recombinant receptor.

57. The method of any one of claims 1 to 45 and 48 to 56, wherein the test engineered cell composition is a cell composition produced for use as a cell therapy for treating a subject with a disease or condition.

58. The method of claim 57, wherein the disease or condition is a cancer or is an autoimmune or inflammatory disease or condition.

59. The method of claim 57 or claim 58, wherein the cells of the cell therapy are primary cells that are autologous to the subject to be treated.

60. The method of claim 57 or claim 58, wherein the cells of the cell therapy are primary cells that are allogeneic to the subject to be treated.

61. The method of any one of claims 10 to 60, wherein the reference standard is a reference engineered cell composition comprising a reference recombinant receptor.

62. The method of claim 61, wherein the reference recombinant receptor expressed by the reference engineered cell composition differs from the recombinant receptor expressed by the test engineered cell composition.

63. The method of claim 61, wherein the reference recombinant receptor expressed by the reference engineered cell composition is the same as the recombinant receptor expressed by the test engineered cell composition.

64. The method of any one of claims 10 to 63, wherein the reference standard is a reference engineered cell composition having a validated cell proliferation.111MF-36448161473504-202964065. The method of claim 64, wherein the validated proliferation index of the reference standard is determined by:(a) culturing cells of the reference engineered cell composition with a recombinant receptorstimulating agent for a period of time to generate a stimulated reference engineered cell composition;(b) after the period of time, adding a luciferase enzyme and a substrate or pro-substrate of the luciferase enzyme to the stimulated reference engineered cell composition and incubating the stimulated cell composition with the luciferase enzyme and the substrate or the pro-substrate to generate a luminescent signal;(c) equilibrating the stimulated reference engineered cell composition to room temperature;(d) detecti ng the luminescent signal from the stimulated reference engineered cell composition; and(e) determining cell proliferation of the reference standard by calculati ng the difference between (A) the luminescent signal detected from the stimulated reference engineered cell composition and (B) the luminescent signal detected from unstimulated cells of the reference engineered cell composition, wherein the unstimulated cells of the reference engineered cell composition have been cultured in the absence of the recombinant receptor-stimulating agent.

66. The method of any one of claims 61 to 65, wherein the reference engineered cell composition is produced ex vivo from primary cells from a subject by a cell engineering manufacturing process to comprise the recombinant receptor, wherein the subject is different from the subject used to produce the test engineered cell composition.

67. The method of claim 66, wherein the subject is a healthy subject not known or suspected of having a disease or condition.

68. The method of any one of claims 56 to 67, wherein the manufacturing process used to manufacture the test engineered cell composition differs from the manufacturing process used to manufacture the reference cell composition.

69. The method of any one of claims 56 to 67, wherein the manufacturing process used to manufacture the test engineered cell composition is the same as the manufacturing process used to manufacture the reference cell composition.

70. The method of any one of claims 1 to 45 and 48 to 69, wherein the recombinant receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).112MF-36448161473504-202964071. The method of claim 70, wherein the recombinant receptor is a CAR and the CAR comprises an extracellular antigen binding domain specific for a target antigen, a transmembrane domain, and an intracellular signaling domain comprising a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta, and a signaling domain from a T cell costimulatory molecule, optionally wherein the T cell costimulatory molecule is 4-1BB and / or CD28.

72. The method of claim 71 , wherein the extracellular antigen binding domain comprises a antibody variable heavy chain domain and variable light chain domain specific for the target antigen.

73. The method of claim 71 or claim 72, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv) specific for the target antigen.

74. The method of claim 71 , wherein the extracellular antigen binding domain comprises a VHH domain.

75. The method of any one of claims 70 to 74, wherein the CAR is monospecific to the target antigen.

76. The method of any one of claims 70 to 74, wherein the CAR is bispecific and the target antigen is a first target antigen and the extracellular binding domain is further specific for a second target antigen.

77. The method of claim 76, wherein the extracellular antigen binding domain comprises a first antigen binding domain comprising a first variable heavy (VH) chain domain and a first variable light (VL) chain domain specific for the first target antigen and a second antigen binding domain comprising a second VH chain domain and a second VL chain domain specific for the second target antigen.

78. The method of claim 77, wherein the extracellular antigen binding domain has a tandem structure comprising a sequence comprising in order the first antigen binding domain and the second antigen binding domain.

79. The method of claim 77, wherein the extracellular antigen binding domain has a loop structure comprising a sequence comprising in order, N- to C-terminal: the first VH chain or first VL chain of the first antigen binding domain, the second VH chain or second VL chain of the second113MF-36448161473504-2029640 antigen binding domain, the other of the second VH chain and second VL chain of the second antigen binding domain, and the other of the first VH chain and second VL chain of the second antigen binding domain.

80. The method of claim 76, wherein the extracellular antigen binding domain comprises a first VHH specific to the first target antigen and a second VHH specific to the second target antigen.

81. The method of any one of claims 71 to 80, further comprising a hinge spacer sequence between the extracellular antigen binding domain and the transmembrane domain.

82. The method of any one of claims 1 to 45 and 49 to 81, wherein the recombinant receptor-stimulating agent is or comprises a binding moiety recognized by or specific to the recombinant receptor, optionally to the extracellular antigen binding domain of the CAR.

83. The method of claim 82, wherein the binding moiety is a target antigen or an extracellular domain binding portion thereof of the recombinant receptor, optionally wherein the extracellular domain binding portion of the target antigen comprises an epitope recognized by the recombinant receptor.

84. The method of claim 82, wherein the binding moiety is an antibody specific to an extracellular binding domain of the recombinant receptor.

85. The method of claim 82 or claim 84, wherein the binding moiety is an anti -idiotypic antibody specific to an extracellular antigen binding domain of the recombinant receptor.

86. The method of any one of claims 82 to 85, wherein the binding moiety is immobilized or attached to a solid support during the culturing.

87. The method of any one of claims 1 to 86, wherein the culturing is initiated when cells of the test engineered cell composition or test cell composition are added to a solid support.

88. The method of claim 86 or claim 87, wherein the solid support is a surface of a culture vessel.

89. The method of claim 86 or claim 87, wherein the solid support is a bead.114MF-36448161473504-202964090. The method of claim 89, wherein the culturing is initiated when cells of the test engineered cell composition or test cell composition and the binding moiety-immobilized beads are contacted in a culture vessel.

91. The method of claim 88 or claim 90, wherein the culture vessel is a multi-well plate, optionally a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96 well -plate or a 384- well plate.

92. The method of claim 91 , wherein the culture vessel is a 96-well plate.

93. The method of any one of claims 88 and 90 to 92, wherein the cells are added to the culture vessel at a cell density of between about 6.7 x 104viable cells / mL and about 3 x 106viable cells / mL.

94. The method of any one of claims 88 and 90 to 93, wherein the number of cells present in the culture vessel at the time the detecting is performed is not more than 6 x 106viable cells / mL.

95. The method of any one of claims 88 and 90 to 94, wherein the number of cells present in the culture vessel at the time the detecting is performed is not less than 3.3 x 104viable cells / mL.

96. The method of any one of claims 1 to 95, wherein the cells of the test engineered cell composition or the test cell composition comprise a T cell.

97. The method of claim 96, wherein the T cells are primary cells.

98. The method of claim 96 or claim 97, wherein the T cells are autologous cells.

99. The method of claim 96 or claim 97, wherein the T cells are allogeneic cells.

100. The method of any one of claims 96 to 99, wherein the T cells are CD3+.

101. The method of any one of claims 96 to 100, wherein the T cells are CD4+ and / or CD8+ T cells.

102. The method of any one of claims 96 to 101, wherein the T cells are CD4+ and CD8+ T cells.115MF-36448161473504-2029640103. The method of any one of claims 1 to 102, wherein the substrate or pro-substrate is cell permeable and is able to enter cells104. The method of any one of claims 1 to 103, wherein the substrate or pro-substrate added to the cells is a luciferin-D substrate or analog or derivative thereof, or is a pro-substrate of any of the foregoing.

105. The method of any one of claims 1 to 103, wherein the substrate or pro-substrate added to the cells is a coelenterazine substrate or analog or derivative thereof, or is a pro-substrate of any of the foregoing.

106. The method of any one of claims 1 to 103, wherein the substrate or pro-substrate added to the cells is a furimazine substrate or analog or derivative thereof, or is a pro-substrate of any of the foregoing.

107. The method of any one of claims 1 to 106, wherein the substrate or pro-substrate added to the cells is a pro-substrate, wherein the pro-substrate is able to be modified by the actions of a cellular enzyme present in the cell to release the substrate inside the cell.

108. The method of claim 107, wherein the cellular enzyme is an esterase.

109. The method of any one of claims 1 to 108, wherein, when present in the cells, the substrate is able to be reduced by molecular oxygen and released from the cells as a substrate for the luciferase.

110. The method of any one of claims 1 to 109, wherein the luciferase enzyme is cell- impermeable and does not enter the cell, wherein the luciferase binds the reduced substrate after it exits the cell.

111. The method of any one of claims 1 to 110, wherein the luciferase is from Oplophorus (OLuc), Gaussia (GLuc), Renilla (RLuc), Pyrophorus, or Photinus or is a variant thereof that is able to produce luminescence when bound to substrate.

112. The method of any one of claims 1 to 111, wherein the luciferase is from Oplophorus (OLuc) or is a variant thereof that is able to produce luminescence when bound to substrate.116MF-36448161473504-2029640113. The method of any one of claims 1 to 112, wherein the luciferase is a OLuc variant that is the variant C1A4E comprising at least 8 substitutions selected from the group consisting of A4E, QI 1R, Q18L, L27V, A33K or A33N, K43R, V44I, A54F or A54I, F68Y or F68D, L72Q, M75K, I90V, P115E, Q124K, and Y138IP, optionally 8, 9, 10, 11, 12, 13, 14 or 15 substitutions.

114. The method of any one of claims 1 to 113, wherein the luciferase is NanoLuc™.

115. The method of any one of claims 1 to 114, wherein the luminescent signal is generated after the luciferase binds to the reduced substrate.

116. The method of any one of claims 1 to 115, wherein the luminescent signal is detected by a luminometer.

117. The method of any one of claims 1 to 116, wherein the method is used as a release assay.

118. The method of any one of claims 1 to 117, wherein the method is used to assess stability.

119. The method of any one of claims 1 to 118, wherein the method has a recovery rate of between about 80% and about 120%.

120. The method of any one of claims 1 to 119, wherein the method has a coefficient of determination of at least 0.97.

121. The method of any one of claims 1 to 120, wherein the method has an intermediate precision of less than 20%.117MF-364481614

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