Analyzing and producing immune cells in bioreactors

Abstract

Data-driven bioreactor cultivation of immune cells may be provided by dividing a first sample of immune cells into first and second populations; cultivating the first and second populations in respective first and second bioreactor to produce respective first and second cultivated populations; measuring a respective values for a potency parameter, a metabolic parameter, or both, at a harvest time for each cultivated population of immune cells; and after the harvest time: collecting a second sample of the immune cells; selecting one of the first bioreactor or the second bioreactor based on which of the respective cultivated populations was measured with a higher value for the potency parameter, a higher value for the metabolic parameter, or both; and cultivating the second sample of the immune cells in the one of the first bioreactor and the second bioreactor selected to produce a cultivated second sample.

Description

ANALYZING AND PRODUCING IMMUNE CELLS IN BIOREACTORSPRIORITY

[0001] The present disclosure claims the benefit of and priority to U.S. Provisional Patent Application No.: 63 / 602,109 titled “ANALYZING AND PRODUCING IMMUNE CELLS IN BIOREACTORS” and filed on November 22, 2023, which is incorporated herein it its entirety.BACKGROUND

[0002] Bioreactors are tools used to cultivate cells harvested from a biological subject, providing an artificial environment in which the cells may grow or be manipulated outside of the body of a biological subject. The cultivation of cells may be performed for research into cell metabolism or for use in the treatment or prophylaxis of various medical conditions; resulting in the cultured cells being harvested from the bioreactor and supplied to a biological subject (e.g., injected into the blood stream or a target location). Culturing the cells may be performed to increase a quantity of the cells or allow for the cells to be modified (e.g., via gene editing) before being supplied to the biological subject. Thus, there is a need to develop methods and systems to analyze and produce immune cells in bioreactors.SUMMARY

[0003] The present disclosure provides for methods and systems for analyzing and producing immune cells in bioreactors. In some embodiments, the disclosure provides for data-driven bioreactor cultivation of immune cells via various methods and apparatuses. Various collected and calculated data to drive how immune cells are cultivated can include various potency and metabolic fitness parameters, such as, immune cell killing activity, basal bioenergetic phenotype (basal mitochondrial ATP Production Ratc-i- glycolytic ATP Production Rate), Mitochondrial Maximal Respiratory Capacity, Spare Respiratory Capacity, and combinations thereof. These data may be collected from various assays taken at different times, and from different bioreactors, to control and inform how current and future cultivation processes are performed, with resulting higher metabolic fitness, killing capacity, speed of production, and combinations thereof for cultivating immune cells.

[0004] An embodiment provided includes, a method of producing immune cells, comprising: dividing a first sample of immune cells into a first population and a second population; cultivating the first population of the immune cells in a first bioreactor to produce a first cultivated population of immune cells; cultivating the second population of the immune cells in a second bioreactor of a different design than the first bioreactor to produce a second cultivated population of immune cells; measuring a first value for a potency parameter, a first value for a metabolic parameter, or both, at a harvest time for the first cultivated population of immune cells, and a second value for the potency parameter, a second value for the metabolic parameter, or both, at the harvest time for the second cultivated population of immune cells; and after the harvest time: collecting a second sample of the immune cells; selecting one of the first bioreactor or the second bioreactor based on which of the respective first cultivated population of immune cells and the second cultivated population of immune cells was measured with a higher value for the potency parameter, a higher value for the metabolic parameter, or both; and cultivating the second sample of the immune cells in the one of the first bioreactor and the second bioreactor selected to produce a cultivated second sample.

[0005] An embodiment provided includes, wherein the first sample and the second sample were collected from the same biological subject.

[0006] An embodiment provided includes, wherein the first sample and the second sample were collected from different biological subjects.

[0007] An embodiment provided includes, administering cultivated immune cells from the cultivated second sample to a biological subject.

[0008] An embodiment provided includes, wherein the biological subject is the same subject from which the second sample was collected.

[0009] An embodiment provided includes, wherein the biological subject is a different subject from which the second sample was collected.

[0010] An embodiment provided includes, wherein the potency parameter comprises a plurality of potency sub-parameters, wherein each potency sub-parameter of the plurality of potency sub-parameters satisfies a corresponding potency threshold.

[0011] An embodiment provided includes, wherein measuring the first value, the second value, or both, for the potency parameter is performed via a potency assay, e.g., an XCELLIGENCE® potency assay (offered by Agilent Technologies Tnc. of Santa Clara California).

[0012] An embodiment provided includes, wherein measuring the first value, the second value, or both, for the potency parameter is performed via an instrument analysis comprising at least one of an impedance measurement or image analysis of killing efficacy.

[0013] An embodiment provided includes, wherein the metabolic parameter comprises a plurality of metabolic sub-parameters, wherein each metabolic sub-parameter of the plurality of metabolic sub-parameters satisfies a corresponding metabolic threshold.

[0014] An embodiment provided includes, wherein measuring the first value, the second value, or both, for the metabolic par ameter is performed via a metabolic assay, e.g., a SEAHORSE XF™ T cell Metabolic Fitness assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0015] An embodiment provided includes, wherein measuring the first value, the second value, or both, for the metabolic parameter is performed via an instrument analysis comprising a measurement of mitochondrial respiration, glycolysis, ATP production, or a combination thereof.

[0016] An embodiment provided includes, further comprising measuring a first value for an expression parameter, at the harvest time for the first cultivated population of immune cells, and a second value for the expression parameter, at the harvest time for the second cultivated population of immune cells.

[0017] An embodiment provided includes, wherein the expression parameter comprises a plurality of expression sub-parameters, wherein each expression sub-parameter of the plurality of expression sub-parameters satisfies a corresponding expression threshold.

[0018] An embodiment provided includes, wherein measuring the first value, the second value, or both, for the expression parameter is performed via flow cytometry.

[0019] An embodiment provided includes, wherein the first bioreactor and the second bioreactor are controlled during cultivation according to one or more (e.g., 2, 3, 4, 5, 6, 7, or all) operational settings for: nutrient and gas exchange; oxygen control; pH control; a feeding regime of media exchange versus media addition; mixing or shear force; device O2 permeability; vessel size; removal of activation beads; or enrichment / selection of a particular subpopulation.

[0020] An embodiment provided includes, wherein selecting the one of the first bioreactor or the second bioreactor is further based on which of the first bioreactor and the second bioreactor reaches a desired threshold for the potency parameter, the metabolic parameter, or both, and optionally the expression parameter, faster.

[0021] An embodiment provided includes, wherein the potency parameter, the metabolic parameter, or both, and optionally the expression parameter, is associated with one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or all) of: clonal abundance; clonal expansion; transduction efficiency; proliferation capacity; cytotoxicity capacity; persistence; sternness; or immune cell exhaustion.

[0022] An embodiment provided includes, wherein the higher value for the potency parameter, the higher value for the metabolic parameter, or both, and optionally a higher value for the expression parameter, indicates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or all) of: an increased clonal abundance; an increased clonal expansion; an increased transduction efficiency; an increased proliferation capacity; an increased cytotoxicity capacity; an increased persistence; an increased metabolic fitness; an increased sternness; or an reduced immune cell exhaustion.

[0023] An embodiment provided includes, wherein the harvest time occurs at a predefined interval after initiation of cultivation, e.g., is at least 12 hours (e.g., at least 24, 36, 48, 60, or 72 hours), e.g., 12-72 hours, 24-60 hours, 36-48 hours, 12-60 hours, 12-48 hours, 12-36 hours, 12-24 hours, 60-72 hours, 48-72 hours, 36-72 hours, 24-72 hours, 24-48 hours, 36-60 hours, or 48-72 hours, or at least 1 day (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), e.g., 1-14 days, 2-13 days, 3-12 days, 4-11 days, 5-10 days, 6-9 days, 7-8 days, 1-12 days, 1-10 days, 1-8 days, 1- 6 days, 1-4 days, 1-2 days, 12-14 days, 10-14 days, 8-14 days, 6-14 days, 4-14 days, 2-14 days, 1- 3 days, 2-4 days, 3-5 days, 4-6 days, 5-7 days, 6-8 days, 7-9 days, 8-10 days, 9-11 days, 10-12 days, 11-13 days, or 2-3 days from initiating cultivation.

[0024] An embodiment provided includes, a method of producing immune cells, comprising: acquiring a value for a potency parameter, a value for a metabolic parameter, or both, from a population of immune cells being cultivated in a bioreactor; monitoring the value for the potency parameter, the value for the metabolic parameter, or both, during a predefined period of time; and in response to identifying a change in the value for the potency parameter, a change in the value for the metabolic parameter, or both, outside of a harvesting threshold before an end of the period of time, harvesting the population of immune cells from the bioreactor before the predefined period of time expires.

[0025] An embodiment provided includes, wherein monitoring the value for the potency parameter, the value for the metabolic parameter, or both, comprises acquiring one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for the potency parameter, one or more (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for the metabolic parameter, or both, from the population of immune cells being cultivated in the bioreactor.

[0026] An embodiment provided includes, wherein the value for the potency parameter, the value for the metabolic parameter, or both, is monitored at least once every 1, 2, 3, 6, 9, 12, 18, or 24 hours, or at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0027] An embodiment provided includes, wherein the potency parameter comprises a plurality of potency sub-parameters, wherein each potency sub-parameter of the plurality of potency sub-parameters satisfies a corresponding potency threshold.

[0028] An embodiment provided includes, wherein acquiring the value for the potency parameter is performed via a potency assay, e.g., an XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0029] An embodiment provided includes, wherein acquiring the value for the potency parameter is performed via an instrument analysis comprising at least one of an impedance measurement or image analysis of killing efficacy.

[0030] An embodiment provided includes, wherein the metabolic parameter comprises a plurality of metabolic sub-parameters, wherein each metabolic sub-parameter of the plurality of metabolic sub-parameters satisfies a corresponding metabolic threshold.

[0031] An embodiment provided includes, wherein acquiring the value for the metabolic parameter is performed via a metabolic assay, e.g., SEAHORSE XF™ T cell Metabolic Fitness assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0032] An embodiment provided includes, wherein acquiring the value for the metabolic parameter is performed via an instrument analysis comprising a measurement of mitochondrial respiration, glycolysis, ATP production, or a combination thereof.

[0033] An embodiment provided includes, further comprising acquiring a value for an expression parameter from the population of immune cells being cultivated in the bioreactor, and monitoring the value for the expression value during the predefined period of time.

[0034] An embodiment provided includes, wherein monitoring the value for the expression parameter comprises acquiring one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for the expression parameter from the population of immune cells being cultivated in the bioreactor.

[0035] An embodiment provided includes, wherein the value for the expression parameter is monitored at least once every 1, 2, 3, 6, 9, 12, 18, or 24 hours, or at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0036] An embodiment provided includes, wherein the expression parameter comprises a plurality of expression sub-parameters, wherein each expression sub-parameter of the plurality of expression sub-parameters satisfies a corresponding expression threshold.

[0037] An embodiment provided includes, wherein acquiring the value for the expression parameter is performed via flow cytometry.

[0038] An embodiment provided includes, wherein the bioreactor is controlled during cultivation according to one or more (e.g., 2, 3, 4, 5, 6, 7, or all) operational settings for: nutrient and gas exchange; oxygen control; pH control; a feeding regime of media exchange versus media addition; mixing or shear force; device O2 permeability; vessel size; removal of activation beads; or cnrichmcnt / sclcction of a particular subpopulation.

[0039] An embodiment provided includes, wherein the potency parameter, the metabolic parameter, or both, and optionally the expression parameter, is associated with one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or all) of: clonal abundance; clonal expansion; transduction efficiency; proliferation capacity; cytotoxicity capacity; persistence; sternness; or immune cell exhaustion.

[0040] An embodiment provided includes, wherein the higher value for the potency parameter, the higher value for the metabolic parameter, or both, and optionally a higher value for the expression parameter, indicates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or all) of: an increased clonal abundance; an increased clonal expansion; an increased transduction efficiency; an increased proliferation capacity; an increased cytotoxicity capacity; an increased persistence; an increased sternness; or a reduced immune cell exhaustion.

[0041] An embodiment provided includes, wherein the predefined period of time is at least 12 hours (e.g., at least 24, 36, 48, 60, or 72 hours), e.g., 12-72 hours, 24-60 hours, 36-48 hours, 12- 60 hours, 12-48 hours, 12-36 hours, 12-24 hours, 60-72 hours, 48-72 hours, 36-72 hours, 24-72 hours, 24-48 hours, 36-60 hours, or 48-72 hours, or at least 1 day (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), e.g., 1-14 days, 2-13 days, 3-12 days, 4-11 days, 5-10 days, 6-9 days, 7-8 days, 1-12 days, 1-10 days, 1-8 days, 1-6 days, 1-4 days, 1-2 days, 12-14 days, 10-14 days, 8- 14 days, 6-14 days, 4-14 days, 2-14 days, 1-3 days, 2-4 days, 3-5 days, 4-6 days, 5-7 days, 6-8days, 7-9 days, 8-10 days, 9-11 days, 10-12 days, 11-13 days, or 2-3 days from initiating cultivation.

[0042] An embodiment provided includes, wherein the change in the value for the potency parameter, the value for the metabolic parameter, or both, and optionally the expression parameter, outside of the harvesting threshold is identified any time within 72 hours (e.g., within 60, 48, 36, 24, or 12 hours), e.g., 12-72 hours, 24-60 hours, 36-48 hours, 12-60 hours, 12-48 hours, 12-36 hours, 12-24 hours, 60-72 hours, 48-72 hours, 36-72 hours, 24-72 hours, 24-48 hours, 36-60 hours, or 48-72 hours, or within 14 days (e.g., within 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day), e.g., 1-14 days, 2-13 days, 3-12 days, 4-11 days, 5-10 days, 6-9 days, 7-8 days, 1-12 days, 1-10 days,1-8 days, 1-6 days, 1-4 days, 1-2 days, 12-14 days, 10-14 days, 8-14 days, 6-14 days, 4-14 days,2-14 days, 1-3 days, 2-4 days, 3-5 days, 4-6 days, 5-7 days, 6-8 days, 7-9 days, 8-10 days, 9-11 days, 10-12 days, 11-13 days, or 2-3 days from initiating cultivation.

[0043] An embodiment provided includes, wherein harvesting the population of immune cells from the bioreactor, before the predefined period of time expires, removes less than 100% of the population of immune cells from the bioreactor, the method further comprising: cultivating a remainder of the population of the immune cells not harvested from the bioreactor until the predefined period of time expires; and harvesting the remainder of the population of the immune cells from the bioreactor in response to the predefined period of time expiring.

[0044] An embodiment provided includes, wherein in response to not identifying the change in the value for the potency parameter, the change in the value for the metabolic parameter, or both, and optionally the change in the value for the expression parameter, outside of the harvesting threshold before the end of the period of time, harvesting the population of immune cells from the bioreactor at the end of the predefined period of time.

[0045] An embodiment provided includes, a method of evaluating an optimal therapeutic harvest time of an immune cell from a bioreactor, comprising: acquiring a value for each of a plurality of parameters from a population of immune cells in the bioreactor, wherein the plurality of parameters comprises two or more of: an expression parameter measured by flow cytometry; a potency parameter measured by impedance, imaging, or both; or a metabolic parameter comprising mitochondrial respiration, glycolysis, ATP production, bioenergetic capacity or a combination thereof, wherein the plurality of parameters are associated with one or more of properties comprising: clonal abundance; clonal expansion; transduction efficiency; proliferation capacity;cytotoxicity capacity; persistence; sternness; or exhaustion, wherein a change in the value for each of the plurality of parameters, or a combination thereof, is indicative of the optimal therapeutic harvest time of the immune cell.

[0046] An embodiment provided includes, wherein the change in the value for each of the plurality of parameters, or a combination thereof, indicates one or more of properties comprising: an increased clonal abundance; an increased clonal expansion; an increased transduction efficiency; an increased proliferation capacity; an increased cytotoxicity capacity; an increased persistence; an increased sternness; or a reduced exhaustion.

[0047] An embodiment provided includes, wherein the optimal therapeutic harvest time provides (a) the optimal expression of a molecule by the immune cells, (b) the optimal killing potency of the immune cells, (c) the optimal bioenergetic profile and spare respiratory capacity of the immune cells, or any combination of (a), (b), or (c).

[0048] An embodiment provided includes, further comprising acquiring one or more (c.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for each of the plurality of parameters from the population of immune cells.

[0049] An embodiment provided includes, wherein the one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for each of the plurality of parameters is acquired at least once every 1, 2, 3, 6, 9, 12, 18, or 24 hours, or at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, from the population of immune cells.

[0050] An embodiment provided includes, wherein the value for the potency parameter is acquired via XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0051] An embodiment provided includes, wherein the value for the metabolic parameter is acquired via SEAHORSE XF® T cell Metabolic Fitness assay.

[0052] An embodiment provided includes, a method of optimizing a control parameter of a bioreactor, comprising: acquiring a value for each of a plurality of parameters from a population of immune cells in the bioreactor, wherein the plurality of parameters comprises two or more of: an expression parameter measured by flow cytometry; a potency parameter measured by impedance, imaging, or both; or a metabolic parameter comprising mitochondrial respiration, glycolysis, ATP production, bioenergetic capacity or a combination thereof, wherein the plurality of parameters are associated with one or more of properties comprising: clonal abundance; clonalexpansion; transduction efficiency; proliferation capacity; cytotoxicity capacity; persistence; sternness; or exhaustion, wherein a change in the value for each of the plurality of parameters, or a combination thereof, is indicative of a control parameter that requires a modification; and further modifying the control parameter, thereby optimizing the control parameter of the bioreactor.

[0053] An embodiment provided includes, wherein the control parameter is associated with one or more (e.g., 2, 3, 4, 5, 6, 7, or all) operational settings for: nutrient and gas exchange; oxygen control; pH control; a feeding regime of media exchange versus media addition; mixing or shear force; device O2 permeability; vessel size; removal of activation beads; or enrichment / selection of a particular subpopulation.

[0054] An embodiment provided includes, wherein the change in the value for each of the plurality of parameters, or a combination thereof, indicates one or more of properties comprising: an increased clonal abundance; an increased clonal expansion; an increased transduction efficiency; an increased proliferation capacity; an increased cytotoxicity capacity; an increased persistence; an increased metabolic fitness; an increased sternness; or a reduced exhaustion.

[0055] An embodiment provided includes, wherein the modification of the control parameter provides (a) the optimal expression of a molecule by the immune cells, (b) the optimal killing potency of the immune cells, (c) the optimal bioenergetic profile and spare respiratory capacity of the immune cells, or any combination of (a), (b), or (c).

[0056] An embodiment provided includes, further comprising acquiring one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for each of the plurality of parameters from the population of immune cells.

[0057] An embodiment provided includes, wherein the one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for each of the plurality of parameters is acquired at least once every 1, 2, 3, 6, 9, 12, 18, or 24 hours, or at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, from the population of immune cells.

[0058] An embodiment provided includes, wherein the value for the potency parameter is acquired via XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0059] An embodiment provided includes, wherein the value for the metabolic parameter is acquired via SEAHORSE XF™ T cell Metabolic Fitness assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0060] An embodiment provided includes, a method of determining an optimal bioreactor for producing immune cells, comprising: cultivating a first population of immune cells from a subject in a first bioreactor; obtaining a first sample of the first population of immune cells from the first bioreactor; subjecting the first sample to an impedance-based analysis and a live cell imaging based analysis to determine an activity of immune cell killing, thereby obtaining a first potency value for the first population of immune cells in the first bioreactor; cultivating a second population of immune cells from the subject in a second bioreactor; obtaining a second sample of the second population of immune cells from the second bioreactor; subjecting the second sample to the impedance-based analysis and the live cell imaging based analysis to determine the activity of immune cell killing, thereby obtaining a second potency value for the second population of immune cells in the second bioreactor; and comparing the first potency value with the second potency value, wherein a higher potency value is indicative of the optimal bioreactor for producing immune cells.

[0061] An embodiment provided includes, wherein the first and second potency values are obtained by an XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0062] An embodiment provided includes, a method of determining an optimal bioreactor for producing immune cells, comprising: cultivating a first population of immune cells from a subject in a first bioreactor; obtaining a first sample of the first population of immune cells from the first bioreactor; subjecting the first sample to an instrument that determines oxygen consumption rate (OCR), proton efflux rate (PER), extracellular acidification rate (ECAR), ATP production rate, or a combination thereof, thereby obtaining a first metabolic value for the first population of immune cells in the first bioreactor; cultivating a second population of immune cells from the subject in a second bioreactor; obtaining a second sample of the second population of immune cells from the second bioreactor; subjecting the first sample to an instrument that determines oxygen consumption rate (OCR), proton efflux rate (PER), extracellular acidification rate (ECAR), ATP production rate, or a combination thereof, thereby obtaining a second metabolic value for the second population of immune cells in the second bioreactor; and comparing the first metabolic value with the second metabolic value, wherein a higher value is indicative of the optimal bioreactor for producing immune cells.

[0063] An embodiment provided includes, the first and second metabolic values are obtained by a SEAHORSE XF™ T cell Metabolic Fitness assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0064] An embodiment provided includes, a method of evaluating immune cells in a bioreactor, comprising: obtaining a first sample of a population of immune cells from the bioreactor; determining a percentage of immune cells that express a molecule (e.g., a CAR), in the first sample, thereby obtaining an expression value for the population of immune cells; obtaining a second sample of the population of immune cells from the bioreactor; subjecting the second sample to an impedance-based analysis and / or a live cell imaging based analysis to determine an activity of immune cell killing, thereby obtaining a potency value for the population of immune cells; and converting the obtained potency value to a normalized potency value based on the expression value.

[0065] An embodiment provided includes, wherein the expression value is obtained by flow cytometry.

[0066] An embodiment provided includes, wherein the potency value is obtained by an XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0067] An embodiment provided includes, further comprising cultivating the population of immune cells in the bioreactor.

[0068] An embodiment provided includes, a method of evaluating immune cells in a bioreactor, comprising: obtaining a first sample of a population of immune cells from the bioreactor; determining a percentage of immune cells that express a molecule (e.g., a CAR) in the first sample, thereby obtaining an expression value for the population of immune cells; adjusting an effector (E) to target (T) ratio for a potency assay of the population of immune cells based on the obtained expression value; obtaining a second sample of the population of immune cells from the bioreactor; subjecting the second sample to the potency assay using the adjusted E to T ratio.

[0069] An embodiment provided includes, the potency assay comprises an impedance-based analysis and a live cell imaging based analysis to determine an activity of immune cell killing, e.g., by an XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0070] An embodiment provided includes, wherein the expression value is obtained by flow cytometry.

[0071] An embodiment provided includes, wherein the adjusted E to T ratio is 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1: 1.

[0072] An embodiment provided includes, further comprising cultivating the population of immune cells in the bioreactor.

[0073] An embodiment provided includes, comprising: culturing immune cells in the bioreactor; extracting an immune cell sample, e.g., by a sample extraction module, from the bioreactor; preparing the extracted cell sample, e.g., by a sample preparation module, to produce a prepared sample; analyzing of a parameter described herein; transmitting a signal for an analyzed parameter to a controller, e.g., a controller module; responsive to the signal for an analyzed parameter, transmitting a second signal.

[0074] An embodiment provided includes, wherein acquiring or measuring a metabolic parameter comprises evaluating the bioenergetic poise and bioenergetic capacity of the sample by a method comprising: acquiring a reference value for oxygen consumption (VOCRef); acquiring a reference value for proton efflux (VPERef); contacting the cell sample with an ATP synthase inhibitor, a mitochondrial uncoupling agent, and an electron transport chain (ETC) inhibitor, sequentially, partly simultaneously, or simultaneously, each contacting forming a reaction mixture; acquiring a value for oxygen consumption for each reaction mixture (VOC\i,x); and acquiring a value for proton efflux for each reaction mixture (VPEviix), thereby evaluating the bioenergetic poise and bioenergetic capacity of the sample.

[0075] An embodiment provided includes, wherein the immune cells, or the population of immune cells, comprise T cells, natural killer (NK) cells, macrophages, or a combination thereof.

[0076] An embodiment provided includes, wherein the immune cells, or the population of immune cells, comprise genetically engineered immune cells targeting an antigen (e.g., tumor antigen), e.g., chimeric antigen receptor (CAR)-immune cells, e.g., CAR-T cells, CAR-NK cells, CAR-macrophages, or a combination thereof.

[0077] An embodiment provided includes, wherein the population of immune cells comprise chimeric antigen receptor (CAR) immune cells, e.g., CAR-T cells or CAR-NK cells.

[0078] An embodiment provided includes, wherein the system comprises one or more processors and a memory storing machine readable instructions, wherein the system is configuredto allow for: culture of immune cells in a bioreactor, e.g., a bioreactor comprising a bioreactor module; extraction of a sample, e.g., by a sample extraction module, from the bioreactor; optionally, preparation of the extracted cell sample, e.g., by a sample preparation module, to produce a prepared sample; analysis of a parameter described herein; transmission of a signal for an analyzed parameter to a controller, e.g., a controller module; responsive to the signal for an analyzed parameter, transmission of a second signal from the controller to the bioreactor; and responsive to the second signal from the controller, maintenance or alteration of a bioreactor module parameter.

[0079] Additional features and advantages of the disclosed method and apparatus are described in, and will be apparent from, the following Detailed Description and the Figures. The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the figures and description. Moreover, it should be noted that the language used in the specification has been principally selected for readability and instructional purposes, and not to limit the scope of the inventive subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 illustrates an example workflow for cell cultivation setup and analysis, according to embodiments of the present disclosure.

[0081] Figure 2 illustrates the effect of cell growth on interwell impedance, as may be measured by an impedance and imaging analysis instrument, according to embodiments of the present disclosure.

[0082] Figure 3 is a flowchart of an example method for data-driven bioreactor cultivation of immune cells, according to embodiments of the present disclosure.

[0083] Figure 4 is a flowchart of an example method for data-driven bioreactor cultivation of immune cells, according to embodiments of the present disclosure.

[0084] Figure 5 illustrates a computing device, according to embodiments of the present disclosure.

[0085] Figures 6-2 ID illustrate data and the analytics performed thereon using the concepts described in the present disclosure.DETAILED DESCRIPTION

[0086] The present disclosure provides for the data-driven bioreactor cultivation of immune cells, in which bioreactors are selected for the cultivation of various cell populations according to measured or derived values for potency and metabolic fitness of the cultured cells, rather than set “recipes” or instructions for operating the bioreactor to produce cultured cells.

[0087] Metabolic Fitness and Potency Assays are two different types of critical quality attributes(CQA) of a cell that can be measured to ensuring consistency and reproducibility during cell therapy production. . Metabolic Fitness can be measured directly by measuring the rate of ATP production using extracellular flux analysis of oxygen consumption (OCR) and extracellular acidification (ECAR) rates or by indirect methods to assess the metabolism of cells such as Flow Cytometry (e.g., Mitochondrial Membrane Potential, glucose analogue incorporation, glutamine analogue incorporation, Scenith-rate of protein translation), Metabolomics, expression level of genes associated with metabolic pathways, etc. Potency assay can be measured as impedancebased killing assays of target cells, image-based killing assays of target cells, chromium or luminescent probe release assay of target cells, interferon-gamma release after cell reactivation, etc.

[0088] As bioreactors are used for the cultivation of living cells, the output of the bioreactor may differ based on the qualities of the cells being cultivated. This variability in output from the bioreactors leads to unpredictability in the quantity and quality of the cultured cells that can be used for treatment or prophylaxis of a target condition in a biological subject. Accordingly, to account for this unpredictability, operators may frequently over-harvest the initial population of cells from the biological subject to cultivate to prepare for potential failures in producing the final cultivated cell product. When cultivation occurs as expected, however, this over-harvesting can result in wasting resources in preparing more of the final cultivated cell product than can be used and negatively affect the biological subject from which the initial sample was taken.

[0089] In one aspect, the disclosure provides a method and system for determining the optimal time for harvesting immune cells during manufacturing of an immunotherapy product. For example, off-line in process measurements can be used to assess the optimal time of immune cell harvest.

[0090] In certain embodiments, the measurement comprises an image-based analysis (e.g., live cell imaging) or an impedance-based analysis, e.g., to obtain a highly sensitive, multiplexed andquantitative measurement of immune cell killing. In some embodiments, the measurement includes the XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0091] In other embodiments, the measurement comprises an evaluation of glycolytic and mitochondrial activity in the immune cells. For example, one or more of the mitoATP production rate, glycoATP production rate, totalATP production rate, percent of ATP from glycolysis (basal), spare respiratory capacity, spare respiratory capacity (%), or maximal respiration can be measured. In some embodiments, mitochondrial respiration, glycolysis, and ATP production are measured. In some embodiments, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells are measured in a multi-well plate. In some embodiments, the measurement includes SEAHORSE XF® T Cell Metabolic Fitness Assay (offered by Agilent Technologies Inc. of Santa Clara California).

[0092] Without wishing to be bound by theory, it is believed that in some embodiments, the methods and systems described herein can be used to determine the optimal time of harvest in a patient specific manner for autologous therapies, the donor- specific time of harvest for allogenic therapies, or both.

[0093] In another aspect, the disclosure provides a method and system for determining the optimal bioreactor for producing (e.g., growing and / or expanding) immune cells for manufacturing an immunotherapy product. Without wishing to be bound by theory, it is believed that in some embodiments the specifications of each bioreactor may be unique and can have an impact on the quality and / or potency of the immune cells being produced. For example, off-line in process measurements can be used to assess the optimal bioreactor for producing immune cells.

[0094] In certain embodiments, the measurement comprises an image-based analysis (e.g., live cell imaging) and an impedance-based analysis, e.g., to obtain a highly sensitive, multiplexed and quantitative measurement of immune cell killing. In some embodiments, the measurement includes the XCELLIGENCE® potency assay.

[0095] In other embodiments, the measurement comprises an evaluation of glycolytic and mitochondrial activity in the immune cells. For example, one or more of the mitoATP production rate, glycoATP production rate, totalATP production rate, percent of ATP from glycolysis (basal), spare respiratory capacity, spare respiratory capacity (%), or maximal respiration can be measured. In some embodiments, mitochondrial respiration, glycolysis, and ATP production are measured.In some embodiments, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells are measured in a multi-well plate. In some embodiments, the measurement includes SEAHORSE XF™ T Cell Metabolic Profiling.

[0096] In yet another aspect, the disclosure provides a method or system for normalizing immune cell potency of a population of immune cells in a bioreactor. Without wishing to be bound by theory, it is believed that in some embodiments, normalizing potency results based on the percentage of cells that are actually transduced is more meaningful comparison between samples. For example, the potency result can be converted to an effective potency based on percent transduction of immune cells based on CAR expression. In some embodiments, the potency is dependent on the time of the harvest of the cells.

[0097] In still another aspect, the disclosure provides a method or system for optimizing a potency assay for a population of immune cells in a bioreactor. In some embodiments, the potency assay is optimized by modifying the effector (E) to target (T) ratio for immune cell killing. In some embodiments, the method comprises an impedance based analysis. In some embodiments, the E to T ratio is adjusted based on the percent of immune cells expressing a chimeric antigen receptor (CAR) or transduced with a vector encoding a CAR, e.g., prior to the performance of a potency assay.

[0098] The present disclosure uses methodologies that provide a more complete bioenergetic picture compared to traditional methods. For example, by combining the measurements of bioenergetic work (e.g., the amount of ATP being generated by the cell), bioenergetic poise (e.g., the proportion of ATP generate by glycolysis of oxidative phosphorylation), and bioenergetic capacity (e.g., the level of increase in glycolytic and mitochondrial activity that the cell can affect in response to increased energy demand), the present disclosure can provide a more complete analysis of metabolic fitness.

[0099] Traditional methods of evaluating metabolic / bioenergetic poise for cell samples typically rely on measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which is a less specific measure of glycolytic activity. Without wishing to be bound by theory, it is believed that the methods described herein may (i) provide a more complete bioenergetic picture, (ii) use more accurate glycolytic parameters, or (iii) use an uncoupler more suited to the measurement of immune cells, compared to traditional methods. In various embodiments, the present disclosure provides a more complete bioenergetic picture and uses moreaccurate glycolytic parameters, for example, for immune cells. The methods described herein are based, at least in part, on the discovery that mitochondrial uncoupling agent, BAM 15, demonstrates more robust performance than Carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), in immune cells, which contributes to the profound improvement of the methods described herein over traditional methods.Definitions

[0100] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0101] As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0102] ’About" and "approximately" as the term used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent(%), typically, within 10%, and more typically, within 5% of a given value or range of values.

[0103] "Acquire" or "acquiring" as the term used herein refers to obtaining possession of a physical entity, or a value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a stalling material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical analysis"), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragmentor other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the reagent. In an embodiment, directly acquiring encompasses a direct measurement. In an embodiment, indirectly acquiring encompasses an inference.

[0104] "Acquiring a sample" as the term used herein refers to obtaining possession of a sample, e.g., a sample described herein, by "directly acquiring" or "indirectly acquiring" the sample. "Directly acquiring a sample" means performing a process (e.g., performing a physical method such as a surgery or extraction) to obtain the sample. "Indirectly acquiring a sample" refers to receiving the sample from another party or source (e.g., a third-party laboratory that directly acquired the sample). Directly acquiring a sample includes performing a process that includes a physical change in a physical substance, e.g., a starting material, such as a tissue, e.g., a tissue in a human patient or a tissue that has was previously isolated from a patient. Exemplary changes include making a physical entity from a starting material; dissecting or scraping a tissue; separating or purifying a substance; combining two or more separate entities into a mixture; or performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond.

[0105] 'Basal mitochondrial ATP production rate" as the term used herein refers to the rate of ATP production by mitochondria in a cell sample before the cell sample is contacted with an ATP synthase inhibitor, a mitochondrial uncoupling agent, and an electron transport chain (ETC) inhibitor to form a reaction mixture. In an embodiment, the basal mitochondrial ATP production rate is calculated by subtracting the minimum oxygen consumption rate (oligo OCR) to a measurement (e.g., the last measurement, or an average of a number of measurements) of oxygen consumption rate before the first contacting of the cell sample with any of the ATP synthase inhibitor, mitochondrial uncoupling agent, or ETC inhibitor (basal OCR) and multiplying by a constant between 2.45 and 2.86 (called P / O ratio) *2 (to convert oxygen atoms to oxygen molecules). In an embodiment, the constant is 2.75.

[0106] "Bioenergetic capacity" as the term used herein refers to the level of increase in glycolytic and / or mitochondrial activity that a cell can affect, utilize, and / or induce. In an embodiment, the bioenergetic capacity is determined in response to increased energy demandand / or in response to inhibition / perturbation of energy-generation. In an embodiment, the bioenergetic capacity comprises a value for oxygen consumption (e.g., an oxygen consumption rate (OCR)) and a value for proton efflux (e.g., a proton efflux rate (PER)). In an embodiment, the value for oxygen consumption (e.g., OCR) is in response to mitochondrial uncoupling. In an embodiment, the value for proton efflux (e.g., PER) in in response to ATPase inhibition. In various embodiments, the PER is glycolytic PER (glycoPER), which mathematically removes the contribution of CO2.

[0107] “Biological subject” as the term used herein refers to a human or non-human animal from which a sample is obtained, or to which a cultured cell population is delivered as part of a therapy for the treatment or prophylaxis of a medical condition, such as a cancer or a tumor. In various embodiments, the biological subject from which the sample is obtained may be the same or different than the biological subject to which a cultured cell population derived from the sample is provided.

[0108] 'Basal glycolytic ATP production rate" as the teim used herein refers to the rate of ATP production by glycolysis (e.g., when glucose is converted into lactate) in a cell sample before the cell sample is contacted with an ATP synthase inhibitor, a mitochondrial uncoupling agent, and an electron transport chain (ETC) inhibitor to form a reaction mixture. In an embodiment, the basal glycolytic ATP production rate is calculated using the measurements of extracellular acidification rate (ECAR) before contacting the cell sample with an ATP synthase inhibitor, and converting the rate of proton efflux considering buffer capacity of media employed in the assay and the microchamber volume of the multiwell plate and discounting the contribution of extracellular CO2 production that is calculated from the measurements of the basal oxygen consumption prior to, and subsequent to, contacting the cell sample with an ETC inhibitor, delivering a values that informs CO2 contribution to measured acidification which in turn is transposed into an ATP production rate.

[0109] "Bioenergetic poise" as the term used herein refers to the balance between aerobic and glycolytic energy production, hi an embodiment, the bioenergetic poise describes the proportion of ATP generated by glycolysis of oxidative phosphorylation. In an embodiment, the bioenergetic poise comprises a relationship, e.g., a ratio, between ATP made by mitochondria and ATP made by glycolysis, between ATP made by mitochondria and total ATP production, between ATP made by glycolysis and total ATP production, or any combination thereof.

[0110] "Bioenergetic work" as the term used herein refers to the amount of ATP being generated by a cell.

[0111] ' ’Cancer" and "tumor" as the terms used interchangeably herein refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell, such as a leukemia cell. These terms include a solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term "cancer" includes premalignant, as well as malignant cancers.

[0112] "CAR NK cell therapy" as the term used herein refers to a therapy that uses a CAR NK cell. "CART cell therapy" as the term used herein refers to a therapy that uses a CART cell.

[0113] "Cell sample" as the term used herein refers to a sample that comprises a cell. In an embodiment, the cell sample comprises a plurality of cells. In various embodiments, the cell is disposed in a medium.

[0114] "Chimeric antigen receptor " or "CAR" as the term used herein refers to a recombinant polypeptide comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain. Chimeric antigen receptors can redirect immune cells toward cells expressing target antigens.

[0115] 'Chimeric antigen receptor NK cell" or "CAR NK cell" as the term used herein refers to an NK cell that has been genetically engineered to express a chimeric antigen receptor (CAR).

[0116] "Chimeric antigen receptor T cell" or "CART cell" as the term used herein and refers to a T cell that has been genetically engineered to express a chimeric antigen receptor (CAR).

[0117] "Compensatory glycolytic capacity" and "maximal glycolytic capacity" as the terms used interchangeably herein refer to the capacity of a cell sample to compensate energy production through glycolysis after inhibition of mitochondrial ATP production and / or increasing energetic demand. Compensatory glycolytic capacity can be expressed as a percentage of basal glycolysis. In an embodiment, the compensatory glycolytic capacity is calculated using the maximal measurement of proton efflux rate (PER) after the cell sample is contacted with an ETC inhibitor or an ionophore.

[0118] 'Critical Quality attribute", “Quality attribute”, “Quality parameter” and similar terminology as the terms used interchangeably herein to describe a measurable characteristic ofthe cell product that can include potency, metabolic fitness or any other quantifiable parameters of the cell.

[0119] Immune cell” as the term used herein refers to cells of an immune system of a biological subject, which may include, but are not limited to: an immune effector cell, a primary immune cell, an immortalized immune cell (e.g., a THP1 cell), a genetically engineered immune cell, a Natural Killer (NK) cell (e.g., a primary NK cell, a naive NK cell, a primary naive NK cell, etc.), a T cell (e.g., a primary T cell, a naive T cell, a T helper cell, cytotoxic T cell, memory T cell, virtual memory T cell, regulatory T cell, innate-like T cell, NK T cell, mucosal associated in variant T cell, gamma delta T cell, etc.) e.g., as the term used herein and refers to an NK cell that has been genetically engineered to express a T cell receptor (TCR).

[0120] "Maximal respiratory capacity" as the term used herein refers to the theoretical capacity of a cell sample to produce ATP by oxidative phosphorylation. In an embodiment, the maximal respiratory capacity is calculated using the maximal measurement of oxygen consumption rate ( OCR) after the cell sample is contacted with a mitochondrial uncoupling agent and discounting the minimum measurement of oxygen consumption rate (OCR) after the cell sample is contacted with an ETC inhibitor, and before any further injections.

[0121] Or" is used herein to mean, and is used interchangeably with, the teim "and / or", unless context clearly indicates otherwise. The use of the term "and / or" in some places herein does not mean that uses of the term "or" are not interchangeable with the term "and / or" unless the context clearly indicates otherwise.

[0122] "Primary cell" as the term used herein refers to a cell isolated or harvested directly from a subject, organ, or tissue. For example, primary cells can be isolated from blood obtained from a living subject. Primary cells can be isolated or harvested using enzymatic or mechanical methods. Once isolated or harvested, primary cells can be cultured in media containing essential nutrients and growth factors to support proliferation. Primary cells can be suspension cells that do not require attachment for growth (e.g., anchorage-independent cells) or adherent cells that require attachment for growth (e.g., anchorage-dependent cells).

[0123] 'Reserve aerobic capacity" and "spare respiratory capacity" as the terms used herein refer to the capacity of a cell sample to produce the extra amount of ATP by oxidative phosphorylation, for example, in case of an acutely increased energy demand. In an embodiment, the reserve aerobic capacity or spare respiratory capacity is calculated from the difference betweenthe maximal measurement of oxygen consumption rate (OCR) after the cell sample is contacted with a mitochondrial uncoupling agent and discounting the basal measurement of oxygen consumption rate before injection of any reagent and can be expressed in multiple units including, for example, units of oxygen consumption or ATP production.

[0124] "Reserve glycolytic capacity" as term used herein refers to the capacity of a cell sample to produce the extra amount of ATP by glycolysis, for example, in case of an acutely increased energy demand. In an embodiment, the reserve glycolytic capacity is calculated as the difference between the maximal glycolytic capacity and basal glycolytic ATP production.

[0125] "Sample" as the term used herein refers to a biological sample obtained or derived from a source of interest. In an embodiment, the source of interest comprises an organism, such as an animal or human. The source of the sample can be blood or a blood constituent; a bodily fluid; a solid tissue as from a fresh, frozen and / or preserved organ, tissue, biopsy, resection, smear, or aspirate; or cells from any time in gestation or development of a subject. In an embodiment, the source of the sample is blood or a blood constituent. In an embodiment, the sample is a primary sample, e.g., obtained directly from a source of interest by any appropriate means. In an embodiment, the sample is a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample.

[0126] "T cell receptor NK cell" or "TCR NK cell" as the term used herein and refers to an NK cell that has been genetically engineered to express a T cell receptor (TCR).

[0127] "T cell receptor T cell" or "TCR T cell" as the term used herein and refers to a T cell that has been genetically engineered to express a T cell receptor (TCR).

[0128] TCR NK cell therapy" as the term used herein refers to a therapy that uses a TCR NK cell.

[0129] "TCR T cell therapy" as the term used herein refers to a therapy that uses a TCR T cell.

[0130] As used herein, the term “optimize” and variations thereof, is used in a sense understood by data scientists to refer to actions taken for continual improvement of a system relative to a goal. An optimized value will be understood to represent “near-best” value for a given reward framework, which may oscillate around a local maximum or a global maximum for a “best” value or set of values, which may change as the goal changes or as input conditions change. Accordingly, an optimal solution for a first goal at a given time may be suboptimal for a second goal at that time or suboptimal for the first goal at a later time.

[0131] As used herein, various chemical compounds are referred to by associated element abbreviations set by the International Union of Pure and Applied Chemistry (IUPAC), which one of ordinary skill in the relevant ail will be familiar with. Similarly, various units of measure may be used herein, which are referred to by associated short forms as set by the International System of Units (SI), which one of ordinary skill in the relevant ail will be familial’ with.

[0132] As used herein, various terms provided with reference to the body of a biological subject are to be understood with reference to the standard anatomical position of that biological subject using anatomical terms of location e.g., as set by the International Federation of Associations of Anatomists or the World Associate of Veterinary Anatomists that will be understood by the person on ordinary skill in the relevant art without further explanation.

[0133] As used herein, “about,” “approximately” and “substantially” are understood to refer to numbers in a range of the referenced number, for example the range of - 10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1% to +1% of the referenced number, most preferably -0.1% to +0.1% of the referenced number.

[0134] Furthermore, all numerical ranges herein should be understood to include all integers, whole numbers, or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of a range from 1 to 10 should be construed as supporting ranges of any two numbers that fall into the initial range of from 1 to 10 (e.g., from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, from X to Y where X > 1 and Y < 10).

[0135] As used in the present disclosure, a phrase referring to “at least one of’ a list of items refers to any set of those items, including sets with a single member, and every potential combination thereof. For example, when referencing “at least one of A, B, or C” or “at least one of A, B, and C”, the phrase is intended to cover the sets of: A, B, C, A-B, B-C, and A-B-C, where the sets may include one or multiple instances of a given member (e.g., A-A, A-A-A, A- A-B, A- A-B-B-C-C-C, etc.) and any ordering thereof. For avoidance of doubt, the phrase “at least one of A, B, and C” shall not be interpreted to mean “at least one of A, at least one of B, and at least one of C”.

[0136] As used in the present disclosure, the term “determining” encompasses a variety of actions that may include calculating, computing, processing, deriving, investigating, looking up (e.g., via a table, database, or other data structure), ascertaining, receiving (e.g., receivinginformation), accessing (e.g., accessing data in a memory), retrieving, resolving, selecting, choosing, establishing, and the like.

[0137] Operational Setup and Practice

[0138] Figure 1 illustrates an example cell cultivation setup 100, according to embodiments of the present disclosure. Sample cells 110 are extracted from a biological subject and loaded into bioreactor 150, where the sample cells 110 are encouraged to multiply in a cultivation process to produce cultivated cells 130. In various embodiments, the cultivation process may include various gene editing or gene expression / activation operations to affect the ability of the sample cells 110 to treat, prevent, or mitigate various medical conditions when the cultivated cells 130 are introduced to a biological subject (which may be the same or a different biological subject from which the cells 110 were initially extracted). To avoid confusion, as cells are understood to grow, divide, and die off during cultivation, the cells placed in the bioreactor are referred to as reactor cells 120, which may display the properties of the sample cells 110 (as input to the biorcactor 150), the cultivated cells 130 (as extracted from the bioreactor 150), or properties intermediate thereto.

[0139] In various embodiments, the sample cells 110 may be divided into two or more populations, which are then loaded into a corresponding number of two or more bioreactors 120. These separate populations may be used to evaluate different cultivation processes (e.g., different growth media, media exchange policies, temperatures for growth, atmospheres of growth, the application of different pharmaceutical compounds, the effect of different bioreactors 120, etc.), to provide a backup population for creating cultured cells 130 for treating a medical condition (e.g., in case of error or failure in one population), or to produce different sets of cultured cells 130 with different properties from one another. In various embodiments, during the cultivation process, the bioreactors 150 may control for one or more of: nutrient and gas exchange, oxygen (O2) control, pH control, a feeding regime of media exchange, media addition during cultivation, mixing or shear force applied to the reactor cells 120, O2 permeability of the bioreactor 150 or wells therein, a vessel size (e.g., of a well in a wellplate used in the bioreactor 150), and whether activation beads are removed during cultivation, automated cell population selection or enrichment during cultivation process.

[0140] During cultivation of CAR cells, the reactor cells 120 may undergo several changes in the genome such as the introduction of CAR constructs or other accessory genes or modifications to key genes. For example, an operator may cultivate the reactor cells 120 to include genes forglucose metabolism, include genes for mitochondrial respiration, include genes for cytokines (e.g., IL15 or IL21), turn on glucose metabolism genes (e.g., STAT5), or target various regulatory pathways (e.g., mTOR, PI3K, AKT, etc.) or checkpoint inhibitors that target proteins that regulate immune activation.

[0141] In various embodiments, the reactor cells 120 are preconditioned (e.g., using cytokines) prior to cultivation and various genetic modification to result in improved potency in the cultured cells 130 by enhancing mitochondrial bioenergetic capacity. For example, preincubating TCR / CAR T cells in vitro under reduced glucose or elevated arginine conditions can improve potency and ability to clear tumors in xenograft-bearing mice. Indeed, preconditioning in either low glucose or high arginine alters bioenergetics (glycolysis and oxidative phosphorylation), which affects differentiation, persistence, tumor infiltration, and resistance to exhaustion. Collectively, these preconditioning results highlight the diverse means by which T cell fitncss / function can be enhanced. Because preconditioning is both inexpensive and technically facile, precondition is likely to become integral to CAR / TCR T cell manufacturing in the future. Accordingly, a comprehensive, robust, real-time workflow, where T cell potency as measured by immune cell killing is combined with biogenetics and persistence parameters, is provided herein for the example setup 100. This workflow provides two independent perspectives from the same experiment that can replace several time-consuming assays performed in series during cell therapy process development.

[0142] To monitor growth and potency of the reactor cells 120, or to determine whether to harvest the reactor cells 120 (e.g., as cultured cells 130), the bioreactor 150 is configured to measure various potency parameters for the reactor cells 120, which may be tested against target cells 140 (e.g., tumor cells extracted from a biological subject, which may be the same or a different biological subject from which the sample cells 110 were extracted or to which the cultured cells 130 will be delivered). These measurements may be performed against one or more extracted samples of the reactor cells 120 or may be performed in real-time against one or more sub-populations of the reactor cells 120 (e.g., in a well of a wellplate held within the bioreactor 150) or against different densities of the reactor cells 120 (E:T).

[0143] In some embodiments, the metabolic fitness of the cells 120 is monitored to determine whether to harvest the reactor cells 120. The metabolic fitness assay measures one or both of the two major cellular metabolic pathways that generate ATP molecules: glycolysis and mitochondrialrespiration. Glycolysis leads to excretion of protons into the media, leading to extra cellular acidification, which is measured by the metabolic analyzer instrument 160. Mitochondrial respiration, an key parameters thereof, is measured as the oxygen consumption rate (OCR) of the cells by the metabolic analyzer instrument 160.

[0144] In the metabolic fitness assay, oligomycin is injected to the cells under analysis, after basal respiration is measured. Oligomycin inhibits ATP synthase (complex V), and impacts or decreases electron flow through the ETC, resulting a reduction in mitochondrial respiration or OCR. This decrease in OCR is linked to cellular ATP production. Following injection of the Oligomycin, BAM15 is injected to the cells under analysis. BAM15 is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential. As a result, electron flow through the ETC is uninhibited, and oxygen consumption by complex IV reaches the maximum. The BAM15-stimulated OCR can then be used to calculate spare respiratory capacity, defined as the difference between maximal respiration and basal respiration. Sparc respiratory capacity is a measure of the ability of the cell to respond to increased energy demand or under stress. A mixture of a complex I inhibitor and a complex III inhibitor (e.g., rotenone and antimycin A) is then injected to shut down mitochondrial respiration and thereby enable calculation of nonmitochondrial respiration driven by processes outside the mitochondria. OCR- rates can be converted in mitochondrial ATP production rate considering P / 0 ratio.

[0145] An impedance and imaging analysis instrument 170, which may be included in the bioreactor 150 or a separate instrument, provides for impedance measurement of the cellular activity and growth of the reactor cells 120 and analysis of the killing capacity of the reactor cells 120 relative to target cells 140 via visual confirmation of the number, density, adhesion, and longitudinal presence of target cells 140 and / or reactor cells 120 within a well of a wellplate.

[0146] Figure 2 illustrates the effect of cell growth on interwell impedance, as may be measured by an impedance and imaging analysis instrument 170, according to embodiments of the present disclosure. As target cells 140 adhere to a surface a well 220 that includes electrodes 230, the cells 210 block a current path 250 through the media 240 in the well 220, resulting in a decreased current flow (e.g., an increased impedance). As time progresses after immune cells 210 are introduced to the well 220, the target cells 140 are killed off - re-opening current paths through the media 240 between the electrodes, resulting in an increased current flow (e.g., a decreased impedance). The impedance and imaging analysis instrument 170 measures these changes inimpedance over time to assess the speed and efficacy of the immune cells 210 in killing off the target cells 140.

[0147] Figure 3 is a flowchart of an example method 300 for data-driven bioreactor cultivation of immune cells, according to embodiments of the present disclosure, in which a bioreactor with improved probability of producing high-potency cultured cells is selected from among a plurality of available bioreactors. Method 300 begins at block 310, where an operator divides a first sample of immune cells into at least a first population and a second population. In various embodiments, these immune cells may be taken from a biological subject in anticipation of modifying these cells for process development without returning the cells to a biological subject, for return to that biological subject or for delivery to a different biological subject. Although generally discussed in relation to a first to two populations, the present disclosure contemplates that the operator may divide the collected sample of cells into any number of populations and that these populations may be the same or different in initial size.

[0148] At block 320, an operator cultivates the populations of immune cells in corresponding different bioreactors to produce respective cultivated cell populations. For example, the first population of the immune cells is cultivated in a first bioreactor to produce a first cultivated cell population, the second population of the immune cells is cultivated in a second bioreactor to produce a second cultivated cell population, ... and the nth population of the immune cells is cultivated in an nth bioreactor to produce an nth cultivated cell population. Each of the bioreactors used for producing the cultivated cells populations may be controlled for various operational settings, including: nutrient and gas exchange, oxygen control, pH control, a feeding regime of media exchange versus media addition during cultivation, mixing or shear force during cultivation, device O2 permeability, vessel size, removal of activation beads during cultivation or enrichment of a particular subpopulation.

[0149] At block 330, the operator measures respective values for critical quality attributes such as potency parameter or metabolic fitness parameter at harvest time for each cultivated cell population to determine which bioreactor (and associated operational settings) produced a cultivated cell population with a most desirable profile.

[0150] In various embodiments, the quality attribute parameter may be a single measured value (e.g., time to kill-50) or the quality parameter is based on a combination of a plurality of subparameters, wherein each sub-parameter of the plurality of sub-parameters each satisfies acorresponding potency threshold. For example, when measuring a combination of sub-parameters 1-n, in order to select the bioreactor, an operator may compare sub-parameter 1 against quality attribute threshold 1, sub-parameter 2 against quality attribute threshold 2, ... and sub-parameter n against potency threshold n. Then, for each population that satisfies quality attribute thresholds 1-n, the operator may judge the combination that yields an optimal combination of the subparameters. The present disclosure contemplates that the treatment of various medical conditions using cell therapies requires various different combinations of quality attributes based on the condition, type of cell being used, quality of the cell type being used, aggressiveness of the medical condition, health of the biological subject, genetic factors of the biological subject, etc. and that the identification of such parameters and the weighting thereof is within the normal and ordinary amount of experimentation that one of ordinary skill in the art is expected to perform. Stated differently, although each therapy is unique, persons of ordinary skill in the relevant art perform this level of experimentation and selection for each therapy, and will be able to uniquely identify the optimal combination of quality attribute parameters without undue experimentation.

[0151] In various embodiments, the potency parameters may be measured by performing an instrument analysis using target cells and impedance and / or image analysis to determine an efficacy of the cultured cells in killing the target cells. In various embodiments, the potency parameters may be measured by performing a potency assay that measures the products and byproducts of the cultured cells or target cells in the presence of cultured cells, which may include cytokines measured by flow cytometry. In various embodiments, different parameters may be collected via different measurement techniques, and may be used in assessing the potency of the cultured cells in parallel or in combination. For example, an operator may acquire a second value of a second parameter of the population of immune cells, different than the first parameter, wherein the first parameter is acquired via flow cytometry and the second parameter is acquired via impedance measurement, measuring potency of the immune cells, image analysis of the effectiveness of the immune cells, etc.

[0152] In various embodiments, the key parameters indicate at least one of: clonal abundance or clonal expansion, transduction efficiency, increased proliferation capacity, increased cytotoxicity capacity, increased persistence, increased metabolic fitness, increased sternness, and reduced exhaustion.

[0153] The present disclosure contemplates that other operations such as immune cell activation and engineering may be included as pail of “cultivation”. Accordingly, "cultivation" may refer to unmanipulated immune cells, or immune cells that have been activated and engineered (e.g., via Lentivirus or other virus or by electroporation of genes or CRISPR reagents).

[0154] At block 340, the operator harvests one or more of the cultivated cells types that have been produced from the initial populations. In some embodiments, the harvested cultivated cell populations may be used to test the quality of various bioreactors and operational settings therefor by performing potency assays or metabolic assays and may be discarded. In some embodiments, a small portion of the harvested cultivated cell types may be used to both test the quality of various bioreactors and operational settings therefor by performing potency assays and / or metabolic assays and for treatment of a medical condition in a biological subject, and method 300 may proceed to block 370 to use one or more of the cells types that satisfy potency and / or metabolic fitness thresholds in treating a medical condition in a biological subject.

[0155] In various embodiments, harvest time occurs at a predefined interval after initiation of cultivation. In some embodiments, the operator may periodically or continuously measure values for one or more quality attributes during cultivation, and harvest time occurs when a change in a potency value outside of a harvesting threshold occurs, which may occur before an end of a predefined period of time. In such embodiments, block 340 may be performed to harvest one or more populations of immune cells from respective bioreactors before the predefined period of time expires. Accordingly, in some embodiments, selecting a bioreactor to perform a subsequent cultivation (as per block 360) may be further based on which bioreactor reaches a desired threshold for the quality attribute faster.

[0156] At block 350, after harvest time of the initial populations of immune cells, the operator collects a second sample of a new population of the immune cells. In various embodiments, this second sample may be collected from the same biological subject from which the samples collected were collected for use in block 310, or a different biological subject.

[0157] At block 360, the operator selects a bioreactor to perform a subsequent cultivation for the second sample of the immune cells based on which of the respective cultivated cell populations was measured (per block 330) with a higher value for the quality attribute, and cultivates the subsequent sample of the immune cells in the selected bioreactor. This cultivation is used to produce a therapeutic using an optimal bioreactor, and operational setting therefor, to improve thespeed of production, potency of the therapeutic agent, reliability of production of the therapeutic agents, and combinations thereof; yielding an improved product and an improved treatment for a medical condition addressed via the therapeutic. As each therapeutic is unique, selection of a given bioreactor does not necessarily translate to using that same bioreactor for another therapy. Stated differently, despite the level of skill of the operator, the operator cannot know a priori which bioreactor to use due to the variability of the starting material and the nature of engineered live cells, which are inherently unpredictable. Therefore, even if after running hundreds of experiments, the skilled artisan accumulates some know-how into which bioreactor be better for a given therapeutic, there is no guarantee that the selected bioreactor would actually be the optimal bioreactor due to the unpredictability of manufacturing therapies based on immune cells, unless the methodologies of the present disclosure were performed.

[0158] At block 370, the operator treats a medical condition in a biological subject by supplying cultivated immune cells from a cultivated cell population. In various embodiments, the cultivated cell population may be produced by one or more of the initial samples (e.g., from block 340) or from the subsequent cultivation (e.g., from block 360). Various treatments may include CAR-T cell therapies used to treat cancers.

[0159] Figure 4 is a flowchart of an example method 400 for data-driven bioreactor cultivation of immune cells, according to embodiments of the present disclosure, in which an optimal harvest time for increased efficacy of engineered immune cells may be determined. Method 400 begins at block 410, where an operator acquires a first value of a first parameter from a population of immune cells being cultivated in a bioreactor. In various embodiments, these parameters may indicate one or more of: clonal abundance or clonal expansion, transduction efficiency, increased proliferation capacity, increased cytotoxicity capacity, increased persistence, increased metabolic fitness, increased sternness, and reduced exhaustion.

[0160] In various embodiments, the potency parameters may be measured by performing an instrument analysis using target cells and impedance and / or image analysis to determine an efficacy of the cultured cells. In various embodiments, the quality of the cells may be tested by performing an assay that measures the cytokines products and byproducts of the cultured cells or target cells in the presence of cultured cells, which may include flow cytometry. The type of measurements collected could be i) potency (measured by impedance, image, or cytokine release, or some probe release form target cells to the medium) or ii) metabolic fitness (measured as XFanalyzer measured as bioenergetic work, capacity, metabolic poise or by other methodologies like mitochondrial membrane potential, etc.). In various embodiments, different parameters may be collected via different measurement techniques, and may be used in assessing the potency of the cultured cells in parallel or in combination. For example, an operator may acquire a second value of a second parameter of the population of immune cells, different than the first parameter, wherein the first parameter is acquired via flow cytometry and the second parameter is acquired via impedance measurement, measuring metabolism of the immune cells, image analysis of the effectiveness of the immune cells, etc.

[0161] In various embodiments, the potency parameter may be a single measured value (e.g., time to kill-50) or the potency parameter is based on a combination of a plurality of subparameters, wherein each sub-parameter of the plurality of sub-parameters each satisfies a corresponding quality attribute threshold. For example, when measuring a combination of subparameters 1-zi, in order to select the biorcactor, an operator may compare sub-parameter 1 against quality attribute threshold 1, sub-parameter 2 against quality attribute threshold 2, ... and subparameter n against quality attribute threshold n. Then, for each population that satisfies quality attribute thresholds 1-n, the operator may judge the combination that yields an optimal combination of the sub-parameters. The present disclosure contemplates that the treatment of various medical conditions using cell therapies requires various different combinations of parameters based on the condition, type of cell being used, aggressiveness of the medical condition, health of the biological subject, genetic factors of the biological subject, etc. and that the identification of such parameters and the weighting thereof is within the normal and ordinary amount of experimentation that one of ordinary skill in the art is expected to perform. Stated differently, although each therapy is unique, persons of ordinary skill in the relevant art perform this level of experimentation and selection for each therapy, and will be able to uniquely identify the optimal combination of quality attribute parameters without undue experimentation.

[0162] At block 420, the operator monitors the first value during a predefined period of time. In various embodiments, the value may be measured periodically (e.g., once every n hours), in response to an external signal generated from another operational setting or parameter (e.g., in response to an O2 content value in a bioreactor dropping below a threshold), or continuously during cultivation. In various embodiments, the predefined period of time may be set via a regulatory agency as at least 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, or 168 hours (or any timetherebetween or subdivision therein) based on guidelines or production procedures for various engineered cell populations approved for use in medical applications.

[0163] At block 430, the operator determines whether a change in the measured and monitored parameter satisfies a harvesting threshold. In response to the change not satisfying the harvesting threshold, method 400 proceeds to block 440. In response to identifying a change in the value outside of a harvesting threshold (before an end of the predefined period of time) method 400 proceeds to block 450 for harvesting the population of immune cells from the bioreactor before the predefined period of time expires. In various embodiments, the early harvest of cells (relative to the predefined period of time) may occur at time between 10 hours and 5 days before the predefined period of time would otherwise indicated for the harvesting of the engineered cells from the bioreactor.

[0164] At block 440, the operator determines whether the harvest time - the predefined period of time - has been reached. In response to the harvest time not being reached, method 400 returns to block 420 to continue monitoring the parameter during cultivation. In response to the harvest time being reached, method 400 proceeds to block 450 for harvesting the population of immune cells at the end of predefined period of time.

[0165] At block 450, the operator harvests the cultured cells from the bioreactor. In various embodiments, harvesting the population of immune cells from the bioreactor before the predefined period of time expires (e.g., after proceeding to block 450 from block 430) removes less than 100% of the population of immune cells from the bioreactor, and method 400 may therefore return to block 420 from block 450 to continue cultivating a remainder of the population of the immune cells not harvested from the bioreactor until the predefined period of time expires or the remaining population again satisfied the harvesting threshold. After satisfying such thresholds with a remainder population, an operator may then return to block 450 to harvest the remainder of the population of the immune cells from the bioreactor.

[0166] In addition to final (or intermediate, based on harvesting thresholds) harvest of engineered cells for use in treating a medical condition, the present disclosure contemplates that partial harvests of the engineered cells from the bioreactor may occur during the cultivation process, which may be used for testing or monitoring the potency parameters of the cells being cultured (e.g., be lock 420). Accordingly, an operator may forego (or forestall) waiting for an expansion stage in the production process of the therapeutic.

[0167] At block 460, the operator treats a medical condition in a biological subject by supplying cultivated immune cells from a cultivated cell population that has been harvested from the bioreactor. In various embodiments, the cultivated cell population may be delivered to a biological subject from which an initial cell sample was taken, or to a different biological subject. In some embodiments, the immune cells can be used for optimization of bioreactor conditions without being used to treat biological subject. Various treatments may include CAR-T cell therapies used to treat cancers.

[0168] Figure 5 illustrates a computing device 500, as may be used to collect or process data in a system for data-driven bioreactor cultivation of immune cells, according to embodiments of the present disclosure. The computing device 500 may include at least one processor 510, a memory 520, and a communication interface 530. In various embodiments, the computing device 500 may be configured for processing method 300 (described in greater detail in regard to Figure 3) and / or method 400 (described in greater detail in regard to Figure 4) and / or interfacing with the various components of the cell cultivation setup 100 (described in greater detail in regard to Figure 1), e.g., by sending command to, and transmitting / receiving data between the impedance and imaging analysis instrument, potency instrument 160, and bioreactor 150.

[0169] The processor 510 may be any processing unit capable of performing the operations and procedures described in the present disclosure. In various embodiments, the processor 510 can represent a single processor, multiple processors, a processor with multiple cores, and combinations thereof.

[0170] The memory 520 is an apparatus that may be either volatile or non-volatile memory and may include RAM, flash, cache, disk drives, and other computer readable memory storage devices. Although shown as a single entity, the memory 520 may be divided into different memory storage elements such as RAM and one or more hard disk drives. As used herein, the memory 520 is an example of a device that includes computer-readable storage media, and is not to be interpreted as transmission media or signals per se.

[0171] As shown, the memory 520 includes various instructions that are executable by the processor 510 to provide an operating system 522 to manage various features of the computing device 500 and one or more programs 524 to provide various functionalities to users of the computing device 500, which include one or more of the features and functionalities described in the present disclosure. One of ordinary skill in the relevant art will recognize that differentapproaches can be taken in selecting or designing a program 524 to perform the operations described herein, including choice of programming language, the operating system 522 used by the computing device 500, and the architecture of the processor 510 and memory 520. In various embodiments, the program 524 may include or make use of a machine learning model 526 that is trained to make determinations as set forth in the present disclosure, and may be retrained or updated based on data collected as set forth in the present disclosure. Accordingly, the person of ordinary skill in the relevant art will be able to select or design an appropriate program 524 based on the details provided in the present disclosure.

[0172] The communication interface 530 facilitates communications between the computing device 500 and other devices, which may also be computing devices as described in relation to Figure 5. In various embodiments, the communication interface 530 includes antennas for wireless communications and various wired communication ports. The computing device 500 may also include or be in communication, via the communication interface 530, one or more input devices (e.g., a keyboard, mouse, pen, touch input device, etc.) and one or more output devices (e.g., a display, speakers, a printer, etc.).

[0173] Although not explicitly shown in Figure 5, it should be recognized that the computing device 500 may be connected to one or more public and / or private networks via appropriate network connections via the communication interface 530. It will also be recognized that software instructions may also be loaded into a non-transitory computer readable medium, such as the memory 520, from an appropriate storage medium or via wired or wireless means.

[0174] Accordingly, the computing device 500 is an example of a system that includes a processor 510 and a memory 520 that includes instructions that (when executed by the processor 510) perform various embodiments of the present disclosure. Similarly, the memory 520 is an apparatus that includes instructions that, when executed by a processor 510, perform various embodiments of the present disclosure.Experimental Results

[0175] The concepts described in the present disclosure are generally counter-intuitive to the current understanding for using bioreactors. Accordingly, the present disclosure supports the position that otherwise identical bioreactors using otherwise identical growth media (and other reagents and pharmaceutical compounds) on a same of type cell from the same biological subject can reliably produce different outcomes in cell culturing with experimental results shown inFigures 6-21D. The present disclosure supports the position that harvesting a cell culture before an allotted cultivation time has passed based on various potency and / or metabolic measurements can result in more effective cell treatments in biological subjects with experimental results shown in Figures 6-2 ID.EXAMPLESExample 1:

[0176] Metabolic Fitness of T cells is a broad concept used to describe, for example, the optimal metabolic phenotype of immunotherapy cell products for increased anti-tumor potency. Metabolic Fitness parameters can include, for example, basal bioenergetic phenotype (basal mitochondrial ATP Production Rate + glycolytic ATP Production Rate), Metabolic Poise, Mitochondrial Maximal Respiratory Capacity, Spare Respiratory Capacity and Glycolytic Capacity. For example, as shown in Table 1, various assay outputs may be correlated with different parameters to measure metabolic fitness of various cells.

[0177] Table 1

[0178] The present disclosure combines the measurements of bioenergetic work (e.g., the amount of ATP being generated by the cell), bioenergetic poise (e.g., the proportion of ATP generated by glycolysis or oxidative phosphorylation), and bioenergetic capacity (e.g., the level of increase in glycolytic and mitochondrial activity that the cell can affect in response to increased energy demand) to optimize cell culture medium conditions during (CAR)T cell therapy manufacture.

[0179] The present disclosure uses ATP production rate as a parameter to describe the metabolic activity and metabolic poise as opposed to the OCR kinetic plot. The method described herein can generate bioenergetic work (ATP Production Rate), metabolic poise and maximal respiration and / or reserve capacity (aerobic and / or glycolytic) from the same well.

[0180] The present disclosure uses a mitochondrial uncoupling agent suitable for a more accurate estimation of maximal and / or reserve capacity for the cells described compared to previously used FCCP

[0181] The sample(s) collected from the expansion system (at least 1-2 x 106cells per sample) were centrifuged (10 min x 1000 g) and cells were resuspended in an appropriate volume of assay media to reach the recommended cell density for the cell type of analysis (for example, for preactivated T cells, a cell density of 2 mill total cells / mL is recommended). A sample (typically 50 pL) of the cell suspension was added to same volume of buffer containing the viability dye 7- A AD (2X) and the total and live cell numbers in the sample were counted using a Flow cytometer. If cell density was higher or less than 60% of the recommended density, cell suspension volume was adjusted to reach the desired cell density range, and the cell sample was counted again to confirm the final cell density. A sample (typically 50 uL) of the cell suspension are seeded in multiwell plates pre-coated with PDL and prewarmed overnight at 37C (in general 3 or more replicate wells per cell sample). Multiwell plates are centrifuged, (1 min, 100g), assay media is added to complete the recommended volume for the particular’ plate type (typically 200 p L) and incubated at 37C in a non-CCh incubator for 45 min.

[0182] The appropriate metabolic Analyzer (e.g., instrument 160) is programmed with command instructions to inject sequentially the solution from the ports in a cartridge disposed above the cell sample in a well and conduct three measurements after each injection.

[0183] The following metabolic modulators working solutions were prepared: Oligomycin A stock solution is prepared to a working concentration of 13.5 pM in assay media. BAM15 stock solutions is prepared at an optimized concentration (generally 25 pM for human T cells) and rotenone plus antimycin A mix stock solution is prepared to a working concentration of 5.5 pM each.

[0184] A sufficient volume of each modulator working solution is added to an assay cartridge such that upon injection the working solution is diluted into the assay medium to the final desired concentration. For example, in human T cells, the final desired concentration is 1.5 pM ofoligomycin A, 2.5 pM of BAM15 and 0.5 pM of Rotenone plus Antimycin A mixture. These concentrations were determined by titration for optimal effectiveness.

[0185] The hydrated assay cartridge containing the indicated reagent is loaded into the instrument.

[0186] The metabolic profile of the cell sample may be determined by calculating: i) Basal mitochondrial ATP production rate, calculated subtracting the minimum oxygen consumption rate after injection of oligomycin A and before any other injection (oligo OCR) to a measurement (e.g., the last measurement, or an average of a number of measurements) of oxygen consumption rate before the first injection of reagents (basal OCR) and multiplying by a constant between 2.45 and 2.86, e.g., the constant 2.75 (called average P / O Ratio, i.e., yield of ATP / O consumed) * 2 (to convert oxygen atoms to oxygen molecules); ii) Basal glycolytic ATP Production Rate is calculated using the measurements of extracellular acidification rate before the Oligomycin Injection and converting the rate of Proton Efflux (considering Buffer Capacity of media employed in the assay and the microchamber volume of the multiwell plate) and discounting the contribution of extracellular CO2 production that is calculated from the measurements of the basal rate of Oxygen Consumption rate and the minimum measurement after the injection of rotenone / antimycin A and before any following injection and using a conversion factor that accounts for a number of CO2 molecules produced in the mitochondria per O2 consumed; iii) Maximal Respiratory Capacity is calculated using the maximal measurement of oxygen consumption rate after BAM 15 injection and discounting the minimum measurement of Oxygen consumption rate after the injection of rotenone / antimycin A and before any following injection; iv) Reserve Aerobic Capacity (also known as Spare Respiratory Capacity) is calculated as the difference between maximal measurement of oxygen consumption rate after BAM 15 injection and a measurement (e.g., the last measurement, or an average of a number of measurements) of oxygen consumption rate before the first injection. Aerobic Reserve Capacity can be expressed in units of Rates of ATP production multiplying by the P / O ratio * 2; v) Maximal Mitochondrial Bioenergetic capacity is calculated using the maximal measurement of oxygen consumption rate after BAM 15 injection and discounting the minimum measurement after the injection of Oligomycin A and before any following injection and Multiplying by P / O Ratio *2; vi) Compensatory (or Maximal Glycolytic Capacity) is calculated using the maximal measurement of glycolytic ATP Production after oligomycin injection and before BAM15 injection, vii) Reserve Glycolytic Capacity.Calculated as the difference between Maximal Glycolytic Capacity and basal glycoATP Production.

[0187] The real-time measurement of impedance is initiated right after the seeding of target cells in the impedance-measuring wells. However, the changes in impedance are reported as Cell Index (CI). The CI is further normalized to the time point right before CAR T-cell addition, named Normalized Cell Index (NCI).

[0188] The functional parameters used for the functional potency assay include:

[0189] Percentage of cytolysis: to determine the level of immune-cell-mediated killing. The percentage of cytolysis utilizes the Normalized Cell Index from the samples (NCI_s) and the average Normalized Cell Index from the target alone control (NCI_t) according to the following equation, % Cytolysis = [1- NCI_s / NCI_t] *100.

[0190] Area Under the Curve (AUC): to convert real-time kinetic data to an endpoint readout. AUC of the percentage of cytolysis is to calculate the area under the time course of % cytolysis, starting from the Normalized Time point that corresponds to the time when effectors are added to the target cells to the selected time point on the curve. The larger the AUC of % cytolysis more potent the effectors would be.

[0191] KT: to determine the speed of the immune cell-mediated cell killing. KT is measured as the time of killing and is calculated starting from the Normalization Time Point that corresponds to the time when effectors and other relevant conditions are added to samples. The impedance / imaging instrument software provides the options for KT of 20, 40, 50, 60, and 80% cytolysis.

[0192] The Slope: to describe the steepness, incline, gradient, or changing rate of a curve within a given time window. For each selected well, the Software calculates the Slope of the Cell Index (or Normalized Cell Index) curve over a chosen Time frame. Data points within this time frame arc fit to a straight line.

[0193] Example data from these functional parameters are shown in Figure 6.

[0194] Figure 7A-7G illustrate assay results in epCAM CART production (CD3 T cells + EpCAM Virus), using a G-REX device, XV1VO medium + IL-2. Figures 7A-7B illustrate bioenergetics work, Figure 7C illustrates metabolic poise, Figure 7D illustrates mitochondrial bioenergetic capacity, Figure 7E illustrates glycolytic bioenergetic capacity, Figure 7F illustrates spare respiratory capacity, and Figure 7G illustrate total bioenergetic capacity.

[0195] Figures 8A-8B illustrate data collected and analyzed for Immunophenotypic Characterization of EpCam CART Production (CD3 T cells + EpCAM Virus), using a G-REX Device, XVIVO Medium + IL-2, with Figure 8 A showing CART immunophenotyping, and Figure 8B showing Activation / Exhaustion levels.

[0196] Figures 9A-9C illustrate data collected and analyzed for Killing Potency of EpCam CART Production (CD3 T cells + EpCAM Virus), using a G-REX Device, XVIVO Medium + IL- 2 after 6, 9, and 10 days, respectively. Figures 9D-9F illustrate data collected and analyzed for Killing Potency of EpCam CART Production (CD3 T cells + EpCAM Virus), using a G-REX Device, XVIVO Medium + IL-2 across the three time periods in which KT50, slope, and AUC of % cytolysis were derived from the time course of %cytolysis shown in Figures 9A-9C. The killing potency of the EpCAM CAR transduced samples gradually decreased over time, as demonstrated by an increase in KT50, a decrease in the slope, and in the AUC of % cytolysis

[0197] Figure 10A-10G illustrate assay results in Metabolic Characterization of PBMCs expansion (PBMCs no virus transduction), using a G-REX Device, XVIVO Medium + IL-2. Figures 10A-10B illustrate bioenergetics work, Figure 10C illustrates metabolic poise, Figure 10D illustrates mitochondrial bioenergetic capacity, Figure 10E illustrates glycolytic bioenergetic capacity, Figure 10F illustrates spare respiratory capacity, and Figure 10G illustrates total bioenergetic capacity.

[0198] Figures 11 A- 11C illustrate Immunophenotypic Characterization of PBMCs expansion (PBMCs no virus transduction), using a G-REX Device, XVIVO Medium + IL-2. The illustrated graphs in Figures 11A-11C show that during the 10 days of expansion in the G-REX device, the monocytes, B cells, and NK cells in PBMCs on day 0 gradually decreased. CD3+ T-cells almost took over the whole culture on day 10. Additionally, the data show that effector memory T-cells (Tern) gradually increased. However, stem cell-like T-cells (Tscm), central memory T-cells (Tern), and fully differentiated effector T-cells (Temra) gradually decreased

[0199] Figures 12A-12C illustrate Killing Potency of PBMCs expansion (PBMCs no virus transduction), using a G-REX Device, XVIVO Medium + IL-2 after 6, 9, and 10 days, respectively.

[0200] Figure 13 illustrates NK cell killing potency of NK cells that were expanded in a T-75 flask according to the procedures described herein.

[0201] Figure 14 illustrates a number of human NK cells expanded in 6 multiwell plate using culture medium supplemented with IL2 using the procedures described herein.

[0202] Figures 15A-15G illustrate assay results in Metabolic Profile of human NK cells expanded in a 6 multiwell plate using Stem Cell NK cell expansion system. Figures 15A-15B illustrate bioenergetics work, Figure 15C illustrates metabolic poise, Figure 15D illustrates mitochondrial bioenergetic capacity, Figure 15E illustrates glycolytic bioenergetic capacity, Figure 15F illustrates spare respiratory capacity, and Figure 15G illustrates total bioenergetic capacity.

[0203] Figures 16A-16G illustrate assay results in Metabolic Profile of human NK cells expanded in 6 multi well plate using culture medium supplemented with IL2. Figures 16A-16B illustrate bioenergetics work, Figure 16C illustrates metabolic poise, Figure 16D illustrates mitochondrial bioenergetic capacity, Figure 16E illustrates glycolytic bioenergetic capacity, Figure 16F illustrates spare respiratory capacity, and Figure 16G illustrates total bioenergetic capacity.Example 2:

[0204] The maximal therapeutic efficacy of T cells depends on cell potency and metabolic fitness. Various instrument based analyses are shown to have ability to screen in real time for T cells with high potency after preconditioning with amino acids under a range of concentrations. Using real-time potency assays in parallel to the instrument assays, the metabolic fitness of these potent arginine preconditioned T cells may be associated with increased oxidative phosphorylation (see e.g., Figures 17A-17B). Figure 17A illustrates the impact of arginine precondition using a mitochondrial stress test in which a kinetic trace of OCR in basal conditions and after injection of Oligomycin (1.5 pM), FCCP (1 pM) , and rotenone / Antimycin A (0.5 pM each) are compared, while Figure 17B illustrates SRC of nontransdudcd, expanded in RPMI, or preconditioned in 6 mM arginine for seven days.

[0205] T cell persistence and memory phenotypes are associated with higher SRC, which provides valuable information about the bioenergetics of a T cell, which can be correlated with T cell potency using the visual or impedance analyses. The combination of these techniques supplies valuable information about the critical parameters of T cell function relevant to cell therapy. The data from both assays provide a comprehensive view of potency and metabolic fitness that can enable testing critical process parameters during CAR T cell manufacturing.

[0206] Despite minimal hands-on time required for assay set up using the described technologies, the wealth of kinetic information generated would be difficult to acquire usingtraditional methods. Figure 18D illustrates this fact, where the % cytolysis traces increase up to ~80 hours before plateauing or, in some cases, decrease in value. This change could either indicate T cell exhaustion or death not confirmed experimentally. While these conclusions may be apparent in real-time impedance and imaging data, these conclusions are less so when using endpoint assays. Figures 18A-18D illustrate the impact of elevated concentrations of arginine have on MART-1 TCR T cell killing efficacy. Figure 18A-18B provide impedance traces for Mel-624.38 target cell cytotoxicity assays treated with MART-1 TCR T cells preconditioned for 7 days in RPMI medium supplemented with an additional 0, 1.5, 3, or 6 mM arginine with an E:T ratio of 5 in Figure 18A and 10 in Figure 18B, and the black arrow denotes time of T cell addition (24 hours). Figure 18C illustrates several images from the 60-hour time point, highlighting the stimulatory effect 6 mM arginine preconditioning has on killing efficacy in which the scale bars = 200 pm. Figure 18D illustrated real-time % cytolysis calculated from impedance data in Figure 18A.

[0207] There arc several mechanisms by which T cells can augment cytotoxic potential, through increased production of perforin / granzyme / cytokines or proliferation. For example, T cells grown in 6 mM arginine- supplemented media consistently grew more slowly than their counterparts grown in regular RPMI measured at the end of 7-day preconditioning by total live cell count (MART-1 TCR T cells in RPMI = 6.5 million and MART-1 TCR T cells in 6 mM arginine = 1.9 million).

[0208] Additionally, the present disclosure reiterates the importance of making comparisons within a single assay versus between assays run on different days. Table 2 shows % cytolysis data for MART-1 TCR T cells produced from different donors and for different transductions of cells from the same donor. While the % cytolysis can vary significantly for assays run on different days, by comparing regular versus supplemented samples within the same assay the results are clear that preconditioning in high arginine always results in a stimulation of killing efficacy. Access to this type of information was much more difficult in the past. By eliminating the need for tedious, less informative endpoint analyses, the presently described assays significantly lower the barrier to systematically evaluate different preconditioning protocols.

[0209] Table 2

[0210] Cells

[0211] Human melanoma target cells Mel-624.38 (HLA-A*0201+) and Mel-624.28 (HLA- A*0201-), as well as PG 13 retrovirus producing cells were used in the analysis provided in Example 2. Each of these cell populations were grown at 37 °C / 5% CO2 in a growth medium containing 10% heat inactivated Fetal Bovine Serum (FBS) and 1% Pen / Strep. The Mel-624.38- Red and Mel-624.28-Red cell populations, which stably expresses nuclear-localized red fluorescent protein (RFP), were produced by transduction with a Lentivirus reagent for nuclear labeling of live cells at a multiplicity of infection of one. Cells were subsequently grown in the presence of puromycin (2 pg / mL) for 14 days to select for stable clones expressing RFP. The PG13 cell population used in this example produces the SAMEN-DMF5 retrovirus, which encodes a T cell receptor (TCR) that recognizes a fragment of MART-1 (27-AAGIGILTV-35) when it is displayed on cells’ surface by HLA-A*0201. To characterize transduction efficiency, this TCR is fused to a CD34 tag. Frozen CD3+ T cells from three different donors for evolution in this example. After thawing, T cells were added to 10 mL of RPMI containing 10% heat inactivated FBS and 1% Pen / Strep. Cells were immediately pelleted at 37 °C and 300 x g for 5 minutes, then resuspended in 10 mL of the same medium supplemented with 300 KJ of rhIL-2 and 25 pL / mL of a T cell Activator. T cells were then incubated in a 6-well plate for 3 or 5 days. Although cells were not counted during this incubation period, fresh media were added as needed to maintain neutral pH. Cells were then subjected to the transduction protocol described below.

[0212] T cell transduction

[0213] 3 x 106PG 13 retrovirus-producing cells in complete growth medium were seeded in aT75 flask. After 24 hours, the growth medium was supplemented with 10 mM sodium butyrate (which aids retrovirus production). After 8 to 10 hours of incubation with sodium butyrate, this medium was replaced with fresh DMEM + 10% FBS + 1% Pen / Strep. The following day, the retrovirus-containing medium was collected and passed through a 0.45 pm cellulose acetate filter. Then, 2 mL of the retrovirus-containing medium was centrifuged onto the retronectin-coated (13 pg / cm2) wells of a 24-well plate at 37 °C and 2,000 x g for 2 hours. Subsequently, media wereaspirated from these wells and 1 x 106 activated T cells were added along with 1 mL of the retrovirus-containing medium. After centrifugation at 37 °C and 2,000 x g for 10 minutes, the medium was supplemented with 600 IU of rhIL2 / mL. The T cells were then incubated at 37 °C overnight. The following day, T cells were collected and centrifuged at 37 °C and 300 x g for 5 minutes and were then resuspended in fresh RPMI + 10% FBS +1% Pen / Strep supplemented with 600 IU of rhIL2 / mL. After a 2-day rest / recovery period, the T cells were subjected to a second round of the same transduction protocol to increase transduction efficiency.

[0214] Metabolic preconditioning

[0215] Immediately after the second round of transduction, T cells were transferred to RPMI + 10% FBS + 1% Pen / Strep supplemented with varying concentrations (0, 1.5, 3, or 6 mM) of arginine, glutamine, or leucine. Because the concentrations of arginine, glutamine, and leucine in the base RPMI medium are 1.15, 2.06, and 0.38 mM, respectively, the final amino acid concentrations evaluated during this prcincubation step were: 1.15 / 2.65 / 4.15 / 7.15 mM (arginine), 2.06 / 3.56 / 5.06 / 8.06 (glutamine), and 0.38 / 1.88 / 3.38 / 6.38 (leucine). T cells were cultured in supplemented media at a density of 1 x 106cell / mL for 7 days and base RPMI groups were considered a baseline control sample. The cells were counted and volume was adjusted daily to maintain the cell density. After the seventh day, the killing capacity and bioenergetics of the T cells were evaluated.

[0216] Cytotoxicity assay

[0217] T cell killing efficacy was evaluated using an impedance and visual scanning instrument. To each well of a wellplate, 50 pL of complete growth medium (DMEM + 10% FBS; no antibiotics) was added. After recording the background impedance signal, 8,000 target cells (Mel-624.38-Red or Mel-624.28-Red) were added to each well in a volume of 50 pL (bringing the total volume to 100 pL / well). After allowing the cells to settle for 30 minutes at room temperature, the plate was transferred to the instrument and data acquisition was initiated. While impedance readings were collected every 15 minutes, photos were taken every 60 minutes. In each well, four fields of view were captured for each channel (brightfield and red fluorescence). Exposure times were as follows: brightfield = automatically adjusted by the instrument software, and ed = 400 ms. T cells subjected to metabolic preconditioning were prepared by pelleting at 37 °C and 300 x g for 5 minutes, then resuspending them in DMEM + 10% FBS. After 24 hours, 100 pL of preconditioned T cells were added to each well (bringing the total volume to 200 pL / well). T celldensities were varied to achieve E:T ratios of 5:1 or 10:1. Data acquisition was then resumed for an additional 4 days.

[0218] Flow cytometry

[0219] Validation of HLA-A*2:01 expression levels in the Mel-624.38 and Mel-624.28 cell populations was performed using a flow cytometer and APC-antihuman HLA-A2 Clone BB 7.2 Transduction efficiency for the DMF5 receptor in T cells was assessed using an APC-conjugated anti-CD34 antibody.

[0220] Metabolic assay

[0221] T cells subjected to the preconditioning protocol described in the present example were resuspended in a metabolic measurement medium supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine. T cells (150,000 cells) were then seeded into each well of a microplate. T cell bioenergetics were analyzed in six replicate wells using a Mito Stress Test kit on a metabolic analyzer. In some embodiments, the stress test kit uses an optimized uncoupled reagent for T cells BAM 15 in addition to or alternatively to FCCP, which can provide a more robust response and allows for obtaining additional information about the glycolytic activity of T cells in one assay.

[0222] Preliminary validation of TCR activity and specificity

[0223] Before interrogating the impact of varying preconditioning parameters, the function and specificity of the transduced receptor should be validated. Towards this end, wells with included impedance sensing electrodes were seeded with 8,000 Mel-624.38 melanoma target cells. Figures 19A-19E show preliminary validation of DMF5 TCR activity and specificity. Figures 19A-19B illustrate using impedance to track MART- 1 TCR T cell-mediated killing of Mel-624.38 (in Figure 19A) and Mel-624.28 (in Figure 19B) target cells that display the MART-1 peptide on their surface. The black arrow at 24 hours denotes the time when T cells were added. Figures 19C- 19D illustrate composite brightfield + red fluorescence photos corroborate killing kinetics observed in impedance traces. Figure 19E illustrates a flow-based validation of transduction efficiency using APC labeled anti-CD34 antibody. Transduction efficiency varied between 15 and 30%.

[0224] After allowing cells to adhere and proliferate for 24 hours, a T cell cytotoxicity assay was performed using an E:T ratio of 5:1. Addition of mock transduced T cells had minimal impact on the growth and proliferation of Mel-624.38 target cells over the 75-hour window (black tracein Figure 19A). In contrast, MART-1 TCR T cells led to an immediate and sustained decrease in impedance, consistent with target cell death (aqua trace in Figure 19A). The killing observed in the present example study was expected considering the previously characterized activity of the DMF5 receptorl l, and the fact that Mel-624.38 cells express HLA-A*02:01 - which is required for displaying the MART- 127-35 peptide that DMF5 recognizes (Figure 19A). When the same assay is repeated with Mel-624.28 cells, which express significantly less HLA-A*02:01 and therefore present less MART-127-35 peptide on their surface, no killing is observed (Figure 19B). This conclusion is corroborated by the real-time imaging time course shown in Figures 19C and 19D. Transduction efficiency was measured using anti-CD34 APC. Figure 19E shows CD34 staining of T cells after transduction to be approximately 30%. Besides confirming the efficacy and specificity of the DMF5 TCR, these data provide a reference point to inform setup of future assay iterations. Specifically, this data set demonstrates that when seeding 8,000 melanoma target cells, using an E: T of 5 yields robust killing within a reasonable time frame.

[0225] Arginine preconditioning enhances T cell cytotoxicity and mitochondrial respiration

[0226] Differentiation of T cell fitness and function are intimately linked to their metabolism. Preconditioning TCR T cells in vitro in an elevated concentration of arginine shifted bioenergetics (increasing oxidative phosphorylation), and was associated with increased killing efficacy once T cells were transfused into tumor bearing mice. Unfortunately, screening a broad array of T cell preconditioning parameters using mouse xenograft models is both costly and time-consuming. For this reason, an in vitro assay to evaluate the functional impact of diverse preconditioning parameters would be advantageous. As a demonstration of an impedance / visual assay instrument’ s ability to address these types of questions, the killing efficacy of MART-1 TCR T cells was evaluated after being preconditioned in three different concentrations of arginine, glutamine, and leucine. Since glutamine and leucine feed into the TCA cycle and have a large impact on T cell metabolism and activation, their concentration was also titrated. A metabolic assay was performed simultaneously with the impedance and visual assays to assess the relationship between cytotoxicity and bioenergetics under different conditions.

[0227] After transduction, T cells were metabolically preconditioned for 7 days in RPMI supplemented with 1.5, 3, and 6 mM arginine, and base RPMI media. The killing assay was set up at two E:T ratios, 5:1 and 10:1 , to demonstrate the impedance / visual assay instrument’s capabilityto distinguish differences in potency using impedance curves (Figures 18A and 18B). To distinctly monitor killing kinetics during a more extended period of time and between samples, the 5:1 E:T ratio was better than 10:1 (Figures 18A and 18B). All further killing assays were performed at a 5:1 E:T ratio. While preconditioning MART-1 TCR T cells in RPMI supplemented with 1.5 or 3 mM arginine had minimal impact, 6 mM arginine stimulated killing efficacy substantially (Figures 18A and 18B). This is highlighted in the real-time % cytolysis plots of Figure 18D, and is corroborated using impedance / visual assay instrument’s imaging capabilities in Figure 18C. At the 60-hour time point, target cells with no T cells added proliferated to confluence and displayed a spread out morphology. In contrast, in the presence of MART-1 TCR T cells preconditioned in regular RPMI, the melanoma target cells were rounded and displayed significant clustering (Figure 18C). This death phenotype was more robust when using MART-1 TCR T cells preincubated in RPMI supplemented with 6 mM arginine.

[0228] Screening preconditioning parameters: temporal duration

[0229] For autologous T cell therapies, a significant emphasis is currently placed on reducing processing time between T cell isolation and patient reinfusion. The benefits of 6 mM arginine shown above were observed after a 7-day preconditioning step after T cell transduction. An obvious question is raised: can similar gains be achieved using protocols of shorter duration? To address this, MART-1 TCR T cells were preconditioned in RPMI supplemented with 6 mM arginine for either 2, 4, or 7 days before being used in a killing assay (Figures 18A to 18C). The differences in killing efficacy evident in these real-time plots are brought into greater focus by plotting the % specific cytolysis at the 60-hour time point (Figure 18D). After a preconditioning step of only 2 days, T cells grown in elevated arginine have a killing efficacy that is >2x higher compared to their counterparts grown in regular RPMI. Extending the duration of preconditioning from 2 to 4 days has minimal impact on the RPMI control cells but more than doubles the killing efficacy of the high arginine cells. Upon increasing the preconditioning duration from 4 to 7 days, something interesting happens; the RPMI control T cells display a ~6x increase in killing efficacy while the high arginine T cells improve only minimally. It is well known that the length of time T cells are allowed to recover post transduction can have an impact on their fitness / function. This data set highlights that the gains achieved via a preconditioning step are most likely attributable to multiple parameters, including both the medium composition and the protocol’s duration.

[0230] Glutamine and leucine preconditioning suppressed

[0231] Figures 20A-20E illustrate that T Glutamine and leucine preconditioning suppresses T cell mediated killing. Figure 20A shows impedance data show the effect of glutamine concentration (O to 6 mM) after 7 days of preconditioning with 5:1 E:T ratio. Figure 20B Impedance data show the effect of leucine concentration (0 to 6 mM) after 7 days of preconditioning with 5:1 E:T ratio. Figure 20C is an impedance graph showing simultaneous assessment of three amino acids together in one assay. Figure 20D illustrates image analysis using where red total integrated intensity is plotted against time. Figure 20E illustrate representative images from each group. The color of each photo’s outline corresponds to the plot colors in Figure 20C and the scale bars = 200 pm.

[0232] Figures 21 A-21 D illustrate that the duration of preconditioning in elevated arginine has a significant impact on MART-1 TCR T cell killing efficacy. 24 hours after seeding Mel-624.38 target cells, the cytotoxicity assay was performed using the MART-1 TCR T cells (E:T = 5) preconditioned in RPMI or RPMI + 6 mM arginine for cither 2 days (shown in Figure 21A), 4 days (shown in Figure 21B), or 7 days (shown in Figure 21C), in which the black arrows denote time of T cell addition. Figure 21D shows calculations using impedance data from Figures 11A-11C for % specific cytolysis calculated for the 60-hour time point.

[0233] T cell mediated killing of 624.38 HLA-A*2:01+ melanoma cells compared to RPMI preconditioned T cells. Glutamine and leucine feed into the TCA cycle and are important amino acids involved in T cell activation and proliferation. Figures 20A and 20B show impedance killing curves for Mel-624.38 target cells using MART-1 TCR T cells that had been preconditioned for 7 days in RPMI supplemented with 0, 1.5, 3, and 6 mM leucine and glutamine. Preconditioning with leucine and glutamine were found to decrease killing efficacy. In Figures 21A and 21B, while the T cells preconditioned in unsupplemented RPMI (aqua trace) do indeed cause a drop in impedance relative to the target cell control (black trace), the extent of killing is less substantial than what was observed in Figure 19A. This interassay variability is a reflection of the biology, not the instrumentation. Although this MART-1 TCR construct always affects a killing response, the magnitude varies when using T cells from different donors (expected), and even between rounds of transduction using T cells from the same donor. This difference highlights the necessity of examining the impact of different preconditioning parameters in parallel rather than across different assays. Because the basal killing efficacy of the MART-1 TCR T cells can vary from one preparation to the next, an additional assay was conducted with all three amino acids at 6 mM toensure that the impact of 6 mM arginine supplementation is reproducible (Figure 20C). Similarly to the previous results, preconditioning with 6 mM arginine was found to stimulate killing efficacy while preconditioning with 6 mM of leucine and glutamine were found to decrease killing efficacy. These conclusions are corroborated by quantifying the total integrated intensity of target cells’ red fluorescent signal (Figure 20D).[02341 Note that although the impedance and imaging data lead to similar conclusions, they do so with differing degrees of sensitivity. For the 6 mM arginine-supplemented sample, target cell death is detectable by the ~35-hour time point using impedance (Figure 20C) but is not detectable until the ~50-hour time point using imaging (Figure 20D). This is a reflection of what each readout is detecting. Whereas impedance detects even subtle changes in attachment strength occurring in the very early stages of the target cell death cascade, the image -based readout does not register target cell death until much later when the nuclear-localized red fluorescent protein is degraded or target cells arc lysed. This emphasizes the importance of using both impedance and imaging methods for information richness, validation, and mechanism of action.

[0235] Certain terms are used throughout the description and claims to refer to particular features or components. As one skilled in the art will appreciate, different persons may refer to the same feature or component by different names. This document does not intend to distinguish between components or features that differ in name but not function.

[0236] Without further elaboration, it is believed that one skilled in the ait can use the preceding description to use the claimed inventions to their fullest extent. The examples and aspects disclosed herein are to be construed as merely illustrative and not a limitation of the scope of the present disclosure in any way. It will be apparent to those having skill in the art that changes may be made to the details of the above-described examples without departing from the underlying principles discussed. In other words, various modifications and improvements of the examples specifically disclosed in the description above are within the scope of the appended claims. For instance, any suitable combination of features of the various examples described is contemplated.

[0237] Within the claims, reference to an element in the singular is not intended to mean “one and only one” unless specifically stated as such, but rather as “one or more” or “at least one”. Unless specifically stated otherwise, the term “some” refers to one or more. No claim element is to be construed under the provision of 35 U.S.C. § 112(f) unless the element is expressly recited using the phrase “means for” or “step for”. All structural and functional equivalents to the elementsof the various embodiments described in the present disclosure that are known or come later to be known to those of ordinary skill in the relevant art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, nothing disclosed in the present disclosure is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

Claims

CLAIMSWhat is claimed is:

1. A method of producing immune cells, comprising: dividing a first sample of immune cells into a first population and a second population; cultivating the first population of the immune cells in a first bioreactor to produce a first cultivated population of immune cells; cultivating the second population of the immune cells in a second bioreactor of a different design than the first bioreactor to produce a second cultivated population of immune cells; measuring a first value for a potency parameter, a first value for a metabolic parameter, or both, at a harvest time for the first cultivated population of immune cells, and a second value for the potency parameter, a second value for the metabolic parameter, or both, at the harvest time for the second cultivated population of immune cells; and after the harvest time: collecting a second sample of the immune cells; selecting one of the first bioreactor or the second bioreactor based on which of the respective first cultivated population of immune cells and the second cultivated population of immune cells was measured with a higher value for the potency parameter, a higher value for the metabolic parameter, or both; and cultivating the second sample of the immune cells in the one of the first biorcactor and the second bioreactor selected to produce a cultivated second sample.

2. The method of claim 1, wherein the first sample and the second sample were collected from the same biological subject.

3. The method of claim 1, wherein the first sample and the second sample were collected from different biological subjects.

4. The method of any of claims 1-3, further comprising administering cultivated immune cells from the cultivated second sample to a biological subject.

5. The method of claim 4, wherein the biological subject is the same biological subject from which the second sample was collected.

6. The method of claim 4, wherein the biological subject is a different subject from which the second sample was collected.

7. The method of any of claims 1-6, wherein the potency parameter comprises a plurality of potency sub-parameters, wherein each potency sub-parameter of the plurality of potency subparameters satisfies a corresponding potency threshold.

8. The method of any of claims 1-7, wherein measuring the first value, the second value, or both, for the potency parameter is performed via a potency assay, e.g., an XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

9. The method of any of claims 1-8, wherein measuring the first value, the second value, or both, for the potency parameter is performed via an instrument analysis comprising at least one of an impedance measurement or image analysis of killing efficacy.

10. The method of any of claims 1-9, wherein the metabolic parameter comprises a plurality of metabolic sub-parameters, wherein each metabolic sub-parameter of the plurality of metabolic sub-parameters satisfies a corresponding metabolic threshold.

11. The method of any of claims 1-10, wherein measuring the first value, the second value, or both, for the metabolic parameter is performed via a metabolic assay, e.g., a SEAHORSE XF™ T cell Metabolic Fitness assay (offered by Agilent Technologies Inc. of Santa Clara California).

12. The method of any of claims 1-11, wherein measuring the first value, the second value, or both, for the metabolic parameter is performed via an instrument analysis comprising a measurement of mitochondrial respiration, glycolysis, ATP production, or a combination thereof.

13. The method of any of claims 1-12, further comprising measuring a first value for an expression parameter, at the harvest time for the first cultivated population of immune cells, and a second value for the expression parameter, at the harvest time for the second cultivated population of immune cells.

14. The method of claim 13, wherein the expression parameter comprises a plurality of expression sub-parameters, wherein each expression sub-parameter of the plurality of expression sub-parameters satisfies a corresponding expression threshold.

15. The method of claim 13 or 14, wherein measuring the first value, the second value, or both, for the expression parameter is perfoimed via flow cytometry.

16. The method of any of claims 1-15, wherein the first biorcactor and the second biorcactor are controlled during cultivation according to one or more (e.g., 2, 3, 4, 5, 6, 7, or all) operational settings for: nutrient and gas exchange; oxygen control; pH control; a feeding regime of media exchange versus media addition; mixing or shear force; device O2 permeability; vessel size; removal of activation beads; or enrichment / selection of a particular subpopulation.

17. The method of any of claims 1-16, wherein selecting the one of the first bioreactor or the second bioreactor is further based on which of the first bioreactor and the second bioreactor reaches a desired threshold for the potency parameter, the metabolic parameter, or both, and optionally the expression parameter, faster.

18. The method of any of claims 1-17, wherein the potency parameter, the metabolic parameter, or both, and optionally the expression parameter, is associated with one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or all) of: clonal abundance; clonal expansion; transduction efficiency; proliferation capacity; cytotoxicity capacity; persistence; sternness; or immune cell exhaustion.

19. The method of any of claims 1-18, wherein the higher value for the potency parameter, the higher value for the metabolic parameter, or both, and optionally a higher value for the expression parameter, indicates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or all) of: an increased clonal abundance; an increased clonal expansion; an increased transduction efficiency; an increased proliferation capacity; an increased cytotoxicity capacity; an increased persistence; an increased metabolic fitness; an increased sternness; or an reduced immune cell exhaustion.

20. The method of any of claims 1-19, wherein the harvest time occurs at a predefined interval after initiation of cultivation, e.g., is at least 3 hours, 6 hours (e.g. at least 6. 12, 18, 24, 30, 36, 42, 48, 60, 72 hours), 9 hours, 12 hours (e.g., at least 24, 36, 48, 60, or 72 hours), e.g., 12- 72 hours, 24-60 hours, 36-48 hours, 12-60 hours, 12-48 hours, 12-36 hours, 12-24 hours, 60-72 hours, 48-72 hours, 36-72 hours, 24-72 hours, 24-48 hours, 36-60 hours, or 48-72 hours, or at least 1 day (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, or 14 days), e.g., 1-14 days, 2-13days, 3-12 days, 4-11 days, 5-10 days, 6-9 days, 7-8 days, 1-12 days, 1-10 days, 1-8 days, 1-6 days, 1-4 days, 1-2 days, 12-14 days, 10-14 days, 8-14 days, 6-14 days, 4-14 days, 2-14 days, 1-3 days, 2-4 days, 3-5 days, 4-6 days, 5-7 days, 6-8 days, 7-9 days, 8-10 days, 9-11 days, 10-12 days, 11-13 days, or 2-3 days from initiating cultivation.

21. A method of producing immune cells, comprising: acquiring a value for a potency parameter, a value for a metabolic parameter, or both, from a population of immune cells being cultivated in a bioreactor; monitoring the value for the potency parameter, the value for the metabolic parameter, or both, during a predefined period of time; and in response to identifying a change in the value for the potency parameter, a change in the value for the metabolic parameter, or both, satisfying a harvesting threshold before an end of the period of time, harvesting the population of immune cells from the biorcactor before the predefined period of time expires.

22. The method of claim 21, wherein monitoring the value for the potency parameter, the value for the metabolic parameter, or both, comprises acquiring one or more (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, or more) additional value for the potency parameter, one or more (e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, or more) additional value for the metabolic parameter, or both, from the population of immune cells being cultivated in the bioreactor.

23. The method of claim 21 or 22, wherein the value for the potency parameter, the value for the metabolic parameter, or both, is monitored at least once every 1, 2, 3, 6, 9, 12, 18, or 24 hours, or at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

24. The method of any of claims 21-23, wherein the potency parameter comprises a plurality of potency sub-parameters, wherein each potency sub-parameter of the plurality of potency subparameters satisfies a corresponding potency threshold.

25. The method of any of claims 21-24, wherein acquiring the value for the potency parameter is performed via a potency assay, e.g., an XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

26. The method of any of claims 21-25, wherein acquiring the value for the potency parameter is performed via an instrument analysis comprising at least one of an impedance measurement or image analysis of killing efficacy.

27. The method of any of claims 21-26, wherein the metabolic parameter comprises a plurality of metabolic sub-parameters, wherein each metabolic sub-parameter of the plurality of metabolic sub-parameters satisfies a corresponding metabolic threshold.

28. The method of any of claims 21-27, wherein acquiring the value for the metabolic parameter is performed via a metabolic assay, e.g., SEAHORSE XF™ T cell Metabolic Fitness assay (offered by Agilent Technologies Inc. of Santa Clara California).

29. The method of any of claims 21-28, wherein acquiring the value for the metabolic parameter is performed via an instrument analysis comprising a measurement of mitochondrial respiration, glycolysis, ATP production, or a combination thereof.

30. The method of any of claims 21-29, further comprising acquiring a value for an expression parameter from the population of immune cells being cultivated in the bioreactor, and monitoring the value for the expression value during the predefined period of time.

31. The method of claim 30, wherein monitoring the value for the expression parameter comprises acquiring one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for the expression parameter from the population of immune cells being cultivated in the bioreactor.

32. The method of claim 30 or 31, wherein the value for the expression parameter is monitored at least once every 1, 2, 3, 6, 9, 12, 18, or 24 hours, or at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, or 14 days.

33. The method of any of claims 30-32, wherein the expression parameter comprises a plurality of expression sub-parameters, wherein each expression sub-parameter of the plurality of expression sub-parameters satisfies a corresponding expression threshold.

34. The method of any of claims 30-33, wherein acquiring the value for the expression parameter is performed via flow cytometry.

35. The method of any of claims 21-34, wherein the bioreactor is controlled during cultivation according to one or more (e.g., 2, 3, 4, 5, 6, 7, or all) operational settings for: nutrient and gas exchange; oxygen control; pH control; a feeding regime of media exchange versus media addition; mixing or shear force; device O2 permeability; vessel size; removal of activation beads; or enrichment / selection of a particular subpopulation.

36. The method of any of claims 21-35, wherein the potency parameter, the metabolic parameter, or both, and optionally the expression parameter, is associated with one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or all) of: clonal abundance; clonal expansion; transduction efficiency; proliferation capacity; cytotoxicity capacity; persistence; sternness; or immune cell exhaustion.

37. The method of any of claims 21-36, wherein the higher value for the potency parameter, the higher value for the metabolic parameter, or both, and optionally a higher value for the expression parameter, indicates one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or all) of: an increased clonal abundance; an increased clonal expansion; an increased transduction efficiency; an increased proliferation capacity; an increased cytotoxicity capacity; an increased persistence; an increased sternness; or a reduced immune cell exhaustion.

38. The method of any of claims 21-37, wherein the predefined period of time is 3 hours, 6 hours (e.g. at least 6. 12, 18, 24, 30, 36, 42, 48, 60, 72 hours), 9 hours, at least 12 hours (e.g., at least 24, 36, 48, 60, or 72 hours), e.g., 12-72 hours, 24-60 hours, 36-48 hours, 12-60 hours, 12-48 hours, 12-36 hours, 12-24 hours, 60-72 hours, 48-72 hours, 36-72 hours, 24-72 hours, 24-48 hours, 36-60 hours, or 48-72 hours, or at least 1 day (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), e.g., 1-14 days, 2-13 days, 3-12 days, 4-11 days, 5-10 days, 6-9 days, 7-8 days, 1-12 days, 1-10 days, 1-8 days, 1-6 days, 1-4 days, 1-2 days, 12-14 days, 10-14 days, 8-14 days, 6-14 days, 4-14 days, 2-14 days, 1-3 days, 2-4 days, 3-5 days, 4-6 days, 5-7 days, 6-8 days, 7-9 days, 8-10 days, 9-11 days, 10-12 days, 11-13 days, or 2-3 days from initiating cultivation.

39. The method of any of claims 21-38, wherein the change in the value for the potency parameter, the value for the metabolic parameter, or both, and optionally the expression parameter, outside of the harvesting threshold is identified any time within 72 hours (e.g., within 60, 48, 36, 24, or 12 hours), e.g., 12-72 hours, 24-60 hours, 36-48 hours, 12-60 hours, 12-48 hours, 12-36 hours, 12-24 hours, 60-72 hours, 48-72 hours, 36-72 hours, 24-72 hours, 24-48 hours, 36-60 hours, or 48-72 hours, or within 14 days (e.g., within 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day), e.g., 1-14 days, 2-13 days, 3-12 days, 4-11 days, 5-10 days, 6-9 days, 7-8 days, 1- 12 days, 1 -10 days, 1-8 days, 1 -6 days, 1-4 days, 1 -2 days, 12-14 days, 10-14 days, 8-14 days, 6-14 days, 4-14 days, 2-14 days, 1-3 days, 2-4 days, 3-5 days, 4-6 days, 5-7 days, 6-8 days, 7-9 days, 8-10 days, 9-11 days, 10-12 days, 11-13 days, or 2-3 days from initiating cultivation.

40. The method of any of claims 21-39, wherein harvesting the population of immune cells from the bioreactor, before the predefined period of time expires, removes less than 100% of the population of immune cells from the bioreactor, the method further comprising: cultivating a remainder of the population of the immune cells not harvested from the bioreactor until the predefined period of time expires; and harvesting the remainder of the population of the immune cells from the bioreactor in response to the predefined period of time expiring.41 . The method of any of claims 21-40, wherein in response to not identifying the change in the value for the potency parameter, the change in the value for the metabolic parameter, or both, and optionally the change in the value for the expression parameter, satisfying the harvesting threshold before the end of the period of time, harvesting the population of immune cells from the bioreactor at the end of the predefined period of time.

42. A method of evaluating an optimal therapeutic harvest time of an immune cell from a bioreactor, comprising: acquiring a value for each of a plurality of parameters from a population of immune cells in the bioreactor, wherein the plurality of parameters comprises two or more of: an expression parameter measured by flow cytometry; a potency parameter measured by impedance, imaging, or both; or a metabolic parameter comprising mitochondrial respiration, glycolysis, ATP production, bioenergetic capacity or a combination thereof, wherein the plurality of parameters are associated with one or more of properties comprising: clonal abundance; clonal expansion; transduction efficiency; proliferation capacity;cytotoxicity capacity; persistence; sternness; or exhaustion, wherein a change in the value for each of the plurality of parameters, or a combination thereof, is indicative of the optimal therapeutic harvest time of the immune cell.

43. The method of claim 42, wherein the change in the value for each of the plurality of parameters, or a combination thereof, indicates one or more of properties comprising: an increased clonal abundance; an increased clonal expansion; an increased transduction efficiency; an increased proliferation capacity; an increased cytotoxicity capacity; an increased persistence; an increased sternness; or a reduced exhaustion.

44. The method of claim 42 or 43, wherein the optimal therapeutic harvest time provides (a) the optimal expression of a molecule by the immune cells, (b) the optimal killing potency of the immune cells, (c) the optimal bioenergetic profile and spare respiratory capacity of the immune cells, or any combination of (a), (b), or (c).

45. The method of any of claims 42-44, further comprising acquiring one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for each of the plurality of parameters from the population of immune cells.

46. The method of claim 45, wherein the one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for each of the plurality of parameters is acquired at least once every 1, 2, 3, 6, 9, 12, 18, or 24 hours, or at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, from the population of immune cells.

47. The method of any of claims 42-46, wherein the value for the potency parameter is acquired via XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

48. The method of any of claims 42-47, wherein the value for the metabolic parameter is acquired via SEAHORSE XF® T cell Metabolic Fitness assay.

49. A method of optimizing a control parameter of a bioreactor, comprising: acquiring a value for each of a plurality of parameters from a population of immune cells in the bioreactor, wherein the plurality of parameters comprises two or more of: an expression parameter measured by flow cytometry; a potency parameter measured by impedance, imaging, or both; or a metabolic parameter comprising mitochondrial respiration, glycolysis, ATP production, bioenergetic capacity or a combination thereof, wherein the plurality of parameters are associated with one or more of properties comprising: clonal abundance; clonal expansion; transduction efficiency; proliferation capacity; cytotoxicity capacity; persistence; sternness; or exhaustion, wherein a change in the value for each of the plurality of parameters, or a combination thereof, is indicative of a control parameter that requires a modification; and further modifying the control parameter, thereby optimizing the control parameter of the bioreactor.

50. The method of claim 49, wherein the control parameter is associated with one or more (e.g., 2, 3, 4, 5, 6, 7, or all) operational settings for: nutrient and gas exchange; oxygen control; pH control; a feeding regime of media exchange versus media addition; mixing or shear force; device O2 permeability; vessel size; removal of activation beads; or enrichment / selection of a particular subpopulation.

51. The method of claim 49 or 50, wherein the change in the value for each of the plurality of parameters, or a combination thereof, indicates one or more of properties comprising: an increased clonal abundance; an increased clonal expansion; an increased transduction efficiency; an increased proliferation capacity; an increased cytotoxicity capacity; an increased persistence; an increased metabolic fitness; an increased sternness; or a reduced exhaustion.

52. The method of any of claims 49-51, wherein the modification of the control parameter provides (a) the optimal expression of a molecule by the immune cells, (b) the optimal killing potency of the immune cells, (c) the optimal bioenergetic profile and spare respiratory capacity of the immune cells, or any combination of (a), (b), or (c).

53. The method of any of claims 49-52, further comprising acquiring one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for each of the plurality of parameters from the population of immune cells.

54. The method of claim 53, wherein the one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional value for each of the plurality of parameters is acquired at least once every 1, 2, 3, 6, 9, 12, 18, or 24 hours, or at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, from the population of immune cells.

55. The method of any of claims 49-54, wherein the value for the potency parameter is acquired via XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

56. The method of any of claims 49-55, wherein the value for the metabolic parameter is acquired via SEAHORSE XF™ T cell Metabolic Fitness assay (offered by Agilent Technologies Inc. of Santa Clara California).

57. A method of determining an optimal bioreactor for producing immune cells, comprising: cultivating a first population of immune cells from a subject in a first bioreactor; obtaining a first sample of the first population of immune cells from the first bioreactor; subjecting the first sample to an impedance-based analysis and / or a live cell imaging based analysis to determine an activity of immune cell killing, thereby obtaining a first potency value for the first population of immune cells in the first bioreactor; cultivating a second population of immune cells from the subject in a second bioreactor; obtaining a second sample of the second population of immune cells from the second bioreactor; subjecting the second sample to the impedance-based analysis and the live cell imaging based analysis to determine the activity of immune cell killing, thereby obtaining a second potency value for the second population of immune cells in the second bioreactor; and comparing the first potency value with the second potency value, wherein a higher potency value is indicative of the optimal bioreactor for producing immune cells.

58. The method of claim 57, wherein the first and second potency values are obtained by anXCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

59. A method of determining an optimal bioreactor for producing immune cells, comprising: cultivating a first population of immune cells from a subject in a first bioreactor; obtaining a first sample of the first population of immune cells from the first bioreactor; subjecting the first sample to an instrument that determines oxygen consumption rate(OCR), proton efflux rate (PER), extracellular acidification rate (ECAR), ATP production rate, or a combination thereof, thereby obtaining a first metabolic value for the first population of immune cells in the first bioreactor; cultivating a second population of immune cells from the subject in a second biorcactor; obtaining a second sample of the second population of immune cells from the second bioreactor; subjecting the first sample to an instrument that determines oxygen consumption rate (OCR), proton efflux rate (PER), extracellular acidification rate (ECAR), ATP production rate, or a combination thereof, thereby obtaining a second metabolic value for the second population of immune cells in the second bioreactor; and comparing the first metabolic value with the second metabolic value, wherein a higher value is indicative of the optimal bioreactor for producing immune cells.

60. The method of claim 59, wherein the first and second metabolic values are obtained by a SEAHORSE XF™ T cell Metabolic Fitness assay (offered by Agilent Technologies Inc. of Santa Clara California).

61. A method of evaluating immune cells in a bioreactor, comprising: obtaining a first sample of a population of immune cells from the bioreactor; determining a percentage of immune cells that express a molecule (e.g., a CAR), in the first sample, thereby obtaining an expression value for the population of immune cells; obtaining a second sample of the population of immune cells from the bioreactor;subjecting the second sample to an impedance-based analysis and / or a live cell imaging based analysis to determine an activity of immune cell killing, thereby obtaining a potency value for the population of immune cells; and converting the obtained potency value to a normalized potency value based on the expression value.

62. The method of claim 61, wherein the expression value is obtained by flow cytometry.

63. The method of claim 61 or 62, wherein the potency value is obtained by an XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

64. The method of any of claims 61-63, further comprising cultivating the population of immune cells in the bioreactor.

65. A method of evaluating immune cells in a bioreactor, comprising: obtaining a first sample of a population of immune cells from the bioreactor; determining a percentage of immune cells that express a molecule (e.g., a CAR) in the first sample, thereby obtaining an expression value for the population of immune cells; adjusting an effector (E) to target (T) ratio for a potency assay of the population of immune cells based on the obtained expression value; obtaining a second sample of the population of immune cells from the bioreactor; subjecting the second sample to the potency assay using the adjusted E to T ratio.

66. The method of claim 65, wherein the potency assay comprises an impedance-based analysis and / or a live cell imaging based analysis to determine an activity of immune cell killing, e.g., by an XCELLIGENCE® potency assay (offered by Agilent Technologies Inc. of Santa Clara California).

67. The method of claim 65 or 66, wherein the expression value is obtained by flow cytometry.

68. The method of any of claims 65-67, wherein the adjusted E to T ratio is 25: 1, 20: 1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.5:1, 0.25:1, or 0.1:1.

69. The method of any of claims 65-68, further comprising cultivating the population of immune cells in the bioreactor.

70. The method of any of claims 1-69, comprising; culturing immune cells in the bioreactor; extracting an immune cell sample, e.g., by a sample extraction module, from the bioreactor; preparing the extracted cell sample, e.g., by a sample preparation module, to produce a prepared sample; analyzing of a parameter described herein; transmitting a signal for an analyzed parameter to a controller, e.g., a controller module; responsive to the signal for an analyzed parameter, transmitting a second signal.

71. The method of any of claims 1-70, wherein acquiring or measuring a metabolic parameter comprises evaluating a bioenergetic poise and a bioenergetic capacity of the sample by a method comprising: acquiring a reference value for oxygen consumption ( VOCRCI); acquiring a reference value for proton efflux (VPERef); contacting the cell sample with an ATP synthase inhibitor, a mitochondrial uncoupling agent, and an electron transport chain (ETC) inhibitor, sequentially, partly simultaneously, or simultaneously, each contacting forming a reaction mixture; acquiring a value for oxygen consumption for each reaction mixture (VOCMi ); and acquiring a value for proton efflux for each reaction mixture ( VPEMIX), thereby evaluating the bioenergetic poise and the bioenergetic capacity of the sample.

72. The method of any of claims 1-71, wherein the immune cells, or the population of immune cells, comprise T cells, natural killer (NK) cells, macrophages, or a combination thereof.

73. The method of any of claims 1-72, wherein the immune cells, or the population of immune cells, comprise genetically engineered immune cells targeting an antigen (e.g., tumor antigen), e.g., chimeric antigen receptor (CAR)-immune cells, e.g., CAR-T cells, CAR-NK cells, CAR-macrophages, or a combination thereof.

74. The method of any of claims 1-73, wherein the population of immune cells comprise chimeric antigen receptor (CAR) immune cells, e.g., CAR-T cells or CAR-NK cells.

75. A system for performing a method of any of claims 1-71, wherein the system comprises one or more processors and a memory storing machine readable instructions, wherein the system is configured to allow for: culture of immune cells in a biorcactor, e.g., a biorcactor comprising a biorcactor module; extraction of a sample, e.g., by a sample extraction module, from the bioreactor; optionally, preparation of the extracted cell sample, e.g., by a sample preparation module, to produce a prepared sample; analysis of a parameter described herein; transmission of a signal for an analyzed parameter to a controller, e.g., a controller module; responsive to the signal for an analyzed parameter, transmission of a second signal from the controller to the bioreactor; and responsive to the second signal from the controller, maintenance or alteration of a bioreactor module parameter.

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