Cytotoxic assay for evaluating the efficacy of therapeutic cell compositions

Abstract

This disclosure relates to a method for determining the potency of an effector cell composition, such as a therapeutic cell composition, for use in conjunction with cell therapy, based on cytotoxicity. The cells of the cell composition may express recombinant receptors, such as chimeric receptors, such as chimeric antigen receptors (CARs), or other transgenic receptors such as T cell receptors (TCRs). The method provides a cytotoxicity assay for identifying the potency of the cell composition, including relative potency.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 460,146, filed on 18 April 2023, and U.S. Provisional Application No. 63 / 567,922, filed on 20 March 2024, both entitled "CYTOTOXICITY ASSAY FOR ASSESSING POTENCY OF THERAPEUTIC CELL COMPOSITIONS," the disclosures of which are incorporated herein by reference in their entirety.

[0002] Reference to electronic sequence listings This application is accompanied by an electronic sequence listing. The sequence listing is provided as a file titled 735042027340SEQLIST.xml, created on April 17, 2024, and is 122,311 bytes in size. The electronic format information of the sequence listing is incorporated in its entirety by reference.

[0003] This disclosure relates to a method for determining the potency of an effector cell composition, such as a therapeutic cell composition, for use in conjunction with cell therapy, based on cytotoxicity. The cells of the cell composition may express recombinant receptors, such as chimeric receptors, such as chimeric antigen receptors (CARs), or other transgenic receptors such as T cell receptors (TCRs). The method provides a cytotoxicity assay for determining the potency of the cell composition, including relative potency. [Background technology]

[0004] Various immunotherapies and / or cell therapies are available to treat diseases and conditions. For example, adoptive cell therapies (including the administration of cells expressing chimeric receptors specific to the disease or disorder of interest, e.g., chimeric antigen receptors (CARs) and / or other recombinant antigen receptors, as well as other adoptive immunotherapy, e.g., T-cell therapy) may be effective in treating cancer or other diseases or disorders. Improved approaches are needed for characterizing effective therapeutic cell compositions, such as in relation to methods for ex vivo production of compositions, or for treating targets using cell therapies. Methods addressing such needs are provided herein. [Overview of the project] [Means for solving the problem]

[0005] In some embodiments, a method for determining the efficacy of an effector cell composition is provided, comprising (a) performing a plurality of incubations, each of which incubations comprising culturing cells of the effector cell composition together with target cells expressing a cell surface target antigen and a reporter molecule stably expressed by the target cells, wherein the cells of the effector cell composition to be cultured include cells engineered to express recombinant receptors that specifically bind to the target antigen and cells that do not express recombinant receptors, and each of the plurality of incubations comprising different adjusted ratios (titrated) of cells of the effector cell composition to target cells. A method is provided herein that includes: (a) measuring the cytotoxic activity from each of a plurality of incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity; and (c) determining an adjusted ratio that yields up to half the amount of cytotoxic activity of the effector cell composition based on the cytotoxic activity measured for each of the plurality of incubations.

[0006] In some parts of any embodiment, the method further includes determining the relative potency of an effector cell composition by comparing a modified ratio that yields up to half the cytotoxic activity of the effector cell composition to a modified ratio that yields up to half the cytotoxic activity of a reference standard.

[0007] In some embodiments, a method for determining the potency of an effector cell composition comprises (a) performing a plurality of incubations, each of which comprises culturing cells of the effector cell composition together with target cells expressing a cell surface target antigen and a reporter molecule stably expressed by the target cells, wherein the cells of the effector cell composition cultured therein include cells engineered to express recombinant receptors that specifically bind to the target antigen and cells that do not express recombinant receptors, and each of the plurality of incubations is performed in different adjusted ratios of cells of the effector cell composition to target cells, and different adjustments A method is provided herein that includes: (b) measuring the cytotoxic activity from each of a plurality of incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity; and (c) determining the relative potency of the effector cell composition by comparing the cytotoxic activity of up to half of the effector cell composition to the cytotoxic activity of up to half of a reference standard, based on the cytotoxic activity measured for each of the plurality of incubations.

[0008] In some of the embodiments, a series of incubations involves culturing cells expressing a certain number of recombinant receptors of the effector composition together with different numbers of target cells. In some embodiments, different numbers of target cells are obtained by serial dilution of the target cells. In some embodiments, the serial dilution is a magnification change between 2x and 8x, 2x and 6x, or 2x and 4x, including the boundary, or between approximately 2x and approximately 8x, approximately 2x and approximately 6x, or approximately 2x and approximately 4x. In some embodiments, the serial dilution is a magnification change between 2x and 8x, including the boundary, or between approximately 2x and approximately 8x. In some embodiments, the serial dilution is a magnification change between 2x and 6x, including the boundary, or between approximately 2x and approximately 6x. In some embodiments, the serial dilution is a magnification change between 2x and 4x, including the boundary, or between approximately 2x and approximately 4x.

[0009] In some of the embodiments, multiple incubations include culturing a certain number of target cells together with cells expressing different numbers of recombinant receptors of the effector composition. In some embodiments, cells expressing different numbers of recombinant receptors of the effector composition are obtained by serial dilution of the cells of the effector cell composition. In some embodiments, the serial dilution is by a magnification change between 2x and 8x, 2x and 6x, or 2x and 4x, including the boundary, or between approximately 2x and approximately 8x, approximately 2x and approximately 6x, or approximately 2x and approximately 4x. In some embodiments, the serial dilution is by a magnification change between 2x and 8x, including the boundary, or between approximately 2x and approximately 8x. In some embodiments, the serial dilution is by a magnification change between 2x and 6x, including the boundary, or between approximately 2x and approximately 6x. In some embodiments, the serial dilution is by a magnification change between 2x and 4x, including the boundary, or between approximately 2x and approximately 4x.

[0010] In some of the embodiments, in each of the multiple incubations, including their respective boundaries, between 1,000 to 80,000 target cells, 5,000 to 15,000 target cells, 8,000 to 12,000 target cells, or 9,000 to 10,000 target cells, or between approximately 1,000 to approximately 80,000 target cells, approximately 5,000 to approximately 15,000 target cells, approximately 8,000 to approximately 12,000 target cells, or between approximately 9,000 to approximately 10,000 target cells, are incubated with the cells of the effector cell composition to be cultured. In some of the optional embodiments, in each of the multiple incubations, between 1,000 and 80,000 target cells, or between approximately 1,000 and 80,000 target cells, including the boundaries, are incubated with the cells of the effector cell composition to be cultured. In some of the optional embodiments, in each of the multiple incubations, between 5,000 and 15,000 target cells, or between approximately 5,000 and 15,000 target cells, including the boundaries, are incubated with the cells of the effector cell composition to be cultured. In some of the optional embodiments, in each of the multiple incubations, between 8,000 and 12,000 target cells, or between approximately 8,000 and 12,000 target cells, including the boundaries, are incubated with the cells of the effector cell composition to be cultured. In some of the optional embodiments, in each of the multiple incubations, between 9,000 and 10,000 target cells, including the boundaries, or between approximately 9,000 and 10,000 target cells, are incubated together with the cells of the effector cell composition to be cultured.

[0011] In some of the embodiments, target cells before multiple incubations are each 0.1 × 10⁶ including their boundaries. 6 From 0.3 × 10 6 Target cells / mL, 0.15 × 10⁶ 6 From 0.25 × 10 6 Target cells / mL or 0.18 × 10⁶ 6 From 0.22 × 10 6between cells / mL of target cells or about 0.1×10 6 cells to about 0.3×10 6 cells / mL, about 0.15×10 6 cells to about 0.25×10 6 cells / mL or about 0.18×10 6 cells to about 0.22×10 6 cells / mL. In some embodiments, the target cells prior to multiple incubations are, including the boundaries, between 0.1×10 6 cells and 0.3×10 6 cells / mL or about 0.1×10 6 cells to about 0.3×10 6 cells / mL. In some embodiments, the target cells prior to multiple incubations are, including the boundaries, between 0.15×10 6 cells and 0.25×10 6 cells / mL or about 0.15×10 6 cells to about 0.25×10 6 cells / mL. In some embodiments, the target cells prior to multiple incubations are, including the boundaries, between 0.18×10 6 cells and 0.22×10 6 cells / mL or about 0.18×10 6 cells to about 0.22×10 6 cells / mL.

[0012] In some of the embodiments, the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 120:1 to 1:1, 50:1 to 2:1, 18:1 to 6:1 and 9:1 to 3:1, or approximately 120:1 to approximately 1:1, approximately 50:1 to approximately 2:1, approximately 18:1 to approximately 6:1 and approximately 9:1 to approximately 3:1, including the boundary. In some of the embodiments, the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 120:1 to 1:1 or approximately 120:1 to approximately 1:1, including the boundary. In some of the embodiments, the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 50:1 to 2:1 or approximately 50:1 to approximately 2:1, including the boundary. In some of the embodiments, the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 18:1 and 6:1 or about 18:1 and about 6:1, including the boundary. In some of the embodiments, the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 9:1 and 3:1 or about 9:1 and about 3:1, including the boundary.

[0013] In some of the embodiments, the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 1:120 to 1:1, 1:50 to 1:2, 1:18 to 1:6 and 1:9 to 1:3, or approximately 1:120 to approximately 1:1, approximately 1:50 to approximately 1:2, approximately 1:18 to approximately 1:6 and approximately 1:9 to approximately 1:3, including the boundary. In some of the embodiments, the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 1:120 to 1:1 or approximately 1:120 to approximately 1:1, including the boundary. In some of the embodiments, the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 1:50 to 1:2 or approximately 1:50 to approximately 1:2, including the boundary. In some of the embodiments, the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 1:18 and 1:6 or about 1:18 to about 1:6, including the boundary. In some of the embodiments, the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 1:9 and 1:3 or about 1:9 to about 1:3, including the boundary.

[0014] Furthermore, a method for evaluating a target cell preparation for use in an efficacy assay, comprising (a) performing a plurality of incubations, each of which comprising culturing cells of an effector cell composition together with cells of a test target cell preparation, wherein the cells of the test target cell preparation express a cell surface target antigen and a reporter molecule, and the cells of the effector cell composition include cells engineered to express a recombinant receptor that specifically binds to the target antigen and cells that do not express the recombinant receptor, and each of the plurality of incubations is performed at different adjusted ratios of cells of the effector cell composition to cells of the test target cell preparation, each of which comprises cells of the effector cell composition expressing the recombinant receptor. A method is provided herein that is based on cells, and includes: (b) measuring the cytotoxic activity from each of several incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity; (c) determining an adjusted ratio with test target cells that yields up to half the amount of cytotoxic activity (adjusted ratio of EC50) of the effector cell composition based on the cytotoxic activity measured for each of several incubations; and (d) comparing the adjusted ratio of EC50 with an adjusted ratio of EC50 determined in a reference cytotoxicity assay performed using a reference target cell line and the same effector cells.

[0015] In some embodiments, the adjusted ratio of the EC50 of a reference cytotoxicity assay is determined by (a) performing a plurality of incubations, each of which comprises culturing cells of an effector cell composition together with cells of a reference target cell line, wherein the cells of the reference cell preparation express a cell surface target antigen and a reporter molecule, and each of the plurality of incubations is performed at different adjusted ratios of cells of the effector cell composition to cells of the reference target cell line, with each of the different adjusted ratios being based on cells expressing the recombinant receptor of the effector cell composition; (b) measuring the cytotoxic activity from each of the plurality of incubations based on the expression or activity of the reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity; and (c) determining the adjusted ratio with the reference target cell line that yields the EC50 of the effector cell composition based on the cytotoxic activity measured for each of the plurality of incubations.

[0016] Furthermore, a method for evaluating a target cell preparation for use in an efficacy assay, comprising (a) performing a plurality of incubations, each of which comprising culturing cells of an effector cell composition together with cells of a test target cell preparation, wherein the cells of the test target cell preparation express a cell surface target antigen and a reporter molecule, and the cells of the effector cell composition include cells engineered to express a recombinant receptor that specifically binds to the target antigen and cells that do not express the recombinant receptor, and each of the plurality of incubations is performed with different adjusted ratios of cells of the effector cell composition to cells of the test target cell preparation, each of which expresses the recombinant receptor of the effector cell composition A method is provided herein that is cell-based, and includes: (b) measuring the cytotoxic activity from each of several incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity; (c) determining the relative potency of an effector cell composition to test target cells by comparing the cytotoxic activity of up to half of the effector cell composition to test target cells (adjusted ratio of EC50) to a first reference EC50 based on the cytotoxic activity measured for each of several incubations; and (d) comparing the relative potency of the effector cell composition to test target cells to the relative potency of a control sample.

[0017] In some embodiments, the method includes (a) performing a plurality of incubations, each of which involves culturing cells of an effector cell composition together with cells of a control target cell preparation expressing a cell surface target antigen and a reporter molecule, each of which incubations is performed at different adjusted ratios of cells of the effector cell composition to cells of the control target cell preparation, each of which adjusted ratios is based on cells expressing the recombinant receptor of the effector cell composition; (b) measuring the cytotoxic activity from each of the plurality of incubations based on the expression or activity of the reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity; and (c) determining the relative potency of the control sample by determining the relative potency of the control sample by comparing the EC50 of the control sample to the EC50 of a second reference standard based on the cytotoxic activity measured for each of the plurality of incubations.

[0018] In any embodiment, the method further comprises evaluating the cells of a test target cell preparation for nonspecific stimulation of effector cells, the cells from a negative target cell preparation being evaluated for stimulating cytolytic activity from effector cells, and the cells of the negative test cell preparation being identical to the cells of the test target cell preparation except that the cells of the negative test cell preparation do not express the target antigen.

[0019] In any embodiment, the method further comprises evaluating the cells of the target cell preparation against nonspecific stimulation of effector cells, and optionally the method comprises (a) performing a second plurality of incubations, each of which comprises culturing cells of the effector cell composition together with cells of a negative target cell preparation that stably expresses a reporter molecule, wherein the cells of the negative target cell preparation are identical to the cells of the test target cell preparation except that they do not express the target antigen, and each of the second plurality of incubations comprises evaluating the cells of the effector cell composition against the cells of the negative target cell preparation (b) to perform the procedure in different adjusted ratios, each of which is based on cells expressing the recombinant receptor of the effector cell composition; (c) to measure the cytotoxic activity from each of the second plurality of incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity; and (d) to determine an adjusted ratio of cells of a negative target cell preparation that yields the EC50 of the effector cell composition, based on the cytotoxic activity measured for each of the second plurality of incubations.

[0020] In any embodiment, the method further comprises evaluating the cells of a test target cell preparation for unwanted lymphocyte stimulation, the cells from the test target cell preparation being evaluated for stimulating cytolytic activity from the cells of the lymphocyte cell composition, and the cells of the lymphocyte cell composition being identical to effector cells, except that the cells of the lymphocyte cell composition do not express recombinant receptors.

[0021] In any embodiment, the method further comprises evaluating cells of a test target cell preparation for unwanted lymphocyte stimulation, and optionally includes: (a) performing a third plurality of incubations, each of which comprises culturing cells of a lymphocyte cell composition together with cells of a test target cell preparation expressing a cell surface target antigen and a reporter molecule, wherein the cells of the cultured lymphocyte cell composition include cells that do not express receptors that specifically bind to the target antigen, and each of the third plurality of incubations is performed in different adjusted ratios of cells of the lymphocyte cell composition to cells of the test target cell preparation; (b) measuring cytotoxic activity from each of the third plurality of incubations based on the expression or activity of the reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with cytotoxic activity; and (c) determining an adjusted ratio of cells of the test target cell preparation that yields an EC50 of the lymphocyte cell composition based on the cytotoxic activity measured for each of the third plurality of incubations.

[0022] In some parts of any embodiment, the method further includes determining the growth rate of cells in a test target cell preparation.

[0023] In some parts of any embodiment, the method further includes determining the adhesion of a test target cell preparation to a container in which the cells are growing.

[0024] In some parts of any embodiment, the method further includes determining whether the cells of the test target cell preparation are capable of stably expressing the reporter molecule prior to multiple incubations. In some parts of any embodiment, the method further includes determining whether the cells of the test target cell preparation express an additional reporter molecule prior to multiple incubations.

[0025] In some of the embodiments, the effector cell composition is a therapeutic cell composition. In some of the embodiments, the effector cell composition is a T cell composition. In some of the embodiments, the cells of the effector cell composition are T cells.

[0026] In some of the embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0027] In some of the embodiments, the test target cell preparation is a target cell line that has been cryopreserved, passaged, engineered to express a target cell antigen, and / or engineered to express a reporter molecule.

[0028] In some of the embodiments, the test target cell preparation was a target cell line cryopreserved in DMSO, where the DMSO was, as appropriate, at concentrations of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30%, or approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30%.

[0029] In some of the embodiments, the test target cell preparation is a passaged target cell line, which, as appropriate, has been passaged 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, or 50 times.

[0030] In some of the embodiments, multiple incubations include culturing a certain number of cells of the effector cell composition together with a different number of cells of the test target cell preparation.

[0031] In some of the embodiments, different numbers of cells in the test target cell preparation are obtained by serially diluting the cells of the test target cell preparation.

[0032] In some of the embodiments, serial dilution is by a magnification change between 2x and 8x, 2x and 6x, or 2x and 4x, including the respective boundaries, or between approximately 2x and approximately 8x, approximately 2x and approximately 6x, or approximately 2x and approximately 4x.

[0033] In some of the embodiments, multiple incubations include culturing a certain number of cells of the target cell preparation together with a different number of cells of the effector cell composition.

[0034] In some of the embodiments, different numbers of cells in the effector cell composition are obtained by serially diluting the cells of the effector cell composition.

[0035] In some of the embodiments, serial dilution is by a magnification change between 2x and 8x, 2x and 6x, or 2x and 4x, including the respective boundaries, or between approximately 2x and approximately 8x, approximately 2x and approximately 6x, or approximately 2x and approximately 4x.

[0036] In some of the embodiments, in each of the multiple incubations, including each boundary, between 1,000 to 80,000 viability target cells, 5,000 to 15,000 viability target cells, 8,000 to 12,000 viability target cells or 9,000 to 10,000 viability target cells, or between approximately 1,000 to approximately 80,000 viability target cells, approximately 5,000 to approximately 15,000 viability target cells, approximately 8,000 to approximately 12,000 viability target cells or approximately 9,000 to approximately 10,000 viability target cells, is incubated with the cells of the effector cell composition to be cultured.

[0037] In some of the embodiments, the cells of the test target cell preparation are each 0.1 × 10⁶ before multiple incubations, including the boundaries. 6 From 0.3 × 10 6 Individual target cells / mL, 0.15 × 10⁶ 6 From 0.25 × 10 6 Individual target cells / mL or 0.18 × 10⁶ 6 From 0.22 × 10 6 Between individual test target cells / mL or approximately 0.1 × 10 6 Approximately 0.3 x 10 from each 6 Individual target cells / mL, approximately 0.15 × 10⁶6 Approximately 0.25 × 10 from each 6 Individual target cells / mL or approximately 0.18 × 10⁶ 6 Approximately 0.22 × 10 from each 6 This is the concentration between individual test target cells / mL.

[0038] In some of the embodiments, the maximum adjusted ratios of cells in the effector cell composition to target cells in multiple incubations, including the boundaries, are between 120:1 to 1:1, 50:1 to 2:1, 18:1 to 6:1 and 9:1 to 3:1 or between approximately 120:1 to approximately 1:1, approximately 50:1 to approximately 2:1, approximately 18:1 to approximately 6:1 and approximately 9:1 to approximately 3:1.

[0039] In some of the embodiments, the maximum adjusted ratios of cells in the effector cell composition to cells in the test target cell preparation for multiple incubations are between 1:120 to 1:1, 1:50 to 1:2, 1:18 to 1:6 and 1:9 to 1:3, including the respective boundaries, or between approximately 1:120 to approximately 1:1, approximately 1:50 to approximately 1:2, approximately 1:18 to approximately 1:6 and approximately 1:9 to approximately 1:3.

[0040] In some of the embodiments, multiple incubations are carried out in at least 3, 4, 5, 6, 7, 8, 9, or 10 different adjusted ratios. In some of the embodiments, multiple incubations are carried out in at least 3 different adjusted ratios. In some of the embodiments, multiple incubations are carried out in at least 4 different adjusted ratios. In some of the embodiments, multiple incubations are carried out in at least 5 different adjusted ratios. In some of the embodiments, multiple incubations are carried out in at least 6 different adjusted ratios. In some of the embodiments, multiple incubations are carried out in at least 7 different adjusted ratios. In some of the embodiments, multiple incubations are carried out in at least 8 different adjusted ratios. In some of the embodiments, multiple incubations are carried out in at least 9 different adjusted ratios. In some of the embodiments, multiple incubations are carried out in at least 10 different adjusted ratios.

[0041] In some of the embodiments, multiple incubations are carried out at different adjusted ratios, which are between 0.001 and 15 or approximately between 0.001 and approximately 15, including the boundary.

[0042] In some of the embodiments, each of a plurality of incubations includes culturing cells of a mock cell composition together with cells of an effector cell composition and target cells, wherein the cultured cells of the mock cell composition do not express recombinant receptors or target antigens. In some embodiments, the mock cell composition is produced using a manufacturing process that is identical to the manufacturing process used to produce the effector cell composition, except that the cells of the mock cell composition are not exposed to nucleic acids encoding recombinant receptors.

[0043] In some of the embodiments, each of the multiple incubations includes a different total number of effector composition cells and target cells.

[0044] In some of the embodiments, each of the multiple incubations includes the same total number of cells of the effector composition, target cells, and mock cell composition. In some of the embodiments, each of the multiple incubations includes a different total number of cells of the effector composition, target cells, and mock cell composition.

[0045] In some of the embodiments, each of the multiple incubations is performed at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times with respect to the effector cell composition. In some of the embodiments, each of the multiple incubations is performed at least 2 times with respect to the effector cell composition. In some of the embodiments, each of the multiple incubations is performed at least 3 times with respect to the effector cell composition. In some of the embodiments, each of the multiple incubations is performed at least 4 times with respect to the effector cell composition. In some of the embodiments, each of the multiple incubations is performed at least 5 times with respect to the effector cell composition. In some of the embodiments, each of the multiple incubations is performed at least 6 times with respect to the effector cell composition. In some of the embodiments, each of the multiple incubations is performed at least 7 times with respect to the effector cell composition. In some of the embodiments, each of the multiple incubations is performed at least 8 times with respect to the effector cell composition. In some of the embodiments, each of the multiple incubations is performed at least nine times for the effector cell composition. In some of the embodiments, each of the multiple incubations is performed at least ten times for the effector cell composition.

[0046] In some of the embodiments, each of the multiple incubations is carried out for 24 to 72 hours, 24 to 48 hours, or 48 to 72 hours, including its respective boundary. In some of the embodiments, each of the multiple incubations is carried out for 24 to 72 hours, or approximately 24 to 72 hours, including its boundary. In some of the embodiments, each of the multiple incubations is carried out for 24 to 72 hours, or approximately 24 to 72 hours, including its boundary. In some of the embodiments, each of the multiple incubations is carried out for 24 to 48 hours, or approximately 24 to 48 hours, including its boundary. In some of the embodiments, each of the multiple incubations is carried out for 48 to 72 hours, or approximately 48 to 72 hours, including its boundary.

[0047] In some of the embodiments, each of the multiple incubations is carried out for less than 24 hours. In some of the embodiments, each of the multiple incubations is carried out for 17 to 23 hours, 18 to 22 hours, or 19 to 21 hours, including the respective boundaries, or for approximately 17 to approximately 23 hours, approximately 18 to approximately 22 hours, or for approximately 19 to approximately 21 hours. In some of the embodiments, each of the multiple incubations is carried out for 17 to 23 hours, including the boundaries, or for approximately 17 to approximately 23 hours. In some of the embodiments, each of the multiple incubations is carried out for 18 to 22 hours, including the respective boundaries, or for approximately 18 to approximately 22 hours. In some of the embodiments, each of the multiple incubations is carried out for 19 to 21 hours, including the boundaries, or for approximately 19 to approximately 21 hours.

[0048] In some of the optional embodiments, each of the multiple incubations is carried out for at least 4 hours.

[0049] In some of the optional embodiments, each of the multiple incubations is carried out at a temperature between 30°C and 39°C or approximately between 30°C and approximately 39°C, including the boundary.

[0050] In some of the optional embodiments, each of the multiple incubations is carried out at a CO2 level between 2.5% and 7.5% or approximately between 2.5% and approximately 7.5%.

[0051] In some of the embodiments, the target cells are cells that have been engineered to express a reporter molecule. In some embodiments, the engineered cells are transduction cells.

[0052] In some of the embodiments, the expression of the reporter molecule by target cells is under the control of the constituent promoter.

[0053] In some of the embodiments, the reporter molecule is a detectable protein.

[0054] In some of the embodiments, the reporter molecule is an enzyme. In some embodiments, the enzyme is luciferase, β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), alkaline phosphatase, or secretory embryonic alkaline phosphatase (SEAP).

[0055] In some of the embodiments, the enzyme is luciferase. In some embodiments, the luciferase is firefly luciferase, click beetle luciferase, sea mushroom (Renilla) luciferase, Gaussia luciferase, Gaussia-Dura luciferase, Oplophorus luciferase, bacterial luciferase, sea firefly (Cypridina) luciferase, Polichthys luciferase, dinoflagellate luciferase, krill luciferase, or fungal luciferase. In some of the embodiments, the luciferase is firefly luciferase.

[0056] In some of the embodiments, the measurement of cytotoxic activity includes adding a detection reagent to each of several incubations.

[0057] In some of the embodiments, the measurement of cytotoxic activity includes, before addition, equilibrating each of the multiple incubations, including the boundary, for 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, or for about 10 to about 120 minutes, about 20 to about 90 minutes, or about 30 to about 60 minutes. In some of the embodiments, the measurement of cytotoxic activity includes, before addition, equilibrating each of the multiple incubations, including the boundary, for 10 to 120 minutes, or for about 10 to about 120 minutes. In some of the embodiments, the measurement of cytotoxic activity includes, before addition, equilibrating each of the multiple incubations, including the boundary, for 20 to 90 minutes, or for about 20 to about 90 minutes. In some of the embodiments, the measurement of cytotoxic activity includes, before addition, equilibrating each of several incubations, including their boundaries, to room temperature for 30 to 60 minutes or about 30 to about 60 minutes.

[0058] In some of the embodiments, the measurement of cytotoxic activity includes, before addition, allowing the detection reagent to equilibrate to room temperature for 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, or approximately 10 to approximately 120 minutes, approximately 20 to approximately 90 minutes, or approximately 30 to approximately 60 minutes, including the boundary. In some of the embodiments, the measurement of cytotoxic activity includes, before addition, allowing the detection reagent to equilibrate to room temperature for 10 to 120 minutes, or approximately 10 to approximately 120 minutes, including the boundary. In some of the embodiments, the measurement of cytotoxic activity includes, before addition, allowing the detection reagent to equilibrate to room temperature for 20 to 90 minutes, or approximately 20 to approximately 90 minutes, including the boundary. In some of the embodiments, the measurement of cytotoxic activity includes, before addition, allowing the detection reagent to equilibrate to room temperature for 30 to 60 minutes, or approximately 30 to approximately 60 minutes, including the boundary.

[0059] In some of the embodiments, the reporter molecule is an enzyme, and the detection reagent is a substrate of the enzyme. In some of the embodiments, the detection reagent is a luciferase substrate. In some of the embodiments, the detection reagent is luciferin or a luciferin analog. In some of the embodiments, the detection reagent is a luciferin analog. In some embodiments, the luciferin analog is 5'-fluoroluciferin.

[0060] In some of the embodiments, the measurement of cytotoxic activity includes incubating each of several incubations, including the boundary, in the presence of a detection reagent, for a period of 5 to 240 minutes, 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, or for approximately 5 to 240 minutes, approximately 10 to 120 minutes, approximately 20 to 90 minutes, or approximately 30 to 60 minutes. In some of the embodiments, the measurement of cytotoxic activity includes incubating each of several incubations, including the boundary, for a period of 5 to 240 minutes, or for approximately 5 to 240 minutes, in the presence of a detection reagent. In some of the embodiments, the measurement of cytotoxic activity includes incubating each of several incubations, including the boundary, for a period of 10 to 120 minutes, or for approximately 10 to 120 minutes, in the presence of a detection reagent. In some of the embodiments, the measurement of cytotoxic activity includes incubating each of the multiple incubations, including the boundary, for 20 to 90 minutes or about 20 to about 90 minutes, in the presence of the detection reagent. In some of the embodiments, the measurement of cytotoxic activity includes incubating each of the multiple incubations, including the boundary, for 30 to 60 minutes or about 30 to about 60 minutes, in the presence of the detection reagent.

[0061] In some of the embodiments, the cytotoxic activity of each of the multiple incubations is measured by luminescence imaging.

[0062] In some of the embodiments, the reporter molecule is a fluorescent protein, and the cytotoxic activity of each of the multiple incubations is measured by fluorescence imaging.

[0063] In some of the embodiments, cytotoxic activity is cytolytic activity.

[0064] In some of the optional embodiments, cytotoxic activity is measured after the termination of each of the multiple incubations.

[0065] In some of the embodiments, cytotoxic activity is measured over time during each of multiple incubations.

[0066] In some of the embodiments, the cytotoxic activity measured for each of the multiple incubations is determined based on the lysis percentage of target cells.

[0067] In some of the embodiments, the adjusted ratio that yields up to half the cytotoxic activity of the effector cell composition is the EC50 of the effector cell composition.

[0068] In some of the embodiments, the adjusted ratio that yields up to half the cytotoxic activity of the reference standard is the EC50 of the reference standard.

[0069] In some of the embodiments, the reference standard is an effector cell composition having a validated and adjusted ratio that yields up to half the amount of cytotoxic activity, a commercially available effector cell composition, an effector cell composition produced using a manufacturing process identical to the manufacturing process used to produce the effector cell composition, an effector cell composition produced using a manufacturing process different from the manufacturing process used to produce the effector cell composition, an effector cell composition comprising cells expressing the same recombinant receptor as the effector cell composition, or an effector cell composition comprising cells expressing a different recombinant receptor than the effector cell composition.

[0070] In some of the embodiments, the reference standard is an effector cell composition produced using a manufacturing process identical to the manufacturing process used to produce the effector cell composition. In some of the embodiments, the reference standard is an effector cell composition having a verified and adjusted ratio that yields up to half the amount of cytotoxic activity.

[0071] In some of the embodiments, the reference standard includes primary cells obtained from a subject. In some embodiments, the subject is a healthy subject. In some embodiments, the subject has a disease or condition. In some embodiments, the disease or condition is cancer.

[0072] In some of the embodiments, the target cells are clones from a cell line or primary cells isolated from a subject. In some of the embodiments, the target cells are derived from a cell line. In some embodiments, the cell line is a tumor cell line.

[0073] In some of the embodiments, the target cells are cells that have been engineered to express the target antigen. In some embodiments, the engineered cells are transduced.

[0074] In some of the embodiments, the target antigen is a first tumor antigen, and the target cells are cells engineered not to express a second tumor antigen.

[0075] In some of the embodiments, the cytotoxic activity measured for each of the multiple incubations achieves a dose-response curve of the cytotoxic activity of the effector cell composition, which includes a linear dose-response range. In some embodiments, the dose-response curve includes a lower asymptote and an upper asymptote of the cytotoxic activity of the effector cell composition.

[0076] In some of the embodiments, the effector cell composition comprises single-cell subtypes concentrated or purified from a biological sample. In some of the embodiments, the effector cell composition comprises a population of mixed cell subtypes obtained by mixing cell subtypes concentrated or purified from a biological sample. In some of the embodiments, the biological sample comprises whole blood samples, buffy coat samples, peripheral blood mononuclear cell (PBMC) samples, unfractionated cell samples, lymphocyte samples, leukocyte samples, apheresis products, or leukocyte apheresis products.

[0077] In some of the embodiments, the effector cell composition includes primary cells obtained from the subject.

[0078] In some embodiments, the reference standard is an effector cell composition prepared from cells obtained from the same subject. In some embodiments, the reference standard is an effector cell composition prepared from cells obtained from different subjects.

[0079] In some of the embodiments, the subject has a disease or condition. In some embodiments, the disease or condition is cancer. In some of the embodiments, the effector cell composition includes autologous cells obtained from the subject to be treated.

[0080] In some of the embodiments, the subject is a healthy subject. In some of the embodiments, the effector cell composition includes allogeneic cells.

[0081] In some of the embodiments, the effector cell composition includes CD3+ T cells. In some of the embodiments, the effector cell composition includes CD4+ T cells and CD8+ T cells.

[0082] In some of the embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0083] In some of the embodiments, multiple incubations are carried out in a flask, tube, or multiwell plate. In some of the embodiments, each of the multiple incubations is carried out individually in a well of the multiwell plate. In some embodiments, the multiwell plate is a 96-well plate, a 48-well plate, a 12-well plate, or a 6-well plate.

[0084] In any embodiment, the method further includes selecting an effector composition for administration to a subject requiring it, based on a modified ratio that yields up to half the cytotoxic activity of the effector cell composition.

[0085] In some of the embodiments, the selection of an effector composition for administration to a target requiring it is based on relative potency.

[0086] In any embodiment, the method further includes determining a dose of cells of the effector cell composition to be administered to a subject requiring it, based on a modified ratio that yields up to half the cytotoxic activity of the effector cell composition.

[0087] In any part of the embodiment, the method further includes determining a cellular dose of the effector composition to be administered to a subject requiring it, based on relative potency.

[0088] In some embodiments, the subject has a disease or condition. In some embodiments, the disease or condition is cancer.

[0089] In some of the optional embodiments, each of the multiple incubations is carried out in serum-free medium.

[0090] In some embodiments, a method for evaluating the efficacy of an effector cell composition is provided herein, comprising (a) incubating cells of the effector cell composition together with (i) target cells expressing a cell surface target antigen and a reporter molecule stably expressed by the target cells and (ii) cells of a mock cell composition, wherein the cells of the effector cell composition to be cultured include cells engineered to express recombinant receptors that specifically bind to the target antigen and cells that do not express recombinant receptors, and the cells of the mock cell composition to be cultured do not express recombinant receptors or target antigens, and (b) measuring cytotoxic activity from incubation based on the expression or activity of the reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with cytotoxic activity.

[0091] In some of the embodiments, between 1,000 to 80,000 target cells, 5,000 to 15,000 target cells, 8,000 to 12,000 target cells, or 9,000 to 10,000 target cells, including their respective boundaries, or between approximately 1,000 to approximately 80,000 target cells, approximately 5,000 to approximately 15,000 target cells, approximately 8,000 to approximately 12,000 target cells, or between approximately 9,000 to approximately 10,000 target cells, incubated with the cells of the effector cell composition to be cultured. In some embodiments, between 1,000 to 80,000 target cells, including their boundaries, or between approximately 1,000 to approximately 80,000 target cells, incubated with the cells of the effector cell composition to be cultured. In some embodiments, between 5,000 and 15,000 target cells, including the boundary, or between approximately 5,000 and 15,000 target cells, the cells of the effector cell composition to be cultured are incubated together. In some embodiments, between 8,000 and 12,000 target cells, including the boundary, or between approximately 8,000 and 12,000 target cells, the cells of the effector cell composition to be cultured are incubated together. In some embodiments, between 9,000 and 10,000 target cells, including the boundary, or between approximately 9,000 and 10,000 target cells, the cells of the effector cell composition to be cultured are incubated together.

[0092] In some of the embodiments, the target cells are each 0.1 × 10⁶ before incubation, including their boundaries. 6 From 0.3 × 10 6 Target cells / mL, 0.15 × 10⁶ 6 From 0.25 × 10 6 Target cells / mL or 0.18 × 10⁶ 6 From 0.22 × 10 6 Between individual target cells / mL or approximately 0.1 × 10⁶ 6 Approximately 0.3 x 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.25 × 10 from each 6Target cells / mL or approximately 0.18 × 10⁶ 6 Approximately 0.22 × 10 from each 6 The concentration is between 0.1 × 10¹ target cells / mL. In some of the embodiments, the target cells, including the boundary, are 0.1 × 10¹ before incubation. 6 From 0.3 × 10 6 Between individual target cells / mL or approximately 0.1 × 10⁶ 6 Approximately 0.3 x 10 from each 6 The concentration is between 10⁴ target cells / mL. In some of the embodiments, the target cells are 0.15 × 10⁶ including the boundary before incubation. 6 From 0.25 × 10 6 Between individual target cells / mL or approximately 0.15 × 10⁶ 6 Approximately 0.25 × 10 from each 6 The concentration is between 10⁴ target cells / mL. In some of the embodiments, the target cells are 0.18 × 10⁶ including the boundary before incubation. 6 From 0.22 × 10 6 Between individual target cells / mL or approximately 0.18 × 10⁶ 6 Approximately 0.22 × 10 from each 6 This is the concentration between individual target cells / mL.

[0093] In some of the embodiments, the mock cell composition is produced using a manufacturing process that is identical to the manufacturing process used to produce the effector cell composition, except that the cells of the mock cell composition are not exposed to nucleic acids encoding recombinant receptors.

[0094] In some of the embodiments, incubation is performed at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times for the effector cell composition. In some of the embodiments, incubation is performed at least 2 times for the effector cell composition. In some of the embodiments, incubation is performed at least 3 times for the effector cell composition. In some of the embodiments, incubation is performed at least 4 times for the effector cell composition. In some of the embodiments, incubation is performed at least 5 times for the effector cell composition. In some of the embodiments, incubation is performed at least 6 times for the effector cell composition. In some of the embodiments, incubation is performed at least 7 times for the effector cell composition. In some of the embodiments, incubation is performed at least 8 times for the effector cell composition. In some of the embodiments, incubation is performed at least 9 times for the effector cell composition. In some of the optional embodiments, incubation is performed at least 10 times for the effector cell composition.

[0095] In some of the optional embodiments, incubation is carried out for 24 to 72 hours, 24 to 48 hours, or 48 to 72 hours, including the boundary, or for approximately 24 to approximately 72 hours, approximately 24 to approximately 48 hours, or approximately 48 to approximately 72 hours. In some of the optional embodiments, incubation is carried out for 24 to 72 hours, including the boundary, or for approximately 24 to approximately 72 hours. In some of the optional embodiments, incubation is carried out for 24 to 48 hours, including the boundary, or for approximately 24 to approximately 48 hours. In some of the optional embodiments, incubation is carried out for 48 to 72 hours, including the boundary, or for approximately 48 to approximately 72 hours.

[0096] In some of the optional embodiments, incubation is carried out for less than 24 hours. In some of the optional embodiments, incubation is carried out for 17 to 23 hours, 18 to 22 hours, or 19 to 21 hours, or approximately 17 to approximately 23 hours, approximately 18 to approximately 22 hours, or approximately 19 to approximately 21 hours, including the boundary. In some of the optional embodiments, incubation is carried out for 17 to 23 hours, or approximately 17 to approximately 23 hours, including the boundary. In some of the optional embodiments, incubation is carried out for 18 to 22 hours, or approximately 18 to approximately 22 hours, including the boundary. In some of the optional embodiments, incubation is carried out for 19 to 21 hours, or approximately 19 to approximately 21 hours, including the boundary.

[0097] In some of the optional embodiments, incubation is carried out at a temperature between 30°C and 39°C or approximately between 30°C and approximately 39°C, including the boundary.

[0098] In some of the optional embodiments, incubation is carried out at CO2 levels between 2.5% and 7.5% or approximately between 2.5% and approximately 7.5%.

[0099] In some of the embodiments, the target cells are cells that have been engineered to express a reporter molecule. In some embodiments, the engineered cells are transduction cells.

[0100] In some of the embodiments, the expression of the reporter molecule by target cells is under the control of the constituent promoter.

[0101] In some of the embodiments, the reporter molecule is a detectable protein.

[0102] In some of the embodiments, the reporter molecule is an enzyme. In some embodiments, the enzyme is luciferase, β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), alkaline phosphatase, or secretory embryonic alkaline phosphatase (SEAP).

[0103] In some of the embodiments, the enzyme is luciferase. In some embodiments, the luciferase is firefly luciferase, click mushroom luciferase, sea oyster luciferase, Gausia luciferase, Gausia-dural luciferase, Oprophorus luciferase, bacterial luciferase, sea firefly luciferase, Polychthys luciferase, dinoflagellate luciferase, krill luciferase, or fungal luciferase. In some of the embodiments, the luciferase is firefly luciferase.

[0104] In some of the embodiments, the measurement of cytotoxic activity includes adding a detection reagent to the incubation.

[0105] In some embodiments, the measurement of cytotoxic activity includes, before addition, equilibrating the incubation to room temperature for 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, including the boundary, or for about 10 to about 120 minutes, about 20 to about 90 minutes, or about 30 to about 60 minutes, respectively. In some embodiments, the measurement of cytotoxic activity includes, before addition, equilibrating the incubation to room temperature for 10 to 120 minutes, including the boundary, or for about 10 to about 120 minutes, respectively. In some embodiments, the measurement of cytotoxic activity includes, before addition, equilibrating the incubation to room temperature for 20 to 90 minutes, including the boundary, or for about 20 to about 90 minutes, respectively. In some embodiments, the measurement of cytotoxic activity includes, before addition, equilibrating the incubation to room temperature for 30 to 60 minutes, including the boundary, or for about 30 to about 60 minutes, respectively.

[0106] In some of the embodiments, the measurement of cytotoxic activity includes, before addition, allowing the detection reagent to equilibrate to room temperature for 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, including the boundary, or for approximately 10 to approximately 120 minutes, approximately 20 to approximately 90 minutes, or approximately 30 to approximately 60 minutes, respectively. In some of the embodiments, the measurement of cytotoxic activity includes, before addition, allowing the detection reagent to equilibrate to room temperature for 10 to 120 minutes, including the boundary, or for approximately 10 to approximately 120 minutes, respectively. In some of the embodiments, the measurement of cytotoxic activity includes, before addition, allowing the detection reagent to equilibrate to room temperature for 20 to 90 minutes, including the boundary, or for approximately 20 to approximately 90 minutes, respectively. In some of the embodiments, the measurement of cytotoxic activity includes, before addition, allowing the detection reagent to equilibrate to room temperature for 30 to 60 minutes, including the boundary, or for approximately 30 to approximately 60 minutes, respectively.

[0107] In some of the embodiments, the reporter molecule is an enzyme, and the detection reagent is a substrate of the enzyme. In some of the embodiments, the detection reagent is a luciferase substrate. In some of the embodiments, the detection reagent is luciferin or a luciferin analog. In some of the embodiments, the detection reagent is a luciferin analog. In some of the embodiments, the luciferin analog is 5'-fluoroluciferin.

[0108] In some of the embodiments, the measurement of cytotoxic activity includes incubation in the presence of a detection reagent for between 5 to 240 minutes, 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, including the boundary, or between approximately 5 to approximately 240 minutes, approximately 10 to approximately 120 minutes, approximately 20 to approximately 90 minutes, or approximately 30 to approximately 60 minutes. In some of the embodiments, the measurement of cytotoxic activity includes incubation in the presence of a detection reagent for between 5 to 240 minutes, including the boundary, or between approximately 5 to approximately 240 minutes. In some of the embodiments, the measurement of cytotoxic activity includes incubation in the presence of a detection reagent for between 10 to 120 minutes, including the boundary, or between approximately 10 to approximately 120 minutes. In some of the embodiments, the measurement of cytotoxic activity includes incubation in the presence of a detection reagent for 20 to 90 minutes or about 20 to about 90 minutes, including the boundary. In some of the embodiments, the measurement of cytotoxic activity includes incubation in the presence of a detection reagent for 30 to 60 minutes or about 30 to about 60 minutes, including the boundary.

[0109] In some of the embodiments, the cytotoxic activity of the incubation is measured by luminescence imaging.

[0110] In some of the embodiments, the reporter molecule is a fluorescent protein, and the cytotoxic activity of the incubation is measured by luminescence imaging.

[0111] In some of the embodiments, cytotoxic activity is cytolytic activity.

[0112] In some of the optional embodiments, cytotoxic activity is measured after the end of incubation.

[0113] In some of the embodiments, cytotoxic activity is measured over time during incubation.

[0114] In some of the embodiments, the cytotoxic activity measured for incubation is determined based on the lysis percentage of target cells.

[0115] In some of the embodiments, the target cells are clones from a cell line or primary cells isolated from a subject. In some of the embodiments, the target cells are derived from a cell line. In some embodiments, the cell line is a tumor cell line.

[0116] In some of the embodiments, the target cells are cells that have been engineered to express the target antigen. In some embodiments, the engineered cells are transduced.

[0117] In some of the embodiments, the target antigen is a first tumor antigen, and the target cells are cells engineered not to express a second tumor antigen.

[0118] In some of the embodiments, the effector cell composition comprises single-cell subtypes concentrated or purified from a biological sample. In some of the embodiments, the effector cell composition comprises a population of mixed cell subtypes obtained by mixing cell subtypes concentrated or purified from a biological sample. In some of the embodiments, the biological sample comprises whole blood samples, buffy coat samples, peripheral blood mononuclear cell (PBMC) samples, unfractionated cell samples, lymphocyte samples, leukocyte samples, apheresis products, or leukocyte apheresis products.

[0119] In some of the embodiments, the effector cell composition includes primary cells obtained from the subject.

[0120] In some embodiments, the subject has a disease or condition. In some embodiments, the disease or condition is cancer. In some of the embodiments, the effector cell composition includes autologous cells obtained from the subject to be treated.

[0121] In some of the embodiments, the subject is a healthy subject. In some of the embodiments, the effector cell composition includes allogeneic cells.

[0122] In some of the embodiments, the effector cell composition includes CD3+ T cells. In some of the embodiments, the effector cell composition includes CD4+ T cells and CD8+ T cells.

[0123] In some of the embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0124] In some of the embodiments, incubation is carried out in a flask, tube, or multiwell plate. In some of the embodiments, incubation is carried out in a multiwell plate. In some of the embodiments, the multiwell plate is a 96-well plate, a 48-well plate, a 12-well plate, or a 6-well plate.

[0125] In some parts of any embodiment, the method further includes selecting an effector cell composition for administration to a target requiring it, based on the cytotoxic activity of the effector cell composition.

[0126] In any embodiment, the method further includes determining a dose of cells of the effector cell composition to be administered to a subject requiring it, based on the cytotoxic activity of the effector cell composition.

[0127] In some of the embodiments, the subject has a disease or condition. In some embodiments, the disease or condition is cancer.

[0128] In some of the embodiments, the target cells are ready-to-use (RTU) cells. In some of the embodiments, the cells in the test target cell preparation are ready-to-use (RTU) cells. In some of the embodiments, the RTU cells are cells that are cryopreserved and used immediately after thawing.

[0129] In some of the embodiments, the effector cell composition is a therapeutic cell composition.

[0130] In some of the embodiments, the incubation in step (a) is carried out in serum-free medium. [Brief explanation of the drawing]

[0131] [Figure 1A] Dose-response curves were constructed using cytotoxic assays to evaluate the efficacy of the therapeutic cell compositions. Figure 1A shows the dose-response curves constructed for three anti-BCMA therapeutic cell compositions. Figure 1B shows the dose-response curves constructed for five anti-GPRC5D therapeutic cell compositions. Figure 1C shows the dose-response curves constructed for five anti-BCMA therapeutic cell compositions. [Figure 1B] Same as above. [Figure 1C] Same as above.

[0132] [Figure 2] Figure 2 shows dose-response curves prepared for anti-BCMA therapeutic cell compositions containing two cell lines with little or no BCMA expression (Raji and K562 cells) and one cell line with high BCMA expression (MM.1S cells).

[0133] [Figure 3A] Figure 3A shows the dose-response curves of 12 independent replications of the reference anti-BCMA therapeutic cell composition. Figure 3B shows the dose-response curve of one analyst performing 10 independent replications of the reference anti-GPRC5D therapeutic cell composition. [Figure 3B] Same as above.

[0134] [Figure 4] Figure 4A shows the experimental conditions used to evaluate the ability of a cytotoxic assay to detect changes in potency due to possible ice crystal growth during cryopreservation. Figure 4B shows the dose-response curve created according to the experimental conditions shown in Figure 4A.

[0135] [Figure 5] Figure 5 shows dose-response curves of cytotoxic assays prepared using four cryogenic storage conditions that lead to varying levels of possible ice crystal growth, compared to a reference standard that was not exposed to cryogenic storage.

[0136] [Figure 6A] Figure 6A shows dose-response curves prepared using eight different upper limit dilutions of various effector cell concentrations prepared for the anti-GPRC5D therapeutic cell composition. Figure 6B shows the predicted relative potency percentage versus the observed relative potency percentage of the dose-response curve. Figure 6C shows dose-response curves prepared using ten different upper limit dilutions of various effector cell concentrations prepared for the anti-BCMA therapeutic cell composition. [Figure 6B] Same as above. [Figure 6C] Same as above.

[0137] [Figure 7] Figure 7 shows the linearity of therapeutic cell compositions across various formulation concentrations, including traditional linearity evaluation (top) and linearity with suitability mock evaluation (bottom), for formulated cells (cells expressing and non-expressing recombinant receptors) and donor-matched mock cells. Since the operational process for producing therapeutic cell compositions may not be 100% efficient (see the square on the right), therapeutic cell compositions and effector cell compositions prepared therefrom are generally expected to contain a certain percentage of non-recombinant receptor cells in addition to effector cells expressing recombinant receptors.

[0138] [Figure 8A] Figure 8A shows the results of traditional linearity assessments of cytotoxic assays. Figure 8B shows the results of linearity assessments, including fit mock assessments, of cytotoxic assays. [Figure 8B] Same as above.

[0139] [Figure 9A]Figures 9A-9C compare the results of traditional linearity assessments of cytotoxicity assays with linearity results including fit mock assessments. Figure 9A compares the results under 200% formulation concentration conditions. Figure 9B compares the results under 100% formulation concentration conditions. Figure 9C compares the results under 50% formulation concentration conditions. [Figure 9B] Same as above. [Figure 9C] Same as above.

[0140] [Figure 10A] Figures 10A–10B show dose-response curves of different effector cell preparations against serially cultured or ready-to-use (RTU) target cells, which were cryopreserved in 5%, 10%, or 20% DMSO and then immediately thawed in the assay before use. Figure 10A shows relative luminescence as a measure of luciferase activity in the target cells of each group across the effector cell versus target cell preparations. Figure 10B shows the results from Figure 10A as dose-response curves plotted as lysis percentages by dividing the RLU of each preparation by the maximum RLU detected within each preparation curve. [Figure 10B] Same as above.

[0141] [Figure 11A] Figures 11A–11D compare linearity assessments using mock effector cells or effector cells at different concentrations against any of the RTU target cells used, after being continuously cultured (Figure 11A) or cryopreserved in 5% DMSO (Figure 11B), 10% DMSO (Figure 11C), or 20% DMSO (Figure 11D), and then immediately thawed in the assay. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above.

[0142] [Figure 12A]Figures 12A-12D compare mock effector cells (Sample 2) or different test sample effector cells to either continuously cultured target cells (Figure 12A), RTU target cells pre-frozen after 5 passages (Figure 12B), RTU target cells pre-frozen after 25 passages (Figure 12C), or RTU target cells pre-frozen after 40 passages (Figure 12D). [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above.

[0143] [Figure 13] Figure 13 compares the viable cell count (VCC) and survival rate (%) of MM.1S cells in complete XVIVO medium (cXVIVO prepared in X-Vivo 15 medium supplemented with 5% human AB serum and 1% GlutaMAX®), XVIVO + AB serum (X-Vivo 15 medium supplemented with 5% human AB serum), XVIVO + 1% GlutaMAX® (X-Vivo 15 medium supplemented with 1% GlutaMAX®), and XVIVO alone (X-Vivo 15 medium without additives).

[0144] [Figure 14] Figure 14 shows the luminescence units (RLUs) of target cells in complete XVIVO medium (cXVIVO prepared in X-Vivo 15 medium supplemented with 5% human AB serum and 1% GlutaMAX®), XVIVO + AB serum (X-Vivo 15 medium supplemented with 5% human AB serum), XVIVO + 1% GlutaMAX® (X-Vivo 15 medium supplemented with 1% GlutaMAX®), and XVIVO alone (X-Vivo 15 medium without additives).

[0145] [Figure 15A]Figures 15A-15C compare the dose-response curves of cXVIVO medium (X-Vivo 15 medium supplemented with 5% human AB serum and 1% GlutaMAX®) versus XVIVO + AB serum (X-Vivo 15 medium supplemented with 5% human AB serum) (Figure 15A), cXVIVO medium versus XVIVO + Glutamax (X-Vivo 15 medium supplemented with 1% GlutaMAX®) (Figure 15B), and cXVIVO medium versus XVIVO alone (X-Vivo 15 medium without additives) (Figure 15C). [Figure 15B] Same as above. [Figure 15C] Same as above. [Modes for carrying out the invention]

[0146] Methods for evaluating or determining the potency, e.g., relative potency, of effector cell compositions are provided herein. In certain embodiments, the effector cell composition is a therapeutic cell composition intended for or manufactured for administration to a subject, including engineered T cell therapy. In some embodiments, the methods are for use in connection with monitoring ex vivo processes for generating therapeutic cell compositions, selecting therapeutic cell compositions for administration (e.g., as part of a post-manufacturing release assay), and / or determining doses for the treatment of diseases or conditions, including various cancers. The embodiments provided relate to effector cell compositions (e.g., therapeutic cell compositions) containing engineered cells, such as recombinant receptors, engineered to express recombinant proteins, which recognize and / or specifically bind to molecules associated with a disease or condition, and are designed to produce a response to such molecules, e.g., an immune response, upon binding to such molecules. The receptors may include chimeric receptors, e.g., chimeric antigen receptors (CARs) and other transgenic antigen receptors, including transgenic T cell receptors (TCRs).

[0147] Several advantages relate to using the provided methods to evaluate or determine the potency of effector cell compositions, e.g., therapeutic cell compositions. The methods provided herein are designed to provide a more comprehensive evaluation of the function (e.g., behavior, activity) of therapeutic cell compositions. The methods provided herein are designed to provide a more biologically relevant measure of the potency of therapeutic cell compositions. In some embodiments, the potency of an effector cell composition (e.g., therapeutic cell composition) determined according to the methods described herein may correlate more strongly with the safety and efficacy of the therapeutic cell composition. In some embodiments, the potency of a therapeutic cell composition determined according to the methods described herein may provide an improved measure of manufacturing control and / or variability, which can then enable an improved evaluation of the stability and activity of the manufactured therapeutic cell composition.

[0148] In some embodiments, cells of an effector cell composition, e.g., a therapeutic cell composition, are incubated with target cells that stably express a reporter molecule. In some embodiments, the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition) is measured based on the expression or activity of the reporter molecule. In some embodiments, the use of target cells that stably express the reporter molecule eliminates the need for additional steps prior to the assay, such as labeling the target cells with a dye and any related steps of washing or centrifugating the target cells. Thus, the method provided is more efficient and has enhanced utility in some embodiments. Furthermore, the use of labeling agents such as dyes can lead to the spontaneous release of the labeling agent from the target cells, and such spontaneous release does not occur as a result of incubation with the cells of the effector cell composition (e.g., a therapeutic cell composition). By avoiding such labeling agents, the method provided is not only more specific in some embodiments, but also frees the incubation time from being limited based on the predicted time at which spontaneous release may occur.

[0149] Furthermore, since the manipulation process for generating effector cell compositions (e.g., therapeutic cell compositions) may not be 100% efficient, it is generally expected that effector cell compositions (e.g., therapeutic cell compositions) will contain a certain percentage of non-recombinant receptor cells in addition to effector cells expressing recombinant receptors. As demonstrated herein, the method provided is sensitive to the level or percentage of non-recombinant receptor cells during incubation with target cells, and the addition of such cells results in increased cytotoxic activity. These results are consistent with changes due to bystander effects that may result from the presence of non-recombinant receptor cells (see, for example, Klampatsa et al. (2020) Molecular Therapy: Oncolytics 18:360-371; Jin et al. (2022) Nature Biomedical Engineering 6:830-841; and Gerdemann et al. (2022) Blood 140(Supplement 1):1167-1168). Furthermore, as demonstrated herein, such bystander effects were not observed in alternative assays used to measure recombinant receptor-dependent activity in response to antigen-presenting target cells, where secreted interferon-gamma (IFNγ) levels were measured. These results indicate that the provided methods for evaluating or determining potency provide a better assessment of the integrity of the functional activity of heterogeneous effector cell compositions (e.g., therapeutic cell compositions) containing both recombinant receptor-expressing and non-expressing cells.

[0150] The method provided relates to a direct method for comparing how effector cell compositions (e.g., therapeutic cell compositions) respond to an antigen. Unlike existing methods that often compare activity to a single-target antigen stimulus, which is often the greatest possible stimulus, the method provided modulates the ratio of target cells to effector cells (cells expressing the recombinant receptor of the therapeutic composition). For example, this ratio can be controlled by maintaining a constant number of effector cells in the assay and by varying the number of target cells. For example, the method provided makes it possible to determine the number of target cells required to reach a particular level of cytotoxic activity. In some embodiments, the target antigen is the antigen of the recombinant receptor. Therefore, in some cases, the target cells are antigen-expressing cells. In some embodiments, a particular level of cytotoxic activity is 50% of the maximum cytotoxic activity. In some embodiments, a particular level of cytotoxic activity is 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the maximum cytotoxic activity. In some embodiments, a particular level of cytotoxic activity is within a range of any of the values ​​disclosed herein. In some embodiments, the provided method allows for the comparison of effector cell compositions (e.g., therapeutic cell compositions). For example, effector cell compositions (e.g., therapeutic cell compositions) can be evaluated by the method provided herein to determine their relative potency, for example, as described herein.

[0151] In some embodiments, the methods provided herein include assay forms comprising a series of incubations in which cells of a therapeutic cell composition in different adjusted ratios and target cells are cultured. In the provided embodiments, the target cells express a target antigen that induces or is signalable via the intracellular signaling domain of a recombinant receptor, e.g., a cell surface target antigen (a target antigen expressed on the surface of the target cell). For example, the target cells may contain an antigen of a recombinant receptor, e.g., a purified antigen or recombinant antigen, an antigen-expressing cell, or an anti-idiotype antibody specific to the extracellular antigen-binding domain of a recombinant receptor (e.g., scFv). In some embodiments, the methods comprising assay forms provided herein are designed to measure the sensitivity of an effector cell composition (e.g., a therapeutic cell composition) by measuring or determining the number of target cells required to stimulate cytotoxic activity in manipulated cells of the therapeutic cell composition. For example, the methods provided herein can determine the number of target cells that stimulate quantifiable and detectable cytotoxic activity of a therapeutic cell composition. In some embodiments, the sensitivity measure includes measurements of cytotoxic activity stimulated by a target antigen expressed on target cells bound to recombinant receptors across multiple adjusted ratios. The ability of methods to assess recombinant receptor-dependent activity at different adjusted ratios allows for the determination, estimation, and / or extrapolation of the general activity or behavior of therapeutic cell compositions to recombinant receptor-specific stimulation.

[0152] The methods provided herein include assays that enable the evaluation of the potency of an effector cell composition (e.g., a therapeutic cell composition) by measuring the cytotoxic activity of cells expressing a recombinant receptor, e.g., any of the recombinant receptors described herein, such as any of those described in Section III, in response to stimulation of the recombinant receptor in a series of controlled incubations. For example, the series of incubations may include culturing engineered cells of an effector cell composition (e.g., a therapeutic cell composition) expressing a recombinant receptor together with target cells expressing a target antigen that stimulates cytotoxic activity upon binding to the recombinant receptor, wherein the culturing is performed in different adjusted ratios of cells of the therapeutic cell composition to target cells, and each incubation is performed in different adjusted ratios. In some embodiments, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more incubations are performed, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more incubations are performed, each incubation containing different ratios of cells from the effector cell composition (e.g., therapeutic cell composition) to the target cells. In some embodiments, 3 or at least 3 incubations are performed, each incubation containing different adjusted ratios of cells from the effector cell composition (e.g., therapeutic cell composition) to the target cells. In some embodiments, 6 or at least 6 incubations are performed, each incubation containing different adjusted ratios of cells from the effector cell composition (e.g., therapeutic cell composition) to the target cells. In some embodiments, 10 or at least 10 incubations are performed, each incubation containing different adjusted ratios of cells from the effector cell composition (e.g., therapeutic cell composition) to the target cells.

[0153] In some embodiments, a certain number of cells of a therapeutic composition are cultured with a different number of target cells to yield a range of (e.g., multiple) different adjusted ratios. Alternatively, in some embodiments, a certain number of target cells may be incubated with a different number of cells of a therapeutic cell composition to yield a range of (e.g., multiple) different adjusted ratios. Regardless of how the different adjusted ratios are achieved, a range of (e.g., multiple) adjusted ratios can be used to evaluate cytotoxic activity across a range of stimulation conditions, for example, by varying the total number of cells of the therapeutic cell composition or the number of target cells. In some embodiments, the range of measurements can be used to extract, estimate, and / or determine how manipulated cells of a particular therapeutic cell composition respond to different levels of recombinant receptor stimulation.

[0154] From the measured cytotoxic activity generated according to the methods described herein, any number of measures can be determined, extracted, extrapolated, estimated, and / or inferred. Examples of measures include adjusted ratios that produce the maximum, minimum, and maximum half (50%) cytotoxic activity; adjusted ratios that produce a specific percentage of the maximum cytotoxic activity (e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90%); and adjusted ratios that encompass a certain range of cytotoxic activity, e.g., 10%–90%, 20%–80%, 30%–70%, and 40%–60% of the maximum cytotoxic activity. In some embodiments, the measured cytotoxic activity of an effector cell composition (e.g., a therapeutic cell composition) is curve-fitted to generate a cytotoxic activity curve. In some embodiments, the curve is similar to a dose-response curve. In some embodiments, the measure of cytotoxic activity and / or ratio at which a particular cytotoxic activity occurs is extrapolated and / or estimated from the curve. In some embodiments, the cytotoxic activity curve can be used to extrapolate the value or measure of the therapeutic cell composition and / or target cells at which a particular cytotoxic activity occurs. In some embodiments, for example, when the cell count of the therapeutic cell composition is kept constant and the number of target cells is changed, the number of target cells is used to determine the maximum, minimum, maximum half, and range at which cytotoxic activity occurs. In some embodiments, for example, when the cell count of the therapeutic cell composition is changed and the number of target cells is kept constant, the number of cells in the therapeutic cell composition (e.g., count, total) is used to determine the maximum, minimum, maximum half, and range at which cytotoxic activity occurs. In some embodiments, a adjusted ratio is used to determine the maximum, minimum, maximum half, and range at which cytotoxic activity occurs. In some embodiments, the number of target cells is used to determine the maximum half of cytotoxic activity. In some embodiments, the number of cells in the effector cell composition (e.g., therapeutic cell composition) is used to determine the maximum half of cytotoxic activity. In some embodiments, adjusted ratios are used to determine the maximum half of the cytotoxic activity.These exemplified measures, as well as others not listed, provide a quantitative description of therapeutic cell compositions that may be used to determine the potency and / or relative potency (e.g., potency relative to a reference standard) of a therapeutic cell composition.

[0155] In some embodiments, the potency of an effector cell composition (e.g., a therapeutic cell composition) is expressed as a modified ratio value or scale, the number of cells in the therapeutic cell composition, and / or the number of target cells, determined based on cytotoxic activity. In some embodiments, the potency of an effector cell composition (e.g., a therapeutic cell composition) is a modified ratio value or scale, the number of cells in the therapeutic cell composition, and / or the number of target cells, at which the maximum half-value of cytotoxic activity (e.g., 50% of maximum activity) occurs. In some embodiments, the potency of an effector cell composition (e.g., a therapeutic cell composition) is a modified ratio at which the maximum half-value of cytotoxic activity (e.g., 50% of maximum activity) occurs. In some embodiments, the potency of a therapeutic cell composition is the concentration of target cells at which the maximum half-value of cytotoxic activity occurs. In some embodiments, according to the measured cytotoxic activity, the maximum half-value of cytotoxic activity at which the 50% effective stimulus (ES50) of the therapeutic cell composition occurs reflects a modified ratio, the concentration of target cells, and / or the cell count.

[0156] In some embodiments, the potency of the therapeutic cell composition is relative. For example, a modified ratio from which up to half the cytotoxic activity is measured for an effector cell composition (e.g., the therapeutic cell composition) can be compared to a modified ratio from which up to half the cytotoxic activity is measured for a reference standard. Naturally, where appropriate, the number of target cells may be used instead of the modified ratio. In some embodiments, the reference standard is an effector cell composition (e.g., the therapeutic cell composition) having a known and / or validated modified ratio that produces up to half the cytotoxic activity. In some embodiments, the reference standard is a commercially available therapeutic cell composition from which the modified ratio producing up to half the cytotoxic activity is determined, for example, by a method as described herein. In some embodiments, the reference standard is a different therapeutic cell composition from which the modified ratio producing up to half the cytotoxic activity is determined, for example, by a method as described herein. In some embodiments, the different therapeutic cell composition contains cells expressing recombinant receptors that bind to the same antigen as the test therapeutic cell composition but have a different receptor structure. In some embodiments, different therapeutic cell compositions express the same recombinant receptor as the experimental therapeutic cell composition, but contain cells produced using a process different from the process used to produce the experimental therapeutic cell composition. In some embodiments, relative potency is a ratio determined by dividing the adjusted ratio yielding up to half the value of the experimental therapeutic cell composition by the adjusted ratio yielding up to half the value of the reference standard. In some embodiments, relative potency is a percentage determined by dividing the adjusted ratio yielding up to half the value of the experimental therapeutic cell composition by the adjusted ratio yielding up to half the value of the reference standard and multiplying by 100.

[0157] In some cases, normalizing the cytotoxic activity of effector cell compositions (e.g., therapeutic cell compositions) is useful for determining whether the cytotoxic activity of two or more effector cell compositions (e.g., therapeutic cell compositions) can be compared. For example, if the cytotoxic activity of two or more therapeutic compositions is determined, the maximum and / or minimum cytotoxic activity of each tested effector cell composition (e.g., therapeutic cell composition) will be different, and normalizing the cytotoxic activity of each composition relative to its own maximum value may allow for an assessment of the validity of comparing recombinant receptor-dependent activity.

[0158] In some embodiments, cytotoxic activity is normalized to the measured maximum cytotoxic activity value. In some embodiments, the cytotoxic activity curve is normalized to the measured maximum cytotoxic activity. In some embodiments, when the cytotoxicity curve is normalized, the maximum activity value is the average over the upper asymptote of the curve. In some embodiments, normalizing the cytotoxic activity of the therapeutic cell composition and the reference standard by their respective maximum values ​​facilitates comparison between the test therapeutic cell composition and the reference standard. In some embodiments, normalizing the cytotoxic activity of the therapeutic cell composition and the reference standard by their respective maximum values ​​facilitates the calculation of relative potency.

[0159] In some embodiments, parallel line testing can be performed by normalizing the cytotoxic activity of the therapeutic cell composition and the reference standard by their respective maximum values. In some embodiments, the results of parallel line testing demonstrate the ability to compare the therapeutic cell composition and the reference standard.

[0160] Methods including assays provided herein for evaluating the efficacy of effector cell compositions (e.g., therapeutic cell compositions) allow for the comparison of different effector cell compositions (e.g., therapeutic cell compositions) including a reference standard. The ability to compare effector cell compositions (e.g., therapeutic cell compositions) not only identifies effector cell compositions (e.g., therapeutic cell compositions) with improved, optimal, and / or consistent potency, but also identifies candidate effector cell compositions (e.g., therapeutic cell compositions) for further development and / or analysis; identifies manufacturing processes and procedures that result in improved or optimally potent effector cell compositions (e.g., therapeutic cell compositions); identifies manufacturing procedures or processes that result in consistently potent effector cell compositions (e.g., therapeutic cell compositions); and / or estimates the variability inherent in manufacturing procedures; determines the dose of an effector cell composition (e.g., therapeutic cell composition) to be administered to a target requiring it, e.g., a dose that produces a clinical response without the occurrence of toxicity; and / or provides a method for comparing the potency of allogeneic effector cell compositions (e.g., therapeutic cell compositions) to autologous effector cell compositions (e.g., therapeutic cell compositions). The methods provided herein are designed to conform to a relative potency format that is independent of whether the test therapeutic composition or reference standard originates from a different donor (e.g., subject), manufacturing process, and / or therapeutic product.

[0161] All publications, including patent documents, scientific papers, and databases, referenced in this application are incorporated herein by reference in whole for any purpose to the same extent as if each individual publication were incorporated by reference individually. If any definition contained herein contradicts or otherwise conflicts with any definition contained in a patent, application, published application, or other publication incorporated herein by reference, the definition contained herein shall prevail over the definition incorporated herein by reference.

[0162] The chapter headings used herein are for systematization purposes only and should not be construed as limiting the subjects described.

[0163] I. Cell efficacy assay Methods for evaluating or determining the efficacy of an effector cell composition (e.g., a therapeutic cell composition) are provided herein. In some embodiments, the effector cell composition (e.g., a therapeutic cell composition) is a therapeutic immune cell composition. In some embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains B cells, T cells and / or natural killer (NK) cells.

[0164] In some embodiments, the effector cell composition (e.g., therapeutic cell composition) is a therapeutic T cell composition. In some embodiments, the effector cell composition (e.g., therapeutic cell composition) contains CD3+ T cells. In some embodiments, the effector cell composition (e.g., therapeutic cell composition) contains CD4+ T cells and / or CD8+ T cells. In some embodiments, the effector cell composition (e.g., therapeutic cell composition) contains CD4+ T cells and CD8+ T cells.

[0165] In some embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains cells engineered to express a recombinant receptor. In some embodiments, the recombinant receptor is any of those described herein, for example, those described in Section III. In some embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains T cells engineered to express a recombinant receptor. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor is a T cell receptor (TCR).

[0166] In some embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains cells that do not express recombinant receptors. In some embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains T cells that do not express recombinant receptors. In some embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains cells that have been manipulated to express recombinant receptors, e.g., T cells, and cells that do not express recombinant receptors, e.g., T cells. In some embodiments, cells that do not express recombinant receptors, e.g., T cells, do not express any recombinant receptors.

[0167] In some embodiments, the provided method includes incubating cells of an effector cell composition (e.g., a therapeutic cell composition) together with target cells.

[0168] In some embodiments, the provided method includes performing multiple incubations. In some embodiments, each of the multiple incubations includes culturing cells of an effector cell composition (e.g., a therapeutic cell composition) together with target cells.

[0169] In some embodiments, the cells of the cultured effector cell composition (e.g., a therapeutic cell composition) include cells that have been engineered to express recombinant receptors, such as T cells. In some embodiments, the cells of the cultured effector cell composition (e.g., a therapeutic cell composition) include cells that do not express recombinant receptors, such as T cells. In some embodiments, the cells of the cultured effector cell composition (e.g., a therapeutic cell composition) include cells that have been engineered to express recombinant receptors, such as T cells, and cells that do not express recombinant receptors, such as T cells.

[0170] In some embodiments, target cells express a reporter molecule. In some embodiments, target cells stably express the reporter molecule. In some embodiments, target cells are cells that express the reporter molecule, for example, cells that have been engineered to stably express it. In some embodiments, the manipulation is performed by transduction. In some embodiments, the manipulation is performed by any of the manipulation methods described herein, for example, any of those described in Section II-C.

[0171] In some embodiments, the expression of the reporter molecule by target cells is controlled by a constituent promoter. In some embodiments, the promoter is a human β-actin, human elongation factor-1α, chicken β-actin combined with a cytomegalovirus initial enhancer, cytomegalovirus (CMV), monkey virus 40, or herpes simplex virus thymidine kinase promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is any of those described herein, for example, those described in Section II-C-1-a.

[0172] In some embodiments, target cells express a target antigen. In some embodiments, the target antigen is a cell surface target antigen (a target antigen expressed on the surface of a target cell). In some embodiments, the target antigen is recognized or bound by a recombinant receptor. In some embodiments, the target antigen is specifically bound by a recombinant receptor.

[0173] In some embodiments, each of the multiple incubations contains different adjusted ratios of cells from the effector cell composition (e.g., therapeutic cell composition) to the target cells. In some embodiments, each of the different adjusted ratios is based on cells expressing the recombinant receptor of the effector cell composition (e.g., therapeutic cell composition).

[0174] In some embodiments, the provided method includes incubating cells of an effector cell composition (e.g., a therapeutic cell composition) together with target cells and cells of a mock cell composition. In some embodiments, the cultured cells of the mock cell composition do not express recombinant receptors. In some embodiments, the cultured cells of the mock cell composition do not express target antigens. In some embodiments, the cultured cells of the mock cell composition do not express recombinant receptors or target antigens.

[0175] In some embodiments, the methods provided are for evaluating or determining bystander effect changes in cells that do not express recombinant receptors.

[0176] In some embodiments, the mock cell composition is produced using any of the manufacturing processes described herein, for example, any of those described in Section II. In some embodiments, the mock cell composition is produced using a manufacturing process that is identical to the manufacturing process used to produce an effector cell composition (e.g., a therapeutic cell composition), except that the cells of the mock cell composition are not exposed to nucleic acids encoding recombinant receptors.

[0177] In some embodiments, the provided method includes measuring cytotoxic activity from incubation.

[0178] In some embodiments, the provided method includes measuring cytotoxic activity from each of a plurality of incubations.

[0179] In some embodiments, cytotoxic activity is measured based on the expression or activity of the reporter molecule. In some embodiments, cytotoxic activity is measured based on the expression of the reporter molecule. In some embodiments, cytotoxic activity is measured based on the activity of the reporter molecule.

[0180] In some embodiments, the expression or activity of the reporter molecule is directly correlated with cytotoxic activity. In some embodiments, the expression or activity of the reporter molecule is inversely correlated with cytotoxic activity.

[0181] In some embodiments, the provided method includes determining a modified ratio that yields a specific level of cytotoxic activity in an effector cell composition (e.g., a therapeutic cell composition). In some embodiments, the modified ratio is determined based on the cytotoxic activity measured for each of several incubations. In some embodiments, the specific level of cytotoxic activity is 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the maximum cytotoxic activity. In some embodiments, the specific level of cytotoxic activity is half the maximum cytotoxic activity.

[0182] In some embodiments, the provided method includes determining the relative potency of an effector cell composition (e.g., a therapeutic cell composition). In some embodiments, the relative potency is determined by comparing a specific level of cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition), for example, a modified ratio that yields up to half the amount of cytotoxic activity, with a reference standard, for example, a modified ratio that yields up to half the amount of cytotoxic activity.

[0183] In some embodiments, relative potency is determined based on cytotoxic activity measured for each of multiple incubations. In some embodiments, relative potency is determined by comparing a specific level of cytotoxic activity of an effector cell composition (e.g., a therapeutic cell composition), e.g., up to half the amount of cytotoxic activity, to the same specific level of cytotoxic activity of a reference standard, e.g., up to half the amount of cytotoxic activity. In some embodiments, the specific level of cytotoxic activity is 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the maximum cytotoxic activity. In some embodiments, the specific level of cytotoxic activity is up to half the amount of cytotoxic activity.

[0184] In some embodiments, the method provided includes selecting a therapeutic composition to be administered to a target requiring it, based on a specific level of cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition), for example, a modified ratio that yields up to half the amount of cytotoxic activity. In some embodiments, the method provided includes selecting a cell dose of the therapeutic composition to be administered to a target requiring it, based on a specific level of cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition), for example, a modified ratio that yields up to half the amount of cytotoxic activity.

[0185] In some embodiments, the method provided includes selecting a therapeutic composition to be administered to a subject requiring it, based on its relative potency. In some embodiments, the method provided includes selecting a cellular dose of the therapeutic composition to be administered to a subject requiring it, based on its relative potency.

[0186] In some embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains primary cells obtained from a subject. In some embodiments, the reference standard contains primary cells obtained from a subject.

[0187] In some embodiments, the subject has a disease or condition. In some embodiments, the disease or condition is one of those described herein. In some embodiments, the disease or condition is cancer.

[0188] In some embodiments, cytotoxic activity is induced in response to stimulation of recombinant receptors. In some embodiments, cytotoxic activity is cytolytic activity.

[0189] In some embodiments, cytotoxic activity occurs in cells expressing the recombinant receptor of the effector cell composition (e.g., a therapeutic cell composition). In some embodiments, cytotoxic activity is recombinant receptor-dependent. In some embodiments, binding of the target antigen to the recombinant receptor stimulates cytotoxic activity.

[0190] In some embodiments, cytotoxic activity is increased in the presence of cells from an effector cell composition (e.g., a therapeutic cell composition) that does not express recombinant receptors.

[0191] In some embodiments, multiple incubations involve culturing cells expressing a certain number of recombinant receptors of the therapeutic composition together with different numbers of target cells. In some embodiments, different numbers of target cells are obtained by serially diluting the target cells.Some explanatory notes indicate that serial dilutions, including the respective boundaries, are defined as follows: changes between 2x and 10x, changes between 2x and 9x, changes between 2x and 8x, changes between 2x and 7x, changes between 2x and 6x, changes between 2x and 5x, changes between 2x and 4x, changes between 2x and 3x, changes between 3x and 10x, changes between 3x and 9x, changes between 3x and 8x, changes between 3x and 7x, changes between 3x and 6x, changes between 3x and 5x, changes between 3x and 4x, changes between 4x and 10x, changes between 4x and 9x, changes between 4x and 8x, and 4x and 7x. Changes between 2x, changes between 4x and 6x, changes between 4x and 5x, changes between 5x and 10x, changes between 5x and 9x, changes between 5x and 8x, changes between 5x and 7x, changes between 5x and 6x, changes between 6x and 10x, changes between 6x and 9x, changes between 6x and 8x, changes between 6x and 7x, changes between 7x and 10x, changes between 7x and 9x, changes between 7x and 8x, changes between 8x and 10x, changes between 8x and 9x or changes between 8x and 10x or changes between approximately 2x and approximately 10x, changes between approximately 2x and approximately 9x Changes between approximately 2x and 8x, changes between approximately 2x and 7x, changes between approximately 2x and 6x, changes between approximately 2x and 5x, changes between approximately 2x and 4x, changes between approximately 2x and 3x, changes between approximately 3x and 10x, changes between approximately 3x and 9x, changes between approximately 3x and 8x, changes between approximately 3x and 7x, changes between approximately 3x and 6x, changes between approximately 3x and 5x, changes between approximately 3x and 4x, changes between approximately 4x and 10x, changes between approximately 4x and 9x, changes between approximately 4x and 8x, changes between approximately 4x and 7x, changes between approximately 4x and 6x This is due to changes in magnification between approximately 4x and 5x, between approximately 5x and 10x, between approximately 5x and 9x, between approximately 5x and 8x, between approximately 5x and 7x, between approximately 5x and 6x, between approximately 6x and 10x, between approximately 6x and 9x, between approximately 6x and 8x, between approximately 6x and 7x, between approximately 7x and 10x, between approximately 7x and 9x, between approximately 7x and 8x, between approximately 8x and 10x, between approximately 8x and 9x, or between approximately 8x and 10x.

[0192] In some embodiments, multiple incubations include culturing a certain number of target cells together with cells expressing different numbers of recombinant receptors of the therapeutic composition. In some embodiments, cells expressing different numbers of recombinant receptors of the therapeutic composition are obtained by serially diluting cells of an effector cell composition (e.g., the therapeutic cell composition).Some explanatory notes indicate that serial dilutions, including the respective boundaries, are defined as follows: changes between 2x and 10x, changes between 2x and 9x, changes between 2x and 8x, changes between 2x and 7x, changes between 2x and 6x, changes between 2x and 5x, changes between 2x and 4x, changes between 2x and 3x, changes between 3x and 10x, changes between 3x and 9x, changes between 3x and 8x, changes between 3x and 7x, changes between 3x and 6x, changes between 3x and 5x, changes between 3x and 4x, changes between 4x and 10x, changes between 4x and 9x, changes between 4x and 8x, and 4x and 7x. Changes between 2x, changes between 4x and 6x, changes between 4x and 5x, changes between 5x and 10x, changes between 5x and 9x, changes between 5x and 8x, changes between 5x and 7x, changes between 5x and 6x, changes between 6x and 10x, changes between 6x and 9x, changes between 6x and 8x, changes between 6x and 7x, changes between 7x and 10x, changes between 7x and 9x, changes between 7x and 8x, changes between 8x and 10x, changes between 8x and 9x or changes between 8x and 10x or changes between approximately 2x and approximately 10x, changes between approximately 2x and approximately 9x Changes between approximately 2x and 8x, changes between approximately 2x and 7x, changes between approximately 2x and 6x, changes between approximately 2x and 5x, changes between approximately 2x and 4x, changes between approximately 2x and 3x, changes between approximately 3x and 10x, changes between approximately 3x and 9x, changes between approximately 3x and 8x, changes between approximately 3x and 7x, changes between approximately 3x and 6x, changes between approximately 3x and 5x, changes between approximately 3x and 4x, changes between approximately 4x and 10x, changes between approximately 4x and 9x, changes between approximately 4x and 8x, changes between approximately 4x and 7x, changes between approximately 4x and 6x This is due to changes in magnification between approximately 4x and 5x, between approximately 5x and 10x, between approximately 5x and 9x, between approximately 5x and 8x, between approximately 5x and 7x, between approximately 5x and 6x, between approximately 6x and 10x, between approximately 6x and 9x, between approximately 6x and 8x, between approximately 6x and 7x, between approximately 7x and 10x, between approximately 7x and 9x, between approximately 7x and 8x, between approximately 8x and 10x, between approximately 8x and 9x, or between approximately 8x and 10x.

[0193] In some embodiments, in each of the multiple incubations, including their respective boundaries, there are 1,000 to 80,000 target cells, 1,000 to 70,000 target cells, 1,000 to 60,000 target cells, 1,000 to 50,000 target cells, 1,000 to 40,000 target cells, 1,000 to 30,000 target cells, 1,000 to 20,000 target cells, 1,000 to 15,000 target cells, 1,000 to 14,000 target cells, and 1,000 to 13,000 target cells. , 1,000 to 12,000 target cells, 1,000 to 11,000 target cells, 1,000 to 10,000 target cells, 1,000 to 9,000 target cells, 1,000 to 8,000 target cells, 1,000 to 7,000 target cells, 1,000 to 6,000 target cells, 1,000 to 5,000 target cells, 1,000 to 4,000 target cells, 1,000 to 3,000 target cells, 1,000 to 2,000 target cells, 5,000 to 80,000 target cells, 5,000 0 to 70,000 target cells, 5,000 to 60,000 target cells, 5,000 to 50,000 target cells, 5,000 to 40,000 target cells, 5,000 to 30,000 target cells, 5,000 to 20,000 target cells, 5,000 to 15,000 target cells, 5,000 to 14,000 target cells, 5,000 to 13,000 target cells, 5,000 to 12,000 target cells, 5,000 to 11,000 target cells, 5,000 to 10,000 target cells, 5 ,000 to 9,000 target cells, 5,000 to 8,000 target cells, 5,000 to 7,000 target cells, 5,000 to 6,000 target cells, 8,000 to 80,000 target cells, 8,000 to 70,000 target cells, 8,000 to 60,000 target cells, 8,000 to 50,000 target cells, 8,000 to 40,000 target cells, 8,000 to 30,000 target cells, 8,000 to 20,000 target cells, 8,000 to 15,000 target cells, 8,000 to 14,000 target cells, 8,000 to 13,000 target cells, 8,000 to 12,000 target cells, 8,000 to 11,000 target cells, 8,000 to 10,000 target cells, 8,000 to 9,000 target cells, 9,000 to 80,000 target cells, 9,000 to 70,000 target cells, 9,000 to 60,000 target cells, 9,000 to 50,000 target cells, 9,000 to 40,000 target cells, 9,000 to 30,000 target cells, 9,000 to 20,000 target cells, 9,000 to 15,000 target cells, 9,000 to 14,000 target cells, 9,000 to 13,000 target cells, 9,000 to 12,000 target cells, 9,000 to 11,000 target cells or between 9,000 and 10,000 target cells, or approximately 1,000 to approximately 80,000 target cells, approximately 1,000 to approximately 70,000 target cells, approximately 1,000 to approximately 60,000 target cells, approximately 1,000 to approximately 50,000 target cells, approximately 1, 000 to approximately 40,000 target cells, approximately 1,000 to approximately 30,000 target cells, approximately 1,000 to approximately 20,000 target cells, approximately 1,000 to approximately 15,000 target cells, approximately 1,000 to approximately 14,000 target cells, approximately 1,000 to approximately 13,000 target cells, approximately 1,000 to approximately 12,000 target cells, approximately 1,000 to approximately 11,000 target cells, approximately 1,000 to approximately 10,000 target cells, approximately 1,000 to approximately 9,000 target cells, approximately 1,000 to approximately 8,000 target cells, approximately 1,000 to approximately 7,000 target cells, approximately 1,000 to approximately 6,000 target cells, approximately 1,000 to approximately 5,000 target cells, approximately 1,000 to approximately 4,000 target cells, approximately 1,000 to approximately 3,000 target cells, approximately 1,000 to approximately 2,000 target cells, approximately 5,000 to approximately 80,000 target cells, approximately 5,000 to approximately 70,000 target cells, approximately 5,000 to approximately 60,000 target cells, approximately 5,000 to approximately 50,000 target cells, approximately 5,000 to approximately 40,000 target cells, approximately 5,000 to approximately 30,000 target cells, approximately 5,000 to approximately 20,000 target cells, approximately 5,000 to approximately 15,000 target cells, approximately 5,000 to approximately 14,000 target cells, approximately 5,000 to approximately 13,000 target cells, approximately 5,000 to approximately 12,000 target cells, approximately 5,000 to approximately 11,000 target cells, approximately 5,000 to approximately 10,000 target cells, approximately 5,000 to approximately 9,000 target cells, approximately 5,000 to approximately 8,000 target cells Cells, approximately 5,000 to 7,000 target cells, approximately 5,000 to 6,000 target cells, approximately 8,000 to 80,000 target cells, approximately 8,000 to 70,000 target cells, approximately 8,000 to 60,000 target cells, approximately 8,000 to 50,000 target cells, approximately 8,000 to 40,000 target cells, approximately 8,000 to 30,000 target cells, approximately 8,000 to 20,000 target cells, approximately 8,000 to 15,000 Target cells, approximately 8,000 to 14,000 target cells, approximately 8,000 to 13,000 target cells, approximately 8,000 to 12,000 target cells, approximately 8,000 to 11,000 target cells, approximately 8,000 to 10,000 target cells, approximately 8,000 to 9,000 target cells, approximately 9,000 to 80,000 target cells, approximately 9,000 to 70,000 target cells, approximately 9,000 to 60,000 target cells, approximately 9,000 to 5 Between 0,000 target cells, approximately 9,000 to 40,000 target cells, approximately 9,000 to 30,000 target cells, approximately 9,000 to 20,000 target cells, approximately 9,000 to 15,000 target cells, approximately 9,000 to 14,000 target cells, approximately 9,000 to 13,000 target cells, approximately 9,000 to 12,000 target cells, approximately 9,000 to 11,000 target cells, or approximately 9,000 to 10,000 target cells.

[0194] In some embodiments, the target cells are contained in the target cell composition prior to the plurality of incubations. In some embodiments, the target cell composition includes, including boundaries, from 0.1×10 6 to 0.3×10 6 target cells / mL, from 0.1×10 6 to 0.28×10 6 target cells / mL, from 0.1×10 6 to 0.26×10 6 target cells / mL, from 0.1×10 6 to 0.25×10 6 target cells / mL, from 0.1×10 6 to 0.24×10 6 target cells / mL, from 0.1×10 6 to 0.22×10 6 target cells / mL, from 0.1×10 6 to 0.2×10 6 target cells / mL, from 0.1×10 6 to 0.18×10 6 target cells / mL, from 0.1×10 6 to 0.15×10 6 target cells / mL, from 0.1×10 6 to 0.16×10 6 target cells / mL, from 0.1×10 6 to 0.14×10 6 target cells / mL, from 0.1×10 6 to 0.12×10 6 target cells / mL, from 0.15×10 6 to 0.3×10 6 target cells / mL, from 0.15×10 6 to 0.28×10 6 target cells / mL, from 0.15×10 6 to 0.26×10 6 target cells / mL, from 0.15×10 6 to 0.25×10 6 target cells / mL, from 0.15×10 6 to 0.24×10 6 target cells / mL, from 0.15×10 6 to 0.22×106 target cells / mL, 0.15×10 6 to 0.2×10 6 target cells / mL, 0.15×10 6 to 0.18×10 6 target cells / mL, 0.18×10 6 to 0.3×10 6 target cells / mL, 0.18×10 6 to 0.28×10 6 target cells / mL, 0.18×10 6 to 0.26×10 6 target cells / mL, 0.18×10 6 to 0.25×10 6 target cells / mL, 0.18×10 6 to 0.24×10 6 target cells / mL, 0.18×10 6 to 0.22×10 6 target cells / mL or 0.18×10 6 to 0.2×10 6 between target cells / mL or about 0.1×10 6 to about 0.3×10 6 target cells / mL, about 0.1×10 6 to about 0.28×10 6 target cells / mL, about 0.1×10 6 to about 0.26×10 6 target cells / mL, about 0.1×10 6 to about 0.25×10 6 target cells / mL, about 0.1×10 6 to about 0.24×10 6 target cells / mL, about 0.1×10 6 to about 0.22×10 6 target cells / mL, about 0.1×10 6 to about 0.2×10 6 target cells / mL, about 0.1×10 6 to about 0.18×10 6 target cells / mL, about 0.1×10 6 to about 0.15×10 6 target cells / mL, about 0.1×106 Approximately 0.16 × 10 from each 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.14 × 10 from each 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.12 × 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.3 x 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 From each individual, approximately 0.28 × 10 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.26 × 10 from each individual 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.25 × 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.24 × 10 from each individual 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.22 × 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.2 × 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.18 × 10 from each 6 Target cells / mL, approximately 0.18 × 10⁶ 6 Approximately 0.3 x 10 from each 6 Target cells / mL, approximately 0.18 × 10⁶ 6 From each individual, approximately 0.28 × 10 6 Target cells / mL, approximately 0.18 × 10⁶ 6 Approximately 0.26 × 10 from each individual 6 Target cells / mL, approximately 0.18 × 10⁶ 6 Approximately 0.25 × 10 from each 6 Target cells / mL, approximately 0.18 × 10⁶ 6 Approximately 0.24 × 10 from each individual 6 Target cells / mL, approximately 0.18 × 10⁶ 6 Approximately 0.22 × 10 from each 6 Target cells / mL or approximately 0.18 × 10⁶ 6 Approximately 0.2 × 10 from each 6 It has a concentration between 1 target cells / mL.

[0195] In some embodiments, target cells before multiple incubations are each 0.1 × 10⁶ including their boundaries. 6 From 0.3 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.28 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.26 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.25 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.24 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.22 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.2 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.18 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.15 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.16 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.14 × 10 6 Target cells / mL, 0.1 × 10⁶ 6 From 0.12 × 10 6 Target cells / mL, 0.15 × 10⁶ 6 From 0.3 × 10 6 Target cells / mL, 0.15 × 10⁶ 6 From 0.28 × 10 6 Target cells / mL, 0.15 × 10⁶ 6 From 0.26 × 10 6 Target cells / mL, 0.15 × 10⁶ 6 From 0.25 × 10 6 Target cells / mL, 0.15 × 10⁶ 6 From 0.24 × 10 6 Target cells / mL, 0.15 × 10⁶ 6 From 0.22 × 10 6Target cells / mL, 0.15 × 10⁶ 6 From 0.2 × 10 6 Target cells / mL, 0.15 × 10⁶ 6 From 0.18 × 10 6 Target cells / mL, 0.18 × 10⁶ 6 From 0.3 × 10 6 Target cells / mL, 0.18 × 10⁶ 6 From 0.28 × 10 6 Target cells / mL, 0.18 × 10⁶ 6 From 0.26 × 10 6 Target cells / mL, 0.18 × 10⁶ 6 From 0.25 × 10 6 Target cells / mL, 0.18 × 10⁶ 6 From 0.24 × 10 6 Target cells / mL, 0.18 × 10⁶ 6 From 0.22 × 10 6 Target cells / mL or 0.18 × 10⁶ 6 From 0.2 × 10 6 Between individual target cells / mL or approximately 0.1 × 10⁶ 6 Approximately 0.3 x 10 from each 6 Target cells per mL, approximately 0.1 × 10⁶ 6 From each individual, approximately 0.28 × 10 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.26 × 10 from each individual 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.25 × 10 from each 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.24 × 10 from each individual 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.22 × 10 from each 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.2 × 10 from each 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.18 × 10 from each 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.15 × 10 from each 6 Target cells per mL, approximately 0.1 × 10⁶ 6Approximately 0.16 × 10 from each 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.14 × 10 from each 6 Target cells per mL, approximately 0.1 × 10⁶ 6 Approximately 0.12 × 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.3 x 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 From each individual, approximately 0.28 × 10 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.26 × 10 from each individual 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.25 × 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.24 × 10 from each individual 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.22 × 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.2 × 10 from each 6 Target cells per mL, approximately 0.15 × 10⁶ 6 Approximately 0.18 × 10 from each 6 Target cells / mL, approximately 0.18 × 10⁶ 6 Approximately 0.3 x 10 from each 6 Target cells / mL, approximately 0.18 × 10⁶ 6 From each individual, approximately 0.28 × 10 6 Target cells / mL, approximately 0.18 × 10⁶ 6 Approximately 0.26 × 10 from each individual 6 Target cells / mL, approximately 0.18 × 10⁶ 6 Approximately 0.25 × 10 from each 6 Target cells / mL, approximately 0.18 × 10⁶ 6 Approximately 0.24 × 10 from each individual 6 Target cells / mL, approximately 0.18 × 10⁶ 6 Approximately 0.22 × 10 from each 6 Target cells / mL or approximately 0.18 × 10⁶ 6 Approximately 0.2 × 10 from each 6 This is the concentration between individual target cells / mL.

[0196] In some embodiments, multiple incubations have the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition (e.g., therapeutic cell composition) (e.g., CAR+ cells) to the target cells. In some embodiments, the maximum adjusted ratios, including the boundaries, are 120:1 to 1:1, 110:1 to 1:1, 100:1 to 1:1, 90:1 to 1:1, 80:1 to 1:1, 70:1 to 1:1, 60:1 to 1:1, 50:1 to 1:1, 40:1 to 1:1, 30:1 to 1:1, 20:1 to 1:1, 18:1 to 1:1, 16:1 to 1:1, 14:1 to 1:1, 12:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, and 7:1 to 1:1. , 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, 120:1 to 2:1, 110:1 to 2:1, 100:1 to 2:1, 90:1 to 2:1, 80:1 to 2:1, 70:1 to 2:1, 60:1 to 2:1, 50:1 to 2:1, 40:1 to 2:1, 30:1 to 2:1, 20:1 to 2:1, 18:1 to 2:1, 16:1 to 2:1, 14:1 to 2:1, 12:1 to 2:1, 10:1 to 2:1, 9:1 to 2:1, 8: 1 to 2:1, 7:1 to 2:1, 6:1 to 2:1, 5:1 to 2:1, 4:1 to 2:1, 3:1 to 2:1, 120:1 to 6:1, 110:1 to 6:1, 100:1 to 6:1, 90:1 to 6:1, 80:1 to 6:1, 70:1 to 6:1, 60:1 to 6:1, 50:1 to 6:1, 40:1 to 6:1, 30:1 to 6:1, 20:1 to 6:1, 18:1 to 6:1, 16:1 to 6:1, 14:1 to 6:1, 16:1 to 6:1, 10:1 to 6:1, 9:1 to 6:1, 8:1 to 6:1, 7:1 to 6:1, 120:1 to 3:1, 110:1 to 3:1, 100:1 to 3:1, 90:1 to 3:1, 80:1 to 3:1, 70:1 to 3:1, 60:1 to 3:1, 50:1 to 3:1, 40:1 to 3:1, 30:1 to 3:1, 20:1 to 3:1, 18:1 to 3:1, 16:1 to 3:1, 14:1 to 3:1, 13:1 to 3:1, 10:1 to 3:1, 9:1 to 3:1, 8:1 to 3:1, 7:1 to 3:1, 6:1 to 3:1Between 5:1 and 3:1 or 4:1 and 3:1, or approximately 120:1 to 1:1, approximately 110:1 to 1:1, approximately 100:1 to 1:1, approximately 90:1 to 1:1, approximately 80:1 to 1:1, approximately 70:1 to 1:1, approximately 60:1 to 1:1, approximately 50:1 to 1:1, approximately 40:1 to 1:1, approximately 30:1 to 1:1, approximately 20:1 to 1:1, approximately 18:1 to 1:1, approximately 16:1 to 1:1, approximately 14:1 to 1:1, approximately 12:1 to 1:1, approximately 10:1 to 1:1, approximately 9:1 to 1:1, approximately 8:1 to 1:1, or approximately 7:1. From approximately 1:1, from approximately 6:1 to approximately 1:1, from approximately 5:1 to approximately 1:1, from approximately 4:1 to approximately 1:1, from approximately 3:1 to approximately 1:1, from approximately 2:1 to approximately 1:1, from approximately 120:1 to approximately 2:1, from approximately 110:1 to approximately 2:1, from approximately 100:1 to approximately 2:1, from approximately 90:1 to approximately 2:1, from approximately 80:1 to approximately 2:1, from approximately 70:1 to approximately 2:1, from approximately 60:1 to approximately 2:1, from approximately 50:1 to approximately 2:1, from approximately 40:1 to approximately 2:1, from approximately 30:1 to approximately 2:1, from approximately 20:1 to approximately 2:1, from approximately 18:1 to approximately 2:1, from approximately 16:1 to approximately 2:1, from approximately 14:1 to approximately 2:1, from approximately 12:1 to approximately 2:1, approximately 10: 1 to approximately 2:1, approximately 9:1 to approximately 2:1, approximately 8:1 to approximately 2:1, approximately 7:1 to approximately 2:1, approximately 6:1 to approximately 2:1, approximately 5:1 to approximately 2:1, approximately 4:1 to approximately 2:1, approximately 3:1 to approximately 2:1, approximately 120:1 to approximately 6:1, approximately 110:1 to approximately 6:1, approximately 100:1 to approximately 6:1, approximately 90:1 to approximately 6:1, approximately 80:1 to approximately 6:1, approximately 70:1 to approximately 6:1, approximately 60:1 to approximately 6:1, approximately 50:1 to approximately 6:1, approximately 40:1 to approximately 6:1, approximately 30:1 to approximately 6:1, approximately 20:1 to approximately 6:1, approximately 18:1 to approximately 6:1, approximately 16:1 to approximately 6:1, approximately 14: 1 to approximately 6:1, approximately 16:1 to approximately 6:1, approximately 10:1 to approximately 6:1, approximately 9:1 to approximately 6:1, approximately 8:1 to approximately 6:1, approximately 7:1 to approximately 6:1, approximately 120:1 to approximately 3:1, approximately 110:1 to approximately 3:1, approximately 100:1 to approximately 3:1, approximately 90:1 to approximately 3:1, approximately 80:1 to approximately 3:1, approximately 70:1 to approximately 3:1, approximately 60:1 to approximately 3:1, approximately 50:1 to approximately 3:1, approximately 40:1 to approximately 3:1, approximately 30:1 to approximately 3:1, approximately 20:1 to approximately 3:1, approximately 18:1 to approximately 3:1, approximately 16:1 to approximately 3:1, approximately 14:1 to approximately 3:1, approximately 13:1 to approximately 3:1,The ratios are approximately between 10:1 and 3:1, 9:1 and 3:1, 8:1 and 3:1, 7:1 and 3:1, 6:1 and 3:1, 5:1 and 3:1, or 4:1 and 3:1.

[0197] In some embodiments, multiple incubations have the maximum adjusted ratio of target cells to cells expressing the recombinant receptor of the effector cell composition (e.g., therapeutic cell composition). In some embodiments, the maximum adjusted ratios, including the boundaries, are 120:1 to 1:1, 110:1 to 1:1, 100:1 to 1:1, 90:1 to 1:1, 80:1 to 1:1, 70:1 to 1:1, 60:1 to 1:1, 50:1 to 1:1, 40:1 to 1:1, 30:1 to 1:1, 20:1 to 1:1, 18:1 to 1:1, 16:1 to 1:1, 14:1 to 1:1, 12:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, and 7:1 to 1:1. , 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, 120:1 to 2:1, 110:1 to 2:1, 100:1 to 2:1, 90:1 to 2:1, 80:1 to 2:1, 70:1 to 2:1, 60:1 to 2:1, 50:1 to 2:1, 40:1 to 2:1, 30:1 to 2:1, 20:1 to 2:1, 18:1 to 2:1, 16:1 to 2:1, 14:1 to 2:1, 12:1 to 2:1, 10:1 to 2:1, 9:1 to 2:1, 8: 1 to 2:1, 7:1 to 2:1, 6:1 to 2:1, 5:1 to 2:1, 4:1 to 2:1, 3:1 to 2:1, 120:1 to 6:1, 110:1 to 6:1, 100:1 to 6:1, 90:1 to 6:1, 80:1 to 6:1, 70:1 to 6:1, 60:1 to 6:1, 50:1 to 6:1, 40:1 to 6:1, 30:1 to 6:1, 20:1 to 6:1, 18:1 to 6:1, 16:1 to 6:1, 14:1 to 6:1, 16:1 to 6:1, 10:1 to 6:1, 9:1 to 6:1, 8:1 to 6:1, 7:1 to 6:1, 120:1 to 3:1, 110:1 to 3:1, 100:1 to 3:1, 90:1 to 3:1, 80:1 to 3:1, 70:1 to 3:1, 60:1 to 3:1, 50:1 to 3:1, 40:1 to 3:1, 30:1 to 3:1, 20:1 to 3:1, 18:1 to 3:1, 16:1 to 3:1, 14:1 to 3:1, 13:1 to 3:1, 10:1 to 3:1, 9:1 to 3:1, 8:1 to 3:1, 7:1 to 3:1, 6:1 to 3:1Between 5:1 and 3:1 or 4:1 and 3:1, or approximately 120:1 to 1:1, approximately 110:1 to 1:1, approximately 100:1 to 1:1, approximately 90:1 to 1:1, approximately 80:1 to 1:1, approximately 70:1 to 1:1, approximately 60:1 to 1:1, approximately 50:1 to 1:1, approximately 40:1 to 1:1, approximately 30:1 to 1:1, approximately 20:1 to 1:1, approximately 18:1 to 1:1, approximately 16:1 to 1:1, approximately 14:1 to 1:1, approximately 12:1 to 1:1, approximately 10:1 to 1:1, approximately 9:1 to 1:1, approximately 8:1 to 1:1, or approximately 7:1. From approximately 1:1, from approximately 6:1 to approximately 1:1, from approximately 5:1 to approximately 1:1, from approximately 4:1 to approximately 1:1, from approximately 3:1 to approximately 1:1, from approximately 2:1 to approximately 1:1, from approximately 120:1 to approximately 2:1, from approximately 110:1 to approximately 2:1, from approximately 100:1 to approximately 2:1, from approximately 90:1 to approximately 2:1, from approximately 80:1 to approximately 2:1, from approximately 70:1 to approximately 2:1, from approximately 60:1 to approximately 2:1, from approximately 50:1 to approximately 2:1, from approximately 40:1 to approximately 2:1, from approximately 30:1 to approximately 2:1, from approximately 20:1 to approximately 2:1, from approximately 18:1 to approximately 2:1, from approximately 16:1 to approximately 2:1, from approximately 14:1 to approximately 2:1, from approximately 12:1 to approximately 2:1, approximately 10: 1 to approximately 2:1, approximately 9:1 to approximately 2:1, approximately 8:1 to approximately 2:1, approximately 7:1 to approximately 2:1, approximately 6:1 to approximately 2:1, approximately 5:1 to approximately 2:1, approximately 4:1 to approximately 2:1, approximately 3:1 to approximately 2:1, approximately 120:1 to approximately 6:1, approximately 110:1 to approximately 6:1, approximately 100:1 to approximately 6:1, approximately 90:1 to approximately 6:1, approximately 80:1 to approximately 6:1, approximately 70:1 to approximately 6:1, approximately 60:1 to approximately 6:1, approximately 50:1 to approximately 6:1, approximately 40:1 to approximately 6:1, approximately 30:1 to approximately 6:1, approximately 20:1 to approximately 6:1, approximately 18:1 to approximately 6:1, approximately 16:1 to approximately 6:1, approximately 14: 1 to approximately 6:1, approximately 16:1 to approximately 6:1, approximately 10:1 to approximately 6:1, approximately 9:1 to approximately 6:1, approximately 8:1 to approximately 6:1, approximately 7:1 to approximately 6:1, approximately 120:1 to approximately 3:1, approximately 110:1 to approximately 3:1, approximately 100:1 to approximately 3:1, approximately 90:1 to approximately 3:1, approximately 80:1 to approximately 3:1, approximately 70:1 to approximately 3:1, approximately 60:1 to approximately 3:1, approximately 50:1 to approximately 3:1, approximately 40:1 to approximately 3:1, approximately 30:1 to approximately 3:1, approximately 20:1 to approximately 3:1, approximately 18:1 to approximately 3:1, approximately 16:1 to approximately 3:1, approximately 14:1 to approximately 3:1, approximately 13:1 to approximately 3:1,The ratios are approximately between 10:1 and 3:1, 9:1 and 3:1, 8:1 and 3:1, 7:1 and 3:1, 6:1 and 3:1, 5:1 and 3:1, or 4:1 and 3:1.

[0198] In some embodiments, each of the multiple incubations is carried out at a different adjusted ratio, which is between 0.001 and 15 or about 0.001 and about 15, including the boundary.

[0199] In some embodiments, multiple incubations are performed in different adjusted ratios of at least 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, 3 or at least 3 incubations are performed. In some embodiments, 6 or at least 6 incubations are performed. In some embodiments, 10 or at least 10 incubations are performed.

[0200] In some embodiments, each of a plurality of incubations includes culturing cells of a mock cell composition together with cells of an effector cell composition (e.g., a therapeutic cell composition) and target cells. In some embodiments, the cultured cells of the mock cell composition do not express recombinant receptors. In some embodiments, the cultured cells of the mock cell composition do not express target antigens. In some embodiments, the cultured cells of the mock cell composition do not express recombinant receptors or target antigens.

[0201] In some embodiments, the mock cell composition is produced using any of the manufacturing processes described herein, for example, any of those described in Section II. In some embodiments, the mock cell composition is produced using a manufacturing process that is identical to the manufacturing process used to produce an effector cell composition (e.g., a therapeutic cell composition), except that the cells of the mock cell composition are not exposed to nucleic acids encoding recombinant receptors.

[0202] In some embodiments, each of the multiple incubations contains a different total number of cells of the therapeutic composition and target cells.

[0203] In some embodiments, each of the multiple incubations contains the same total number of cells for the therapeutic composition, target cells, and mock cell composition. In some embodiments, the cells for the mock cell composition are added to normalize the total number of cells across the multiple incubations.

[0204] In some embodiments, each of the multiple incubations contains a different total number of cells for the therapeutic composition, target cells, and mock cell composition.

[0205] In some embodiments, cytotoxic activity is measured from each of several incubations. In some embodiments, cytotoxic activity from each of several incubations is measured, for example, by fluorescence or luminescence. In some embodiments, the measured cytotoxic activity values ​​are fitted by a curve to generate a cytotoxic activity curve. In some embodiments, a tuned ratio that yields the maximum half of the cytotoxic activity is determined based on the cytotoxic activity measured from each of several incubations. In some embodiments, the tuned ratio that yields the maximum half of the cytotoxic activity is inferred, extrapolated, or estimated from the cytotoxic activity curve. In some embodiments, the cytotoxic activity curve is normalized to the measured maximum cytotoxic activity. In some embodiments, the tuned ratio that yields the maximum half of the cytotoxic activity is the potency of the effector cell composition (e.g., a therapeutic cell composition). In some embodiments, where appropriate, the number of target cells may be reported instead of the tuned ratio.

[0206] In some embodiments, a modified ratio that yields up to half the amount of cytotoxic activity is compared to a modified ratio that yields up to half the amount of cytotoxicity in a reference standard. For example, a modified ratio of an effector cell composition (e.g., a therapeutic cell composition) that yields up to half the amount of cytotoxic activity is divided by a modified ratio that yields up to half the amount of cytotoxic activity in a reference standard, determined, for example, according to the method described herein, to obtain a relative potency. In some embodiments, the relative potency is expressed as a ratio. In some embodiments, the relative potency is expressed as a percentage.

[0207] The methods provided herein for determining potency may be carried out using replication. For example, an assay may be performed two, three, four, five times or more times. In some embodiments, the replications are used to verify the accuracy and / or precision of the assay, including the consistency of the measured and / or determined cytotoxic activity and / or relative potency. Any of the multiple incubations performed with cells versus target cells of a particular adjusted ratio of therapeutic composition may be carried out by replication, for example, at least two, three, four, five, six, seven, eight, nine, or ten times. In some embodiments, one or more of the multiple incubations include performing the incubation at least two, three, four, five, six, seven, eight, nine, or ten times with the same adjusted ratio. In some embodiments, each of the multiple incubations is performed by replication at least two, three, four, five, six, seven, eight, nine, or ten times. In some embodiments, a single assay is performed by performing the assay in two or three series with a particular effector cell composition (e.g., therapeutic cell composition). In some embodiments, the assay is performed in a two-run cycle. In some embodiments, the assay is performed in a three-run cycle.

[0208] In some cases where the assay is performed in, for example, two or three series, the measured cytotoxic activity from each of the replicas is used to provide a statistical measure of cytotoxic activity. For example, in some cases, the mean, median, standard deviation, and / or variance of each measure of cytotoxic activity are determined. In some embodiments, the mean of each measure of cytotoxic activity is determined. In some embodiments, the standard deviation of each measure of cytotoxic activity is determined. In some embodiments, the mean measure of cytotoxic activity is fitted using a mathematical model to generate a cytotoxic activity curve. In some embodiments, the curve is normalized to the mean maximum. In some embodiments, the adjusted ratio of the mean that yields the maximum half of the cytotoxic activity is the potency of the effector cell composition (e.g., therapeutic cell composition). In some embodiments, where appropriate, the number of target cells may be reported instead of the adjusted ratio. In some embodiments, the potency of an effector cell composition (e.g., a therapeutic cell composition) is a relative potency determined by taking an average adjusted ratio that yields the maximum half amount of cytotoxic activity and comparing the average adjusted ratio to a single or average adjusted ratio that yields the maximum half amount of cytotoxic activity in a reference standard. In some embodiments, the relative potency is the average potency of the effector cell composition (e.g., a therapeutic cell composition) divided by the single or average potency of the standard reference. In some embodiments, the relative potency is expressed as a ratio. In some embodiments, the relative potency is expressed as a percentage.

[0209] The assays provided herein may be carried out in any container suitable for multiple incubations. In some embodiments, the assay is carried out in a flask. In some embodiments, the assay is carried out in a tube, such as a microcentrifuge tube or a PCR tube. In some embodiments, the assay is carried out in a multiwell plate. For example, the multiwell plate may be a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate. In certain embodiments, the assay is carried out or performed in a 96-well plate. In some embodiments, the multiwell plate is a white multiwell plate.

[0210] The conditions under which incubations using effector cell compositions (e.g., therapeutic cell compositions) and target cells are cultured may include specific media, temperature, oxygen content, carbon dioxide content, time, and / or drugs, such as nutrients, amino acids, antibiotics, and ions, one or more of these. In some embodiments, multiple incubations using effector cell compositions and target cells are cultured in serum-free media. In some embodiments, multiple incubations using effector cell compositions and target cells are cultured in serum-containing media. In some embodiments, multiple incubations including effector cell compositions and target cells are cultured in Glutamax-containing media. The duration of the multiple incubations is intended to balance with at least the minimum amount of time required for possible cytotoxic activity to be detected (e.g., measured). In some embodiments, the multiple incubations are carried out for 1, 2, or 3 days, about 1, 2, or 3 days, or at least 1, 2, or 3 days. In some embodiments, the multiple incubations are carried out for 1 or 2 days, about 1 or 2 days, or at least 1 or 2 days. In some embodiments, multiple incubations are performed for 24, 36, 48, 60, or 72 hours, approximately 24, 36, 48, 60, or 72 hours, or at least 24, 36, 48, 60, or 72 hours. In some embodiments, multiple incubations are performed for 24 or 48 hours, approximately 24 or 48 hours, or at least 24 or 48 hours. In some embodiments, multiple incubations are performed between 24 and 72 hours, or approximately 24 and approximately 72 hours. In some embodiments, multiple incubations are performed between 24 and 48 hours, or approximately 24 and approximately 48 hours. In some embodiments, multiple incubations are performed for 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.In some embodiments, multiple incubations are performed for 30 minutes, about 30 minutes, or at least 30 minutes. In some embodiments, multiple incubations are performed for 60 minutes, about 60 minutes, or at least 60 minutes. In some embodiments, multiple incubations are performed between 10 and 60 minutes, 20 and 60 minutes, 30 and 60 minutes, 40 and 60 minutes, 50 and 60 minutes, or between about 10 and 60 minutes, 20 and 60 minutes, 30 and 60 minutes, 40 and 60 minutes, or 50 and 60 minutes. In some embodiments, multiple incubations are performed for 3 hours, about 3 hours, or at least 3 hours. In some embodiments, multiple incubations are performed for 4 hours, about 4 hours, or at least 4 hours. In some embodiments, multiple incubations are performed for 5 hours, about 5 hours, or at least 5 hours. In some embodiments, multiple incubations are performed for 6 hours, about 6 hours, or at least 6 hours.

[0211] In some embodiments, each of the multiple incubations is carried out for less than 24 hours. In some embodiments, each of the multiple incubations is between 17 and 23 hours, 17 and 22 hours, 17 and 21 hours, 17 and 20 hours, 17 and 19 hours, 17 and 18 hours, 18 and 23 hours, 18 and 22 hours, 18 and 21 hours, 18 and 20 hours, 18 and 19 hours, 19 and 23 hours, 19 and 22 hours, 19 and 21 hours, 19 and 20 hours, 20 and 23 hours, 20 and 22 hours, 20 and 21 hours, 21 and 23 hours, 21 and 22 hours, or 22 and 23 hours, including each boundary. The implementation will take place between approximately 23 hours, 17 to 22 hours, 17 to 21 hours, 17 to 20 hours, 17 to 19 hours, 17 to 18 hours, 18 to 23 hours, 18 to 22 hours, 18 to 21 hours, 18 to 20 hours, 18 to 19 hours, 19 to 23 hours, 19 to 22 hours, 19 to 21 hours, 19 to 20 hours, 19 to 19 hours, 19 to 23 hours, 19 to 22 hours, 19 to 21 hours, 19 to 20 hours, 20 to 23 hours, 20 to 22 hours, 20 to 21 hours, 21 to 23 hours, or 22 to 23 hours.

[0212] In some embodiments, each of the multiple incubations is carried out for 24 to 72 hours, 24 to 48 hours, or 72 hours, including each boundary, or for approximately 24 to approximately 72 hours, approximately 24 to approximately 48 hours, or approximately 72 hours. In some embodiments, each of the multiple incubations is carried out for 3 to 8 hours or for approximately 3 to approximately 8 hours. In some embodiments, each of the multiple incubations is carried out for 4 to 6 hours or for approximately 4 to approximately 6 hours.

[0213] In some embodiments, multiple incubations are carried out at temperatures of approximately 25 to approximately 38°C, for example, approximately 30 to approximately 37°C, for example, 37°C ± 2°C or approximately 37°C ± 2°C. In some embodiments, multiple incubations are carried out at CO2 levels of approximately 2.5% to approximately 7.5%, for example, approximately 4% to approximately 6%, for example, 5% ± 0.5% or approximately 5% ± 0.5%. In some embodiments, multiple incubations are carried out at temperatures of 37°C or approximately 37°C and / or at CO2 levels of 5% or approximately 5%.

[0214] A. Effector cell composition The methods provided herein are directed to evaluate the potency of effector cell compositions, such as therapeutic cell compositions, such as therapeutic T cell compositions, manufactured by any process. In some embodiments, the methods provided herein may be used to evaluate effector cell compositions (e.g., therapeutic cell compositions) manufactured according to a process described herein (e.g., Section II). The methods provided may be used to evaluate the potency of any effector cell composition containing cells capable of cytotoxic activity against target cells. In certain embodiments, the cells of the cell composition are T cells. In some embodiments, the effector cell composition is a therapeutic cell composition manufactured for administration as cell therapy, for example, for adoptive cell therapy methods. In some embodiments, the potency and / or relative potency of multiple effector cell compositions (e.g., therapeutic cell compositions) manufactured by any process may be evaluated according to the methods provided herein. In some embodiments, the multiple effector cell compositions (e.g., therapeutic cell compositions) to be evaluated are produced by the same manufacturing process. In some embodiments, multiple effector cell compositions (e.g., therapeutic cell compositions) are produced by the same manufacturing process but contain different recombinant receptors. In some embodiments, the target is the antigen of the recombinant receptor. Therefore, in some cases, the target-expressing cells are antigen-expressing cells. In some embodiments, all different recombinant receptors bind to the same target, e.g., the target antigen. In some embodiments, different recombinant receptors bind to different targets, e.g., the target antigen. In some embodiments, multiple effector cell compositions (e.g., therapeutic cell compositions) to be evaluated are produced by different manufacturing processes. In some embodiments, multiple effector cell compositions (e.g., therapeutic cell compositions) are produced by different manufacturing processes but contain the same recombinant receptor. In some embodiments, multiple effector cell compositions (e.g., therapeutic cell compositions) are produced by different manufacturing processes and contain different recombinant receptors. In some embodiments, all different recombinant receptors bind to the same antigen.In some embodiments, multiple effector cell compositions (e.g., therapeutic cell compositions) are produced from a single subject. In some embodiments, multiple effector cell compositions (e.g., therapeutic cell compositions) are produced from different subjects. In some embodiments, the subject is a healthy donor. In some embodiments, the subject has a disease or condition, such as cancer. The methods provided herein make it possible to compare the potency and / or relative potency between effector cell compositions (e.g., therapeutic cell compositions) including a reference standard, which is an effector cell composition (e.g., therapeutic cell composition), regardless of the method of production.

[0215] In some embodiments, an effector cell composition (e.g., a therapeutic cell composition) is generated or manufactured in connection with a process for generating or producing an effector cell composition (e.g., a therapeutic cell composition) containing selected or enriched, engineered cells derived from, for example, an input population, obtained from one or more input populations, e.g., a single biological sample (see Section II-A, e.g.). In certain embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains cells expressing recombinant receptors, e.g., CARs or TCRs. In certain embodiments, the cells of the effector cell composition (e.g., a therapeutic cell composition) are suitable for administration to a subject as a therapy, e.g., autologous cell therapy or allogeneic cell therapy. Methods provided herein may be used to evaluate the potency and / or relative potency of an effector cell composition (e.g., a therapeutic cell composition) for cell therapy.

[0216] In some embodiments, the process for producing or generating an effector cell composition of engineered cells (e.g., a therapeutic cell composition) includes some or all of the following steps: collecting or obtaining a biological sample; isolating, selecting or concentrating input cells from the biological sample; cryopreserving and storing the input cells, and then thawing them; selecting and stimulating input cells of interest, e.g., T cells, e.g., CD3+, CD4+, CD8+ T cells; genetically engineering stimulated cells to express or contain recombinant polynucleotides, e.g., polynucleotides encoding recombinant receptors such as CARs; formulating cultured cells into an output composition; and cryopreserving and storing the formulated output cells until the cells are released for injection and / or administration to a subject. In some embodiments, the method for producing an effector cell composition (e.g., a therapeutic cell composition) does not include steps of expanding or increasing the number of cells during the process, such as by culturing cells in a bioreactor under conditions that the cells expand to a threshold amount, which is at least 2, 3, 4, 5 times, or greater than, the number, level, or concentration of cells compared to the input population. In some embodiments, a method for producing an effector cell composition (e.g., a therapeutic cell composition) includes a step of expanding or increasing the number of cells during the process, such as by incubating or culturing the cells in a bioreactor under conditions such as the cells expanding to a threshold amount, which is at least 2, 3, 4, 5 times or greater than the input population in terms of cell volume, level, or concentration. In some embodiments, genetic engineering of cells is or includes a step of transducing a viral vector into the cells, such as by spinocuring the cells in the presence of viral particles and then incubating the cells under static conditions in the presence of viral particles. See, for example, Section II-C.

[0217] In certain embodiments, the total duration of the process for producing the manipulated cells, from the start of stimulation to cell collection, harvesting, or formulation, is approximately 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours, or less than 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours. In some embodiments, the total duration of the provided process for producing engineered cells, from the start of stimulation to cell collection, harvesting, or formulation, including the boundaries, is between 36 to 120 hours, 48 ​​to 96 hours, or 48 to 72 hours, or approximately between 36 to approximately 120 hours, approximately 48 to approximately 96 hours, or approximately 48 to approximately 72 hours. In certain embodiments, the amount of time to complete the provided process, as measured from the start of incubation to cell collection, harvesting, or formulation, is 48 hours, 72 hours, or 96 hours, approximately 48 hours, approximately 72 hours, or approximately 96 hours, or less than 48 hours, 72 hours, or 96 hours. In certain embodiments, the amount of time to complete the provided process, as measured from the start of incubation to cell collection, harvesting, or formulation, is 48 hours ± 6 hours, 72 hours ± 6 hours, or 96 hours ± 6 hours.

[0218] In some embodiments, the entire manufacturing process is carried out using a single population of enriched cells, e.g., T cells, e.g., CD3+, CD4+, and CD8+ T cells. In certain embodiments, the manufacturing process is carried out using two or more input populations of enriched cells, e.g., T cells, which are combined before and / or during the process of making or generating a single effector cell composition (e.g., a therapeutic cell composition) (e.g., a therapeutic cell composition containing CD4+ and CD8+ T cells). In some embodiments, the enriched cells, e.g., T cells, are or include engineered cells such as T cells, e.g., T cells transduced to express recombinant receptors.

[0219] In some embodiments, the duration or amount of time required to complete the provided process, such that the isolation, concentration and / or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample is measured up to the time at which the manipulated cells of the effector cell composition (e.g., therapeutic cell composition) are collected, formulated and / or cryoprotected, is 48 hours, 72 hours, 96 hours, 120 hours, 4 days, 5 days, 7 days or 10 days, approximately 48 hours, approximately 72 hours, approximately 96 hours, approximately 120 hours, approximately 4 days, approximately 5 days, approximately 7 days or 10 days, or less than 48 hours, 72 hours, 96 hours, 120 hours, 4 days, 5 days, 7 days or 10 days. In some embodiments, the duration or amount of time required to complete the provided process, measured from the isolation, concentration and / or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time when the manipulated cells are collected, formulated and / or cryoprotected, is 4 to 5 days, or about 4 to 5 days. In some embodiments, the duration or amount of time required to complete the provided process, measured from the isolation, concentration and / or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time when the manipulated cells are collected, formulated and / or cryoprotected, is 5 days, or about 5 days. In some embodiments, the duration or amount of time required to complete the provided process, measured from the isolation, concentration and / or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time when the manipulated cells are collected, formulated and / or cryoprotected, is 5 days, or less than 5 days. In some embodiments, the duration or amount of time required to complete the provided process, such that the isolation, concentration, and / or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample is measured up to the time when the manipulated cells are collected, formulated, and / or cryoprotected, is 4 days or approximately 4 days.In some embodiments, isolated, selected, or concentrated cells are not cryoprotected before stimulation, and the duration or amount required to complete the provided process, such that the time from the isolation, concentration, and / or selection of input cells to the time when the manipulated cells are collected, formulated, and / or cryoprotected, is measured, is 48 hours, 72 hours, 96 hours, or 120 hours, or less than 48 hours, 72 hours, 96 hours, or 120 hours.

[0220] In certain embodiments, the effector cell composition (e.g., therapeutic cell composition) is prepared from a population of cells isolated, concentrated, or selected from a biological sample, such as CD4+ and CD8+ T cells or CD3+ T cells. In some embodiments, the time from the time the biological sample is collected from the subject to the generation or preparation of the effector cell composition (e.g., therapeutic cell composition) is within a time reduction compared to other methods or processes.

[0221] In certain embodiments, an effector cell composition (e.g., a therapeutic cell composition) containing engineered T cells expressing a recombinant receptor (e.g., CAR) is enriched with CD3+ T cells. In some embodiments, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 96% or about 96%, at least 98% or about 98%, at least 98.5% or about 98.5%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, 100% or about 100% of the total cells, total viable cells, total living cells, total T cells, total viable T cells, total living T cells, total living CD45+ cells or their CAR-expressing cells in the composition are CD3+, e.g., CD3+ T cells or CAR+CD3+ T cells. In some embodiments, between 75% and 80% or about 75% and about 80%, between 80% and 85% or about 80% and about 85% and about 85% and about 90%, between 90% and 95% or about 90% and about 95% and about 99% and about 95% and about 99% of the total cells, total viable cells, total living cells, total T cells, total viable T cells, total living CD45+ cells or CAR-expressing cells in the effector cell composition (e.g., therapeutic cell composition), are CD3+, for example, CD3+ T cells or CAR+CD3+ T cells. In some embodiments, 80% or about 80%, 81% or about 81%, 82% or about 82%, 83% or about 83%, 84% or about 84%, 85% or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, 99% or about 99% of the total living CD45+ cells or their CAR-expressing cells in the effector cell composition (e.g., therapeutic cell composition) are CD3+, for example, CD3+ T cells or CAR+CD3+ T cells.In some embodiments, between about 80% and about 100%, between about 85% and about 99%, between about 88% and about 98%, between about 96% and about 99%, or between about 97% and about 99%, of the total living CD45+ cells or their CAR-expressing cells in the effector cell composition (e.g., therapeutic cell composition) are CD3+, for example, CD3+ T cells or CAR+CD3+ T cells. In some embodiments, the effector cell composition (e.g., therapeutic cell composition) consists of or essentially consists of CD3+ T cells.

[0222] In certain embodiments, an effector cell composition (e.g., a therapeutic cell composition) containing engineered T cells expressing a recombinant receptor (e.g., CAR) is enriched with CD4+ and CD8+ T cells. In some embodiments, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 96% or about 96%, at least 98% or about 98%, at least 98.5% or about 98.5%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, 100% or about 100% of the total cells, total viable cells, total living cells, total T cells, total viable T cells, total living CD45+ cells or their CAR-expressing cells in the effector cell composition (e.g., a therapeutic cell composition) are CD4+ or CD8+. In some embodiments, between 75% and 80% or about 75% and about 80%, between 80% and 85% or about 80% and about 85% and about 85% and about 90% and about 90% and about 90% and about 90% and about 90% and about 95% and about 95% and about 99% and about 95% and about 99% and about 95% and about 99% and about 95% and about 99% and about 95% and about 99% and about 95% and about 99% and about 99% and about 95% and about 99% and about 99% and about 95% and about 99% and about 99% and about 90 In some embodiments, 80% or about 80%, 81% or about 81%, 82% or about 82%, 83% or about 83%, 84% or about 84%, 85% or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, 99% or about 99% of the total living CD45+ cells or their CAR-expressing cells in the effector cell composition (e.g., therapeutic cell composition) are CD4+ or CD8+.In some embodiments, between about 80% and about 100%, between about 85% and about 99%, between about 88% and about 98%, between about 96% and about 99%, or between about 97% and about 99% of the total living CD45+ cells or their CAR-expressing cells in the effector cell composition (e.g., therapeutic cell composition), they are CD4+ or CD8+. In some embodiments, the effector cell composition (e.g., therapeutic cell composition) consists of or essentially consists of CD4+ T cells and CD8+ T cells.

[0223] In certain embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains at least 50% or about 50%, at least 60% or about 60%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 99% or about 99%, or at least 99.9% or about 99.9% of viable cells. In some embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains at least 75% or about 75% of viable cells. In certain embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains at least 85% or about 85%, at least 90% or about 90%, or at least 95% or about 95% of viable cells. In some embodiments, at least 70% of the T cells in the effector cell composition (e.g., a therapeutic cell composition) are viable T cells. In some embodiments, at least 75% of the T cells in the effector cell composition (e.g., therapeutic cell composition) are viable T cells. In some embodiments, at least 80% of the T cells in the effector cell composition (e.g., therapeutic cell composition) are viable T cells. In some embodiments, at least 85% of the T cells in the effector cell composition (e.g., therapeutic cell composition) are viable T cells. In some embodiments, at least 90% of the T cells in the effector cell composition (e.g., therapeutic cell composition) are viable T cells. In some embodiments, the T cells are characterized as CD3+. In some embodiments, the effector cell composition (e.g., therapeutic cell composition) contains at least 50% or about 50%, at least 60% or about 60%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 99% or about 99%, or at least 99.9% or about 99.9% of viable CD3+ T cells.In certain embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains at least 75% or about 75% viable CD3+ T cells. In certain embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains at least 85% or about 85%, at least 90% or about 90%, or at least 95% or about 95% viable CD3+ T cells. In some embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains at least 50% or about 50%, at least 60% or about 60%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 99% or about 99%, or at least 99.9% or about 99.9% viable CD4+ T cells. In certain embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains at least 75% or about 75% viable CD4+ T cells. In certain embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains at least 85% or about 85%, at least 90% or about 90%, or at least 95% or about 95% viable CD4+ T cells. In certain embodiments, the effector cell composition (e.g., a therapeutic cell composition) contains at least 50% or about 50%, at least 60% or about 60%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 99% or about 99%, or at least 99.9% or about 99.9% viable CD8+ T cells. In some embodiments, the effector cell composition (e.g., the therapeutic cell composition) contains at least 75% or about 75% viable CD8+ T cells. In certain embodiments, the effector cell composition (e.g., the therapeutic cell composition) contains at least 85% or about 85%, at least 90% or about 90%, or at least 95% or about 95% viable CD8+ T cells.

[0224] In some of the embodiments, viability is determined by staining with acridine orange (AO) and propidium iodide (PI).

[0225] In certain embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD3+ T cells in the effector cell composition (e.g., therapeutic cell composition) express recombinant proteins (e.g., CARs). In some embodiments, at least 50% of the CD3+ T cells in the effector cell composition (e.g., therapeutic cell composition) express recombinant proteins (e.g., CARs). In certain embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or more than 99% of the CD4+ T cells in the effector cell composition (e.g., therapeutic cell composition) express recombinant proteins (e.g., CAR). In certain embodiments, at least 50% of the CD4+ T cells in the effector cell composition (e.g., therapeutic cell composition) express recombinant proteins (e.g., CAR). In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or more than 99% of the CD8+ T cells in the effector cell composition (e.g., therapeutic cell composition) express recombinant proteins (e.g., CARs). In certain embodiments, at least 50% of the CD8+ T cells in the effector cell composition (e.g., therapeutic cell composition) express recombinant proteins (e.g., CARs).

[0226] In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, at least 95% of the cells in the effector cell composition (e.g., the therapeutic cell composition) express recombinant receptors. In certain embodiments, at least 50% of the cells in the effector cell composition (e.g., the therapeutic cell composition) express recombinant receptors. In certain embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD3+ T cells in the effector cell composition (e.g., the therapeutic cell composition) express recombinant receptors. In some embodiments, at least 50% of the CD3+ T cells in the effector cell composition (e.g., therapeutic cell composition) express recombinant receptors. In specific embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or more than 99% of the CD4+ T cells in the effector cell composition (e.g., therapeutic cell composition) express recombinant receptors. In specific embodiments, at least 50% of the CD4+ T cells in the effector cell composition (e.g., therapeutic cell composition) express recombinant receptors. In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or more than 99% of the CD8+ T cells in the effector cell composition (e.g., therapeutic cell composition) express recombinant receptors.In a particular embodiment, at least 50% of the CD8+ T cells in the effector cell composition (e.g., a therapeutic cell composition) express recombinant receptors.

[0227] In certain embodiments, the majority of cells in the effector cell composition (e.g., a therapeutic cell composition) are naive-like, central memory, and / or effector memory cells. In certain embodiments, the majority of cells in the effector cell composition (e.g., a therapeutic cell composition) are naive-like or central memory cells. In some embodiments, the majority of cells in the effector cell composition (e.g., a therapeutic cell composition) are positive for one or more of CCR7 or CD27 expression. In certain embodiments, the cells in the effector cell composition (e.g., a therapeutic cell composition) have a larger proportion of naive-like or central memory cells that output a population created from an alternative process, such as a process involving expansion and proliferation.

[0228] In certain embodiments, the cells of the effector cell composition (e.g., a therapeutic cell composition) have a low proportion and / or frequency of depleted and / or senescent cells. In certain embodiments, the cells of the output population have a low proportion and / or frequency of depleted and / or senescent cells. In some embodiments, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells of the effector cell composition (e.g., a therapeutic cell composition) are depleted and / or senescent. In certain embodiments, less than 25% of the cells of the effector cell composition (e.g., a therapeutic cell composition) are depleted and / or senescent. In certain embodiments, less than 10% of the cells of the output population are depleted and / or senescent. In certain embodiments, the cells have a low proportion.

[0229] In some embodiments, the cells of the effector cell composition (e.g., a therapeutic cell composition) have a low percentage and / or frequency of cells that are negative for CD27 and CCR7 expression, e.g., surface expression. In certain embodiments, the cells of the effector cell composition (e.g., a therapeutic cell composition) have a low percentage and / or frequency of CD27-CCR7- cells. In some embodiments, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells in the effector cell composition (e.g., a therapeutic cell composition) are CD27-CCR7- cells. In certain embodiments, less than 25% of the cells in the effector cell composition (e.g., a therapeutic cell composition) are CD27-CCR7- cells. In certain embodiments, less than 10% of the cells in the effector cell composition (e.g., a therapeutic cell composition) are CD27-CCR7- cells. In the embodiment, less than 5% of the cells in the effector cell composition (e.g., therapeutic cell composition) are CD27-CCR7- cells.

[0230] In some embodiments, the cells of the effector cell composition (e.g., therapeutic cell composition) have a high percentage and / or frequency of cells that are positive for one or both of CD27 and CCR7 expression, for example, surface expression. In some embodiments, the cells of the effector cell composition (e.g., therapeutic cell composition) have a high percentage and / or frequency of cells that are positive for one or both of CD27 and CCR7. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the cells of the effector cell composition (e.g., therapeutic cell composition) are positive for one or both of CD27 and CCR7. In various embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the CD4+CAR+ cells in the effector cell composition (e.g., therapeutic cell composition) are positive for one or both CD27 and CCR7. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the CD8+CAR+ cells in the effector cell composition (e.g., therapeutic cell composition) are positive for one or both CD27 and CCR7.

[0231] In certain embodiments, the cells of the effector cell composition (e.g., therapeutic cell composition) have a high proportion and / or frequency of cells that are positive for CD27 and CCR7 expression, e.g., surface expression. In some embodiments, the cells of the effector cell composition (e.g., therapeutic cell composition) have a high proportion and / or frequency of CD27+CCR7+ cells. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the cells in the effector cell composition (e.g., therapeutic cell composition) are CD27+CCR7+ cells. In various embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the CD4+CAR+ cells in the effector cell composition (e.g., therapeutic cell composition) are CD27+CCR7+ cells. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the effector cell composition (e.g., therapeutic cell composition) consists of CD8+CAR+ cells, CD27+CCR7+ cells.

[0232] In certain embodiments, the cells of the effector cell composition (e.g., therapeutic cell composition) have a low percentage and / or frequency of cells that are negative for CCR7 and positive for CD45RA expression, e.g., surface expression. In some embodiments, the cells of the effector cell composition (e.g., therapeutic cell composition) have a low percentage and / or frequency of CCR7-CD45RA+ cells. In certain embodiments, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells in the effector cell composition (e.g., therapeutic cell composition) are CCR7-CD45RA+ cells. In some embodiments, less than 25% of the cells in the output population (e.g., effector cell composition (e.g., therapeutic cell composition)) are CCR7-CD45RA+ cells. In certain embodiments, less than 10% of the cells in the output population (e.g., effector cell composition (e.g., therapeutic cell composition)) are CCR7-CD45RA+ cells. In certain embodiments, less than 5% of the cells in the effector cell composition (e.g., therapeutic cell composition) are CCR7-CD45RA+ cells.

[0233] In some embodiments, the therapeutic cell manufacturing process is different, and as a result, alternative manufacturing processes can be compared, for example, by comparing the potency of differently manufactured effector cell compositions (e.g., therapeutic cell compositions). For example, in some embodiments, the alternative process may include a step of cell expansion. In some embodiments, the alternative process may not include a step of cell expansion. In some embodiments, the alternative process includes separate steps for cell selection and stimulation. In some embodiments, the alternative process includes a single step for cell selection and stimulation. In some embodiments, the alternative process may differ in one or more specific embodiments, but if not, it contains similar or identical features, aspects, steps, stages, reagents and / or conditions of the process associated with the provided method. In some embodiments, the alternative process differs in ways including, but not limited to, including, different reagents and / or culture medium formulations; the presence of serum during incubation, transduction, transfection and / or culture; different cell composition of the input population, e.g., the ratio of CD4+ to CD8+ T cells; different stimulation conditions and / or different stimulating reagents; different ratios of stimulating reagents to cells; different vectors and / or transduction methods; different timings or sequences for incubating, transducing and / or transfecting cells; the absence or difference of one or more recombinant cytokines present during incubation or transduction (e.g., different cytokines or different concentrations); or different timings for harvesting or collecting cells.

[0234] In some embodiments, the cells of an effector cell composition (e.g., a therapeutic cell composition) are engineered to express a recombinant receptor, such as a CAR or TCR, that specifically binds to a ligand, such as one associated with a disease or condition, such as one associated with or expressed on tumor or cancer cells (see, for example, Section III). In some embodiments, the recombinant receptor contains an extracellular ligand-binding domain that specifically binds to the antigen. In some embodiments, the recombinant receptor is a CAR containing an extracellular antigen-recognizing domain that specifically binds to the antigen. In some embodiments, the ligand, such as the antigen, is a protein expressed on the surface of a cell. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a treated peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.

[0235] Exemplary recombinant receptors, including CARs and recombinant TCRs, and methods for manipulating and introducing these receptors into cells, are described, for example, in the International Patent Application Publications WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, and the U.S. Patent Application Publications US2002131960, US2013287748, and US201301493. 37, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, as well as those described in European Patent Application No. EP2537416 and / or Sadelain Examples include those described by et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; and Wu et al., Cancer, 2012 March 18(2): 160-75. In some embodiments, genetically engineered antigen receptors include CARs as described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668 A1.

[0236] In some embodiments, the manipulated cells of an effector cell composition (e.g., a therapeutic cell composition) contain recombinant receptors (e.g., CARs) that bind to tumor antigens. In some embodiments, the recombinant receptors specifically recognize and / or target antigens associated with cancer and / or present on a universal tag.In some embodiments, antigens recognized or targeted by recombinant receptors include B cell maturation antigen (BCMA), ROR1, carbonic anhydrase 9 (CAIX), Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimer, and EGFR. vIII, Folate-binding protein (FBP), FCRL5, FCRH5, Fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, Kinase insertion domain receptor (kdr), Kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen in melanoma (PRAME), Survivin, TAG72, B7-H6, IL-13 receptor alpha2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, avb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, dual antigen, cancer-testicular antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms' tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, and any of the above human antigens as appropriate; these are pathogen-specific antigens.In some embodiments, the antigens recognized and / or targeted by recombinant receptors are selected from the group consisting of Notch 1, Notch 2, Notch 3, Notch 4, cell surface-associated mucin 1 (MUC1), ephrin B2, beta-glycan (TGFBR3), CD43, CD44, CSF1R, CX3CR1, CXCL16, Delta 1, E-cadherin, N-cadherin, HLA-A2, IFNaR2, IL1R1, IL1R2, IL6R, and amyloid precursor protein (APP).

[0237] In some embodiments, the antigen recognized and targeted by the recombinant receptor is B cell maturation antigen (BCMA). Exemplary antigen-binding domains and CARs containing such antigen-binding domains that target or specifically bind to BCMA are known; see, for example, WO2016 / 090320, WO2016090327, WO2010104949A2 and WO2017173256. In some embodiments, the antigen-binding domain is an scFv containing VH and VL derived from an antibody or antibody fragment specific to BCMA. In some embodiments, the antibody or antibody fragment that binds to BCMA is or contains VH and VL derived from the antibody or antibody fragment shown in international patent applications, publication numbers WO2016 / 090327 and WO2016 / 090320.

[0238] As described above, the assay may include multiple incubations, each incubation being a culture containing different adjusted ratios of engineered cells of an effector cell composition (e.g., a therapeutic cell composition) to target cells, or vice versa, making it possible to stimulate recombinant receptors of the engineered cells to stimulate cytotoxic activity, e.g., cytolytic activity. The number of cells may vary depending on the specific form of the assay, e.g., the size of the vessel in which the assay is performed. It is understood that the number will be smaller when the assay is performed in a vessel with a smaller surface area than in a vessel with a larger surface area. Typically, the number of cells is sub-confluent, such as 25% confluent or 50% confluent or less. In some embodiments, the ratios include a linear dose-response increase in cytotoxic activity across multiple adjusted ratios of a reference standard. In some embodiments, the ratios also include lower asymptotes and upper asymptotes of cytotoxic activity representing the minimum and maximum responses, respectively, of the reference standard.

[0239] In some embodiments, the number of manipulated cells (e.g., CAR+ cells) in the effector cell composition (e.g., therapeutic cell composition) is changed while the number of target cells remains constant to generate different ratios. In some embodiments, the number of cells (e.g., CAR+ cells) in the effector cell composition (e.g., therapeutic cell composition) changes over incubation, from 1 × 10⁻⁶ to 1 × 10⁻⁶. 4 ~1 × 10 6 Individual cells, 1×10 4 ~9×10 5 Individual cells, 1×10 4 ~8×10 5 Individual cells, 1×10 4 ~7×10 5 Individual cells, 1×10 4 ~6×10 5 Individual cells, 1×10 4 ~5×10 5 Individual cells, 1×10 4 ~4×10 5 Individual cells, 1×10 4 ~3×10 5 Individual cells, 1×104 ~2×10 5 Individual cells, 1×10 4 ~1 × 10 5 Individual cells, 1×10 4 ~9×10 4 Individual cells, 1×10 4 ~8×10 4 Individual cells, 1×10 4 ~7×10 4 Individual cells, 1×10 4 ~6×10 4 Individual cells, 1×10 4 ~5×10 4 Individual cells, 1×10 4 ~4×10 4 Individual cells, 1×10 4 ~3×10 4 Individual cells, 1×10 4 ~2×10 4 Individual cells or approximately 1 × 10⁻⁶ 4 ~Approx. 1×10 6 Individual cells, approximately 1 x 10 4 ~Approx. 9×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 8×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 7×10 5 Individual cells, approximately 1 x 10 4 ~about 6×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 5×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 4×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 3×10 5 Individual cells, approximately 1 x 10 4 ~about 2×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 1×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 9×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 8×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 7×10 4 Individual cells, approximately 1 x 10 4 ~about 6×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 5×10 4Individual cells, approximately 1 x 10 4 ~Approx. 4×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 3×10 4 Individual cells, approximately 1 x 10 4 ~about 2×10 4 Individual cells are prepared. In some embodiments, the number of cells in the effector cell composition (e.g., therapeutic cell composition) (e.g., the number of CAR+ cells) is 1 × 10⁶ over multiple incubations. 4 ~1 × 10 5 Individual cells, 1×10 4 ~8×10 4 Individual cells, 1×10 4 ~6×10 4 Individual cells, 1×10 4 ~4×10 4 Individual cells, 1×10 4 ~2×10 4 Individual cells or approximately 1 × 10⁻⁶ 4 ~Approx. 1×10 5 Individual cells, 1×10 4 ~Approx. 8×10 4 Individual cells, 1×10 4 ~about 6×10 4 Individual cells, 1×10 4 ~Approx. 4×10 4 Individual cells, 1×10 4 ~about 2×10 4The number of cells is adjusted to 10,000 or about 10,000 to 1,000,000 or about 1,000,000 individual cells over multiple incubations. In some embodiments, the number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., the number of CAR+ cells) is adjusted to 10,000 or about 10,000 to 500,000 or about 500,000 individual cells over multiple incubations. In some embodiments, the number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., the number of CAR+ cells) is adjusted to 10,000 or about 10,000 to 250,000 or about 250,000 individual cells over multiple incubations. In some embodiments, the cell count (e.g., the number of CAR+ cells) of the effector cell composition (e.g., a therapeutic cell composition) is adjusted to 10,000 or about 10,000 to 200,000 or about 200,000 cells over multiple incubations. In some embodiments, the cell count (e.g., the number of CAR+ cells) of the effector cell composition (e.g., a therapeutic cell composition) is adjusted to 10,000 or about 10,000 to 150,000 or about 150,000 cells over multiple incubations. In some embodiments, the cell count (e.g., the number of CAR+ cells) of the effector cell composition (e.g., a therapeutic cell composition) is adjusted to 10,000 or about 10,000 to 100,000 or about 100,000 cells over multiple incubations. In some embodiments, the cell count (e.g., the number of CAR+ cells) of an effector cell composition (e.g., a therapeutic cell composition) is adjusted to 10,000 or about 10,000 to 50,000 or about 50,000 cells over multiple incubations. In some embodiments, the cell count of any of the aforementioned effector cell compositions (e.g., a therapeutic cell composition) is the total number of cells, the total number of viable cells, the total number of CAR+ cells, the total number of CD8+ cells, the total number of CD4+ cells, the total number of CD3+ cells, the total number of CD8+ / CAR+ cells, the total number of CD4+ / CAR+ cells, or the total number of CD3+ / CAR+ cells.In some embodiments, the number of cells in any of the effector cell compositions (e.g., therapeutic cell compositions) is the total number of CAR+ cells.

[0240] In some embodiments, the number of cells in the effector cell composition (e.g., therapeutic cell composition) is maintained constant, while the number of target cells is adjusted over multiple incubations. In some embodiments, a certain number of cells in the effector cell composition (e.g., therapeutic cell composition) (e.g., a certain number of CAR+ cells) is maintained over multiple incubations at approximately 1 × 10⁻⁶ 4 ~Approx. 1×10 6 Individual cells, approximately 1 x 10 4 ~Approx. 9×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 8×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 7×10 5 Individual cells, approximately 1 x 10 4 ~about 6×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 5×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 4×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 3×10 5 Individual cells, approximately 1 x 10 4 ~about 2×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 1×10 5 Individual cells, approximately 1 x 10 4 ~Approx. 9×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 8×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 7×10 4 Individual cells, approximately 1 x 10 4 ~about 6×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 5×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 4×10 4 Individual cells, approximately 1 x 10 4 ~Approx. 3×10 4 Individual cells, approximately 1 x 10 4 ~about 2×10 4This is the number between individual cells. In some embodiments, a certain number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., a certain number of CAR+ cells) is 1 × 10⁶ over multiple incubations. 4 ~1 × 10 5 Individual cells, 1×10 4 ~8×10 4 Individual cells, 1×10 4 ~6×10 4 Individual cells, 1×10 4 ~4×10 4 Individual cells, 1×10 4 ~2×10 4 Individual cells or approximately 1 × 10⁻⁶ 4 ~Approx. 1×10 5 Individual cells, 1×10 4 ~Approx. 8×10 4 Individual cells, 1×10 4 ~about 6×10 4 Individual cells, 1×10 4 ~Approx. 4×10 4 Individual cells, 1×10 4 ~about 2×10 4This is the number of individual cells. In some embodiments, a certain number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., a certain number of CAR+ cells) is 10,000 or about 10,000 to 1,000,000 individual cells or about 1,000,000 individual cells over multiple incubations. In some embodiments, a certain number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., a certain number of CAR+ cells) is 10,000 or about 10,000 to 500,000 individual cells or about 500,000 individual cells over multiple incubations. In some embodiments, a certain number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., a certain number of CAR+ cells) is 10,000 or about 10,000 to 250,000 individual cells or about 250,000 individual cells over multiple incubations. In some embodiments, a certain number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., a certain number of CAR+ cells) is 10,000 or about 10,000 to 150,000 or about 150,000 cells over multiple incubations. In some embodiments, a certain number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., a certain number of CAR+ cells) is 10,000 or about 10,000 to 100,000 or about 100,000 cells over multiple incubations. In some embodiments, a certain number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., a certain number of CAR+ cells) is 10,000 or about 10,000 to 50,000 or about 50,000 cells over multiple incubations. In some embodiments, a certain number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., a certain number of CAR+ cells) is 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 cells over multiple incubations, or about 5,000, about 10,000, about 20,000, about 30,000, about 40,000, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000, or about 100,000 cells.In some embodiments, a certain number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., a certain number of CAR+ cells) is 5,000, 10,000, 20,000, 30,000, 40,000, or 50,000 cells or about 5,000, about 10,000, about 20,000, about 30,000, about 40,000, or about 50,000 cells over multiple incubations. In some embodiments, a certain number of cells in an effector cell composition (e.g., a therapeutic cell composition) (e.g., a certain number of CAR+ cells) is 50,000 cells or about 50,000 cells across multiple incubations. In some embodiments, a certain number of cells in any of the aforementioned effector cell compositions (e.g., a therapeutic cell composition) is the total number of cells, the total number of viable cells, the total number of CAR+ cells, the total number of CD8+ cells, the total number of CD4+ cells, the total number of CD3+ cells, the total number of CD8+ / CAR+ cells, the total number of CD4+ / CAR+ cells, or the total number of CD3+ / CAR+ cells. In some embodiments, a certain number of cells in any of the aforementioned effector cell compositions (e.g., a therapeutic cell composition) is the total number of CAR+ cells.

[0241] B. Target cells Methods for evaluating efficacy provided herein include means for stimulating cells of an effector cell composition (e.g., a therapeutic cell composition). For example, it is intended that any means suitable for stimulating cells that can be quantified and delivered may be used to generate different ratios of cells of an effector cell composition (e.g., a therapeutic cell composition) to target cells. In some embodiments, the means of stimulation is achieved by target cells expressing a target antigen that can bind to intracellular signals via a recombinant receptor, stimulate, and produce cytotoxic activity. Exemplary target cells include cells expressing antigens of recombinant receptors (e.g., purified antigens or recombinant antigens) or antibodies such as anti-idiotype antibodies.

[0242] Numerous characteristics can help make cells suitable target cells. Some such characteristics include proliferation dynamics, viability, antigen expression, stability of antigen expression, and low non-CAR activity when used with effector cells. In some embodiments, target cells may have a fast proliferation rate, e.g., a short doubling time. In some embodiments, target cells have low or no adhesion to the vessel in which they are cultured. In some embodiments, target cells express a large amount of antigen or overexpress the antigen. In some embodiments, target cells maintain high viability while cultured. In some embodiments, target cells can proliferate to high densities. In some embodiments, target cells stably express the antigen. In some embodiments, target cells show low reactivity with effector cells, which is not due to the recombinant receptors on the effector cells.

[0243] As described above, in some embodiments, multiple incubations of different adjusted ratios of cells in an effector cell composition (e.g., therapeutic cell composition) to target cells can be achieved by culturing a fixed number of cells (e.g., live cells, CAR+, CD4+, CD8+, CD3+, CD4+ / CAR+, CD8+ / CAR+, CD3+ / CAR+ cells) of the effector cell composition (e.g., therapeutic cell composition) together with a variable or adjusted number of target cells. In some embodiments, the number of target cells is changed or adjusted by 10,000 times, 5,000 times, 1,000 times, 1,500 times, 500 times, 250 times, 200 times, 150 times, 100 times, 75 times, 50 times, 25 times, or 10 times, or by approximately 10,000 times, 5,000 times, 1,000 times, 1,500 times, 500 times, 250 times, 200 times, 150 times, 100 times, 75 times, 50 times, 25 times, or 10 times. In some embodiments, the number of target cells is varied or adjusted between 10,000 and 100 times, 5,000 and 100 times, or 1,000 and 100 times, or approximately 10,000 and 100 times, 5,000 and 100 times, or 1,000 and 100 times, across multiple incubations. In some embodiments, the number of target cells is varied or adjusted between 5,000 times, 1,000 times, 1,500 times, or 500 times, or approximately 5,000 times, 1,000 times, 1,500 times, or 500 times, across multiple incubations. In some embodiments, the number of target cells is varied or adjusted by 5,000 times or approximately 5,000 times, across multiple incubations. In some embodiments, the number of target cells is varied or adjusted by 1,000 times or approximately 1,000 times, across multiple incubations. In some embodiments, the number of target cells is changed or adjusted by a factor of 500 or approximately 500 over multiple incubations.

[0244] In some embodiments, the target cells are modified or adjusted over multiple incubations in a modified ratio compared to a fixed number of cells expressing the recombinant receptor in the therapeutic composition (effector cells). In some embodiments, the adjusted ratio is a target cell to effector cell (T:E) ratio of 100:1 to 0.001, for example, a T:E ratio of 50:1 to 0.050, 25:1 to 0.025, 12:1 to 0.012:1, 10:1 to 0.010, or 5:1 to 0.5. In some embodiments, the ratio is a T:E ratio of 12:1 to 0.012:1, or approximately 12:1 to approximately 0.012:1. The specific range of the ratio may vary depending on the specific target antigen and target cells being employed.

[0245] In certain embodiments, the target cells are exogenous, heterologous, and / or autologous to the subject. In some embodiments, the target cells are exogenous to the subject.

[0246] In certain embodiments, the target cells are tumor cells. In some embodiments, the target cells are primary cells. In some embodiments, the target cells are derived from a cell line. In some embodiments, the cell line is an immortal cell line. In some embodiments, the target cells are derived from cancer cells and / or tumor cells, e.g., human cancer cells and / or human tumor cells. In some embodiments, the target cells are cells derived from a cancer cell line, optionally a human cancer cell line. In some embodiments, the target cells are cells derived from a tumor cell line, optionally a human tumor cell line.

[0247] In certain embodiments, the target cells are tumor cells. In some embodiments, the target cells are circulating tumor cells, such as neoplastic immune cells, such as neoplastic B cells (or cells derived from neoplastic B cells).

[0248] In some embodiments, target cells may be generated from commercially available cell lines. In some embodiments, the cell line is a human cell line. In certain embodiments, the cell line includes isogenic cells, luciferase-labeled cells, cancer cell lines, organoids, stem cells, melanoma cells, or breast cancer cell lines, specifically B lymphoblast cells (e.g., MM.1S or MM.1R).

[0249] In some embodiments, the cell line is a tumor cell line. Any of several tumor cell lines are known and available for use as target cells. Tumor cell lines expressing specific tumor antigens are known, or the surface expression of tumor antigens can be readily determined or measured by those skilled in the art using any of the following techniques, for example, by flow cytometry. Exemplary tumor cell lines include lymphoma cells (Raji; Daudi; Jeko-1; BJAB; Ramos; NCI-H929; BCBL-1; DOHH-2, SC-1, WSU-NHL, JVM-2, Rec-1, SP-53, RL, Granta) 519, NCEP-1, CL-01), leukemia cells (BALL-1, RCH-ACV, SUP-B15); cervical cancer cells (33A; CaSki; HeLa), lung cancer cells (NCI-H358; A5 49, H1355, H1975, Calu-1, H1650 and H727), breast cells, (Hs-578T;ZR-75-1;MCF-7;MCF-7 / HER2;MCF10A;MDA-MB Examples include, but are not limited to, -231;SKBR-3, BT-474, MDA-MB-231); ovarian cells (ES-2;SKOV-3;OVCAR3;HEY1B); and multiple myeloma cells (U266, NCI-H929, RPMI-8226, OPM2, LP-1, L363, MM.1S, MM.1R, MC / CAR, JJN3, KMS11, AMO-1, EJM;MOLP-8).For example, exemplary CD19-expressing cell lines include, but are not limited to, Raji, Daudi, and BJAB; exemplary CD20-expressing cell lines include, but are not limited to, Daudi, Ramos, and Raji; exemplary CD22-expressing cell lines include, but are not limited to, Ramos, Raji, A549, H727, and H1650; exemplary Her2-expressing cell lines include SKOV3, BT-474, and SKBR-3; exemplary BCMA expression Examples of cell lines include, but are not limited to, RPMI-8226, NCI-H929, MM1S, MM1R, and KMS11; examples of GPRC5D-expressing cell lines include, but are not limited to, AMO-1, EJM, NCI-H929, MM.1S, MM1.R, MOLP-8, and OPM-2; and examples of ROR1-expressing cell lines include, but are not limited to, A549, MDA-MB-231, H1975, BALL-1, and RCH-ACV.

[0250] In some embodiments, the cell line may be K562, U937, 721.221, T2, or C1R cells.

[0251] In certain embodiments, the target cells are MM.1S cells that express a target antigen against a recombinant receptor expressed by an effector cell composition (e.g., a therapeutic cell composition).

[0252] In certain embodiments, the target cells are derived from a cell line that has been transduced to express a target antigen. This cell line may be a mammalian cell line, such as a human cell line. In some embodiments, the human cell line may be K562, U937, 721.221, T2, or C1R cells. For example, the K562 chronic myeloid leukemia cell line may be transduced with a nucleic acid encoding a tumor antigen. In some embodiments, the cell line may be manipulated using a plasmid vector or messenger RNA (mRNA) encoding the tumor antigen of interest. In some embodiments, the transduction may be lentivirus-based transduction. In some embodiments, the cell line (e.g., K562 cells) stably expresses an exogenous nucleic acid encoding a tumor antigen. In some embodiments, the exogenous nucleic acid may be integrated into the genome of the cell line (e.g., K562 cells). In some embodiments, the exogenous nucleic acid may be integrated into the genome of the cell line (e.g., K562 cells) at a specific locus. In some embodiments, exogenous nucleic acids can be integrated into the genome of a cell line (e.g., K562 cells) using a genome-safe harbor (GSH). GSHs are sites that support the stable integration and expression of exogenous nucleic acids while minimizing the risk of unwanted interactions with the host cell genome (see, e.g., Sadelain et al., Nat Rev Cancer. (2011) 12(1):51-8). Several safe GSHs have been identified for the stable integration of exogenous nucleic acids in human cells, including AAVS1, a naturally occurring site of AAV virus integration on chromosome 19; the CCR5 gene, a chemokine receptor gene also known as the HIV-1 coreceptor; and a human ortholog of the mouse Rosa26 locus (see, e.g., Papapetrou and Schambach Mol Ther. (2016) 24(4):678-684).

[0253] In some embodiments, target cells are generated from primary cells. In some embodiments, primary cells are obtained from a sample. In some embodiments, the sample is blood or a blood-derived sample, or an apheresis or leukocyte apheresis product, or derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs and / or cells derived therefrom. In some embodiments, target cells are generated from tumor or cancer cells obtained from the sample.

[0254] In certain embodiments, the target cells are human cancer cells or derived therefrom. In some embodiments, target cells include AIDS-related cancers, breast cancer, gastrointestinal cancers, anal cancers, appendiceal cancers, bile duct cancers, colon cancers, colorectal cancers, esophageal cancers, gallbladder cancers, islet cell tumors, pancreatic neuroendocrine tumors, liver cancers, pancreatic cancers, rectal cancers, small intestine cancers, stomach cancers, endocrine cancers, adrenocortical carcinomas, parathyroid cancers, pheochromocytomas, pituitary tumors, thyroid cancers, eye cancers, intraocular melanomas, retinoblastomas, bladder cancers, kidney (renal cell) cancers, penile cancers, prostate cancers, transitional cell renal pelvis and ureteral cancers, testicular cancers, urethral cancers, Wilms' tumor or other pediatric kidney tumors, germ cell cancers, central nervous system cancers, extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, gynecological cancers, cervical cancers, endometrial cancers, gestational trophoblastic tumors, ovarian epithelial carcinomas, uterine sarcomas, vaginal cancers, and vulvar cancers. Head and neck cancer, hypopharyngeal cancer, pharyngeal cancer, lip and oral cancer, metastatic squamous neck cancer, nasopharyngeal cancer, oropharyngeal cancer, sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, throat cancer, musculoskeletal cancer, bone cancer, Ewing's sarcoma, gastrointestinal stromal tumor (GIST), osteosarcoma, malignant fibrous histiocytoma of bone, rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, neurological cancer, brain tumor, astrocytoma, brainstem glioma, central nervous system Atypical teratomas / rhabdoid tumors, central nervous system germ cell tumors, central nervous system germ cell tumors, craniopharyngiomas, ependymomas, medulloblastomas, spinal cord tumors, supratentorial primitive neuroectoderm tumors and pinealoblastomas, neuroblastomas, respiratory cancers, thoracic cancers, non-small cell lung cancers, small cell lung cancers, malignant mesotheliomas, thymomas, thymic carcinomas, skin cancers, Kaposi's sarcoma, melanomas or Merkel cell carcinomas or any equivalent human cancers thereof.

[0255] In certain embodiments, the target cells are derived from non-hematopoietic cancers, e.g., solid tumors. In certain embodiments, the tumor cells are derived from hematological cancers. In certain embodiments, the target cells are derived from cancers that are B-cell malignancies or hematological malignancies. In certain embodiments, the target cells are derived from non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML), or myeloma, e.g., multiple myeloma (MM) or any equivalent human cancer thereto. In some embodiments, the target cells are neoplastic, oncogenic, and / or tumorigenic B cells. Multiple tumor cell lines are known and available and can be selected depending on the antigen recognized by a specific recombinant receptor (e.g., CAR).

[0256] In some embodiments, target cells are engineered to express a reporter molecule.

[0257] Target cells can be manipulated to express a reporter molecule by any method known in the art. Exemplary methods include those mediated by viruses, e.g., retroviruses or lentiviruses, transduction, transposons, and electroporation for nucleic acid transfer. In some embodiments, target cells are manipulated to express a target antigen and a reporter. In some embodiments, the manipulation for expressing the target antigen and reporter is independently selected from any of the manipulation methods described herein. In some embodiments, target cells endogenously express the target antigen.

[0258] In some embodiments, target cells are passaged sequentially to prepare them for use in the assays described herein. In some embodiments, target cells are passaged sequentially by transferring them to new containers and adding or diluting them with new medium. In some embodiments, target cells are passaged 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times in medium and then used for cytotoxic assays. In some embodiments, target cells are passaged once and then used for cytotoxic assays. In some embodiments, target cells are passaged 5 times and then used for cytotoxic assays. In some embodiments, target cells are passaged 10 times and then used for cytotoxic assays. In some embodiments, target cells are passaged 15 times and then used for cytotoxic assays. In some embodiments, target cells are passaged 20 times and then used for cytotoxic assays. In some embodiments, the target cells are passaged 25 times and then used for cytotoxic assays. In some embodiments, the target cells are passaged 30 times and then used for cytotoxic assays. In some embodiments, the target cells are passaged 40 times and then used for cytotoxic assays. In some embodiments, the target cells are passaged 50 times and then used for cytotoxic assays.

[0259] In some embodiments, target cells are frozen and / or cryopreserved before being used in the assays described herein. In some embodiments, target cells are frozen and / or cryopreserved in cryopreservation medium and / or cryopreservative. In some embodiments, the cryopreservation medium and / or cryopreservative may prevent the formation of intracellular crystals during the freezing process. In some embodiments, cryopreserved target cells are thawed and then used in cytotoxic assays. In some embodiments, cryopreserved target cells are washed to remove the cryopreservation medium after they have been thawed and then used in cytotoxic assays.

[0260] In some embodiments, target cells are suspended in cryopreservation medium before cryopreservation. In some embodiments, target cells are suspended in DMSO before cryopreservation. In some embodiments, target cells are thawed before use in any of the assays described herein, and the cryopreservation medium (e.g., DMSO) is removed before use in any of the assays described herein. In some embodiments, target cells are cryopreserved in about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, or about 30% DMSO, or values ​​in any of the above. In some embodiments, target cells are cryopreserved in a medium containing between 1% and 30%, between 1% and 25%, between 1% and 20%, between 1% and 15%, between 5% and 30%, between 5% and 25%, or between 5% and 20% DMSO. In some embodiments, target cells are cryopreserved in about 1% DMSO. In some embodiments, target cells are cryopreserved in about 5% DMSO. In some embodiments, target cells are cryopreserved in about 15% DMSO. In some embodiments, target cells are cryopreserved in about 20% DMSO. In some embodiments, target cells are cryopreserved in about 25% DMSO. In some embodiments, target cells are cryopreserved in about 30% DMSO. In some embodiments, target cells are cryopreserved in a medium containing between 1% and 25% DMSO. In some embodiments, target cells are cryopreserved in a medium containing between 1% and 20% DMSO. In some embodiments, target cells are cryopreserved in a medium containing between 1% and 15% DMSO. In some embodiments, target cells are cryopreserved in a medium containing between 5% and 25% DMSO. In some embodiments, target cells are cryopreserved in a medium containing between 5% and 20% DMSO. In some embodiments, target cells are cryopreserved in a medium containing between 5% and 15% DMSO.

[0261] In some embodiments, target cells are passaged 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times in culture medium and then cryopreserved for future use in any of the assays described herein. In some embodiments, target cells are passaged once in culture medium and then cryopreserved for future use in any of the assays described herein. In some embodiments, target cells are passaged 5 times in culture medium and then cryopreserved for future use in any of the assays described herein. In some embodiments, target cells are passaged 10 times in culture medium and then cryopreserved for future use in any of the assays described herein. In some embodiments, target cells are passaged 15 times in culture medium and then cryopreserved for future use in any of the assays described herein. In some embodiments, target cells are passaged 20 times in culture medium and then cryopreserved for future use in any of the assays described herein. In some embodiments, the target cells are passaged 25 times in culture medium and then cryopreserved for future use in any of the assays described herein. In some embodiments, the target cells are passaged 30 times in culture medium and then cryopreserved for future use in any of the assays described herein. In some embodiments, the target cells are passaged 35 times in culture medium and then cryopreserved for future use in any of the assays described herein. In some embodiments, the target cells are passaged 40 times in culture medium and then cryopreserved for future use in any of the assays described herein. In some embodiments, the target cells are passaged 50 times in culture medium and then cryopreserved for future use in any of the assays described herein.

[0262] 1.Target antigen In some embodiments, the target cells are cells that express a target antigen recognized by a recombinant receptor.

[0263] In certain embodiments, target cells may contain integrins (avb6 integrin), B cell maturation antigens (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (also known as CA9, CAIX, or G250), cancer-testis antigen, cancer / testis antigen 1B (also known as CTAG, NY-ESO-1, and LAGE-2), carcinoembryonic antigen (CEA), cyclins, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), tegmented epidermal growth factor protein (tEGFR), and type III epidermal growth factor receptor mutations (EGFR). vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate-binding protein (FBP), folate receptor alpha, fetal acetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (e rb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-AIA1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insertion domain receptor (kdr), kappa light chain, LI cell adhesion molecule (LI-CAM), LI-CAM CE7 epitope, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-Al, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1),It expresses MUC16, natural killer group 2 member D (KG2D) ligand, melan A (MART-1), neuronal adhesion molecule (NCAM), carcinoembryonic antigen, melanoma preferential expression antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (also known as TRPl, TYRPl, or gp75), tyrosinase-related protein 2 (also known as TRP2, dopachrome, tautomerase, dopachrome delta-isomerase, or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), or a combination thereof. In some embodiments, target cells express pathogen-specific or pathogen-expressed antigens, or antigens associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens. In certain embodiments, target cells express one or more antigens associated with B-cell malignancies, e.g., one of several known B-cell markers. In certain embodiments, target cells express CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, CD30, or a combination thereof. In some embodiments, target cells express CD19, e.g., human CD19.

[0264] In embodiments of the provided method, the cells of the effector cell composition express recombinant receptors, such as CARs, as described in sections IA and III. In some embodiments, the target antigen is a portion of or includes a polypeptide antigen that is recognized or bound by a recombinant receptor such as a CAR. In certain embodiments, the portion of the target antigen is a region containing an epitope recognized or bound by a recombinant receptor, such as a CAR. In certain embodiments, the portion of the polypeptide antigen is about 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 30 of the polypeptide recognized or bound by a recombinant receptor, such as a CAR. It contains 0, 400, or 500 amino acids, or at least 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, or 500 amino acids, and in some cases, consecutive amino acids. In certain embodiments, the polypeptide moiety contains the amino acid sequence of an epitope recognized by a recombinant receptor and / or CAR.

[0265] In certain embodiments, the target antigen or moiety is a polypeptide variant containing about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity with respect to the polypeptide bound to and / or recognized by the recombinant receptor and / or CAR, or containing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity.

[0266] In certain embodiments, the extracellular domain of a recombinant receptor (e.g., CAR) is specific to or binds to BCMA, and the target antigen is BCMA or the extracellular domain portion of BCMA. In some embodiments, the BCMA polypeptide is a mammalian BCMA polypeptide. In certain embodiments, the BCMA polypeptide is a human BCMA polypeptide. In some embodiments, the BCMA antigen is the extracellular domain or a portion thereof of BCMA, which includes an epitope recognized by the antigen receptor, e.g., CAR. In certain embodiments, the BCMA antigen is a polypeptide having an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 13, or a fragment thereof containing at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170 or at least 180 consecutive amino acids of SEQ ID NO: 13, or includes the same. In some embodiments, the BCMA antigen is the sequence or an epitope recognized by an antigen receptor, e.g., CAR, or a portion thereof containing the same, or includes the same.

[0267] In certain embodiments, the extracellular domain of a recombinant receptor (e.g., CAR) is specific to or binds to ROR1, and the target antigen is ROR1 or the extracellular domain portion of ROR1. In certain embodiments, the ROR1 polypeptide is mammalian. In certain embodiments, the ROR1 polypeptide is human. In some embodiments, the antigen is the extracellular domain of ROR1 or a portion thereof containing an epitope recognized by an antigen receptor, e.g., CAR. In some embodiments, the antigen is an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 19, or a polypeptide having a fragment thereof containing at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, or at least 180 consecutive amino acids of SEQ ID NO: 19. In some embodiments, the ROR1 antigen includes the sequence shown in SEQ ID NO: 19 or a portion thereof containing an antigen receptor, such as an epitope recognized by CAR.

[0268] In certain embodiments, the extracellular domain of a recombinant receptor (e.g., CAR) is specific to or binds to CD22, and the target antigen is CD22 or the extracellular domain portion of CD22. In certain embodiments, the CD22 polypeptide is mammalian. In certain embodiments, the CD22 polypeptide is human. In some embodiments, the target antigen is the extracellular domain of CD22 or a portion thereof including an epitope recognized by an antigen receptor, e.g., CAR. In some embodiments, the target antigen is an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with SEQ ID NO: 14, or a polypeptide having a fragment thereof containing at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, or at least 180 consecutive amino acids of SEQ ID NO: 14. In some embodiments, the CD22 antigen includes the sequence shown in SEQ ID NO: 14 or a portion thereof containing an epitope recognized by an antigen receptor, such as CAR.

[0269] In certain embodiments, the extracellular domain of a recombinant receptor (e.g., CAR) is specific to or binds to CD19, and the target antigen is CD19 or the extracellular domain portion of CD19. In certain embodiments, the CD19 polypeptide is mammalian. In certain embodiments, the CD19 polypeptide is human. In some embodiments, the target antigen is the extracellular domain of CD19 or a portion thereof containing an epitope recognized by an antigen receptor, e.g., CAR. In some embodiments, the target antigen is an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 15, or a polypeptide having a fragment of the same sequence containing at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, or at least 180 consecutive amino acids of SEQ ID NO: 15. In some embodiments, the CD19 antigen includes the sequence shown in SEQ ID NO: 15 or a portion of the same sequence containing an antigen receptor, such as an epitope recognized by CAR.

[0270] In some embodiments, the target antigen or portion thereof is combined as a multimer, e.g., a dimer, containing two or more polypeptide antigens or portions or variants that are recognized and / or bound by a recombinant receptor, e.g., an antigen receptor (e.g., CAR). In some embodiments, the polypeptide antigen or portion thereof is identical. In certain embodiments, the polypeptide antigen is directly or indirectly ligated to a region or domain, e.g., a multimerizing domain, which facilitates or stabilizes the interaction between two or more polypeptide antigens through complementary interactions between the domains or regions. In some embodiments, providing the polypeptide antigen as a multimer, e.g., a dimer, provides a polyvalent interaction between the antigen or its extracellular domain portion and the antigen-binding domain of an antigen receptor, e.g., CAR, which, in some embodiments, may increase the binding strength of the interaction. In some embodiments, the increased binding strength by the antigen or its extracellular domain portion conjugated to beads may be advantageous for the stimulating or agonist activity of the antigen receptor, e.g., CAR.

[0271] In some embodiments, polypeptides are directly or indirectly conjugated to a polymerizing domain. Exemplary polymerizing domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The polymerizing domain may be, for example, an immunoglobulin constant region or domain, such as an Fc domain or a portion thereof derived from IgG, including IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, and IgM, and its modifications. In certain embodiments, polypeptide antigens are directly or indirectly conjugated to an Fc domain. In some embodiments, the polypeptide is a fusion polypeptide comprising a polypeptide antigen or portion thereof and an Fc domain.

[0272] In certain embodiments, the target antigen or its extracellular domain portion is a fusion polypeptide comprising an Fc domain. In some embodiments, the Fc domain consists of the second and third constant domains (i.e., CH2 and CH3 domains) of the heavy chain of an IgA or IgD isotype, e.g., CH2 or CH3 of IgG, IgA, and IgD isotypes. In some embodiments, the Fc domain consists of three constant heavy chain domains (i.e., CH2, CH3, and CH4 domains) of an IgM or IgE isotype. In some embodiments, the Fc domain may further include a hinge sequence or a portion thereof. In certain embodiments, the Fc domain contains some or all of the hinge domain of an immunoglobulin molecule, as well as the CH2 and CH3 domains. In some cases, the Fc domain may form a dimer of two polypeptide chains joined by one or more disulfide bonds. In some embodiments, the Fc domain is derived from an immunoglobulin (e.g., IgG, IgA, IgM, or IgE) of a suitable mammal (e.g., human, mouse, rat, goat, sheep, or monkey). In some embodiments, the Fc domain is derived from the C of IgG. H 2 and C H It contains three domains. In certain embodiments, the Fc domain is fused to the C-terminus of the polypeptide antigen. In certain embodiments, the Fc domain is fused to the N-terminus of the polypeptide antigen.

[0273] In some embodiments, the Fc domain is an IgG Fc domain or a portion or variant thereof. In some embodiments, the Fc domain is a human IgG Fc domain or a portion or variant thereof comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 16. In certain embodiments, the Fc domain is a wild-type human IgG Fc domain or a portion or variant thereof. In certain embodiments, the Fc domain is a variant of the wild-type human IgG1 Fc domain.

[0274] In some embodiments, the fusion polypeptide contains a mutant Fc domain. In certain embodiments, the mutant human IgG Fc domain contains mutations, e.g., substitutions, deletions, or insertions, that reduce, decrease, and / or diminish pairing between the Fc domain and the light chain. In some embodiments, the mutant human IgG Fc domain contains mutations that reduce the binding affinity between the Fc domain and the Fc receptor. In certain embodiments, the mutant human IgG Fc domain contains mutations that reduce, decrease, and / or diminish the interaction between the Fc domain and the Fc receptor, or the probability or possibility of such interaction. In some embodiments, the mutant human IgG Fc domain contains mutations that reduce the binding affinity between the Fc domain and proteins in the complement system. In certain embodiments, the mutant human IgG Fc domain contains mutations that reduce, decrease, and / or diminish the interaction between the Fc domain and proteins in the complement system, or the probability or possibility of such interaction.

[0275] In some embodiments, the target antigen or a portion thereof is ligated to a mutant human IgG1 Fc domain. In some embodiments, the mutant human IgG Fc domain contains a substitution of cysteine ​​to serine in the hinge region of the Fc domain. In some embodiments, the mutant human IgG Fc domain contains a substitution of leucine to alanine in the hinge region of the Fc domain. In certain embodiments, the mutant human IgG Fc domain contains a substitution of glycine to alanine in the hinge region. In certain embodiments, the mutant human IgG Fc domain contains a substitution of alanine to serine in the CH2 region of the Fc domain. In some embodiments, the mutant human IgG Fc domain contains a substitution of proline to serine in the CH2 region of the Fc domain. In some embodiments, the mutant human IgG Fc domain contains the amino acid sequence shown in Sequence ID No. 17.

[0276] In some embodiments, the target antigen or its extracellular domain portion is provided as a fusion polypeptide containing an Fc domain, the Fc domain being located at the C-terminus of the fusion polypeptide.

[0277] In some embodiments, the target antigen and a multimerizing domain, such as an Fc domain, are linked by a linker, such as an amino acid linker. In certain embodiments, the target antigen is fused to the N-terminus of the amino acid linker, and a multimerizing domain, such as an Fc domain, is fused to the C-terminus of the linker. The amino acid linker can be of any length and may contain any combination of amino acids, but the linker length may be relatively short (e.g., 10 or fewer amino acids) to reduce interactions between linked domains. The amino acid composition of the linker may also be adjusted to reduce the number of amino acids with bulky side chains or amino acids that are likely to introduce secondary structures. Suitable amino acid linkers may have amino acid lengths of up to 3, 4, 5, 6, 7, 10, 15, 20, or 25 amino acids, but are not limited to these. A typical amino acid linker sequence includes GGGGS (SEQ ID NO: 22), and the linker contains 2, 3, 4, or 5 copies of GGGGS (SEQ ID NO: 22).

[0278] In some embodiments, the target antigen is provided as a BCMA fused to an Fc domain, for example, the extracellular domain of human BCMA (BCMA-Fc). In certain embodiments, the BCMA-Fc antigen contains all or part of the amino acid sequence shown in SEQ ID NO: 18, or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of SEQ ID NO: 18, and an amino acid sequence containing an antigen receptor, for example, an epitope recognized by CAR.

[0279] In some embodiments, the target antigen is provided as ROR1, for example, the extracellular domain of human ROR1 (ROR1-Fc), fused to an Fc domain. In certain embodiments, the ROR-1-Fc antigen contains all or part of the amino acid sequence shown in SEQ ID NO: 20, or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of SEQ ID NO: 20, and an amino acid sequence containing an epitope recognized by an antigen receptor, for example, CAR.

[0280] In certain embodiments, the target antigen is provided as the extracellular domain of CD22, e.g., human CD22 (e.g., CD22-Fc), fused to an Fc domain. In certain embodiments, the CD22-Fc antigen contains all or part of the amino acid sequence shown in SEQ ID NO: 21, or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of SEQ ID NO: 21, and an amino acid sequence containing an epitope recognized by an antigen receptor, e.g., CAR.

[0281] In some embodiments, the Fc fusion of the target antigen or its extracellular binding domain is linked to or attached to a surface support as a dimer formed by two Fc-fusion polypeptides containing the polypeptide antigen or its partial Fc domain. In some embodiments, the resulting polypeptide antigen-Fc fusion protein, e.g., BCMA-Fc, ROR1-Fc, CD22-Fc, or CD19-Fc, may be expressed, for example, in host cells transformed with an expression vector, thereby resulting in an assembly between Fc domains by intramolecular disulfide bonds formed between the Fc portions, yielding a dimer, e.g., a bivalent polypeptide antigen fusion protein. In some embodiments, the host cell is a mammalian cell line. Examples of mammalian cells for recombinant expression of the protein include HEK293 cells or CHO cells or derivatives thereof. In some embodiments, the nucleic acid encoding the Fc fusion protein further comprises a signal peptide for secretion from the cell. In exemplary embodiments, the signal peptide is CD33 (e.g., shown in SEQ ID NO: 12).

[0282] In some embodiments, the cells of an effector cell composition (e.g., a therapeutic cell composition) express a recombinant receptor that binds to or recognizes a universal tag that can be fused to an antibody or a fragment or variant thereof. In certain embodiments, cells expressing such a recombinant receptor can specifically recognize and kill target cells, such as tumor cells, that are bound by an antibody fused to the universal tag. An example is T cells expressing an anti-FITC CAR that can bind to and / or recognize various human cancer cells when bound to a cancer-reactive FITC-labeled antibody. Thus, in some embodiments, the same recombinant receptor that binds to the universal tag is useful for treating various cancers, provided that there is an available antibody that recognizes cancer-associated antigens containing the universal tag. Certain embodiments intend that any polypeptide domain that does not prevent the antibody from binding to its reactive target, and that can be fused to an antibody or its antigen-binding fragment or variant, is suitable for use as a universal tag.

[0283] In some embodiments, the target antigen is pathogen-specific or pathogen-expressing antigen, or includes such antigen. In some embodiments, the target antigen is a viral antigen (such as a viral antigen derived from HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen. In certain embodiments, the target cell is a tumor cell, or derived therefrom. In some embodiments, the tumor cell is cancerous. In certain embodiments, the tumor cell is non-cancerous. In some embodiments, the tumor cell is a circulating B cell, e.g., a circulating B cell capable of forming a tumor in vivo, or derived therefrom. In some embodiments, the tumor cell is a circulating B cell that is neoplastic, tumorigenic, or cancerous B cell, or derived therefrom.

[0284] In certain embodiments, the tumor cells are human cancer cells or derived therefrom. In some embodiments, tumor cells include AID-related cancers, breast cancer, gastrointestinal cancers, anal cancers, appendiceal cancers, bile duct cancers, colon cancers, colorectal cancers, esophageal cancers, gallbladder cancers, islet cell tumors, pancreatic neuroendocrine tumors, liver cancers, pancreatic cancers, rectal cancers, small intestine cancers, stomach cancers, endocrine cancers, adrenocortical carcinomas, parathyroid cancers, pheochromocytomas, pituitary tumors, thyroid cancers, eye cancers, intraocular melanomas, retinoblastomas, bladder cancers, kidney (renal cell) cancers, penile cancers, prostate cancers, transitional cell renal pelvis and ureteral cancers, testicular cancers, urethral cancers, Wilms' tumor or other pediatric kidney tumors, germ cell cancers, central nervous system cancers, extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, gynecological cancers, cervical cancers, endometrial cancers, gestational trophoblastic tumors, ovarian epithelial carcinomas, uterine sarcomas, vaginal cancers, and vulvar cancers. Head and neck cancer, hypopharyngeal cancer, pharyngeal cancer, lip and oral cancer, metastatic squamous neck cancer, nasopharyngeal cancer, oropharyngeal cancer, sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, throat cancer, musculoskeletal cancer, bone cancer, Ewing's sarcoma, gastrointestinal stromal tumor (GIST), osteosarcoma, malignant fibrous histiocytoma of bone, rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, neurological cancer, brain tumor, astrocytoma, brainstem glioma, non-central nervous system cancer Derived from cells of typical teratomas / rhabdoid tumors, central nervous system germ cell tumors, central nervous system germ cell tumors, craniopharyngiomas, ependymomas, medulloblastomas, spinal cord tumors, supratentorial primitive neuroectoderm tumors and pinealoblastomas, neuroblastomas, respiratory cancers, thoracic cancers, non-small cell lung cancers, small cell lung cancers, malignant mesotheliomas, thymomas, thymic carcinomas, skin cancers, Kaposi's sarcoma, melanomas or Merkel cell carcinomas or any equivalent human cancers thereto.

[0285] In certain embodiments, tumor cells are derived from non-hematopoietic cancers, e.g., solid tumors. In certain embodiments, tumor cells are derived from hematological cancers. In certain embodiments, tumor cells are derived from cancers that are B-cell malignancies or hematological malignancies. In certain embodiments, tumor cells are derived from non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML), or myeloma, e.g., multiple myeloma (MM) or any equivalent human cancer thereto. In some embodiments, antigen-expressing cells are neoplastic, oncogenic, and / or tumorigenic B cells. Multiple tumor cell lines are known and available and can be selected depending on the antigen recognized by a specific recombinant receptor (e.g., CAR).

[0286] Several tumor cell lines are known and available. Tumor cell lines expressing specific tumor antigens are known, or the surface expression of tumor antigens can be readily determined or measured by those skilled in the art using any of the various techniques, for example, by flow cytometry. Exemplary tumor cell lines include lymphoma cells (Raji; Daudi; Jeko-1; BJAB; Ramos; NCI-H929; BCBL-1; DOHH-2, SC-1, WSU-NHL, JVM-2, Rec-1, SP-53, RL, Granta) 519, NCEP-1, CL-01), leukemia cells (BALL-1, RCH-ACV, SUP-B15); cervical cancer cells (33A; CaSki; HeLa), lung cancer cells (NCI-H358) ;A549, H1355, H1975, Calu-1, H1650 and H727), breast cells, (Hs-578T;ZR-75-1;MCF-7;MCF-7 / HER2;MCF10A; Examples include MDA-MB-231;SKBR-3, BT-474, MDA-MB-231); ovarian cells (ES-2;SKOV-3;OVCAR3;HEY1B); and multiple myeloma cells (U266, NCI-H929, RPMI-8226, OPM2, LP-1, L363, MM.1S, MM.1R, MC / CAR, JJN3, KMS11, AMO-1, EJM;MOLP-8). For example, exemplary CD19-expressing cell lines include Raji, Daudi, and BJAB; exemplary CD20-expressing cell lines include Daudi, Ramos, and Raji; exemplary CD22-expressing cell lines include, but are not limited to, Ramos, Raji, A549, H727, and H1650; exemplary Her2-expressing cell lines include SKOV3, BT-474, and SKBR-3; exemplary BCMA-expressing cell lines include RPMI-8226, NCI-H929, MM1S, MM1R, and KMS11; exemplary GPRC5D-expressing cell lines include AMO-1, EJM, NCI-H929, MM.1S, MM1.R, MOLP-8, and OPM-2; and exemplary ROR1-expressing cell lines include A549, MDA-MB-231, H1975, BALL-1, and RCH-ACV.

[0287] In some embodiments, the target cells are derived from a cell line that has been transduced to express the target antigen of the recombinant receptor. In some embodiments, the target antigen is a tumor antigen.

[0288] In some embodiments, the target antigen expressed on target cells is an antibody or antigen-binding fragment that specifically recognizes a recombinant receptor such as a CAR. In some embodiments of these embodiments, the antibody or antigen-binding fragment specifically recognizes the extracellular portion of the recombinant receptor, for example, a CAR (for example, specifically, it specifically binds to an extracellular epitope of the recombinant receptor).

[0289] In some embodiments, the target antigen is a recombinant receptor, such as a recombinant receptor as described in Section III, for example, an anti-idiotype antibody or its antigen-binding fragment ("anti-ID") that specifically recognizes a CAR. In particular, the anti-idiotype antibody targets the antigen-binding site of another antibody, for example, the scFv of the extracellular antigen-binding domain of a CAR. In some embodiments, anti-ID can bind to the recombinant receptor and stimulate cytotoxic activity. Exemplary anti-idiotype antibodies against antigen-specific CARs are known. These include, but are not limited to, anti-idiotype antibodies directed against CARs directed by CD22, e.g., see PCT publication number WO2013188864; CARs directed by CD19, e.g., see PCT publication number WO2018 / 023100; CARs directed by GPRC5D, e.g., see PCT application number PCT / US2020 / 063497; and CARs directed by BCMA, e.g., see PCT application number PCT / US2020 / 063492.

[0290] The term “antibody” is used herein in its broadest sense and includes polyclonal and monoclonal antibodies, examples of which include intact antibodies and functional (antigen-binding) antibody fragments, e.g., fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, e.g., single-chain variable fragments (scFv), and single-domain antibody fragments (e.g., sdAb, sdFv, nanobody) fragments. The term also encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, e.g., intrabodies, peptidebodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies, e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem scFv, and tandem triscFv. The term "antibody" should be understood to encompass those functional antibody fragments unless otherwise specified. This term also encompasses intact or full-length antibodies, including any class or subclass of antibodies, such as IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0291] The term “anti-idiotype antibody” refers to an antibody containing an antibody idiotope, such as an antigen-binding fragment that specifically recognizes, is specifically targeted to, and / or specifically binds to. The antibody idiotope may, but may not, include residues in the antibody’s complementarity-determining region (CDR), the antibody’s variable region, and / or one or more partial or partial parts of such a variable region, and / or any combination thereof, of such a CDR. The CDR may be one or more selected from the group consisting of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The antibody’s variable region may be a heavy chain variable region, a light chain variable region, or a combination of a heavy chain variable region and a light chain variable region. A partial fragment or portion of the heavy chain variable region and / or light chain variable region of an antibody may be a fragment containing two or more, five or more, or ten or more consecutive amino acids within the heavy chain variable region or light chain variable region of the antibody, for example, about 2 to about 100, about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 consecutive amino acids; an idiotope may contain multiple discontinuous stretches of amino acids. A partial fragment of the heavy chain variable region and light chain variable region of an antibody may be a fragment containing two or more, five or more, or ten or more consecutive amino acids within the variable region, for example, about 2 to about 100, about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 consecutive amino acids, and in some embodiments, it may contain one or more CDRs or CDR fragments. A CDR fragment can consist of two or more consecutive or discontinuous amino acids, or five or more amino acids. Therefore, the antibody idiotope can consist of approximately 2 to approximately 100, approximately 5 to approximately 100, approximately 10 to approximately 100, approximately 2 to approximately 50, approximately 5 to approximately 50, or approximately 10 to approximately 50 consecutive amino acids containing one or more CDRs or one or more CDR fragments within the heavy chain variable region or light chain variable region of the antibody.In another embodiment, the idiotope may be a single amino acid located in the variable region of the antibody, for example, in the CDR region.

[0292] In some embodiments, an idiotope is any single antigenic determinant or epitope within the variable region of an antibody. In some cases, it may overlap with the actual antigen-binding site of the antibody, and in some cases, it may include a variable region sequence outside the antigen-binding site of the antibody. In some embodiments, a set of individual idiotopes of an antibody is called an "idiotype" of such an antibody.

[0293] The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR," and are known in the art to refer to discontinuous sequences of amino acids within the antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.

[0294] The precise amino acid sequence boundaries of a given CDR or FR can be found in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme), and Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003. This can be easily determined using one of several well-known schemes, including those described by Jan;27(1): 55-77 ("IMGT" numbering scheme) and Honegger A and Plueckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3): 657-70, ("Aho" numbering scheme).

[0295] The boundaries of a given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence length, with insertions corresponding to insertion letters, e.g., "30a," and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("insertions and deletions") in different configurations, resulting in differential numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0296] Table 1 below lists exemplary placement boundaries for CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, and contact schemes, respectively. For CDR-H1, residue numbers are listed using both Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, FR-L1 is located between CDR-L1 and CDR-L2. Since the shown Kabat numbering scheme places insertions at H35A and H35B, it is known that when the ends of a Chothia CDR-H1 loop are numbered using the shown Kabat numbering transformation, the transformation changes between H32 and H34 depending on the length of the loop.

[0297] [Table 1]

[0298] Therefore, unless otherwise specified, a given antibody or region, for example, its variable region's "CDR" or "complementarity-determining region" or individual specific CDRs (e.g., "CDR-H1, CDR-H2"), should be understood to encompass one (or specific) complementarity-determining region as defined by any of the schemes above. For example, a designated CDR (e.g., CDR-H3) is given V Hor V L When it is stated that an amino acid sequence contains the amino acid sequence of a corresponding CDR, such a CDR is understood to have the sequence of a corresponding CDR (e.g., CDR-H3) within a variable region as defined by any of the schemes described above. In some embodiments, a specific CDR sequence is specified.

[0299] Similarly, unless otherwise specified, a given antibody or its region, for example, its variable region FR or individual specific FRs (e.g., FR-H1, FR-H2), should be understood to encompass one (or specific) framework region as defined by any known scheme. In some cases, a scheme is specified for the identification of a specific CDR, FR, or multiple FRs or multiple CDRs, such as a CDR as defined by Kabat, Chothia, or contact method. In other examples, a specific amino acid sequence of the CDR or FR is given.

[0300] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain that is involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (V H and V L Generally, each domain has a similar structure containing four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). Single V H or V L The domain may be sufficient to confer antigen-binding specificity. Furthermore, the V from the antibody that binds to the antigen can be used to confer antigen-binding specificity to an antibody that binds to a specific antigen. H or V L Using the domain, isolate and compare each complementary V L or V HThis allows for screening of domain libraries. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0301] Antibodies contain antibody fragments. An "antibody fragment" refers to a molecule other than the intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment, e.g., scFv, containing heavy chain variable chain regions and / or light chain variable chain regions.

[0302] A single-domain antibody is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody.

[0303] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, as well as production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, for example, a fragment containing arrangements not found in nature, such as two or more antibody regions or chains joined by a synthetic linker, such as a peptide linker, and / or one that cannot be produced by enzymatic digestion of naturally occurring intact antibodies. In some embodiments, the antibody fragment is an scFv.

[0304] A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all framework amino acid residues are derived from human FRs. In some embodiments, the humanized form of a non-human antibody, e.g., a mouse antibody, is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In certain embodiments, the humanized antibody is an antibody from a non-human species having one or more complementarity-determining regions (CDRs) from a non-human species and a framework region (FR) from a human immunoglobulin molecule. In some embodiments, the humanized antibody may also contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of a non-human antibody usually refers to a variant of a non-human antibody that has been humanized to reduce its immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, certain FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived) to restore or improve antibody specificity or affinity (see, for example, Queen, U.S. Patent No. 5,585,089 and Winter, U.S. Patent No. 5,225,539). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0305] In certain embodiments, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the heavy chain variable region of the recipient are replaced by residues from the heavy chain variable region of a non-human species (donor antibody), e.g., mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capabilities. In some cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or the donor antibody. In some embodiments, the nucleic acid sequences encoding the human heavy and light chain variable regions are modified so that one or more CDR sequences in the human (acceptor) sequence are replaced by sequences encoding the respective CDRs in the non-human antibody sequence (donor sequence). In some embodiments, the human acceptor sequence may contain FRs derived from different genes. In certain embodiments, the humanized antibody will contain at least one, usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are from the human immunoglobulin sequence. In some embodiments, the humanized antibody will, as appropriate, include at least a portion of the immunoglobulin constant region (Fc), which is typically that of human immunoglobulin. For further details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patents No. 6,982,321 and 7,087,409, which are incorporated herein by reference.In some embodiments, humanized anti-idiotype antibodies are provided herein.

[0306] In certain embodiments, antibodies, such as anti-idiotype antibodies, are humanized. In certain embodiments, antibodies are humanized by any suitable known means. For example, in some embodiments, a humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often called “implant” residues, which are usually taken from an “implant” variable domain. In certain embodiments, humanization may be carried out essentially by, for example, substituting the hypervariable region sequence of the corresponding sequence of a human antibody, according to the methods of Winter and collaborators (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536). Therefore, such “humanized” antibodies are chimeric antibodies in which substantially fewer than the intact human variable domains are replaced by corresponding sequences from non-human species (U.S. Patent No. 4,816,567). In certain embodiments, the humanized antibody is a human antibody in which some hypervariable region residues and possibly some FR residues are replaced by residues from similar sites in a rodent antibody.

[0307] Sequences encoding full-length antibodies can then be obtained by conjugating given heavy-chain variable and light-chain variable sequences to human constant-state heavy and light-state regions. Suitable human constant-state light-state sequences include kappa and lambda constant-state light-state sequences. Suitable human constant-state heavy-state sequences include sequences encoding IgG1, IgG2, and immunostimulatory IgG1 mutants. Such mutants may have reduced ability to activate complement and / or antibody-dependent cytotoxicity, as described in U.S. Patent No. 5,624,821; WO99 / 58572 and U.S. Patent No. 6,737,056. Other suitable constant-state heavy-state sequences include IgG1 with substitutions E233P, L234V, L235A, A327G, A330S, P331S, and deletion of residue 236. In another embodiment, the full-length antibody includes IgA, IgD, IgE, IgM, IgY, or IgW sequences.

[0308] A suitable human donor sequence can be determined by a sequence comparison of the peptide sequence encoded by the mouse donor sequence against a group of human sequences, preferably sequences encoded by human germline immunoglobulin genes or mature antibody genes. Human sequences with high sequence homology, preferably those with the highest homology determined, may act as acceptor sequences for the humanization process.

[0309] In addition to exchanging human CDRs for mouse CDRs, further manipulation of human donor sequences may be performed to obtain sequences encoding humanized antibodies with optimized properties (e.g., antigen affinity).

[0310] Furthermore, the modified human acceptor antibody variable domain sequence may also be configured to encode one or more amino acids (according to the Kabat numbering system) at positions 4, 35, 38, 43, 44, 46, 58, 62, 64, 65, 66, 67, 68, 69, 73, 85, 98 in the lightly variable region and positions 2, 4, 36, 39, 43, 45, 69, 70, 74, 75, 76, 78, 92 in the heavily variable region, corresponding to a non-human donor (Carter and Presta, U.S. Patent No. 6,407,213).

[0311] In certain embodiments, it is generally desirable that antibodies be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this objective, in some embodiments, humanized antibodies are prepared by a process of analysis of the parent sequence and various conceptual humanization products using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that exemplify and display the highly probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Investigation of these displays allows for the analysis of the likely roles of residues in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and transplanted sequences so that desired antibody characteristics, such as increased affinity for the target antigen(s), are achieved. Generally, hypervariable region residues are most substantially involved in directly influencing antigen binding.

[0312] In certain embodiments, the selection of human variable domains, both light and heavy, to be used in the production of humanized antibodies may be important for reducing antigenicity. Following a so-called "best-fit" method, the sequences of variable domains from rodent antibodies are screened against a complete library of known human variable domain sequences. The human sequence most closely resembling that of rodents is then accepted as the human framework for the humanized antibody. See, for example, Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Alternatively, a specific framework derived from the consensus sequences of all human antibodies in a particular subgroup of the light or heavy chain is used. The same framework may be used for several different humanized antibodies. For example, see Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623.

[0313] Among antibodies are human antibodies. A "human antibody" is an antibody that has the amino acid sequence corresponding to that of an antibody produced by a human or human cell or by a non-human source that utilizes a sequence encoding other human antibodies, including the human antibody repertoire or human antibody library. This term excludes humanized forms of non-human antibodies that include non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.

[0314] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic antigen administration. Such animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci or are randomly integrated outside the chromosome or within the animal's chromosomes. In such transgenic animals, endogenous immunoglobulin loci are generally inactivated. Human antibodies can also be derived from human antibody libraries, including cell-free libraries containing phage displays and sequences encoding antibodies derived from the human repertoire.

[0315] Among antibodies are monoclonal antibodies, which contain monoclonal antibody fragments. The term "monoclonal antibody" as used herein refers to an antibody obtained from or within a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for naturally occurring mutations or potential variants arising during the production of the monoclonal antibody preparation, such variants generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain various antibodies directed against various epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on an antigen. This term should not be interpreted as requiring the production of antibodies by any particular method. Monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, production from hybridomas, recombinant DNA methods, phage display, and other antibody display methods.

[0316] 2. Reporter In some embodiments, target cells express a reporter molecule that can be used to determine whether the target cells are viable and to evaluate the cytotoxicity, e.g., cytolytic activity, of an effector cell composition (e.g., a therapeutic cell composition). In some embodiments, the reporter is a protein. In some embodiments, the reporter is a fluorescent protein or enzyme whose activity can be detected. In some embodiments, the concentration or amount of the reporter is related to the amount of viable target cells. In some embodiments, to establish a baseline for the reporter, a known concentration or amount of target cells that have not been incubated with cells expressing the recombinant receptor, or that have been incubated with cells that do not express the recombinant receptor, is used, and the baseline for the reporter is an amount or concentration of the reporter that indicates little to no cytotoxic activity.

[0317] In some embodiments, the reporter is or contains a detectable protein. In some embodiments, the reporter is a fluorescent protein. In some embodiments, the cytotoxic activity of each of multiple incubations is measured by fluorescence imaging. In some embodiments, the fluorescent protein is green fluorescent protein (GFP), high-sensitivity green fluorescent protein (EGFP), e.g., superfold GFP (sfGFP), red fluorescent protein (RFP), e.g., tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), high-sensitivity blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), as well as species variants of the fluorescent protein, monomeric variants, codon-optimized, stabilized, and / or enhanced variants thereof. In some embodiments, the reporter can be used to count cells. In certain embodiments, a decrease in fluorescence may indicate a decrease in target cell viability and can be used as a reading of the cytotoxic activity of an effector cell composition (e.g., a therapeutic cell composition). In some embodiments, the reporter is or includes enzymes such as luciferase, the lacZ gene derived from Escherichia coli (E. coli), alkaline phosphatase, secretory embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT). Exemplary luminescence reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or their variants.

[0318] In some embodiments, the enzyme is luciferase. In some embodiments, the cytotoxic activity of each of the multiple incubations is measured by luminescence imaging. Luminescence imaging can be performed using any commercially available instrument, for example, a plate reader for doing so, such as a PerkinElmer® EnVision® plate reader.

[0319] In some embodiments, the luciferase is firefly luciferase, click mushroom luciferase, sea oyster luciferase, Gausia luciferase, Gausia-dural luciferase, Oprophorus luciferase, bacterial luciferase, sea firefly luciferase, Polychthys luciferase, dinoflagellate luciferase, krill luciferase, or fungal luciferase. In some embodiments, the luciferase is firefly luciferase.

[0320] In some embodiments, enzyme activity can be detected by adding a substrate that can be detected during enzyme expression and functional activity. In certain embodiments where the reporter is an enzyme, enzyme activity can be measured as a target cell viability reading using the addition of one or more substrates. In some embodiments, measuring cytotoxic activity involves adding a detection reagent to each of several incubations. In some embodiments, the reporter molecule is an enzyme, and the detection reagent is a substrate of the enzyme.

[0321] In some embodiments, the measurement of cytotoxic activity includes, before addition, equilibrating each of several incubations to room temperature for between 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, including the boundaries of each incubation.

[0322] In some embodiments, the measurement of cytotoxic activity includes, before addition, equilibrating the detection reagent to room temperature for 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, including the boundary, respectively, or for about 10 to about 120 minutes, about 20 to about 90 minutes, or about 30 to about 60 minutes.

[0323] In some embodiments, cytotoxic activity is measured in the presence of the detection reagent for each of the multiple incubations, including the boundaries, at intervals of 5 to 240 minutes, 5 to 180 minutes, 5 to 120 minutes, 5 to 90 minutes, 5 to 60 minutes, 5 to 45 minutes, 5 to 30 minutes, 5 to 15 minutes, 10 to 240 minutes, 10 to 180 minutes, 10 to 120 minutes, 10 to 90 minutes, and 10 minutes. 60 minutes, 10 to 45 minutes, 10 to 30 minutes, 10 to 15 minutes, 20 to 240 minutes, 20 to 180 minutes, 20 to 120 minutes, 20 to 90 minutes, 20 to 60 minutes, 20 to 45 minutes, 20 to 30 minutes, 30 to 240 minutes, 30 to 180 minutes, 30 to 120 minutes, 30 to 90 minutes, 30 to 60 minutes, or between 30 to 45 minutes, or approximately 5 minutes to approximately 240 minutes, approximately 5 minutes to approximately 180 minutes, approximately 5 minutes to approximately 120 minutes, approximately 5 minutes to approximately 90 minutes, approximately 5 minutes to approximately 60 minutes, approximately 5 minutes to approximately 45 minutes, approximately 5 minutes to approximately 30 minutes, approximately 5 minutes to approximately 15 minutes, approximately 10 minutes to approximately 240 minutes, approximately 10 minutes to approximately 180 minutes, approximately 10 minutes to approximately 120 minutes, approximately 10 minutes to approximately 90 minutes, approximately 10 minutes to approximately 60 minutes, approximately 10 minutes to approximately 45 minutes, approximately 10 minutes to approximately 30 minutes, approximately 10 minutes to approximately 15 minutes, This includes incubation for approximately 20 to 240 minutes, 20 to 180 minutes, 20 to 120 minutes, 20 to 90 minutes, 20 to 60 minutes, 20 to 45 minutes, 20 to 30 minutes, 30 to 240 minutes, 30 to 180 minutes, 30 to 120 minutes, 30 to 90 minutes, 30 to 60 minutes, or 30 to 45 minutes.

[0324] In some embodiments, the reporter indicates cell viability, and detection of the reporter indicates that the target cells are viable. In some embodiments, the reporter indicates target cell viability, and a decrease in the reporter indicates the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition).

[0325] In certain embodiments, the detection reagent is added before, during, and / or after the target cells are incubated with the effector cell composition (e.g., a therapeutic cell composition). In some embodiments, the reporter is an enzyme capable of producing luminescence in the presence of the detection reagent. In some embodiments, the reporter is luciferase. In certain embodiments, the reporter is firefly luciferase. In some embodiments, the detection reagent is a luciferase substrate. In some embodiments, the detection reagent is luciferin. In some embodiments, the detection reagent is a luciferin analog, which in some embodiments may be any commercially available luciferin analog. In some embodiments, the luciferin analog is 5'-fluoroluciferin. In certain embodiments, the detection reagent is a substrate for ONE-Glo® EX or any other commercially available luciferase. In some embodiments, luminescence is generated in the presence of a reporter and a detection reagent, the luminescence indicates cell viability, and a decrease in luminescence is a reading of the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition).

[0326] In some embodiments, the reporter is or includes a dye or a fluorescent probe. In some embodiments, the cytotoxic activity of each of multiple incubations is measured by fluorescence or emission imaging. In some embodiments, the dye or fluorescent probe indicates cell viability, and the decrease in fluorescence is a reading of the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition).

[0327] In some embodiments, the reporter indicates cell death, and detection of the reporter indicates that the target cells are dying. In some embodiments, the reporter indicates target cell death, and an increase in the reporter indicates the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition).

[0328] In certain embodiments, the reporter is lactase dehydrogenase. In certain embodiments, the substrates are lactate, NAD+, and luciferase, and cell death causes the release of lactase dehydrogenase, which reacts with the substrate to produce luminescence. In certain embodiments, the luminescence can be measured, and an increase in luminescence indicates the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition).

[0329] In an alternative embodiment, luminescence is generated in the presence of a reporter and a detection reagent, the luminescence indicates cell death, and the increase in luminescence is a reading of the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition).

[0330] In some embodiments, the reporter is or includes a dye or a fluorescent probe. In some embodiments, the cytotoxic activity of each of multiple incubations is measured by fluorescence or emission imaging. In some embodiments, the dye or fluorescent probe indicates cell death, and the increase in fluorescence is a reading of the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition).

[0331] C. Measurement of cytotoxic activity The efficacy evaluation method provided herein involves measuring the cytotoxic activity of an effector cell composition (e.g., a therapeutic cell composition) in relation to target cells. As described above, the assay provided enables the measurement of cytotoxic activity, e.g., cytolytic activity, in relation to target antigens expressed by target cells from multiple incubation conditions, each incubation comprising different adjusted ratios of cells in the effector cell composition (e.g., a therapeutic cell composition) to the target cells.

[0332] In some embodiments, cytotoxic activity is measured after the termination of each of the multiple incubations. In some embodiments, cytotoxic activity is measured during each of the multiple incubations. In some embodiments, cytotoxic activity is measured over time during each of the multiple incubations.

[0333] In some embodiments, cytotoxic activity is a response to stimulation using a target antigen expressed by a target cell. In certain embodiments, cells are incubated in the presence of target cells capable of stimulating cytotoxic activity, and cytotoxic activity is or includes at least one aspect of the response to the stimulation.

[0334] In some embodiments, cytotoxic activity is evaluated by measuring, detecting, or quantifying cytotoxic activity in response to a stimulus (e.g., target cells). In certain embodiments, cells of an effector cell composition (e.g., a therapeutic cell composition) are cultured together with target cells, and the interaction or binding of the target cells to the recombinant receptor stimulates, for example, induces, cytotoxic activity. In some embodiments, the cytotoxic activity is specific to cells expressing the recombinant receptor. In some embodiments, cells of an effector cell composition (e.g., a therapeutic cell composition) that do not express the recombinant receptor may also exhibit cytotoxic activity depending on the target cell. In certain embodiments, cytotoxic activity occurs in cells expressing the recombinant receptor, but does not occur or occurs only minimally in cells that do not express the receptor. In some embodiments, the presence of cells that do not express the recombinant receptor may increase the cytotoxic activity of cells expressing the recombinant receptor.

[0335] In some embodiments, cytotoxic activity is measured in an effector cell composition (e.g., a therapeutic cell composition) containing cells expressing recombinant receptors such as CARs, and the measured value is compared to one or more controls. In certain embodiments, the control is a similar or identical composition of unstimulated cells. For example, in some embodiments, cytotoxic activity is measured in the cell composition after or during incubation with target cells, and the obtained measured value is compared to a control measurement of cytotoxic activity from a similar or identical cell composition that has not been incubated with target cells. In some embodiments, both the effector cell composition (e.g., a therapeutic cell composition) and the control cell composition contain cells expressing recombinant receptors. In some embodiments, the control is taken from a similar cell composition that does not contain cells expressing recombinant receptors, such as CAR+ cells. Thus, in some embodiments, an effector cell composition (e.g., a therapeutic cell composition) containing cells expressing recombinant receptors and a control cell composition that does not contain cells expressing recombinant receptors are brought into contact with target cells. In certain embodiments, the control is a measured value from the same cell composition expressing recombinant receptors taken before any stimulation. In certain embodiments, a control measurement is taken to determine the background signal, and the control measurement is subtracted from the activity measurement. In some embodiments, the activity measurement in the cell composition is divided by the control measurement to obtain a value that is a ratio of activity above the control level. In some embodiments, all cytotoxic activity measurements are adjusted or normalized to a control measurement, for example, when the target cells were not cultured together with the cells of the effector cell composition (e.g., therapeutic cell composition). In some embodiments, adjusting or normalizing the measurements to control conditions provides a more accurate measure of cytotoxic activity.

[0336] In some embodiments, target cells are cells that express a target antigen (e.g., an antigen or antibody) recognized by a recombinant receptor. In some embodiments, the recombinant receptor (e.g., CAR) and a certain number of effector cell compositions (e.g., therapeutic cell compositions) are mixed with the cells in a ratio of 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2 or 1:0.1 or about 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, 1: Cells from an effector cell composition (e.g., a therapeutic cell composition), including 15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1, are incubated in multiple ratios to target cells expressing a target antigen (e.g., an antigen or antibody), or in a range between any of the above, for example, between 1:1 to 1:10 or 1:0.2 to 1:12, including the respective boundaries. In some embodiments, the multiple ratios include any and all ratios provided herein.In some embodiments, the recombinant receptor is a CAR, and a controlled number of CAR+ cells in an effector cell composition (e.g., a therapeutic cell composition) are paired with a certain number of target cells expressing a target antigen (e.g., an antigen or antibody) in a ratio of 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.4, 1:0.3, 1:0.2 or 1:0.1 or approximately 1:100, 1: Cells of an effector cell composition (e.g., a therapeutic cell composition) are incubated in multiple ratios to antigen-expressing cells, or in a range between any of the above, for example, between 1:1 and 1:10 or between 1:0.2 and 1:12, including the respective boundaries. In some embodiments, the multiple ratios include any ratio provided herein.

[0337] In some embodiments, each containing a boundary, the effector cell composition (e.g., therapeutic cell composition) is approximately 1 × 10 2 From approximately 1 x 10 4 During that time, approximately 1 x 10 3 From approximately 1 x 10 5 During that time, approximately 1 x 10 4 From approximately 1 x 10 6 During that time, approximately 1 x 10 5 From approximately 1 x 10 7 During that time, approximately 1 x 10 6 From approximately 1 x 10 8 During that time, approximately 1 x 10 7 From approximately 1 x 10 9 Between and about 1 × 10 8 From approximately 1 x 10 10 The cells in between are incubated with a certain number of target cells.

[0338] In some embodiments, the cells of an effector cell composition (e.g., a therapeutic cell composition) are incubated with target cells in a certain volume of cell medium. It is understood that the exact volume may be a function of the surface area of ​​the vessel (e.g., a multiwell plate) in which the assay is being performed. In certain embodiments, the cells of an effector cell composition (e.g., a therapeutic cell composition) are incubated with target cells in a volume of at least or about 1 μL, at least or about 10 μL, at least or about 25 μL, at least or about 50 μL, at least or about 100 μL, at least or about 500 μL, at least or about 1 mL, at least or about 1.5 mL, at least or about 2 mL, at least or about 2.5 mL, at least or about 5 mL, at least or about 10 mL, at least or about 20 mL, at least or about 25 mL, at least or about 50 mL, at least or about 100 mL, or more than 100 mL. In certain embodiments, the cells of an effector cell composition (e.g., a therapeutic cell composition) are incubated with target cells in volumes ranging from about 1 μL to about 100 μL, from about 100 μL to about 500 μL, from about 500 μL to about 1 mL, from about 500 μL to about 1 mL, from about 1 mL to about 10 mL, from about 10 mL to about 50 mL, or from about 10 mL to about 100 mL, each including a boundary. In certain embodiments, the cells of an effector cell composition (e.g., a therapeutic cell composition) are incubated with target cells in volumes ranging from about 100 μL to about 1 mL, each including a boundary. In certain embodiments, the cells of an effector cell composition (e.g., a therapeutic cell composition) are incubated with target cells in a volume of about 500 μL. In some embodiments, the multiwell plate is a 6-well plate, and the volume is 1 mL or about 1 mL to 3 mL or about 3 mL. In some embodiments, the multiwell plate is a 12-well plate with a volume of 1 mL or about 1 mL to 2 mL or about 2 mL. In some embodiments, the multiwell plate is a 24-well plate with a volume of 0.5 mL or about 0.5 mL to 1 mL or about 1 mL.In some embodiments, the multiwell plate is a 48-well plate with a volume of 0.2 mL or about 0.2 mL to 0.4 mL. In some embodiments, the multiwell plate is a 96-well plate with a volume of 0.1 mL or about 0.1 mL to 0.2 mL.

[0339] In some embodiments, a certain number of cells from an effector cell composition (e.g., a therapeutic cell composition) are incubated with target cells at concentrations varying between approximately 1 fmol and approximately 1 pmol, approximately 1 pmol and approximately 1 nmol, approximately 1 nmol and approximately 1 μmol, approximately 1 μmol and approximately 1 mmol, or approximately 1 mmol and 1 mol, each including the boundary. In specific embodiments, a certain number of cells from an effector cell composition (e.g., a therapeutic cell composition) are incubated with target cells at concentrations varying between approximately 1 fM and approximately 1 pM, approximately 1 pM and approximately 1 nM, approximately 1 nM and approximately 1 μM, approximately 1 μM and approximately 1 mM, or approximately 1 mM and 1 mol, each including the boundary. Exemplary units include pg / mL, pg / (mL / hr), pg(mL × cells), pg / (mL × hr × cells), and pg / (mL × hr × 10 6Examples include individual cells. In certain embodiments, cytotoxic activity is the cytolytic activity of the effector cell composition (e.g., therapeutic cell composition). In some embodiments, cytotoxic activity is evaluated by exposing, incubating, and / or contacting cells of the effector cell composition (e.g., therapeutic cell composition) with target cells. Cytotoxic activity can be measured directly or indirectly by measuring the number of target cells over time. For example, target cells may be engineered to stably express a reporter molecule before incubation with cells of the effector cell composition (e.g., therapeutic cell composition), e.g., a reporter molecule detectable when target cells are lysed, or a reporter molecule detectable in viable target cells. In some embodiments, cytotoxic activity is determined based on the expression of the reporter molecule. In some embodiments, cytotoxic activity is determined based on the activity of the reporter molecule. These readings can provide direct or indirect information on the number of target cells and / or target cell death and can be measured at different times between assays. A reduction in the number of target cells and / or an increase in target cell death indicate cytotoxicity of the cells, e.g., cytolytic activity. In some embodiments, the cytotoxic activity of an effector cell composition (e.g., a therapeutic cell composition) is evaluated by reducing the activity of a reporter molecule expressed in target cells.

[0340] In certain embodiments, after incubation, a substrate of the reporter molecule is added for the detection of the reporter. In some embodiments, the amount of reporter is used to determine the number of viable target cells. In some embodiments, the amount of reporter activity is used to determine the number of viable target cells. In certain embodiments, the reporter is luciferase. In certain embodiments, the substrate is luciferin. In some embodiments, the reduction in luminescence emitted by target cells is used as a reading of the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition).

[0341] In certain embodiments, the reporter is a fluorescent protein. In some embodiments, an effector cell composition (e.g., a therapeutic cell composition) is incubated with target cells expressing a fluorescent protein. In certain embodiments, the decrease in fluorescence of the target cells after incubation with the effector cell composition (e.g., a therapeutic cell composition) is used as a reading of the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition).

[0342] In certain embodiments, cells from an effector cell composition (e.g., a therapeutic cell composition) are incubated with target cells for up to or about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours, about 48 hours, or longer than 48 hours. In some embodiments, a certain number of cells from an effector cell composition (e.g., a therapeutic cell composition) are incubated with antigen-expressing cells for about 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, a certain number of cells from an effector cell composition (e.g., a therapeutic cell composition), each containing a boundary, are incubated for about 1 × 10⁶ 2 From approximately 1 x 10 4 During that time, approximately 1 x 10 3 From approximately 1 x 10 5 During that time, approximately 1 x 10 4 From approximately 1 x 10 6 During that time, approximately 1 x 10 5 From approximately 1 x 10 7 During that time, approximately 1 x 10 6 From approximately 1 x 10 8 During that time, approximately 1 x 10 7 From approximately 1 x 10 9 Between or about 1 x 10 8 From approximately 1 x 10 10 The cells in between are incubated with a fluctuating number of target cells to yield multiple ratios. In a particular embodiment, a certain number of cells in the effector cell composition (e.g., therapeutic cell composition), each containing a boundary, are approximately 1 × 10⁶. 2 From approximately 1 x 10 4 During that time, approximately 1 x 10 3 From approximately 1 x 10 5During that time, approximately 1 x 10 4 From approximately 1 x 10 6 During that time, approximately 1 x 10 5 From approximately 1 x 10 7 During that time, approximately 1 x 10 6 From approximately 1 x 10 8 During that time, approximately 1 x 10 7 From approximately 1 x 10 9 Between or about 1 x 10 8 From approximately 1 x 10 10 CAR+ cells are incubated with a variable number of target cells, resulting in multiple ratios.

[0343] In some embodiments, cytotoxic activity measurements are compared to a control. In certain embodiments, the control is a culture of target cells not incubated with cells from an effector cell composition (e.g., a therapeutic cell composition). In some embodiments, the control is a measurement from a control cell composition that does not contain CAR+ cells incubated with target cells in the same proportions.

[0344] In certain embodiments, the measurement of a cell lysis activity assay is the number of viable target cells at a given time during incubation or at the end of incubation for each ratio tested. In some embodiments, the measurement is the amount of target cell death determined by subtracting the number of target cells in a co-incubation at a given time point from the number of control target cells incubated alone. In some embodiments, the measurement is the percentage of surviving target cells at a given time point compared to the starting number of target cells. In certain embodiments, the measurement is the number of cells that have died over a certain amount of time. In certain embodiments, the measurement is the number of dead cells per cell in a cell composition. In some embodiments, the measurement is the number of dead cells per cell, or the number of dead cells per set of cells or reference, for example, 10 per 100 cells in a composition. 3 10 per individual cell 4 10 per individual cell 5 10 per individual cell 6 10 per individual cell 710 per individual cell 8 10 per individual cell 9 Per individual cell, or 10 10 This is the number of dead target cells per individual cell. In certain embodiments, the measurement is the number of dead cells per CAR+ cell or reference number or set number in the cell composition. In certain embodiments, the measurement is the number of dead cells per unit of time in the cell composition. In certain embodiments, the measurement is the number of dead cells per unit of time in the effector cell composition (e.g., therapeutic cell composition) per CAR+ cell. In some embodiments, the measurement is the lysis percentage of target cells at the end of incubation.

[0345] In some embodiments, cytotoxic activity measurements are fitted using a mathematical model to obtain a cytotoxic activity curve. Curve fitting may, in some cases, allow for the estimation or extrapolation of the behavior of an effector cell composition (e.g., a therapeutic cell composition), such as its cytotoxic activity. It is intended that any method known in the art for performing curve fitting may be used. In some embodiments, the curve is S-shaped. In some embodiments, a tuned ratio that yields the maximum half of the cytotoxic activity is determined based on the cytotoxic activity measured from each of several incubations. In some embodiments, the tuned ratio that yields the maximum half of the cytotoxic activity is estimated, extrapolated, or estimated from the cytotoxic activity curve. In some embodiments, the cytotoxic activity curve is normalized to the measured maximum cytotoxic activity. In some embodiments, the cytotoxic activity curve is normalized to the upper asymptote of the curve, and, where appropriate, to the range of values ​​of the upper asymptote.

[0346] In some embodiments, methods including assays as described herein may be performed in 2, 3, or more series to validate the measured values ​​of cytotoxic activity. In some cases where the assay is performed, for example, in 2, 3, or more series, the cytotoxic activity measured from each of the replicates is used to provide a descriptive statistical measure of cytotoxic activity. For example, in some cases, for each of the multiple ratio tests, the mean (e.g., averaging), median, standard deviation, and / or variance of each measure of cytotoxic activity are determined. In some embodiments, the mean of each measure of cytotoxic activity is determined. In some embodiments, the standard deviation of each measure of cytotoxic activity is determined. In some embodiments, the mean measure of cytotoxic activity is fitted using a mathematical model to generate or estimate a cytotoxic activity curve. In some embodiments, the curve is normalized to the mean maximum. In some embodiments, the curve is normalized to an upper asymptote, and, where appropriate, to the mean of the range of values ​​of the upper asymptote.

[0347] The scales described herein may be used with reference to reference standards, such as the reference standard described herein, for example, in Section ID-2-a.

[0348] D. Determination of Effect The methods provided herein make it possible to determine the potency of effector cell compositions (e.g., therapeutic cell compositions). It is intended that the assays described herein may be used to evaluate the potency of effector cell compositions (e.g., therapeutic cell compositions) produced by any other manufacturing process that allows cells of the manufactured therapeutic cell composition to be cultured, in an assay comprising a process such as that described herein (e.g., Section II) and a plurality of incubations, each incubation comprising culturing cells of different proportions of the therapeutic composition together with target cells capable of stimulating cytotoxic activity, e.g., cytolytic activity, in the therapeutic cell composition. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more effector cell compositions (e.g., therapeutic cell compositions) may be evaluated according to the methods provided herein.

[0349] The potency of an effector cell composition (e.g., a therapeutic cell composition) can be determined by taking measurements of cytotoxic activity at each of several ratios tested. In some embodiments, the measurements are composites determined by taking the average or median over two, three, or more replicates. In some embodiments, the standard deviation and / or variance of the measurements can be determined. In some embodiments, one or more measurements, including composite measurements of the cytotoxic activity of the effector cell composition (e.g., a therapeutic cell composition) depending on the target cells, can be used to determine the potency of the effector cell composition (e.g., a therapeutic cell composition). In some embodiments, the target cells can be any of those described in Section IB.

[0350] In some embodiments, multiple incubations at different ratios result in multiple measurements to which curve fitting methods can be applied. In some embodiments, the multiple measurements include composite measurements (e.g., mean or median). For example, cytotoxic activity measurements can be fitted to a curve, e.g., an S-shape, to allow for inference, extrapolation, or estimation of the behavior (e.g., sensitivity) of an effector cell composition (e.g., a therapeutic cell composition). In some embodiments, the curve fitted to the measurements can be used to estimate the behavior (e.g., sensitivity) of a therapeutic composition that was not directly examined during the assay. For example, using a curve, one can estimate the lower asymptote; minimum value; loss of detection of cytotoxic activity; specific percentages of maximum half (10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90%); maximum half value (e.g., 50% cytotoxic activity); ranges of 10%–90%, 20%–80%, 30%–70%, or 40%–60% of maximum cytotoxicity (i.e., maximum activity as described below); upper asymptote; and the ratio at which each of the maximum value and range occurs.

[0351] It is intended that the potency of an effector cell composition (e.g., a therapeutic cell composition) can be determined using any scale (ratio at maximum half, range, maximum, minimum, asymptote, and composite scales). In some embodiments, the potency is relative potency.

[0352] 1. Effect In some embodiments, the potency of an effector cell composition (e.g., a therapeutic cell composition) is defined as the ratio at which one or more cytotoxic activity measurements or a range occur. In some embodiments, one or more measurements or a range are composite measurements, e.g., mean or median, determined from replicated experiments. In some embodiments, the measurements and ratios are determined from a cytotoxic activity curve of the measured cytotoxic activity. In some embodiments, the measured cytotoxic activity is normalized to the maximum activity measured for the therapeutic composition. In some embodiments, the cytotoxic activity curve is normalized to the maximum cytotoxic activity measured for the effector cell composition (e.g., a therapeutic cell composition). In some embodiments, the cytotoxic activity curve is normalized to the upper asymptote of the cytotoxic activity measured for the effector cell composition (e.g., a therapeutic cell composition), and optionally, to the mean of the values ​​measured over the asymptote.

[0353] In some embodiments, the potency of an effector cell composition (e.g., a therapeutic cell composition) is in the range of a ratio that results in 10% to 90% cytotoxic activity, or vice versa. In some embodiments, the range of a ratio that results in 10% to 90% cytotoxic activity is estimated from a cytotoxic activity curve. In some embodiments, for example, when the cytotoxic activity scale or cytotoxic activity curve is normalized, the range of cytotoxic activity values ​​is 0.1 to 0.9 or 10% to 90%.

[0354] In some embodiments, the potency of an effector cell composition (e.g., a therapeutic cell composition) is in the range of a ratio that results in 20% to 80% cytotoxic activity, or vice versa. In some embodiments, the range of a ratio that results in 20% to 80% cytotoxic activity is estimated from a cytotoxic activity curve. In some embodiments, for example, when the cytotoxic activity scale or cytotoxic activity curve is normalized, the range of cytotoxic activity values ​​is 0.2 to 0.8 or 20% to 80%.

[0355] In some embodiments, the potency of an effector cell composition (e.g., a therapeutic cell composition) is in the range of a ratio that produces 30% to 70% cytotoxic activity, or vice versa. In some embodiments, the range of a ratio that produces 30% to 70% cytotoxic activity is estimated from a cytotoxic activity curve. In some embodiments, for example, when the cytotoxic activity scale or cytotoxic activity curve is normalized, the range of cytotoxic activity values ​​is 0.3 to 0.7 or 30% to 70%.

[0356] In some embodiments, the potency of an effector cell composition (e.g., a therapeutic cell composition) is in the range of a ratio that results in 40% to 60% cytotoxic activity, or vice versa. In some embodiments, the range of a ratio that results in 40% to 60% cytotoxic activity is estimated from a cytotoxic activity curve. In some embodiments, for example, when the cytotoxic activity scale or cytotoxic activity curve is normalized, the range of cytotoxic activity values ​​is 0.4 to 0.6 or 40% to 60%.

[0357] In some embodiments, the potency of an effector cell composition (e.g., a therapeutic cell composition) is a range of ratios that produce a maximum half dose of cytotoxic activity. In some embodiments, the maximum half dose value and ratio that produce a maximum half dose value are estimated from a cytotoxic activity curve. In some embodiments, for example, the maximum half dose of cytotoxic activity is 0.5 or 50% when the cytotoxic activity scale or cytotoxic activity curve is normalized.

[0358] In some embodiments, for example, when the cytotoxic activity curve is fitted by an S-shape, the linear portion of the curve is determined. In some embodiments, the potency is the measured value and the corresponding ratio from the linear portion of the curve. In some embodiments, the maximum half-value measured value and the ratio are determined from the linear portion of the curve.

[0359] 2. Relative effect The methods provided herein enable the determination of the potency of effector cell compositions (e.g., therapeutic cell compositions) relative to different effector cell compositions (e.g., therapeutic cell compositions), for example, a reference standard. This type of potency is sometimes referred to as relative potency. For example, effector cell compositions (e.g., therapeutic cell compositions) evaluated according to the methods provided herein can be compared to different effector cell compositions (e.g., therapeutic cell compositions) evaluated according to the methods provided herein (e.g., a reference standard, e.g., as described below) to determine how the potencies of the effector cell compositions (e.g., therapeutic cell compositions) relate to each other. This provides the advantage that multiple effector cell compositions (e.g., therapeutic cell compositions) can be compared to determine which composition has the highest or optimal potency. In some embodiments, optimal potency is the potency that can induce a therapeutic effect in the subject, e.g., a sustained response, progression-free survival. In some embodiments, optimal potency is the potency that does not result in toxicity in the subject. In some embodiments, optimal efficacy is the efficacy that can induce a therapeutic effect in the subject, such as a sustained response or progression-free survival, without causing toxicity.

[0360] In some embodime...

Claims

1. A method for determining the efficacy of an effector cell composition, (a) Performing multiple incubations, each of which involves culturing cells of an effector cell composition together with target cells expressing a cell surface target antigen and a reporter molecule stably expressed by the target cells, The cells in the cultured effector cell composition include cells engineered to express recombinant receptors that specifically bind to the target antigen and cells that do not express recombinant receptors. Each of the multiple incubations is performed with different adjusted ratios of cells from the effector cell composition to the target cells, and each of the different adjusted ratios is based on cells expressing the recombinant receptor of the effector cell composition. (b) Measuring the cytotoxic activity from each of several incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity, and (c) Determine a modified ratio that yields up to half the cytotoxic activity of the effector cell composition, based on the cytotoxic activity measured for each of the multiple incubations. Methods that include...

2. The method according to claim 1, further comprising determining the relative potency of an effector cell composition by comparing a modified ratio that yields up to half the cytotoxic activity of the effector cell composition to a modified ratio that yields up to half the cytotoxic activity of a reference standard.

3. A method for determining the efficacy of an effector cell composition, (a) Performing multiple incubations, each of which involves culturing cells of an effector cell composition together with target cells expressing a cell surface target antigen and a reporter molecule stably expressed by the target cells, The cells in the cultured effector cell composition include cells engineered to express recombinant receptors that specifically bind to the target antigen and cells that do not express recombinant receptors. Each of the multiple incubations is performed with different adjusted ratios of cells from the effector cell composition to the target cells, and each of the different adjusted ratios is based on cells expressing the recombinant receptor of the effector cell composition. (b) Measuring the cytotoxic activity from each of several incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity, and (c) Determining the relative potency of the effector cell composition by comparing the cytotoxic activity of the effector cell composition to the cytotoxic activity of the reference standard at up to half the amount of the effector cell composition, based on the cytotoxic activity measured for each of multiple incubations. Methods that include...

4. The method according to any one of claims 1 to 3, wherein multiple incubations include culturing cells expressing a certain number of recombinant receptors of an effector composition together with different numbers of target cells.

5. The method according to claim 4, wherein different numbers of target cells are obtained by serially diluting the target cells.

6. The method according to claim 5, wherein the serial dilution is by a change in magnification between 2 to 8, 2 to 6, or 2 to 4, or between approximately 2 to approximately 8, approximately 2 to approximately 6, or approximately 2 to approximately 4, including the respective boundaries.

7. The method according to any one of claims 1 to 3, wherein multiple incubations include culturing a certain number of target cells together with cells expressing different numbers of recombinant receptors of the effector composition.

8. The method according to claim 7, wherein cells expressing different numbers of recombinant receptors of the effector composition are obtained by serially diluting the cells of the effector cell composition.

9. The method according to claim 8, wherein the serial dilution is by a change in magnification between 2 to 8, 2 to 6, or 2 to 4, or between approximately 2 to approximately 8, approximately 2 to approximately 6, or approximately 2 to approximately 4, including the respective boundaries.

10. The method according to any one of claims 1 to 9, wherein in each of the multiple incubations, between 1,000 to 80,000 target cells, 5,000 to 15,000 target cells, 8,000 to 12,000 target cells or between 9,000 to 10,000 target cells, including their respective boundaries, or between approximately 1,000 to approximately 80,000 target cells, approximately 5,000 to approximately 15,000 target cells, approximately 8,000 to approximately 12,000 target cells or between approximately 9,000 to approximately 10,000 target cells, is incubated with cells of the effector cell composition to be cultured.

11. The target cells, prior to a plurality of incubations, each including a boundary, are from 0.1 × 10 ６ cells to 0.3 × 10 ６ cells / mL, from 0.15 × 10 ６ cells to 0.25 × 10 ６ cells / mL or from 0.18 × 10 ６ cells to 0.22 × 10 ６ cells / mL, or are at a concentration between about 0.1 × 10 ６ cells and about 0.3 × 10 ６ cells / mL, from about 0.15 × 10 ６ cells to about 0.25 × 10 ６ cells / mL or from about 0.18 × 10 ６ cells to about 0.22 × 10 ６ cells / mL, according to the method according to any one of claims 1 to 10.

12. The method according to any one of claims 1 to 11, wherein the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 120:1 to 1:1, 50:1 to 2:1, 18:1 to 6:1 and 9:1 to 3:1, or between approximately 120:1 to approximately 1:1, approximately 50:1 to approximately 2:1, approximately 18:1 to approximately 6:1 and approximately 9:1 to approximately 3:1, including the boundaries, respectively.

13. The method according to any one of claims 1 to 11, wherein the maximum adjusted ratio of cells expressing the recombinant receptor of the effector cell composition to target cells in multiple incubations is between 1:120 to 1:1, 1:50 to 1:2, 1:18 to 1:6 and 1:9 to 1:3, including the boundary, or between approximately 1:120 to approximately 1:1, approximately 1:50 to approximately 1:2, approximately 1:18 to approximately 1:6 and approximately 1:9 to approximately 1:

3.

14. A method for evaluating target cell preparations for use in efficacy assays, (a) Performing multiple incubations, each of which involves culturing cells of an effector cell composition together with cells of a test target cell preparation, wherein the cells of the test target cell preparation express cell surface target antigens and reporter molecules. The cells in the effector cell composition include cells that have been engineered to express recombinant receptors that specifically bind to the target antigen, and cells that do not express recombinant receptors. Each of the multiple incubations is performed with different adjusted ratios of cells from the effector cell composition to cells from the test target cell preparation, and each of the different adjusted ratios is based on cells expressing the recombinant receptor of the effector cell composition. (b) Measuring the cytotoxic activity from each of several incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity. (c) Based on the cytotoxic activity measured for each of the multiple incubations, determine the adjusted ratio with the test target cells that yields up to half the cytotoxic activity (adjusted ratio of EC50) of the effector cell composition, and (d) Compare the adjusted EC50 ratio to the adjusted EC50 ratio determined in a reference cytotoxicity assay performed using the reference target cell line and the same effector cells. Methods that include...

15. The adjusted ratio of EC50 in the reference cytotoxic assay is (a) Performing multiple incubations, each of which involves culturing cells of an effector cell composition together with cells of a reference target cell line, wherein the cells of the reference cell preparation express cell surface target antigens and reporter molecules. Each of the multiple incubations is performed with different adjusted ratios of cells from the effector cell composition to cells from the reference target cell line, and each of the different adjusted ratios is based on cells expressing the recombinant receptor of the effector cell composition. (b) Measuring the cytotoxic activity from each of several incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity, and (c) Determining an adjusted ratio of the effector cell composition to a reference target cell line that yields an EC50, based on the cytotoxic activity measured for each of the multiple incubations. The method according to claim 14, as determined by...

16. A method for evaluating target cell preparations for use in efficacy assays, (a) Performing multiple incubations, each of which involves culturing cells of an effector cell composition together with cells of a test target cell preparation, wherein the cells of the test target cell preparation express cell surface target antigens and reporter molecules. The cells in the effector cell composition include cells that have been engineered to express recombinant receptors that specifically bind to the target antigen, and cells that do not express recombinant receptors. Each of the multiple incubations is performed with different adjusted ratios of cells from the effector cell composition to cells from the test target cell preparation, and each of the different adjusted ratios is based on cells expressing the recombinant receptor of the effector cell composition. (b) Measuring the cytotoxic activity from each of several incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity. (c) Determining the relative potency of the effector cell composition against test target cells by comparing the cytotoxic activity of up to half of the effector cell composition against test target cells (adjusted ratio of EC50) with a first reference EC50, based on the cytotoxic activity measured for each of multiple incubations, and (d) Compare the relative potency of the effector cell composition against the test target cells with the relative potency of the control sample. Methods that include...

17. (a) Performing multiple incubations, each of which involves culturing cells of an effector cell composition together with cells of a control target cell preparation expressing a cell surface target antigen and a reporter molecule, Each of the multiple incubations is performed with different adjusted ratios of cells from the effector cell composition to cells from the control target cell preparation, and each of the different adjusted ratios is based on cells expressing the recombinant receptor of the effector cell composition. (b) Measuring the cytotoxic activity from each of several incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity, and (c) Determining the relative potency of the control sample by comparing the EC50 of the control sample to the EC50 of a second reference standard based on the cytotoxic activity measured for each of the multiple incubations. The method according to claim 16, further comprising determining the relative potency of a control sample by means of the method.

18. The method according to any one of claims 14 to 17, further comprising evaluating the cells of a test target cell preparation for nonspecific stimulation of effector cells, wherein cells from a negative target cell preparation are evaluated for stimulating cytolytic activity from effector cells, and the cells of the negative test cell preparation are identical to the cells of the test target cell preparation, except that the cells of the negative test cell preparation do not express the target antigen.

19. This further includes evaluating the cells of the target cell preparation for nonspecific stimulation of effector cells, as appropriate. (a) Performing a second plurality of incubations, each of which comprises culturing cells of an effector cell composition together with cells of a negative target cell preparation that stably expresses a reporter molecule, The cells in the negative target cell preparation are identical to those in the test target cell preparation, except that they do not express the target antigen. Each of the second multiple incubations is carried out in different adjusted ratios of cells from the effector cell composition to cells from the negative target cell preparation, and each of the different adjusted ratios is based on cells expressing the recombinant receptor of the effector cell composition. (b) Measuring the cytotoxic activity from each of the second of a plurality of incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity, and (c) Based on the cytotoxic activity measured for each of the second multiple incubations, determine the adjusted ratio of the negative target cell preparation to the cells that yields the EC50 of the effector cell composition. The method according to any one of claims 14 to 18, including the method described above.

20. The method according to any one of claims 14 to 19, further comprising evaluating the cells of a test target cell preparation for unwanted lymphocyte stimulation, wherein the cells from the test target cell preparation are evaluated for stimulating cytolytic activity from the cells of a lymphocyte cell composition, and the cells of the lymphocyte cell composition are identical to effector cells, except that the cells of the lymphocyte cell composition do not express recombinant receptors.

21. This further includes evaluating the cells of the test target cell preparation against unwanted lymphocyte stimulation, as appropriate. (a) Performing a third incubation, each of which comprises culturing cells of a lymphocyte cell composition together with cells of a test target cell preparation expressing a cell surface target antigen and a reporter molecule, The cells in the cultured lymphocyte cell composition include cells that do not express receptors that specifically bind to the target antigen. Each of the third incubations is carried out in different adjusted ratios of cells from the lymphocyte cell composition to cells from the test target cell preparation. (b) Measuring the cytotoxic activity from each of the third of a plurality of incubations based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with the cytotoxic activity, and (c) Based on the cytotoxic activity measured for each of the third incubations, determine the adjusted ratio of the test target cell preparation to the cells that yields the EC50 of the lymphocyte cell composition. The method according to any one of claims 14 to 20, including the method described above.

22. The method according to any one of claims 14 to 21, further comprising determining the growth rate of cells in a test target cell preparation.

23. The method according to any one of claims 14 to 22, further comprising determining the adhesion of a test target cell preparation to a container in which cells are growing.

24. The method according to any one of claims 14 to 23, further comprising determining whether the cells of the test target cell preparation are capable of stably expressing the reporter molecule before multiple incubations.

25. The method according to any one of claims 14 to 24, further comprising determining whether the cells of the test target cell preparation express an additional reporter molecule before multiple incubations.

26. The method according to any one of claims 1 to 25, wherein the effector cell composition is a therapeutic cell composition.

27. The method according to any one of claims 14 to 26, wherein the effector cell composition is a T cell composition.

28. The method according to any one of claims 14 to 27, wherein the cells of the effector cell composition are T cells.

29. The method according to any one of claims 14 to 28, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

30. The method according to any one of claims 14 to 29, wherein the test target cell preparation is a target cell line that has been cryopreserved, passaged, manipulated to express a target cell antigen, and / or manipulated to express a reporter molecule.

31. The method according to any one of claims 1 to 30, wherein the test target cell preparation is a target cell line cryopreserved in DMSO, and the DMSO is optionally in a concentration of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30%, or about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30%.

32. The method according to any one of claims 1 to 31, wherein the test target cell preparation is a subcultured target cell line, and the target cell line has been subcultured 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, or 50 times, as appropriate.

33. The method according to any one of claims 14 to 32, wherein multiple incubations include culturing a certain number of cells of an effector cell composition together with a different number of cells of a test target cell preparation.

34. The method according to claim 33, wherein different numbers of cells of the test target cell preparation are obtained by serially diluting the cells of the test target cell preparation.

35. The method according to claim 34, wherein the serial dilution is by a change in magnification between 2 to 8, 2 to 6, or 2 to 4, or between approximately 2 to approximately 8, approximately 2 to approximately 6, or approximately 2 to approximately 4, including the respective boundaries.

36. The method according to any one of claims 14 to 32, wherein multiple incubations include culturing a certain number of cells of a target cell preparation together with a different number of cells of an effector cell composition.

37. The method according to claim 36, wherein different numbers of cells of the effector cell composition are obtained by serially diluting the cells of the effector cell composition.

38. The method according to claim 37, wherein the serial dilution is by a change in magnification between 2 to 8, 2 to 6, or 2 to 4, or between approximately 2 to approximately 8, approximately 2 to approximately 6, or approximately 2 to approximately 4, including the respective boundaries.

39. The method according to any one of claims 14 to 38, wherein in each of the multiple incubations, between 1,000 to 80,000 viability target cells, 5,000 to 15,000 viability target cells, 8,000 to 12,000 viability target cells or 9,000 to 10,000 viability target cells, including their respective boundaries, or between approximately 1,000 to approximately 80,000 viability target cells, approximately 5,000 to approximately 15,000 viability target cells, approximately 8,000 to approximately 12,000 viability target cells or approximately 9,000 to approximately 10,000 viability target cells, is incubated with cells of the effector cell composition to be cultured.

40. The cells in the test target cell preparation were 0.1 × 10⁶ before each incubation, including the boundaries. ６ From one 0.3 × 10 ６ Test target cells / mL, 0.15 × 10⁶ ６ From 0.25 × 10 ６ Individual test target cells / mL or 0.18 × 10⁶ cells ６ From 0.22 × 10 ６ Between individual test target cells / mL or approximately 0.1 × 10⁶ ６ Approximately 0.3 x 10 from each ６ Target cells per mL, approximately 0.15 × 10⁶ ６ Approximately 0.25 x 10 from each individual ６ Individual target cells / mL or approximately 0.18 × 10⁶ ６ Approximately 0.22 x 10 from each ６ The method according to any one of claims 14 to 39, wherein the concentration is between one test target cell / mL.

41. The method according to any one of claims 14 to 40, wherein the maximum adjusted ratio of cells in the effector cell composition to target cells in multiple incubations is between 120:1 to 1:1, 50:1 to 2:1, 18:1 to 6:1 and 9:1 to 3:1, or between approximately 120:1 to approximately 1:1, approximately 50:1 to approximately 2:1, approximately 18:1 to approximately 6:1 and approximately 9:1 to approximately 3:1, respectively, including the boundaries.

42. The method according to any one of claims 14 to 41, wherein the maximum adjusted ratio of cells in the effector cell composition to cells in the test target cell preparation for multiple incubations is between 1:120 to 1:1, 1:50 to 1:2, 1:18 to 1:6 and 1:9 to 1:3, including the boundaries, respectively, or between approximately 1:120 to approximately 1:1, approximately 1:50 to approximately 1:2, approximately 1:18 to approximately 1:6 and approximately 1:9 to approximately 1:

3.

43. The method according to any one of claims 1 to 42, wherein multiple incubations are carried out in at least 3, 4, 5, 6, 7, 8, 9, or 10 different adjusted ratios.

44. The method according to any one of claims 1 to 43, wherein each of the multiple incubations is carried out at different adjusted ratios, including the boundary, between 0.001 and 15 or between approximately 0.001 and approximately 15.

45. The method according to any one of claims 1 to 13 and 43 to 44, wherein each of a plurality of incubations comprises culturing cells of a mock cell composition together with cells of an effector cell composition and target cells, and the cultured cells of the mock cell composition do not express recombinant receptors or target antigens.

46. The method according to claim 45, wherein the mock cell composition is produced using a manufacturing process that is identical to the manufacturing process used to produce the effector cell composition, except that the cells of the mock cell composition are not exposed to nucleic acids encoding recombinant receptors.

47. The method according to any one of claims 1 to 13 and 43 to 46, wherein each of the multiple incubations comprises a different total number of effector composition cells and target cells.

48. The method according to any one of claims 45 to 47, wherein each of the multiple incubations comprises the same total number of cells of the effector composition, target cells, and mock cell composition.

49. The method according to any one of claims 45 to 48, wherein each of the multiple incubations comprises a different total number of cells of the effector composition, target cells, and mock cell composition.

50. The method according to any one of claims 1 to 49, wherein each of the multiple incubations is carried out at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times with respect to the effector cell composition.

51. The method according to any one of claims 1 to 50, wherein each of the multiple incubations is carried out for a period of 24 to 72 hours, 24 to 48 hours, or 48 to 72 hours, or approximately 24 to approximately 72 hours, approximately 24 to approximately 48 hours, or approximately 48 to approximately 72 hours, including each boundary.

52. The method according to any one of claims 1 to 51, wherein each of the multiple incubations is carried out for less than 24 hours.

53. The method according to any one of claims 1 to 50 and 52, wherein each of the multiple incubations is carried out for 17 to 23 hours, 18 to 22 hours, or 19 to 21 hours, including each boundary, or for approximately 17 to approximately 23 hours, approximately 18 to approximately 22 hours, or approximately 19 to approximately 21 hours.

54. The method according to any one of claims 1 to 50 and 52, wherein each of the multiple incubations is carried out for at least four hours.

55. The method according to any one of claims 1 to 54, wherein each of the multiple incubations is carried out at a temperature between 30°C and 39°C or between approximately 30°C and approximately 39°C, including the boundary.

56. Each of the multiple incubations has a CO2 content between 2.5% and 7.5% or between approximately 2.5% and 7.5%. ２ The method according to any one of claims 1 to 55, implemented at a level.

57. The method according to any one of claims 1 to 13 and 43 to 56, wherein the target cells are cells that have been engineered to express a reporter molecule.

58. The method according to claim 57, wherein the operation is performed by transduction.

59. The method according to any one of claims 1 to 58, wherein the expression of the reporter molecule is under the control of a constituent promoter.

60. The method according to any one of claims 1 to 59, wherein the reporter molecule is a detectable protein.

61. The method according to any one of claims 1 to 60, wherein the reporter molecule is an enzyme.

62. The method according to claim 61, wherein the enzyme is luciferase, β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), alkaline phosphatase, or secretory embryonic alkaline phosphatase (SEAP).

63. The method according to claim 61 or claim 62, wherein the enzyme is luciferase.

64. The method according to claim 63, wherein the luciferase is firefly luciferase, click beetle luciferase, sea mushroom (Renilla) luciferase, Gaussia luciferase, Gaussia-Dura luciferase, Oplophorus luciferase, bacterial luciferase, sea firefly (Cypridina) luciferase, Polychthys luciferase, dinoflagellate luciferase, krill luciferase, or fungal luciferase.

65. The method according to claim 63 or claim 64, wherein the luciferase is firefly luciferase.

66. The method according to any one of claims 1 to 65, wherein the measurement of cytotoxic activity includes adding a detection reagent to each of a plurality of incubations.

67. The method according to claim 66, wherein the measurement of cytotoxic activity includes, before addition, equilibrating each of a plurality of incubations to room temperature for between 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, including the boundaries, or between approximately 10 to approximately 120 minutes, approximately 20 to approximately 90 minutes, or approximately 30 to approximately 60 minutes.

68. The method according to claim 66 or claim 67, wherein the measurement of cytotoxic activity includes, before addition, equilibrating the detection reagent to room temperature for 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, including the boundary, respectively.

69. The method according to any one of claims 66 to 68, wherein the reporter molecule is an enzyme and the detection reagent is a substrate of the enzyme.

70. The method according to any one of claims 66 to 69, wherein the detection reagent is a luciferase substrate.

71. The method according to any one of claims 66 to 70, wherein the detection reagent is luciferin or a luciferin analog.

72. The method according to any one of claims 66 to 71, wherein the detection reagent is a luciferin analog.

73. The method according to claim 72, wherein the luciferin analog is 5'-fluoroluciferin.

74. The method according to any one of claims 65 to 73, wherein the measurement of cytotoxic activity comprises incubating each of a plurality of incubations in the presence of a detection reagent, including each boundary, for a period of 5 to 240 minutes, 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, or for a period of approximately 5 to 240 minutes, approximately 10 to 120 minutes, approximately 20 to 90 minutes, or approximately 30 to 60 minutes.

75. The method according to any one of claims 1 to 74, wherein the cytotoxic activity of each of the multiple incubations is measured by luminescence imaging.

76. The method according to any one of claims 1 to 60, wherein the reporter molecule is a fluorescent protein, and the cytotoxic activity of each of the multiple incubations is measured by fluorescence imaging.

77. The method according to any one of claims 1 to 76, wherein the cytotoxic activity is cytolytic activity.

78. The method according to any one of claims 1 to 77, wherein cytotoxic activity is measured after the termination of each of the multiple incubations.

79. The method according to any one of claims 1 to 78, wherein cytotoxic activity is measured over time during each of a plurality of incubations.

80. The method according to any one of claims 1 to 13 and 43 to 79, wherein the measured cytotoxic activity of each of the multiple incubations is determined based on the lysis percentage of target cells.

81. The method according to any one of claims 1 to 80, wherein the adjusted ratio that yields up to half the cytotoxic activity of the effector cell composition is the EC50 of the effector cell composition.

82. The method according to any one of claims 2 to 13 and 43 to 81, wherein the adjusted ratio that yields up to half the amount of cytotoxic activity of the reference standard is the EC50 of the reference standard.

83. The method according to any one of claims 2 to 13 and 43 to 82, wherein the reference standard is an effector cell composition having a verified adjusted ratio that yields up to half the amount of cytotoxic activity, a commercially available effector cell composition, an effector cell composition produced using a manufacturing process identical to the manufacturing process used to produce the effector cell composition, an effector cell composition produced using a manufacturing process different from the manufacturing process used to produce the effector cell composition, an effector cell composition comprising cells expressing the same recombinant receptor as the effector cell composition, or an effector cell composition comprising cells expressing a different recombinant receptor than the effector cell composition.

84. The method according to any one of claims 2 to 13 and 43 to 83, wherein the effector cell composition is produced using a manufacturing process in which the reference standard is identical to the manufacturing process used to produce the effector cell composition.

85. The method according to any one of claims 2 to 13 and 43 to 84, wherein the reference standard is an effector cell composition having a verified and adjusted ratio that yields up to half the amount of cytotoxic activity.

86. The method according to any one of claims 2 to 13 and 43 to 85, wherein the reference standard includes primary cells obtained from the subject.

87. The method according to claim 86, wherein the subject is a healthy subject.

88. The method according to claim 86, wherein the subject has a disease or condition.

89. The method according to claim 88, wherein the disease or condition is cancer.

90. The method according to any one of claims 1 to 13 and 43 to 89, wherein the target cells are clones from a cell line or primary cells taken from a subject.

91. The method according to any one of claims 1 to 13 and 43 to 90, wherein the target cells are derived from a cell line.

92. The method according to claim 91, wherein the cell line is a tumor cell line.

93. The method according to any one of claims 1 to 13 and 43 to 92, wherein the target cells are cells that have been manipulated to express a target antigen.

94. The method according to claim 93, wherein the operation is performed by transduction.

95. The method according to any one of claims 1 to 13 and 43 to 94, wherein the target antigen is a first tumor antigen, and the target cells are cells that have been engineered not to express a second tumor antigen.

96. The method according to any one of claims 1 to 95, wherein the cytotoxic activity measured for each of the multiple incubations achieves a dose-response curve of cytotoxic activity of the effector cell composition, which includes a linear dose-response range.

97. The method according to claim 96, wherein the dose-response curve includes a lower asymptote and an upper asymptote of the cytotoxic activity of the effector cell composition.

98. The method according to any one of claims 1 to 97, wherein the effector cell composition comprises a single-cell subtype concentrated or purified from a biological sample.

99. The method according to any one of claims 1 to 97, wherein the effector cell composition comprises a population of mixed cell subtypes obtained by mixing cell subtypes concentrated or purified from a biological sample.

100. The method according to claim 98 or claim 99, wherein the biological sample comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product.

101. The method according to any one of claims 1 to 100, wherein the effector cell composition includes primary cells obtained from a subject.

102. The method according to claim 101, wherein the reference standard is an effector cell composition produced from cells obtained from the same subject.

103. The method according to claim 102, wherein the reference standard is an effector cell composition produced from cells obtained from different subjects.

104. The method according to any one of claims 101 to 103, wherein the subject has a disease or condition.

105. The method according to claim 104, wherein the disease or condition is cancer.

106. The method according to any one of claims 1 to 105, wherein the effector cell composition includes autologous cells obtained from a subject to be treated.

107. The method according to any one of claims 101 to 103, wherein the subject is a healthy subject.

108. The method according to any one of claims 1 to 103 and 107, wherein the effector cell composition includes allogeneic cells.

109. The method according to any one of claims 1 to 108, wherein the effector cell composition comprises CD3+ T cells.

110. The method according to any one of claims 1 to 109, wherein the effector cell composition comprises CD4+ T cells and CD8+ T cells.

111. The method according to any one of claims 1 to 110, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

112. The method according to any one of claims 1 to 111, wherein multiple incubations are carried out in a flask, tube, or multiwell plate.

113. The method according to any one of claims 1 to 112, wherein each of the multiple incubations is carried out individually in the wells of a multiwell plate.

114. The method according to claim 113, wherein the multiwell plate is a 96-well plate, a 48-well plate, a 12-well plate, or a 6-well plate.

115. The method according to any one of claims 1 to 114, further comprising selecting an effector composition for administration to a subject requiring it, based on an adjusted ratio that yields up to half the amount of cytotoxic activity of the effector cell composition.

116. The method according to any one of claims 2 to 13 and 43 to 115, further comprising selecting an effector composition to be administered to a subject requiring it, based on relative potency.

117. The method according to any one of claims 1 to 13 and 43 to 116, further comprising determining a dose of cells of the effector cell composition to be administered to a subject requiring it, based on a modified ratio that yields up to half the amount of cytotoxic activity of the effector cell composition.

118. The method according to any one of claims 2 to 13 and 43 to 117, further comprising determining a cellular dose of an effector composition to be administered to a subject requiring it, based on relative potency.

119. The method according to any one of claims 115 to 118, wherein the subject has a disease or condition.

120. The method according to claim 119, wherein the disease or condition is cancer.

121. The method according to any one of claims 1 to 120, wherein each of the multiple incubations is carried out in a serum-free medium.

122. A method for evaluating the efficacy of an effector cell composition, (a) Incubating cells of an effector cell composition together with (i) target cells expressing a cell surface target antigen and a reporter molecule stably expressed by the target cells, and (ii) cells of a mock cell composition, The cells in the cultured effector cell composition include cells engineered to express recombinant receptors that specifically bind to the target antigen and cells that do not express recombinant receptors. The cells of the cultured mock cell composition do not express recombinant receptors or target antigens, and are incubated, and (b) Measuring cytotoxic activity from incubation based on the expression or activity of a reporter molecule, wherein the expression or activity of the reporter molecule is inversely correlated with cytotoxic activity. Methods that include...

123. The method according to claim 122, wherein, including each boundary, between 1,000 to 80,000 target cells, between 5,000 to 15,000 target cells, between 8,000 to 12,000 target cells or between 9,000 to 10,000 target cells, or between approximately 1,000 to approximately 80,000 target cells, between approximately 5,000 to approximately 15,000 target cells, between approximately 8,000 to approximately 12,000 target cells or between approximately 9,000 to approximately 10,000 target cells, is incubated with the cells of the effector cell composition to be cultured.

124. The target cells, including their boundaries, were 0.1 × 10 before incubation. ６ From one 0.3 × 10 ６ Target cells per mL, 0.15 × 10⁶ ６ From 0.25 × 10 ６ Target cells per mL or 0.18 × 10⁶ ６ From 0.22 × 10 ６ Between individual target cells / mL or approximately 0.1 × 10⁶ ６ Approximately 0.3 x 10 from each ６ Target cells per mL, approximately 0.15 × 10⁶ ６ Approximately 0.25 x 10 from each individual ６ Target cells per mL or approximately 0.18 × 10⁶ ６ Approximately 0.22 x 10 from each ６ The method according to claim 122 or claim 123, wherein the concentration is between 1 target cells / mL.

125. The method according to any one of claims 122 to 124, wherein the mock cell composition is produced using a manufacturing process that is identical to the manufacturing process used to produce the effector cell composition, except that the cells of the mock cell composition are not exposed to nucleic acids encoding recombinant receptors.

126. The method according to any one of claims 122 to 125, wherein incubation is performed at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times with respect to the effector cell composition.

127. The method according to any one of claims 122 to 126, wherein the incubation is carried out for 24 to 72 hours, 24 to 48 hours, or 48 to 72 hours, or approximately 24 to approximately 72 hours, approximately 24 to approximately 48 hours, or approximately 48 to approximately 72 hours, including each boundary.

128. The method according to any one of claims 122 to 126, wherein incubation is carried out for less than 24 hours.

129. The method according to any one of claims 122 to 126 and 128, wherein the incubation is carried out for 17 to 23 hours, 18 to 22 hours, or 19 to 21 hours, including each boundary, or for approximately 17 to approximately 23 hours, approximately 18 to approximately 22 hours, or approximately 19 to approximately 21 hours.

130. The method according to any one of claims 122 to 129, wherein the incubation is carried out at a temperature between 30°C and 39°C or between approximately 30°C and approximately 39°C, including the boundary.

131. Incubation is between 2.5% and 7.5% or between approximately 2.5% and 7.5% CO2 ２ The method according to any one of claims 122 to 130, which is carried out at a level.

132. The method according to any one of claims 122 to 131, wherein the target cells are cells that have been engineered to express a reporter molecule.

133. The method according to claim 132, wherein the operation is performed by transduction.

134. The method according to any one of claims 122 to 133, wherein the expression of the reporter molecule by target cells is under the control of a constituent promoter.

135. The method according to any one of claims 122 to 134, wherein the reporter molecule is a detectable protein.

136. The method according to any one of claims 122 to 135, wherein the reporter molecule is an enzyme.

137. The method according to claim 136, wherein the enzyme is luciferase, β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), alkaline phosphatase, or secretory embryonic alkaline phosphatase (SEAP).

138. The method according to claim 136 or claim 137, wherein the enzyme is luciferase.

139. The method according to claim 138, wherein the luciferase is firefly luciferase, click beetle luciferase, sea oat luciferase, Gausia luciferase, Gausia-dural luciferase, Oprophorus luciferase, bacterial luciferase, sea firefly luciferase, Polychthys luciferase, dinoflagellate luciferase, krill luciferase, or fungal luciferase.

140. The method according to claim 138 or claim 139, wherein the luciferase is firefly luciferase.

141. The method according to any one of claims 122 to 140, wherein the measurement of cytotoxic activity includes adding a detection reagent to the incubation.

142. The method according to claim 141, wherein the measurement of cytotoxic activity includes equilibrating the incubation to room temperature for 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, including the boundary, before addition, or for about 10 to about 120 minutes, about 20 to about 90 minutes, or about 30 to about 60 minutes.

143. The method according to claim 141 or claim 142, wherein the measurement of cytotoxic activity includes equilibrating the detection reagent to room temperature for 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, including the boundary, before addition, or for about 10 to about 120 minutes, about 20 to about 90 minutes, or about 30 to about 60 minutes.

144. The method according to any one of claims 141 to 143, wherein the reporter molecule is an enzyme and the detection reagent is a substrate of the enzyme.

145. The method according to any one of claims 141 to 144, wherein the detection reagent is a luciferase substrate.

146. The method according to any one of claims 141 to 145, wherein the detection reagent is luciferin or a luciferin analog.

147. The method according to any one of claims 141 to 146, wherein the detection reagent is a luciferin analog.

148. The method according to claim 147, wherein the luciferin analog is 5'-fluoroluciferin.

149. The method according to any one of claims 141 to 148, wherein the measurement of cytotoxic activity includes incubating in the presence of a detection reagent for between 5 to 240 minutes, 10 to 120 minutes, 20 to 90 minutes, or 30 to 60 minutes, including the boundaries, or between approximately 5 to approximately 240 minutes, approximately 10 to approximately 120 minutes, approximately 20 to approximately 90 minutes, or approximately 30 to approximately 60 minutes.

150. The method according to any one of claims 122 to 149, wherein the cytotoxic activity of the incubation is measured by luminescence imaging.

151. The method according to any one of claims 122 to 135, wherein the reporter molecule is a fluorescent protein, and the cytotoxic activity of the incubation is measured by fluorescence imaging.

152. The method according to any one of claims 122 to 151, wherein the cytotoxic activity is cytolytic activity.

153. The method according to any one of claims 122 to 152, wherein cytotoxic activity is measured after termination of incubation.

154. The method according to any one of claims 122 to 152, wherein cytotoxic activity is measured over time during incubation.

155. The method according to any one of claims 122 to 154, wherein the cytotoxic activity measured for incubation is determined based on the lysis percentage of target cells.

156. The method according to any one of claims 122 to 155, wherein the target cells are clones from a cell line or primary cells taken from a subject.

157. The method according to any one of claims 122 to 156, wherein the target cells are derived from a cell line.

158. The method according to claim 157, wherein the cell line is a tumor cell line.

159. The method according to any one of claims 122 to 158, wherein the target cells are cells that have been manipulated to express a target antigen.

160. The method according to claim 159, wherein the operation is performed by transduction.

161. The method according to any one of claims 122 to 160, wherein the target antigen is a first tumor antigen, and the target cells are cells that have been modified not to express a second tumor antigen.

162. The method according to any one of claims 122 to 161, wherein the effector cell composition comprises a single-cell subtype concentrated or purified from a biological sample.

163. The method according to any one of claims 122 to 161, wherein the effector cell composition comprises a population of mixed cell subtypes obtained by mixing cell subtypes concentrated or purified from a biological sample.

164. The method according to claim 162 or claim 163, wherein the biological sample comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product.

165. The method according to any one of claims 122 to 164, wherein the effector cell composition includes primary cells obtained from a subject.

166. The method according to claim 165, wherein the subject has a disease or condition.

167. The method according to claim 166, wherein the disease or condition is cancer.

168. The method according to any one of claims 122 to 167, wherein the effector cell composition includes autologous cells obtained from a subject to be treated.

169. The method according to any one of claims 122 to 165, wherein the subject is a healthy subject.

170. The method according to any one of claims 1 to 165 and 169, wherein the effector cell composition includes allogeneic cells.

171. The method according to any one of claims 122 to 170, wherein the effector cell composition comprises CD3+ T cells.

172. The method according to any one of claims 122 to 171, wherein the effector cell composition comprises CD4+ T cells and CD8+ T cells.

173. The method according to any one of claims 122 to 172, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

174. The method according to any one of claims 122 to 173, wherein the incubation is carried out in a flask, tube or multiwell plate.

175. The method according to any one of claims 122 to 174, wherein the incubation is carried out in a multiwell plate.

176. The method according to claim 175, wherein the multiwell plate is a 96-well plate, a 48-well plate, a 12-well plate, or a 6-well plate.

177. The method according to any one of claims 122 to 176, further comprising selecting an effector cell composition for administration to a target requiring it, based on the cytotoxic activity of the effector cell composition.

178. The method according to any one of claims 122 to 177, further comprising determining a dose of cells of the effector cell composition to be administered to a subject requiring it, based on the cytotoxic activity of the effector cell composition.

179. The method according to claim 177 or claim 178, wherein the subject has a disease or condition.

180. The method according to claim 179, wherein the disease or condition is cancer.

181. The method according to any one of claims 1 to 13 and 43 to 180, wherein the target cells are ready-to-use (RTU) cells.

182. The method according to any one of claims 14 to 42, wherein the cells of the test target cell preparation are ready-to-use (RTU) cells.

183. The method according to claims 181 and 182, wherein the RTU cells are cells that are cryopreserved and used immediately after thawing.

184. The method according to any one of claims 120 to 181, wherein the effector cell composition is a therapeutic cell composition.

185. The method according to any one of claims 122 to 180, wherein the incubation in step (a) is carried out in a serum-free medium.

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