WO2025097034A1 - Methods for manufacture of engineered t cells from whole blood samples - Google Patents

Abstract

Disclosed herein are methods for the manufacture of engineered T cells that express a heterologous protein, such as a chimeric antigen receptor (CAR), in which T cells are isolated from whole blood samples.

Description

METHODS FOR MANUFACTURE OF ENGINEERED T CELLS FROM WHOLE

BLOOD SAMPLES

TECHNICAL FIELD

[0001] The present disclosure generally relates to methods and compositions for the manufacture of engineered T cells that express a heterologous protein, such as a chimeric antigen receptor (CAR).

INCORPORATION BY REFERENCE

[0002] This patent application incorporates by reference the material included in the amino acid and/or nucleotide sequence listing prepared in compliance with the ST.26 Standard in the .xml file named “KYVA-OOl-OlWO-Sequence-Listing.xml”, prepared on November 1, 2024 and filed herewith.

BACKGROUND

[0003] Chimeric antigen receptor (CAR) T cell therapy has transformative potential in treating many life-altering diseases and conditions, e.g., cancer and immune disorders. As an immunotherapy, CAR T cell treatments have shown promise in their abilities to selectively target and nullify the underlying causes of a disease, while providing a durable response to prevent recurrence.

[0004] Despite its potential, several key challenges remain for the commercial viability of CAR T cells. Presently, manufacture of CAR T cells involves lengthy ex -vivo cell culture procedures that are costly and result in a high product variability. For example, prolonged ex vivo culture is associated with phenotypic changes (differentiation) that are poorly characterized and potentially detrimental to therapeutic efficacy, with these changes becoming more numerous the longer the manufacturing process extends.

[0005] The current methods for manufacturing CAR T cells, which include several steps including an extended ex vivo culture, are costly, both in terms of economics and patient outcomes. This expense begins at the very initial steps in which T cells are collected for engineering into CAR T cells. Primarily, in order to obtain a sufficient number of donor T cells for downstream manufacturing, methods for manufacturing CAR T cells rely on a leukapheresis step and/or peripheral blood mononuclear cell (PBMC) isolation process used on an apheresis sample to produce a product from which the T cells are isolated.

[0006] This initial requirement presents a major delay and expense in producing a CAR T cellular product. In itself, leukapheresis is a time-consuming process, which requires experienced technicians and specialized facilities. Beyond the general expense of the procedure, for many patients, scheduling a leukapheresis procedure is a challenge — patients with certain diseases may require T cell depleting therapies, which precludes obtaining a sufficient number of T cells. Thus, patients are forced to undergo a delay in receiving T cell depleting treatments or forego a CAR T cell therapy. Moreover, if possible, avoiding leukapheresis altogether eliminates the risks inherent with placement of a central venous catheter.

[0007] Eliminating the requirement for leukapheresis/PBMC isolation of the manufacturing process will improve the efficacy and outcomes of CAR T cell therapy.

[0008] Consequently, unless manufacturing processes are developed that reliably and economically produce T cells that are safe and effective, the potential of CAR T cell therapies will never translate into clinical application, as economic and logistical bottlenecks will hinder their development and delivery.

SUMMARY OF THE DISCLOSURE

[0009] Disclosed herein are methods for manufacturing engineered T cells that express a heterologous protein, such as a chimeric antigen receptor (CAR). Exemplary methods for manufacturing CAR T cells of the disclosure may include obtaining T cells from a subject (e.g., a donor or a patient) from whole blood. In some aspects, T cells are obtained from peripheral blood mononuclear cells (PBMC) isolated from the whole blood. In more preferred aspects, the present Inventors have discovered that surprisingly, sufficient numbers of T cells for CAR T cell manufacturing may be obtained directly from whole blood, without the need for a leukapheresis step or PBMC isolation step. This includes when used as the starting material in CAR T cell manufacturing processes of the disclosure that extend for between 6-9 days (generally about 8 days) and the rapid methods, which are able to produce engineered cells in around two-to-three days after receiving a whole blood sample containing donor T cells. [0010] Further, certain methods of the disclosure include a concurrent T cell isolation and activation step. Thus, in certain methods for manufacturing CAR T cells of the disclosure, T cells are isolated from whole blood or PBMCs and concurrently activated. Not only does the concurrent activation/i solation reduce the overall manufacturing time, it also is able to isolate a sufficient number of T cells, at a high purity, to manufacture the CAR T cells from a very small whole blood sample (e.g., samples of 100 mb, or less, of blood).

[0011] Certain exemplary methods for manufacturing CAR T cells of the disclosure are “rapid” processes, and produce CAR T cells significantly faster than other processes. These shortened processes of the disclosure, usually two-to-three days in total, employ a brief culture after T cell isolation/activation before transduction, generally less than or significantly less than a day, and a similarly brief cultivation after transduction before harvesting the desired CAR T cell product. Despite the small starting whole blood samples, the lack of a leukapheresis or other similar T cell isolating step, and the brief cultivation steps, these methods of the disclosure are able to produce a large harvest of CAR T cells — all within 2-3 days of receiving a starting sample. Surprisingly, these two-to-three-day manufacturing process produced a final CAR T cell product of a higher functional activity than other 6 to 9-day processes. CAR T cells manufactured using the methods of the disclosure have shown a superior T cell expansion in the final CAR T product.

[0012] Further, the cells made using the methods of the disclosure unexpectedly show a more favorable phenotype as a result of the condensed processes of the disclosure. The cells were far less differentiated than those using longer processes. When compared to longer processes, the cells produced using the shortened methods of the disclosure included higher levels of stem cell memory T cells (Tscm), which generally only account for 2-3% of circulating T cells and are associated with long-term defensive immunity, anti-tumor activity, and self-renewal. Further, these cells expressed the introduced CARs at a very high percentage, leading to a pure end product that showed targeted cytotoxicity with low T cell exhaustion. Even more surprising, the shorted manufacturing process produces a large number of polyfunctional cells, which simultaneously secrete multiple sets cytokines, chemokines, and/or cytotoxic granules simultaneously. Owing to this polyfunctional behavior, such cells are known to provide a more effective immune response, which is desirable in a therapeutic CAR T cell product. [0013] Similarly, certain methods of the disclosure that use a longer manufacture time (e.g., 6 to 9 days) and a whole blood starting sample, rather than an apheresis sample as used in prior methods, are still able to produce cells with a similar phenotype to those produced from methods using apheresis starting material. Thus, the presently disclosed methods are able to ease the bottlenecks in production associated with obtaining apheresis samples, while delivering an equivalent or better therapeutically effective cellular product.

[0014] Accordingly, in certain aspects, the present disclosure provides methods for producing a population of engineered T cells expressing a heterologous protein, e.g., a chimeric antigen receptor. An exemplary method may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample; after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.

[0015] In certain aspects, the present disclosure provides rapid methods for producing a population of engineered T cells expressing a heterologous protein. An exemplary rapid method may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample; between 10 hours and 25 hours after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum- free cultivation medium for a period of between 24 hours and 60 hours; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.

[0016] In preferred methods of the disclosure, the T cells are isolated from the whole blood sample directly without an intervening T cell isolation step and/or leukapheresis. In some methods of the disclosure, prior to the binding step, the method comprises isolating peripheral blood mononuclear cells (PBMC) comprising the T cells from the whole blood sample without a leukapheresis step.

[0017] In preferred methods of the disclosure, the contacting step occurs between 12 hours and 21 hours after the binding step. In preferred methods of the disclosure, the contacting step occurs between 15 hours and 19 hours after the binding step. In certain preferred aspects, the contacting step occurs between 17 hours and 19 hours after the binding step. In certain methods, the contacting step occurs about 18 hours after the binding step.

[0018] In certain methods of the disclosure, the cultivating step is for a period of between 29 hours and 59 hours. In preferred methods of the disclosure, the cultivating step is for a period of between 36 hours and 52 hours. In more preferred aspects, the cultivating step is for a period of between 46 hours and 50 hours. In certain aspects, the cultivating step is for a period of about 48 hours.

[0019] In certain aspects, the present disclosure provides methods for producing a population of engineered T cells expressing a heterologous protein. An exemplary method may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample; between 20 hours and 28 hours after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium for a period of between 4 days and 9 days; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.

[0020] In alternative methods of the disclosure, the contacting step occurs between about 20 and 28 hours after the binding step and the cultivating step is for a period of 4-9 days. In certain aspects, the contacting step occurs between about 22 and 26 hours after the binding step. In certain aspects, the contacting step occurs between about 23 and 25 hours after the binding step. In certain aspects, the contacting step occurs about 24 hours after the binding step. In certain aspects, the cultivating step is for a period of about 5 to about 7 days. In certain aspects, the cultivating step is for a period of about 6 days. In certain aspects, the cultivating step is for a period of about 8 days.

[0021] In exemplary methods of the disclosure, the harvested T cells comprise between about 10% and 60% CD45RO-/CCR7+ T cells (Tnscm). In more preferred methods, the harvested T cells comprise between about 15% and 60% CD45RO-/CCR7+ T cells (Tnscm). In certain aspects, the harvested T cells comprise at least 18% Tnscm. In certain aspects, the harvested T cells comprise at least 22% Tnscm. In certain aspects, the harvested T cells comprise at least 25% Tnscm.

[0022] The Tnscm may include naive T cells (Tn) and stem cell memory T cells (Tscm). In preferred aspects, the Tnscm comprise more Tscm than Tn. The Tnscm may comprise at least 1.5x more Tscm than Tn; at least twice as many Tscm as Tn; at least 3x more Tscm than Tn; at least 5x more Tscm than Tn; at least lOx more Tscm than Tn; and/or at least 50x more Tscm than Tn.

[0023] In certain methods of the disclosure, the one or more anti-CD3 antibodies and the one or more anti-CD28 antibodies attached to the same support. In certain methods, the one or more anti- CD3 antibodies and/or the one or more anti-CD28 antibodies are attached to the support via a cleavable linker. The linker may be, for example, an enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker. Accordingly, methods of the disclosure may further include contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface.

[0024] In certain aspects, the surface is a solid surface. Preferably, the solid surface is a bead, well, chip, or microfluidic channel. More preferably, the solid surface is a bead. In some aspects, the surface comprises a polymer. In certain aspects, the polymer is a hydrogel. In some methods, the surface comprises a polymer scaffold.

[0025] In preferred methods of the disclosure, the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg. In preferred aspects, at least 1% of the harvested T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MIP-la and MIP-lb. In certain methods, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNgand Granzyme B.

[0026] In preferred methods of the disclosure, the polyfunctional T cells and/or a portion of the population thereof, comprise two or more of: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

[0027] In some methods of the disclosure, the polyfunctional T cells and/or a portion of the population of polyfunctional T cells comprise: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

[0028] In preferred methods, the serum free cultivation medium comprises at least one cytokine. Preferably, the at least one cytokine comprises one or more of IL-2, IL-21, IL-7, and IL-15. In some methods, the at least one cytokine comprises one or more of IL-21, IL-7, and IL-15 and does not comprise IL-2.

[0029] In certain methods of the disclosure, the number of isolated T cells from the whole blood sample is between about IxlO⁶ and about IxlO⁸ total T cells and the number of harvested T cells is between about IxlO⁶ and about 5xl0⁸. In certain methods of the disclosure, the number of isolated T cells from the whole blood sample is between about 1.5xl0⁷ and about IxlO⁸ total T cells and the number of harvested T cells is between about 7.5xl0⁷ and about 1.5xl0⁸. In certain preferred methods, the number of isolated T cells from the whole blood sample is between about 5xl0⁷ and about 7.5xl0⁷ total T cells and the number of harvested T cells is between about 7.5xl0⁷ and about 1.2xl0⁸.

[0030] In certain aspects, the culturing step produces between a 1.0- and a 4-fold expansion of the harvested T cells. In certain aspects, the culturing step produces between a 1.2- and a 4-fold expansion of the harvested T cells.

[0031] The present disclosure also provides methods for producing a population of engineered T cells expressing a heterologous protein comprising the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample without an intervening PBMC isolation step or a leukapheresis step; contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.

[0032] In exemplary methods of the disclosure, the harvested T cells comprise between about 10% and 60% CD45RO-/CCR7+ T cells (Tnscm). In more preferred methods, the harvested T cells comprise between about 15% and 60% CD45RO-/CCR7+ T cells (Tnscm). In certain aspects, the harvested T cells comprise at least 18% Tnscm. In certain aspects, the harvested T cells comprise at least 22% Tnscm. In certain aspects, the harvested T cells comprise at least 25% Tnscm.

[0033] The Tnscm may include naive T cells (Tn) and stem cell memory T cells (Tscm). In preferred aspects, the Tnscm comprise more Tscm than Tn. The Tnscm may comprise at least 1.5x more Tscm than Tn; at least twice as many Tscm as Tn; at least 3x more Tscm than Tn; at least 5x more Tscm than Tn; at least lOx more Tscm than Tn; and/or at least 50x more Tscm than Tn.

[0034] In preferred methods of the disclosure, the contacting step occurs between 12 hours and 21 hours after the binding step. In preferred methods of the disclosure, the contacting step occurs between 15 hours and 19 hours after the binding step. In certain preferred aspects, the contacting step occurs between 17 hours and 19 hours after the binding step. In certain methods, the contacting step occurs about 18 hours after the binding step.

[0035] In certain methods of the disclosure, the cultivating step is for a period of between 29 hours and 59 hours. In preferred methods of the disclosure, the cultivating step is for a period of between 36 hours and 52 hours. In more preferred aspects, the cultivating step is for a period of between 46 hours and 50 hours. In certain aspects, the cultivating step is for a period of about 48 hours.

[0036] In certain methods of the disclosure, the one or more anti-CD3 antibodies and the one or more anti-CD28 antibodies attached to the same support. In certain methods, the one or more anti- CD3 antibodies and/or the one or more anti-CD28 antibodies are attached to the support via a cleavable linker. The linker may be, for example, an enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker. Accordingly, methods of the disclosure may further include contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface. [0037] In certain aspects, the surface is a solid surface. Preferably, the solid surface is ahead, well, chip, or microfluidic channel. More preferably, the solid surface is a bead. In some aspects, the surface comprises a polymer. In certain aspects, the polymer is a hydrogel. In some methods, the surface comprises a polymer scaffold.

[0038] In preferred methods of the disclosure, the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg. In preferred aspects, at least 1% of the harvested T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MIP-la and MIP-lb. In certain methods, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNg and Granzyme B.

[0039] In preferred methods of the disclosure, the polyfunctional T cells and/or a portion of the population thereof, comprise two or more of: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

[0040] In some methods of the disclosure, the polyfunctional T cells and/or a portion of the population of polyfunctional T cells comprise: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

[0041] In preferred methods, the serum free cultivation medium comprises at least one cytokine. Preferably, the at least one cytokine comprises one or more of IL-2, IL-21, IL-7, and IL-15. In some methods, the at least one cytokine comprises one or more of IL-21, IL-7, and IL-15 and does not comprise IL-2. In alternative methods, the serum free cultivation medium comprises no added cytokines.

[0042] In certain methods of the disclosure, the number of isolated T cells from the whole blood sample is between about lxl0⁶ and about IxlO⁸ total T cells and the number of harvested T cells is between about 2.5xl0⁷ and about 5xl0⁸. In certain methods of the disclosure, the number of isolated T cells from the whole blood sample is between about 1.5xl0⁷ and about IxlO⁸ total T cells and the number of harvested T cells is between about 7.5xl0⁷ and about 1.5xl0⁸. In certain preferred methods, the number of isolated T cells from the whole blood sample is between about 5xl0⁷ and about 7.5xl0⁷ total T cells and the number of harvested T cells is between about 7.5xl0⁷ and about 1.2xl0⁸.

[0043] In certain aspects, the culturing step produces between a 1.0- and a 4-fold expansion of the harvested T cells. In certain aspects, the culturing step produces between a 1.2- and a 4-fold expansion of the harvested T cells.

[0044] Alternative methods of the disclosure for producing a population of engineered T cells expressing a heterologous protein, may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD4 antibodies and one or more anti-CD8 antibodies attached to a support, thereby isolating the T cells from the whole blood sample; between 10 hours and 25 hours after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium for a period of between 24 hours and 60 hours; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.

[0045] In certain aspects, the method further includes a step of activating the isolated T cells.

[0046] In preferred methods of the disclosure, the T cells are isolated from the whole blood sample directly without an intervening T cell isolation step and/or leukapheresis. In some methods of the disclosure, prior to the binding step, the method comprises isolating peripheral blood mononuclear cells (PBMC) comprising the T cells from the whole blood sample without a leukapheresis step. [0047] In exemplary methods of the disclosure, the harvested T cells comprise between about 10% and 60% CD45RO-/CCR7+ T cells (Tnscm). In more preferred methods, the harvested T cells comprise between about 15% and 60% CD45RO-/CCR7+ T cells (Tnscm). In certain aspects, the harvested T cells comprise at least 18% Tnscm. In certain aspects, the harvested T cells comprise at least 22% Tnscm. In certain aspects, the harvested T cells comprise at least 25% Tnscm.

[0048] The Tnscm may include naive T cells (Tn) and stem cell memory T cells (Tscm). In preferred aspects, the Tnscm comprise more Tscm than Tn. The Tnscm may comprise at least 1.5x more Tscm than Tn; at least twice as many Tscm as Tn; at least 3x more Tscm than Tn; at least 5x more Tscm than Tn; at least lOx more Tscm than Tn; and/or at least 50x more Tscm than Tn.

[0049] In preferred methods of the disclosure, the contacting step occurs between 12 hours and 21 hours after the binding step. In preferred methods of the disclosure, the contacting step occurs between 15 hours and 19 hours after the binding step. In certain preferred aspects, the contacting step occurs between 17 hours and 19 hours after the binding step. In certain methods, the contacting step occurs about 18 hours after the binding step.

[0050] In certain methods of the disclosure, the cultivating step is for a period of between 29 hours and 59 hours. In preferred methods of the disclosure, the cultivating step is for a period of between 36 hours and 52 hours. In more preferred aspects, the cultivating step is for a period of between 46 hours and 50 hours. In certain aspects, the cultivating step is for a period of about 48 hours.

[0051] In certain methods of the disclosure, the one or more anti-CD4 antibodies and the one or more anti-CD8 antibodies attached to the same support. In certain methods, the one or more anti- CD8 antibodies and/or the one or more anti-CD4 antibodies are attached to the support via a cleavable linker. The linker may be, for example, an enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker. Accordingly, methods of the disclosure may further include contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface.

[0052] In certain aspects, the surface is a solid surface. Preferably, the solid surface is ahead, well, chip, or microfluidic channel. More preferably, the solid surface is a bead. In some aspects, the surface comprises a polymer. In certain aspects, the polymer is a hydrogel. In some methods, the surface comprises a polymer scaffold. [0053] In preferred methods of the disclosure, the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg. In preferred aspects, at least 1% of the harvested T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MLP- la and MIP-lb. In certain methods, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNg and Granzyme B.

[0054] In preferred methods of the disclosure, the polyfunctional T cells and/or a portion of the population thereof, comprise two or more of: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

[0055] In some methods of the disclosure, the polyfunctional T cells and/or a portion of the population of polyfunctional T cells comprise: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

[0056] In preferred methods, the serum free cultivation medium comprises at least one cytokine. Preferably, the at least one cytokine comprises one or more of IL-2, IL-21, IL-7, and IL-15. In some methods, the at least one cytokine comprises one or more of IL-21, IL-7, and IL-15 and does not comprise IL-2.

[0057] In certain methods of the disclosure, the number of isolated T cells from the whole blood sample is between about lxl0⁶ and about IxlO⁸ total T cells and the number of harvested T cells is between about 2.5xl0⁷ and about 5xl0⁸. In certain methods of the disclosure, the number of isolated T cells from the whole blood sample is between about 1.5xl0⁷ and about IxlO⁸ total T cells and the number of harvested T cells is between about 7.5xl0⁷ and about 1.5xl0⁸. In certain preferred methods, the number of isolated T cells from the whole blood sample is between about 5xl0⁷ and about 7.5xl0⁷ total T cells and the number of harvested T cells is between about 7.5xl0⁷ and about 1.2xl0⁸.

[0058] In certain aspects, the culturing step produces between a 1.0- and a 4-fold expansion of the harvested T cells. In certain aspects, the culturing step produces between a 1.2- and a 4-fold expansion of the harvested T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG. 1 provides an exemplary workflow for methods of the disclosure used to produce engineered T cells from a whole blood sample.

[0060] FIG. 2 is a bar graph showing T-cell expansion, represented by the fold increase of the number of CD3+ cells at 72 hours following thawing relative to the number of CD3+ cells upon thawing to compare methods of manufacturing CAR T cells of the disclosure.

[0061] FIG. 3 is a graph depicting percentage of CAR expression at 72 hours following thawing to compare methods of manufacturing CAR T cells of the disclosure.

[0062] FIGs. 4A-4B show percentage of TNSCM (naive and stem cell memory T cells (CD45RO- /CCR7+)) upon thawing. "UNTD" denotes untransduced controls.

[0063] FIGs. 5A-5D are bar graphs depicting memory phenotypes of CAR+ cells upon thawing (FIGS. 5A and 5C) or 72 hours after thawing (FIGS. 5B and 5D). "Post Enrichment" condition was untransduced.

[0064] FIG. 6 shows percentage of cytolysis of NALM6 target cells when incubated with the CAR-T cells produced by Conditions D and E. "D" is KYV 6-day condition, "E" is KYV 8-day condition, and "UNTD" denoted an untransduced condition.

[0065] FIGS. 7A-7D are bar graphs depicting secretion frequency of each of 32 cytokines from cells manufactured using the KYV 3-day v2 protocol ("C") (FIGS. 7A and 7C) or the KYV-9- day protocol ("E") (FIGS. 7B and 7D), after overnight incubation in the presence of CD 19+ NALM6 target cells, "ut" corresponds to untransduced control cells that were otherwise subjected to each of the steps of the 3 -day or 9-day protocol.

[0066] FIG. 8 is a bar graph showing polyfunctionality of CAR-T cells produced by Process C.

[0067] FIG. 9 is a bar graph showing percentage of cells, produced by Process C or E, out of all polyfunctional cells that secreted the cytokines indicated.

[0068] FIG. 10 is a flow chart illustrating the experimental design and conditions of six CAR-T cell manufacturing processes.

[0069] FIG. 11 provides data from same-donor studies validate that the X-LAB system provided superior PBMC enrichment from whole blood samples.

[0070] FIG. 12 provides data showing the CD3+, CD4+, CD8+ cell percentage (T cells) in samples, including whole blood samples.

[0071] FIG. 13 outlines the protocols used to assess a direct-from-blood T cell isolation step. [0072] FIG. 14 provides enrichment and expansion data comparing methods of the disclosure that isolate T cells from whole blood for CAR T cell manufacture.

[0073] FIG. 15 provides CD3+ and CD4+ percentage data comparing methods of the disclosure that isolate T cells from whole blood for CAR T cell manufacture.

[0074] FIG. 16 provides enrichment and expansion data comparing methods of the disclosure that isolate T cells from whole blood for CAR T cell manufacture.

[0075] FIG. 17 provides data showing that cells produced using a 3-day manufacturing protocol of the disclosure to produce CAR T cells from T cells isolated directly from whole blood show a less differentiated phenotype compared to mock (untransduced) control cells.

[0076] FIG. 18 provides data showing the target-specific cytotoxicity of CAR T cells produced using the methods of the disclosure.

[0077] FIG. 19 provides the results for the target-dependent cytokine release by the CAR T cells derived from healthy donor (HD) whole blood, following 24h in vitro co-culture with CD 19+ NALM6 target cells at the indicated E:T (effectortarget) ratios.

[0078] FIG. 20 shows data for CD19-targeted CAR-T cells manufactured using methods of the disclosure and CD19+ NALM6 target cells that were co-cultured at the indicated E:T ratios (CAR-T effector:NALM6 target) and % killing of target cells was measured by flow cytometry at each indicated timepoint. At each timepoint, a fresh round of target cells was added to the coculture to assess the serial, repeated cytotoxicity of the CAR-T over time.

[0079] FIG. 21 provides target-specific expansion data for CAR T cells manufactured using methods of the disclosure.

[0080] FIG. 22 provides TBNK/memory phenotype of pre- and post-enrichment material for cells produced starting with T cells isolated from whole blood.

[0081] FIG. 23 provides TBNK/memory phenotype of a final CAR T cell product (e.g., after expansion) produced using an 8-day process.

[0082] FIG. 24 provides TBNK/memory phenotype of pre- and post-enrichment material for cells produced starting with T cells isolated from whole blood.

[0083] FIG. 25 provides TBNK/memory phenotype of pre- and post-enrichment material for cells produced starting with T cells isolated from whole blood.

[0084] FIG. 26 outlines steps of a three-day method of the disclosure starting from whole blood (WB) starting material (SM).

[0085] FIG. 27A shows the T-cell memory phenotype of cells produced using the three-day methods of the disclosure.

[0086] FIG. 27B shows the cytolytic activity of the cells produced using the the 3-day methods of the disclosure.

[0087] FIG. 27C shows results of a serial rechallenge assay.

[0088] FIG. 27D shows that the 3-day process Ingenui-T cells successfully killed autologous primary B cells in a dose-dependent manner. Ingenui-T cells or untransduced T cells, derived from whole blood, were co-cultured with autologous (donor-matched) PBMCs at the indicated effector to target (E:T) ratios, representing the ratio of CAR⁺ T cells (effector) to total PBMCs (target). Survival of B cells, defined by the expression of CD 19 or CD20 surface markers, was measured by flow cytometry at 48 hours. Data are shown as mean ± SD from 3 technical replicates per condition, from one donor representative of N=2 healthy donors. ****p<0.0001, comparing matched E:T ratios, by 2-way ANOVA (GraphPad Prism).

[0089] FIG. 28A shows fold expansion, viability, and T cell purity of CAR-T cells manufactured in the KYV 3-Day process starting from leukapheresis material. KYV 3-Day Conditions “A”, “B”, “C”, “D” indicate different culture cytokine(s) used. “NT” = non-transduced. N=4 healthy donors per condition. [0090] FIG. 28B shows yield and T cell purity of CAR-T cells manufactured in the KYV 3-Day process starting from freshly collected whole blood from n=5 donors.

[0091] FIG. 29A shows % CAR+ expression analyzed by flow cytometry in CAR-T cells manufactured in the KYV 3-Day process starting from leukapheresis material. CAR expression was analyzed within total CD3+ T cells or within CD4+ or CD8+ T cells, at the time of harvest. KYV 3-Day Conditions “A”, “B”, “C”, “D” indicate different culture cytokine(s) used. N=4 healthy donors per condition.

[0092] FIG. 29B shows % CAR+ expression analyzed by flow cytometry in CAR-T cells manufactured in the KYV 3-Day process starting from freshly collected whole blood. CAR expression was analyzed within total T cells. N=5 donors.

[0093] FIG. 30A shows % CD4+ and CD8+ analyzed within total CD3+ in CAR-T cells manufactured in the KYV 3-Day process starting from leukapheresis material. KYV 3-Day Conditions “Cl”, “C2”, “C3”, “C4” indicate different culture cytokine(s) used. N=4 healthy donors per condition. “NT” = non-transduced controls. “Conv 9-day” refers to donor-matched CAR-T cells manufactured in a conventional 9-day culture process. “Aph SM” refers to the leukapheresis donor starting material prior to the KYV 3-Day process.

[0094] FIG. 30B % CD4+ and CD8+ analyzed within total CD5+ in CAR-T cells manufactured in the KYV 3-Day process starting from freshly collected whole blood. N=7 donors combined. “WB SM” refers to the donor whole blood starting material prior to the KYV 3-Day process. [0095] FIG. 31A-31B shows results of T cell memory phenotype analyzed by flow cytometry within CD4+ or CD8+ T cells, in CAR-T cells manufactured in the KYV 3-Day process starting from leukapheresis material. KYV 3-Day Conditions “Cl”, “C2”, “C3”, “C4” indicate different culture cytokine(s) used. N=4 healthy donors per condition. “NT” = non-transduced controls. “Conv 9-day” refers to donor-matched CAR-T cells manufactured in a conventional 9-day culture process. “Aph SM” refers to the leukapheresis donor starting material prior to the KYV 3-Day process. Tnaive = naive (CCR7+CD45RO-CD95-); Tscm = stem cell memory (CCR7+CD45RO-CD95+); Tern = central memory (CCR7+CD45RO+); Tern = effector memory (CCR7-CD45RO+); Te = effector (CCR7-CD45RO-CD95+).

[0096] FIG. 31C T cell memory phenotype analyzed within total CD3+ T cells, in CAR-T cells manufactured in the KYV 3-Day process starting from freshly collected whole blood, compared to a conventional 9-day process starting from leukapheresis material. N=4 donors combined. “WB SM” and “Aph SM” refer to the donor whole blood or leukapheresis starting materials respectively, prior to the culture process. T cell memory subsets were analyzed as defined in Figs. 31A-31B.

[0097] FIG. 32 shows target-dependent cytotoxic activity of anti-CD19 CAR-T cells manufactured from KYV 3-day process against CD19+ expressing target cells by measuring the % Cytolysis of CD 19+ NALM6 target cells or CD 19- CEM/C1 control cells after co-culture with anti-CD19 CAR-T cells manufactured from KYV 3-Day process, at the indicated E:T (EffectorTarget) ratios. N=2 donors shown. Cytolytic activity was measured by luminescence assay and normalized to target cells alone (0: 1).

[0098] FIG. 33A shows results of killing or outgrowth of CD 19+ NALM6 target cells during 120h co-culture with anti-CD19 CAR-T cells manufactured in the KYV 3 -Day process from leukapheresis starting material, at the indicated E:T (Effector:Target) ratios of 0.3:1 or 1 : 1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized to time = 0. KYV 3-Day Conditions “Cl”, “C2”, “C3”, “C4” indicate different culture cytokine(s) used. “NT” = non-transduced control T cells. One representative donor shown from n=4.

[0099] FIG. 33B shows results of killing or outgrowth of CD19+ NALM6 target cells during 120h co-culture with anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from freshly collected whole blood, at the indicated E:T (Effector: Target) ratios of 0.3 : 1 or 1 : 1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized to time = 0. “NT” = non-transduced control T cells. “Conv 9-day” refers to donor-matched CAR-T cells manufactured in a conventional 9-day culture process, from leukapheresis starting material. One representative donor shown from n=4.

[0100] FIG. 34 shows CAR-mediated cytotoxic activity of anti-CD19 CAR-T cells manufactured from KYV 3-day process against CD 19+ primary human B cells using the measured % Cytolysis of CD 19+ primary human B cells in co-culture with anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from freshly collected whole blood. E:T (Effector: Target) ratios indicate the ratio of CAR+ T cells to total PBMC (peripheral blood mononuclear cells) plated in co-culture. “Conv 9-day” refers to donor-matched CAR-T cells manufactured in a conventional 9-day culture process, derived from leukapheresis starting material. One representative donor shown in each panel from n=5.

[0101] FIG 35 shows target-dependent cytokine release by anti-CD19 CAR-T cells manufactured from KYV 3 -day process, in response to CD 19+ expressing target cells via measured IFN-gamma production by anti -CD 19 CAR-T cells manufactured in the KYV 3 -Day process from freshly collected whole blood, in co-culture with CD 19+ NALM6 target cells or CD 19- CEMC1 control cells at the indicated E:T (Effector: Target) ratios. Culture supernatants were collected and analyzed by ELLA. N=2 donors shown.

[0102] FIG. 36A shows effector dose-dependent, CAR-mediated cytokine release by anti-CD19 CAR-T cells manufactured from KYV 3-day process, in response to CD19+ expressing target cells through measured cytokine release by anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from leukapheresis starting material, in co-culture with CD 19+ NALM6 target cells at the indicated E:T (EffectorTarget) ratios of 0.3 : 1 or 1 : 1. Culture supernatants were collected and the indicated cytokines were analyzed by MSD. KYV 3-Day Conditions “Cl”, “C2”, “C3”, “C4” indicate different culture cytokine(s) used in the manufacturing process. “NT” = non-transduced control T cells. N=4 healthy donors per condition.

[0103] FIG. 36B shows Cytokine release by anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from freshly collected whole blood, in co-culture with CD19+NALM6 target cells at the indicated E:T (EffectorTarget) ratios of 0.3: 1 or 1 : 1. Culture supernatants were collected and the indicated cytokines were analyzed by MSD. “Conv 9-day” refers to donor-matched CAR- T cells manufactured in a conventional 9-day culture process, derived from leukapheresis starting material. N=4 healthy donors per condition.

[0104] FIG. 36C shows cytokine release by anti-CD19 CAR-T cells manufactured in the KYV 3- Day process from freshly collected whole blood, in co-culture with CD 19+ NALM6 target cells at the indicated E:T (EffectorTarget) ratios of 0.3: 1 or 1 : 1. Culture supernatants were collected and the indicated cytokines were analyzed by MSD. “Conv 9-day” refers to donor-matched CAR- T cells manufactured in a conventional 9-day culture process, derived from leukapheresis starting material. N=4 healthy donors per condition.

[0105] FIG. 37 shows results of a long-term serial cytotoxic activity by anti-CD19 CAR-T cells manufactured from KYV 3-day process compared to a conventional 9-day process, in response to CD19+ expressing target cells. Fig. 37 provides the duration of in vitro cytotoxicity by KYV 3- Day or Conv 9-Day anti-CD19 CAR T cells, derived from a healthy donor, in a serial rechallenge assay against CD19+ NALM6 tumor cells. KYV 3-Day CAR T cells were derived from leukapheresis starting material (“APH”, Top panel) or freshly collected whole blood (“WB”, Bottom panel). CAR T cells were co-cultured in triplicate with NALM6 target cells at the indicated Effector: Target (E:T) ratios, and the survival of NALM6 cells was analyzed every 2-3 days by flow cytometry. The time (days) to loss of CAR-mediated cytotoxic activity, defined as the assay timepoint at which >95% survival of target cells was detected, was measured for each individual replicate. Data representative of n=4 donors.

[0106] FIG. 38 shows results of in vitro expansion by anti -CD 19 CAR-T cells manufactured from KYV 3-day process compared to a conventional 9-day process, in response to CD19+ expressing target cells. In vitro expansion of KYV 3-Day or conventional (“Conv”) 9-Day anti -CD 19 CAR T cells in response to repeated stimulation in co-culture with CD 19+ expressing REH target cells. KYV 3 -Day CAR T cells were derived from leukapheresis starting material (“APH”, panel A, n=4) or other freshly collected starting material (“WB”, panel B, n=3), and compared to donor- matched Conv 9-Day CAR T cells derived from leukapheresis material. CAR T cells were cocultured with mitomycin C-treated REH target cells at a 1 : 1 ratio, and cells were re-plated every 3-4 days with new target cells. Total fold expansion of CAR+ T cells (gated by flow cytometry analysis) was measured at day 16.

[0107] FIG. 39A shows in vivo activity of anti-CD19 CAR-T cells manufactured from KYV 3- day process compared to conventional 9-day process, in CD 19+ NALM6 tumor-bearing NSG mice. Mean NALM6 tumor growth in NSG mice treated with the indicated doses of donor- matched anti -CD 19 CAR T cells manufactured from the KYV 3-day process or a conventional (“Conv”) 9-day process, both derived from leukapheresis (“APH”) starting material. NALM6- luciferase tumor cells were injected i.v. into mice at day -7 prior to T cell transfer. On day 0, mice were given a single i.v. injection of the indicated doses of CAR T cells. Tumor burden in each animal was measured twice per week using IVIS bioluminescent imaging and shown as total flux (photons/sec). Data is shown as mean ± SEM of all animals per group. Data is representative of 2 studies using n=2 independent donors. [0108] FIG. 39B shows individual NALM6 tumor growth curves in NSG mice treated with a le6 CAR+ T cell dose of donor-matched anti-CD19 CAR T cells. CAR T cells were manufactured from the KYV 3-day process, derived from freshly collected whole blood (“WB”), or a conventional (“Conv”) 9-day process, derived from leukapheresis starting material. Tumor cells were inoculated and mice were treated and analyzed as described for FIG. 39A. N=5 animals per group.

[0109] FIG. 40 outlines two 9-day processes of the disclosure for manufacturing CAR T cells starting from fresh whole blood starting material.

[0110] FIGS. 41A-41E provide Flow cytometry data characterizing CAR T cells produced using 9-day methods of the disclosure starting from whole blood starting material.

[0111] FIG. 42 provides % cytolysis data characterizing CAR T cells produced using 9-day methods of the disclosure starting from whole blood starting material.

[0112] FIG. 43 provides cytokine secretion data characterizing CAR T cells produced using 9- day methods of the disclosure starting from whole blood starting material.

DETAILED DESCRIPTION

[0113] Disclosed herein are methods for manufacture of engineered T cells that express a heterologous protein, such as a chimeric antigen receptor (CAR), under certain conditions. Also contemplated are similar methods for manufacture of engineered T cells that comprise a heterologous gene, wherein the heterologous gene may or may not encode a protein. Further disclosed are compositions suitable for use in conjunction with the disclosed methods.

[0114] Exemplary methods for manufacturing such cells (e.g., CAR T cells) of the disclosure may include obtaining T cells from a subject (e.g., a donor or a patient) from whole blood. In some aspects, T cells are obtained from peripheral blood mononuclear cells (PBMC) isolated from the whole blood. In more preferred aspects, the present Inventors have discovered that surprisingly, sufficient numbers of T cells for CAR T manufacturing may be obtained directly from whole blood, without the need for a leukapheresis step or PBMC isolation step.

[0115] Methods of the disclosure may include a concurrent T cell isolation and activation step. In such methods, T cells may be isolated from whole blood or PBMCs and concurrently activated. This reduces the overall manufacturing time, while retaining the ability isolate a sufficient number of T cells, at a high purity, to manufacture the CAR T cells from a very small whole blood sample (e.g., samples of less than 100 mb of blood).

[0116] Exemplary methods for manufacturing CAR T cells of the disclosure employ a short culture after T cell isolation/activation before transduction, generally less than or significantly less than a day, and a similarly short cultivation after transduction before harvesting the desired CAR T cell product. Accordingly, methods of the disclosure are able to produce a large harvest of CAR T cells — all within 2-3 days of receiving a starting sample.

[0117] Surprisingly, methods of the disclosure have been shown to produce a final CAR T cell product of a higher quality than existing, longer processes. CAR T cells manufactured using the methods of the disclosure have shown a superior T cell expansion in the final CAR T product; a more favorable phenotype as a result of the condensed processes of the disclosure; and less differentiation than with longer processes. Further, the resulting CAR T cells included a very high percentage of stem cell memory T cells (Tscm), which generally only account for 2-3% of circulating T cells and are associated with long-term defensive immunity, anti-tumor activity, selfrenewal, and immune-regulation.

[0118] Cells made using methods of the disclosure expressed the introduced CARs at a very high percentage, leading to a pure end product with targeted cytotoxicity and low T cell exhaustion. Even more surprising, the shorted manufacturing process produces a large number of polyfunctional cells, which simultaneously secrete multiple sets cytokines, chemokines, and/or cytotoxic granules simultaneously. Owing to this polyfunctional behavior, such cells are known to provide a more effective immune response, which is desirable in a therapeutic CAR T cell product.

Definitions

[0119] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. Generally, nomenclatures utilized in connection with techniques described herein are those well-known and commonly used in the art. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. As used herein, singular forms "a”, “and”, and "the" include plural referents unless the context clearly indicates otherwise. Thus, e.g., reference to "an antibody" includes a plurality of antibodies and reference to "an antibody" in certain embodiments includes multiple antibodies, and so forth.

[0120] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, e.g., reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4% 91.5%, etc., 92.1% 92.2% 92.3% 92.4% 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5-fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5 fold, etc., and so forth.

[0121] "About" a number, as used herein, refers to range including the number and ranging from 10% below that number to 10% above that number. "About" a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.

[0122] As used herein, the terms "activate, activating," "activated," and the like, when used in the context of T cell activation, encompasses various associated biological processes such as induction of intracellular signaling pathways associated with T cell activation, change in expression of cell surface markers, cytokine release, proliferation, and the like). Generally, T cell activation occurs as a result of engagement of a T cell receptor complex or a functional portion thereof (e.g., CD3) and a costimulatory molecule (e.g., CD28) on the T cell by the major histocompatibility complex (MHC) and costimulatory molecules on antigen presenting cells, respectively. Induction of intracellular signaling cascades associated with T cell activation include activation of the P13K pathway, recruitment of PH-domain containing proteins (e.g., PDKI), and eventual cytokine production (e.g., IL-2). Changes in expression of T cell surface markers occur as a result of activation, leading to increases in expression of one or more of CD69, CD71, CD25, CD 137, HLA- DR, CTLA-4, and others. Production and secretion of cytokines, chemokines, and other proteins (e.g., fFNy, Granzyme B, IL-1B, and/or IL-2) may also result from T cell activation.

[0123] As used herein, the term "basal cultivation medium" refers to a culture medium containing a minimal set of ingredients that are essential for the survival of cells (e.g., T cells). A "basal cultivation medium" typically is an aqueous solution including amino acids (e.g., L-racemers of glycine, arginine, asparagine, aspartate, cysteine, glutamine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and/or valine), vitamins (e.g., biotin, choline chloride, D-calcium pantothenate, folate, niacinamide, paraaminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin Bl 2, and/or i-inositol, among others), salts (e.g., calcium nitrate, ferric nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium pyruvate, and/or sodium phosphate, among others), a source of sugar (e.g., D-glucose), and, optionally, a reducing agent (e.g., glutathione). Representative examples of basal cultivation medium include, without limitation, RPM] 1640, Eagle's Minimal Essential Medium (EMEM), Dulbecco¹ s Modified Eagle' s Medium (DMEM), Minimum Essential Medium Eagle (a-MEM), and Glasgow Minimal Essential Medium (Glasgow¹ s MEM), among others. In most cases, a "basal cultivation medium" does not include protein additives (e.g., cytokines, growth factors, and/or albumin). In some embodiments, a "basal cultivation medium" has a pH of between 7.0 and 7.4, such as, 7.0, 7.1, 7.2, 7.3, or 7.4. In some embodiments, a "basal cultivation medium" has an osmolarity of between 290 and 320 mOsmol (e.g., 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 mOsmol). A skilled artisan will appreciate that culture media with other pH and osmolarity values can be used. In some embodiments, a "basal cultivation medium" is hypotonic, isotonic, or hypertonic.

[0124] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a chimeric receptor protein comprising an extracellular domain that has antigen-binding specificity, a transmembrane domain, and an intracellular signaling domain. In some cases, the extracellular domain can comprise an antigen-binding domain. In some cases, the transmembrane domain can comprise a transmembrane domain derived from a natural polypeptide obtained from a membrane-binding or transmembrane protein.

[0125] For example, a transmembrane domain can include, without limitation, a transmembrane domain from a T cell receptor alpha or beta chain, a CD3 zeta chain, a CD28 polypeptide, or a CD8 polypeptide. In some cases, the intracellular domain can comprise a cytoplasmic signaling domain (e.g., any of the cytoplasmic signaling domains described herein) and one or more costimulatory domains (e.g., any of the exemplary co-stimulatory domains described herein). [0126] As used herein, the term “contact”, “contacting”, “contacted” and the like includes exposing one composition (e.g., a cell or a population of cells, such as T cells) to another composition (e.g., a polynucleotide) by any means such that they can be in direct interaction. One of skill in the art will appreciate that an exemplary method of contacting a population of cells with an agent (e.g., a nucleic acid encoding a heterologous protein, such as a CAR) is by mixing an aqueous suspension of the cells with an aqueous solution or suspension of the agent. Although not all individual cells in the population may get in direct interaction with the agent immediately, even if the agent is provided in excess amount relative to the cells, over a period of time (e.g., 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, or longer) according to the method, a substantial amount of the cells (e.g., at least 1%, 5% 10% 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of the cells in the population) will be in direct interaction with a molecule of the agent.

[0127] As used herein, the term "cytotoxicity" refers to the ability of a cell, such as a T cell engineered to express a chimeric antigen receptor (CAR) according to the methods disclosed herein, to cause cell death (e.g., apoptosis or necrosis) of another cell (e.g., a target cell). For example, an engineered T cell expressing a CAR can elicit a cytotoxic response against a target cell expressing a target antigen. Binding between the CAR' s antigen-binding domain and the target antigen can, in certain embodiments, lead to T cell activation and killing of the target cell .Assays for detecting cytotoxicity induced by CAR-T cells include, without limitation, a chromium release assay, bioluminescence assay (e.g., luciferase-mediated bioluminescence imaging), real-time impedance-based analysis, flow cytometry (e.g., in combination with a viability dye, such as CTV), and CFSE/PI assay.

[0128] As used herein, the term "delivery vehicle" refers to any pharmaceutical carrier, diluent, excipient, and the like, which are generally intended for use in connection with administration of biologically active agents, including nucleic acids. For example, a delivery vehicle may include lipid- or polymer-based transfer vehicles for the delivery of nucleic acids, including, but not limited to, a lipid nanoparticle, a liposome, polymer nanoparticle (nanocapsule or nanosphere), and the like. In certain embodiments, a delivery vehicle is a lipid nanoparticle. In the context of nucleic acid delivery to target cells, a "delivery vehicle" can also include any vector (e.g., viral or non-viral vector) capable of delivering the nucleic acid(s) to the target cell(s). In certain embodiments, a delivery vehicle is a viral vector, such as a lentiviral vector.

[0129] As used herein, the term "engineered," when referencing a cell (e.g., T cell) that has been contacted with a nucleic acid encoding a heterologous protein (e.g., a CAR), means that the nucleic acid or a fragment thereof encoding the heterologous protein is stably integrated into the cell ' s genome after the contacting.

[0130] As used herein, the term "harvesting" and the like refers to isolation and/or collection of a cell or population of cells (e.g., T cells) following incubation of said cells under culture conditions. In certain embodiments, harvesting includes change of one or more conditions, such as temperature, cell culture medium, and/or availability of certain agents (e.g., one or more agents that activate CD3 and/or CD28, one or more cytokines, and/or a polynucleotide encoding a heterologous protein) such that the step immediately prior to the harvesting step is discontinued.

[0131] As used herein, the term "heterologous" refers to a nucleic acid or polypeptide sequence or domain which is not present in its native form or amount in its native environment. For example, in some embodiments, a heterologous nucleic acid (e g., gene) is not present between its adjacent flanking sequences, e.g., wherein the heterologous sequence is not found in nature coupled to the nucleic acid or polypeptide sequences occurring at one or both ends. In some embodiments, a heterologous protein is entirely absent from a native cell prior to being engineered to expresses the protein. In some embodiments, a heterologous protein is present with different post-translational modifications from a protein in a native cell prior to being engineered to expresses the protein. In some embodiments, a heterologous protein is present in a substantially lower amount than a protein in a native cell prior to being engineered to expresses the protein.

[0132] As used herein, the term "sample¹ refers to a biological sample, such as a blood sample (e.g., a whole blood sample), obtained from a subject (e.g., a human). In certain embodiments, the sample is a blood sample processed by conventional methods to isolate a desired blood fraction (e.g., serum or plasma) or one or more cell types of interest (e.g., peripheral blood mononuclear cells (PBMCs), a lymphocyte, such as a T lymphocyte). For instance, a "sample" can refer to a leukapheresis sample obtained from the blood of a subject. In certain embodiment, a “sample” refers to a whole blood sample obtained from a subject. [0133] Disclosed herein, in certain embodiments, are methods for the rapid manufacture of engineered T cells that comprise a heterologous gene or express a heterologous protein, such as a chimeric antigen receptor (CAR). Also disclosed are compositions suitable for use in conjunction with the disclosed methods. The methods disclosed herein provide certain advantages over prior CAR-T manufacturing methods, including production of more potent CAR-T cells as compared to CAR-T cells produced with longer manufacturing protocols, thereby facilitating the use of lower CAR-T cell dosages for therapeutic use. Furthermore, the disclosed methods preserve T cell "sternness" (i.e., less differentiated phenotype), thereby producing CAR-T cells with higher potential for proliferation. Shorter CAR-T manufacturing time resulting from the disclosed methods also facilitates scaling down of CAR-T cell manufacturing, resulting in reduced costs, reduced "needle-to-needle" time (i.e., time from harvesting of a patient's T cells to delivering autologous engineered T cells back to the patient), and improved patient access.

CAR-T cells

[0134] Chimeric antigen receptors (CARs) are artificially constructed hybrid receptor proteins or polypeptides containing an antigen-binding domain, e.g., an antigen-binding fragment of an antibody which can take various formats such as a single chain variable fragment (scFv), linked to one or more intracellular signaling or activation domains (optionally including a costimulatory domain) via a transmembrane domain. Autologous T cell-based therapies, such as T cells modified to express CARS, have demonstrated remarkable therapeutic benefit to patients suffering from cancer.

[0135] Without wishing to be bound to a particular theory or mechanism, it is believed that by eliciting an antigen-specific response against a cell expressing a target antigen, CARs provide one or more of the following benefits: targeting and destroying target antigen expressing cells, reducing or eliminating the target cells, facilitating infiltration of immune cells to a target tissue, and enhancing/extending anti-cancer responses. CAR-T cells can also be used to reduce an autoimmune response by targeting cells (e.g., B cells) that mediates the autoimmunity.

[0136] Though heralding an unprecedented therapeutic promise, CAR-T cell production has faced substantial obstacles, including, as is relevant to the scope of the present disclosure, long manufacturing times. Prolongation of the CAR-T cell manufacturing process can disadvantageously lead to batch loss, reduced expansion and persistence of CAR-T cells in vivo, increased batch-to-batch variability of the final cell product, increased differentiation and heterogeneity in the final cell product, increased manufacturing costs, and a prolongation in providing potentially life-saving therapy to patients. Accordingly, the present disclosure provides methods and compositions for the rapid of manufacture of CAR-T cells. It is understood that similar methods and compositions can be used for rapid manufacture of T cells that express other heterologous genes.

Methods of Manufacturing Engineered T Cells

[0137] Disclosed herein, in certain embodiments, are methods of manufacturing a population of T cells engineered to express a heterologous protein. The presently disclosed methods enable rapid manufacture of engineered T cells that express a heterologous protein, such as a chimeric antigen receptor (CAR), under specified conditions.

[0138] T cells are leukocytes that have completed maturation in the thymus, can identify certain foreign antigens, and perform various roles in the immune system, including activation and deactivation of other immune cells. Generally, a T cell can be any T cell such as a cultured T cell, e.g., a primary T cell, or a T cell derived from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or sub-populations thereof. The T cell can be a CD3+ cell. T cells can be CD4+, CD8+, or CD4+ and CD8+. For example, a T cell can be a CD4+ / CD8+ double positive T cell, CD4 + helper T cell (e.g., Thl or Th2 cell), CD8+ T cell (e.g., a cytotoxic T cell), peripheral cell, including but not limited to a blood mononuclear cell (PBMC), peripheral blood leukocyte (PBL), tumor infdtrating lymphocyte (TIL), memory T cell, naive T cell, regulatory T cell, y8 T cell, etc. A T cell can be any T cell at any stage of development. Additional types of helper T cells include Th3 (Treg) cells, Th 17 cells, Th9 cells, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (TEM cells and TEMRA cells). A T cell can also refer to a genetically modified T cell (e.g., an engineered T cell), such as a T cell that has been modified to express a heterologous protein, such as a chimeric antigen receptor (CAR). T cells can also be differentiated from stem cells or progenitor cells.

[0139] In certain embodiments, the disclosed methods relate to the production of engineered T cells expressing a CAR having a binding specificity to a target antigen. The disclosed methods are used, in certain embodiments, to produce a population of engineered T cells from a starting population of cells over a short course (e.g., 2, 3, or 4 days). For instance, the methods of the disclosure are capable of producing a population of engineered T cells expressing a heterologous protein (e.g., a CAR) in about 48 hours (hrs), 49 hrs, 50 hrs, 51 hrs, 52 hrs, 53 hrs, 54 hrs, 55 hrs,

56 hrs, 57 hrs, 58 hrs, 59 hrs, 60 hrs, 61 hrs, 62 hrs, 63 hrs, 64 hrs, 65 hrs, 66 hrs, 67 hrs, 68 hrs,

69 hrs, 70 hrs, 71 hrs, 72 hrs, 73 hrs, 74 hrs, 75 hrs, 76 hrs, 77 hrs, 78 hrs, 79 hrs, 80 hrs, 81 hrs,

82 hrs, 83 hrs, 84 hrs, 85 hrs, 86 hrs, 87 hrs, 88 hrs, 89 hrs, 90 hrs, 91 hrs, 92 hrs, 93 hrs, 94 hrs,

95 hrs, or 96 hrs.

[0140] In certain preferred aspects, the methods allow for the production of engineered T cells (e.g., T cells expressing a CAR) in about 2-3 days.

[0141] Accordingly, the disclosed methods include, in certain embodiments, steps for (1) optionally enriching/isolating a biological sample from a subject (e.g., a human subject) containing a starting population of T cells; (2) activating the starting population of T cells with an agent that binds CD3 and an agent that binds a costimulatory molecule (e.g., CD28/CD3); (3) contacting the T cells with a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein (e.g., a CAR) and cultivating the T cells under conditions and for a brief time suitable to facilitate expression of the heterologous protein by the T cells; and (4) harvesting and, optionally, storing the engineered T cells for later use (e.g., therapeutic use or quality control testing). The sections that follow below describe each of the aforementioned steps in greater detail.

[0142] FIG. 1 provides a schematic for methods of the disclosure used to produce CAR T cells from whole blood samples and/or enriched PBMCs. As will be described in greater detail, the steps of this workflow may include certain variations. One such variation may occur at the isolation and activation step. Certain preferred methods of the disclosure combine these steps in a single, concurrent isolation and activation step using a T cell activating agent (e.g., CD3/CD28 beads) to both isolate and activate the T cells. Sample preparation step

[0143] Disclosed herein, in certain embodiments, are methods for the isolation of a starting population of T cells from a biological sample, such as a sample obtained from a subject (e.g., a human subject). Non-limiting examples of biological sample include cells, tissue (e.g., tissue obtained by biopsy), blood, serum, plasma, or any sample derived therefrom.

[0144] In preferred embodiments, the sample is a whole blood sample obtained from the subject from which T cells are isolated without using an apheresis/leukapheresis step. In such methods, T cells may be isolated directly, in a single step from the whole blood and/or the whole blood undergoes a step of enriching peripheral blood mononuclear cells (PBMC) from which T cells are isolated. In preferred methods, T cells are isolated directly from whole blood without using an apheresis/leukapheresis step and without using a step to isolate or enrich PBMCs from which the T cells are obtained. Surprisingly, the presently disclosed methods are able to obtain a sufficiently pure and numerous T cell population directly from a small sample of whole blood without a series of intervening steps to isolate/enrich the T cells from the whole blood.

[0145] In certain embodiments, the method comprises obtaining the sample from the subject. In certain embodiments, the method comprises having obtained the sample from the subject.

[0146] In certain embodiments, the methods disclosed herein include obtaining a starting population of T cells from a biological sample obtained from a subject. In certain embodiments, the biological sample is a leukapheresis sample. In certain embodiments, the biological sample is a whole blood sample. In certain embodiments, the starting population of T cells includes T helper (Th) cells, cytotoxic T (Tc) cells, memory T (TM) cells, regulatory T (Treg) cells, innate like T cells. In certain embodiments, the Th cells include Thl cells, Th2 cells, Thl7 cells, Th9 cells, Tfh cells, and/or Th22 cells. In certain embodiments, the TM cells include central memory T cells (TCM) cells, effector memory T (TEM) cells, tissue-resident memory T (TR ) cells, and virtual memory T (TVM) cells. In certain embodiments, the innate-like T cells include natural killer T (NKT) cells, mucosal -associated invariant T (MAIT) cells, and y5 T cells.

[0147] In certain embodiments, the method includes isolating the starting population of T cells from the sample. Isolation of T cells may include an initial purification of T cells from a mixture of plasma, lymphocytes, platelets, red blood cells, monocytes, and granulocytes. Methods for isolation of T cells from a biological sample, such as a whole blood sample, enriched PBMC sample, or a leukapheresis sample, are well-known. Exemplary methods may include elutriation, density gradient centrifugation, enrichment by selection, and the like. For example, the method may include obtaining or having obtained a biological sample, such as a fresh, refrigerated, frozen, or cryopreserved product or alternative source of hematopoietic tissue, such as a whole blood sample, bone marrow sample, or a tumor or organ biopsy or removal (e.g., thymectomy) from an entity, such as a laboratory, hospital, or healthcare provider, and performing the aforementioned isolation steps to produce an enriched population of T cells (e.g., starting population of T cells) suitable for expression of a heterologous protein.

[0148] Surprisingly, the brief two-to-three-day manufacturing processes of the disclosure are able to produce CAR T cells directly from a whole blood sample and/or an enriched PBMC sample, without use of an initial leukapheresis. Prior to the present Inventor’s discovery, methods for producing CAR T cells, especially at sufficient throughput and speed, employed leukapheresis to create a concentrated sample of leukocytes from which T cells could be easily isolated. Nevertheless, the present Inventors have demonstrated that the presently disclosed methods are able to isolate a sufficient number of T cells directly from whole blood and/or whole blood enriched for PBMCs to produce CAR T cells. This not only avoids the costs and potential complications associated with leukapheresis, but also shortens the time required to produce the desired CAR T cells while expanding the potential availability of obtaining samples due to the reduced logistical complications required for a simple whole blood draw.

[0149] Furthermore, the purity of the starting population of T cells can be increased by using one or more selection steps, such as negative selection or positive selection. Negative selection typically involves removal of undesired cell types from a mixed population of cells in a sample using one or more agents that selectively bind to the undesired cell type, whereas positive selection typically involves isolation of the desired cell population using one or more agents that selectively bind to the desired cell type. Enrichment of a T cell population by negative selection can be accomplished, for example, with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immuno-adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail can include antibodies to CD 14, CD20, CDb, CD 16, HLA-DR, and CDR

[0150] On the other hand, a positive selection step can be used to specifically select for the desired cell type, including in methods of the disclosure in which it may be used to directly isolate T cells from a whole blood or PBMC sample. Positive selection of T cells can, in certain embodiments, include incubation of a mixed population of cells that contains the T cells (e.g., a whole blood/PBMC sample) with an agent having a CD3-binding moiety (e.g., anti-CD3 antibody- conjugated beads) for a time sufficient for positive selection of the desired T cells. In certain embodiments, the time period is about 30 minutes. In certain embodiments, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In certain embodiments, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In certain embodiments, the time period is 10 to 24 hours, for example, 18 hours. Longer incubation times may be used to isolate T cells in any context where there are few T cells as compared to other cell types.

[0151] In certain embodiments, the starting population of T cells comprise CD8+ T cells (e.g., CD8+ cytotoxic T cells). In certain embodiments, the starting population of T cells further comprise CD4+ T cells (e.g., CD4+ helper T cells). In certain embodiments, the starting population of T cells comprises 1-10% 1-20%, 1-30%, 1-40%, 1-50%, 1-60% 10-20% 10-30%, 10-40%, 10- 50%, 10-60%, 20-30%, 20-40%, 20-50%, 20-60%, 30-40%, 30-50%, or 30-60% of CD8+ T cells (e.g., CD8+ cytotoxic T cells) out of all T cells in the population. In certain embodiments, the starting population of T cells further comprises 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1- 70%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 30-40%, 30-50%, 30-60%, or 30-70% of CD4+ T cells (e.g., CD4+ helper T cells) out of all T cells in the population. In certain embodiments, the starting population of T cells comprise CD8+ T cells (e.g., CD8+ cytotoxic T cells) and CD4+ T cells (e.g., CD4+ helper T cells) at a ratio of 1 :5 to 5: 1, 1 :4 to 4: 1, 1 :3 to 3: 1, or 1 :2 to 2:1.

[0152] In certain embodiments, the starting population of T cells is produced to achieve a desired degree of purity. For example, the starting population of T cells may include T cells in an amount of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, of the total number of cells in the population. Purity of the starting population of T cells may be measured using routine methods, such as fluorescence assisted cell sorting (FACS), immuno-panning, microarray-based methods, and the like. Various known T cell phenotyping methods may also be applied to further increase purity of the starting population of T cells.

[0153] Furthermore, one or more freezing and thawing cycles can be performed on the starting population of T cells to enrich for the desired cell type. For example, freezing and thawing can improve the purity of a population of T cells by further removing granulocytes and, to some degree, monocytes in a mixed population of cells. Routine and conventional methods for freezing and thawing T cells can be used in conjunction with the methods disclosed herewith. In certain embodiments, following freezing, the frozen cells are thawed, washed, and allowed to rest for, e.g., one hour, at room temperature prior to activation using the disclosed methods.

[0154] In certain embodiments, the starting population of T cells may be assayed for viability using known methods. For example, the starting population of T cells may be assayed using one or more known markers of T cell identity and a viability marker (e.g., dye, antibody, and the like), wherein overlap in a signal that indicates both T cell identity and viability is indicative of the viability of the starting population of T cells. In certain embodiments, the starting population includes a percentage of viable T cells that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more of the total number of T cells in the starting population of T cells.

[0155] In certain embodiments, the starting population of T cells may be assayed for exhaustion and/or activation status. For example, the starting population of T cells may be tested for exhaustion status using one or more (e.g., 1, 2, 3, or more) T cell exhaustion markers, including but not limited to overexpression of one or more of LAG-3, PD-1, PD-LI TIM-3, 2B4, CD160, TIGIT, CTLA-4, VISTA, and the like. Activation status of T cells in the starting population can be assessed by testing for overexpression of one or more T cell activation markers (e.g., CD69, CD71, CD25, CD 137, HLA-DR, CTLA-4, L2RA/CD25, IFNy, TNFa, and the like). Additional indicators of T cell activation include, without limitation, T cell proliferation and differentiation. [0156] Following isolation and enrichment of T cells of the starting population of T cells from the biological sample of origin, the starting population of T cells may be incubated under culture conditions suitable for maintaining the T cells in a resting state prior to activation. Such methods generally do not isolate the T cells using an activating agent (e.g., CD3/CD28 beads), but other T cell isolating methods known in the art, e.g., CD4/CD8 beads.

[0157] However, in certain preferred methods of the disclosure, the T cells are concurrently activated when isolated, e.g., through the use of CD3/CD28 beads. In such instances, there is no period of resting prior to activation. However, transduction may occur after a brief culture following activation. In certain aspects, transduction occurs at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours or more after activation.

[0158] In alternative or additional aspects, the step of activation and/or contact with activating stimuli (e.g., CD3/CD28 beads) may occur for a period occurs of at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours, or more. Preferably, the time from isolation/activation to transduction is less than 30 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than

21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than

16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than

11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour. In preferred aspects, the time from isolation/activation to transduction is about 12-24 hours and more preferably about 14-22 hours, and more preferably about 16-22 hours, and more preferably about 18 hours.

[0159] The starting population of T cells may be seeded at a desirable density that facilitates T cell transduction (and/or activation if required in certain methods) with a nucleic acid vector encoding a heterologous protein (e.g., a CAR). For example, the starting population of T cells may be seeded in culture at a concentration of IxlO⁵ cells/mL to l^x10⁷ cells/mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of about 1^X10⁶ cells/mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of l^x10⁶ cells/mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of about 2 *10⁶ cells/mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of 10⁶ cells/mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of about 5^X10⁶ cells/mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of 5xl0⁶ cells/mL.

[0160] In certain methods of the disclosure, the number of isolated T cells from the whole blood sample is between about IxlO⁶ and about IxlO⁸ total T cells and the number of harvested CAR T cells is between about IxlO⁸ and about 5xl0⁸. In certain methods of the disclosure, the number of isolated T cells from the whole blood sample is between about 12.5xl0⁷ and about IxlO⁸ total CAR T cells and the number of harvested T cells is between about 7.5xl0⁷ and about 1.5xl0⁸. In certain preferred methods, the number of isolated T cells from the whole blood sample is between about 5xl0⁷ and about 7.5xl0⁷ total T cells and the number of harvested CAR T cells is between about 7.5xl0⁷ and about 1.2xl0⁸.

[0161] In certain embodiments, prior to activation, the starting population of T cells is incubated in a culture medium containing no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum (e.g., human serum). In certain embodiments, prior to activation, the starting population of T cells is incubated in a culture medium containing 2% serum. In certain embodiments, prior to activation, the starting population of T cells is incubated in a serum-free culture medium.

[0162] In certain embodiments, prior to activation, the starting population of T cells is incubated in a culture medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL- 15, and IL-21. In certain embodiments, the one or more cytokines is IL-2. In certain embodiments, the one or more cytokines is IL-7 and IL-15. In certain embodiments, the one or more cytokines is IL-2, IL-7, and IL-15. In certain embodiments, the one or more cytokines is IL- 21. In certain embodiments, the one or more cytokines is IL-21, IL-7, and IL-15. In certain embodiments, the T cells are contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng/mL of IL-2, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 100 ng/mL of IL -2. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-7, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 10 ng/mL of IL-7. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-15, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 10 ng/mL of IL-15. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-21, either alone or in combination with one or more other cytokines. In certain embodiments, prior to activation, the starting population of T cells is incubated in the absence of any of the cytokines selected from IL-2, IL-7, IL-15, and IL-2L In certain embodiments, prior to activation, the starting population of T cells is incubated in a cytokine-free culture medium.

[0163] T cell activation step

[0164] Disclosed herein, in certain embodiments, are methods of manufacturing a population of engineered T cells expressing a heterologous protein (e.g., a CAR) that include a step for activating a starting population of T cells. In certain embodiments, activation of the starting population of T cells includes contacting the starting population of T cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the T cells. In certain embodiments, the costimulatory molecule is CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In certain embodiments, the agent that stimulates a CD3/TCR complex is an anti-CD3 antibody or an antigen-binding fragment thereof (e g., full-length IgG, Fab fragment, single domain antibody, scFv, diabody, triabody, and the like). In certain embodiments, the agent that stimulates a CD3/TCR complex is small molecule or peptide ligand .In certain embodiments, the costimulatory molecule is CD28. In certain embodiments, the agent that stimulates a costimulatory molecule is an anti-CD28 antibody or an antigen-binding fragment thereof. [0165] In certain embodiments, the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule comprises a bead (e.g., a magnetic bead). In certain embodiments, the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule is a solid surface (e.g., bead) comprising an anti-CD3 antibody and/or an anti-CD28 antibody covalently attached thereto. In certain embodiments, the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead. In certain embodiments, the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule (e.g., CD28) comprise a first agent that stimulates CD3 and a second agent that stimulates the costimulatory molecule. In certain embodiments, the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule (e.g., CD28) are the same agent. In certain embodiments, the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule (e.g., CD28) is a bispecific antibody that specifically binds to CD3 and CD28. In certain embodiments, the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule (e.g., CD28) is a bead (e.g., a magnetic bead) comprising anti-CD3 antibodies and anti-CD28 antibodies covalently attached thereto. In instances where the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule (e.g., CD28) is a bead, it is understood that the bead may remain attached with a T cell at the end of the activation step (i.e., the beginning of the transfection step), and the T cell is detached from the bead prior to harvest as a result of natural degradation of the protein part of the agent and/or through the use of a cleavable linker.

[0166] In certain aspects, the stimulating agent (e.g., anti-CD3 antibody) and costimulatory molecule (e.g., anti-CD28 antibody) are attached to a surface via a cleavable linker. In such methods, one or more of these activating agents may be selectively removed by providing the cells with a stimulus that cleaves the cleavable linker(s).

[0167] For example, in certain methods of the disclosure, the stimulating agent(s) (e.g., anti-CD3 antibodies) and/or the costimulatory molecule(s) (e.g., anti-CD28 antibodies) are attached to a support via a cleavable linker. Similarly, in methods not concurrently isolating and activating the T cells, e.g., using CD4/CD8 beads, the anti-CD4 and anti-CD8 antibodies may likewise be surface attached using cleavable linkers. In such methods, the anti-CD4/CD8 binding may be stopped through linker cleavage, after which, for example, the cells may be contacted with an activating agent(s). In certain methods of the disclosure, the one or more anti-CD3 antibodies and the one or more anti-CD28 antibodies attached to the same support. Alternatively, the antibodies are bound to different supports, e.g., different beads.

[0168] In preferred methods, one or more surface-bound anti-CD3 antibodies and/or the one or more anti-CD28 antibodies are used to concurrently isolate and activate T cells from a whole blood sample. In certain aspects, the anti-CD3 and anti-CD28 antibodies are attached to the support via a cleavable linker. The linker may be, for example, an enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker. Accordingly, methods of the disclosure may further include contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface.

[0169] In certain aspects, the surface is a solid surface. Preferably, the solid surface is a bead, well, chip, or microfluidic channel. More preferably, the solid surface is a bead.

[0170] In some aspects, the surface comprises a polymer. In certain aspects, the polymer is a hydrogel. In some methods, the surface comprises a polymer scaffold. In certain embodiments, the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule comprises an anti-CD3 antibody and/or an anti -costimulatory molecule antibody covalently attached to a colloidal polymeric matrix (e.g., nanomatrix). In certain embodiments, the matrix comprises or consists of a polymeric, for example, biodegradable or biocompatible inert material, for example, which is non-toxic to cells. In certain embodiments, the matrix is composed of hydrophilic polymer chains, which obtain maximal mobility in aqueous solution due to hydration of the chains. In certain embodiments, the mobile matrix may be of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. A polysaccharide may include for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectins, xanthan, guar gum or alginate. Other polymers may include polyesters, polyethers, polyaciylates, polyacrylamides, polyamines, polyethylene imines, polyquatemium polymers, polyphosphazenes, polyvinylalcohols, polyvinylacetates, polyvinylpyrrolidones, block copolymers, or polyurethanes. [0171] In certain embodiments, contacting of the starting population of T cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule is performed once at the start of the activation step. In certain embodiments, contacting of the starting population of T cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule is performed concurrently with T cell isolation. In certain embodiments, contacting of the starting population of T cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule is performed once at the start of the activation step, and one or more (e.g., 1, 2, 3, or more) times throughout the duration of the activation step.

[0172] Without wishing to be bound by any theory, a skilled artisan will appreciate that the duration of binding of the agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule to one or more cells of the starting population of T cells will depend on the specific agent(s) used, the concentration of the agent(s), the concentration of cells seeded in culture, among other factors.

[0173] In certain embodiments, the duration of the activation step is 12-24 hours (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours). In certain embodiments, the duration of the activation step is about 18 hours (e.g., 16, 17, 18, 19, or 20 hours). In certain embodiments, the duration of the activation step is 18 hours. In certain embodiments, after the activation step, the T cells remain associated with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the T cells, such that activation may continue during the subsequent step(s). In certain embodiments, activation effectively continues until detachment of the T cells from the agent (e.g., by natural degradation of the protein part of the agent and/or application of a stimulus that cleaves a cleavable linker).

[0174] In certain embodiments, during the activation step, the starting population of T cells is incubated in a culture medium containing no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum.

[0175] In certain embodiments, during the activation step, the starting population of T cells is incubated in a culture medium 2% serum. In certain embodiments, during the activation step, the starting population of T cells is incubated in a cultivation medium comprising a basal cultivation medium and serum (e.g., 2% serum). In certain embodiments, the cultivation medium does not further comprise any added cytokine or growth factor, other than the proteins from the serum. In certain embodiments, the cultivation medium does not further comprise any added protein (e.g., soluble protein), other than the proteins from the serum.

[0176] In certain embodiments, during the activation step, the starting population of T cells is incubated in a serum-free culture medium. In certain embodiments, during the activation step, the starting population of T cells is incubated in a basal cultivation medium not supplemented by serum. In certain embodiments, the basal cultivation medium does not comprise any cytokine or growth factor. In certain embodiments, the basal cultivation medium does not comprise any added protein. It is understood that during the cell culture, the cells in the cultivation medium may secrete proteins. It is also understood that the agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule may naturally degrade over time, releasing fragments into the cultivation medium. None of these proteins are considered as "added proteins. "

[0177] In certain embodiments, during the activation step, the starting population of T cells is incubated in a culture medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL-15, and IL-21. In certain embodiments, the one or more cytokines is IL-2. In certain embodiments, the one or more cytokines is IL-7 and IL-15. In certain embodiments, the one or more cytokines is IL-2, IL-7, and IL-15. In certain embodiments, the one or more cytokines is IL-2L In certain embodiments, the one or more cytokines is IL-21, IL-7, and IL-15. In certain embodiments, the T cells are contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng/mL of IL-2, either alone or in combination with one or more other cytokines.

[0178] In certain embodiments, the T cells are contacted with 100 ng/mL of IL-2. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-7, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 10 ng/mL of IL-7. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL- 15, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 10 ng/mL of IL-15. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-21, either alone or in combination with one or more other cytokines. In certain embodiments, prior to activation, the starting population of T cells is incubated in the absence of any of the cytokines selected from IL-2, IL-7, IL-15, and IL-21. Either a serum-free cultivation medium (e.g., a basal cultivation medium) or a serum-supplemented cultivation medium can be provided in the presence or absence of the cytokines and combinations thereof disclosed herein.

[0179] In preferred methods, the cultivation medium is a serum free cultivation medium and comprises at least one cytokine. Preferably, the at least one cytokine comprises one or more of IL- 2, IL-21, IL-7, and IL-15. In some methods, the at least one cytokine comprises one or more of IL-21, IL-7, and IL-15 and does not comprise IL-2.

[0180] In certain embodiments, prior to activation, the starting population of T cells is incubated in a cytokine-free culture medium or without added cytokine. In other embodiments, contacting of the starting population of T cells with the agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule (e.g., CD28) is performed simultaneously while contacting the population of cells with the one or more cytokines. In certain embodiments, contacting of the starting population of T cells with the agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule (e.g., CD28) is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hrs or more prior to contacting with the one or more cytokines.

Transfection step: delivery of heterologous nucleic acids to T cells

[0181] Disclosed herein, in certain embodiments, are methods for producing an engineered population of T cells expressing a heterologous protein (e.g., a CAR), including delivering a nucleic acid encoding the heterologous protein to the T cells after the activation step. Also disclosed herein are polynucleotides encoding a heterologous protein (e.g., a CAR).

[0182] In certain embodiments, delivery of the nucleic acid(s) encoding a heterologous protein to the T cells is performed no later than about 18 hours (e.g., no later than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hours or less) after initiating the activation step. In certain embodiments, delivery of the nucleic acid(s) encoding a heterologous protein to the T cells is performed about 18 hours (e.g., 16, 17, 18, 19, or 20) hours after initiating the activation step. In certain embodiments, delivery of the nucleic acid(s) encoding a heterologous protein to the T cells is performed 18 hours after initiating the activation step. Where transfection begins after initiation of the activation step, the cells at the end of the activation step (i.e., the beginning of the transfection step) are referred to herein as an "activated population of T cells," although at least a portion of the cells in the population may not have been fully activated and further activation can occur during the transfection step. In other embodiments, delivery of the nucleic acid(s) encoding a heterologous protein to a starting population of T cells is performed concurrently (e.g., simultaneously) with the activation step.

[0183] In certain embodiments, the disclosed methods include contacting the population of T cells, after the activation step, with an effective amount of a polynucleotide encoding the heterologous protein, such that the cell exhibits stable expression of the heterologous protein for at least 1 hour (hr), 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 12 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, or more.

[0184] In certain embodiments, a polynucleotide encoding a heterologous protein (e.g., a CAR) includes a codon-optimized nucleic acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 55, 65, 75, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) nucleotide differences as compared to the parent (i.e., non-codon optimized) nucleic acid. The nucleic acid sequence can be codon-optimized in accordance with various principles, for example, the principle that the frequency of occurrence of synonymous codons (e.g., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. This process may be performed on any of the sequences described in this specification to enhance expression or stability. Codon optimization may be performed using conventional methods. The sequence surrounding the translational start site can be converted to a consensus Kozak sequence according to known methods.

Nucleic acid vectors

[0185] In addition to achieving high rates of transcription and translation, stable expression of an exogenous gene (e.g., a polynucleotide encoding a heterologous polypeptide or a functional fragment thereof) in a mammalian cell can be achieved by integration of the polynucleotide containing the gene into the nuclear genome of the mammalian cell. A variety of vectors for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of a mammalian cell have been developed. Expression vectors for use in the compositions and methods described herein contain a polynucleotide sequence that encodes a heterologous protein (e.g., a CAR), as well as, e.g., additional sequence elements used for the expression of these agents and/or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of a heterologous protein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of heterologous protein contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5' and 3' untranslated regions, an internal ribosomal entry site ORES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin.

[0186] Expression vectors for use in the compositions and methods described herein may express a heterologous protein (e.g., a CAR) from monocistronic or polycistronic expression cassettes. A monoci stronic expression cassette contains a polynucleotide sequence that encodes a single gene. Host cells described herein can be transfected with multiple vectors, for example, each containing a monocistronic expression cassette, or with a single vector containing more than one monocistronic expression cassette. Polycistronic expression cassettes can be used to simultaneously express two or more proteins from a single transcript. Polycistronic expression cassettes may include bicistronic or tri ci stronic expression cassettes, which can be used to generate two or three proteins, respectively, from a single transcript and may include IRES sequences to recruit ribosomes to initiate translation from a region of the mRNA other than the 5' cap. Alternatively, foot-and-mouth disease virus 2A (FMDV 2A) polynucleotides can be utilized to express two or more genes (e.g., 2 genes, 3 genes, or more), and can be used in polycistronic expression cassettes to produce equimolar levels of multiple genes from the same transcript. FMDV 2A mediates a co-translational cleavage event, which separates proteins linked by 2A sequences, and multiple 2A sequences may be used in one vector. 2A-like sequences from other viruses can also be used in the compositions and methods described herein, including the 2A-like sequences from equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and Thosea asigna virus (T2A).

Viral vectors

[0187] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell. Viral genomes are particularly useful vectors for gene delivery as the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors are a retrovirus (e.g., Retroviridae family viral vector, such as a lentiviral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picomavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types I and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MV A), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses are: avian leukosissarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, alpharetrovirus, gammaretrovirus, and spumavirus. Other examples are murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lenti viruses.

[0188] Exemplary lentiviral vectors that may be used in accordance with the present disclosure include vectors derived from human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV).

[0189] Retroviral vectors typically are constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by a gene of interest or expression cassette of interest (e.g., an engineered nucleic acid as described here). Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal. Accordingly, in some embodiments, a minimum retroviral vector comprises from 5' to 3' a 5' long terminal repeat (LTR), a packaging signal, an optional exogenous promoter and/or enhancer, an exogenous gene of interest (or engineered nucleic acid), and a 3' LTR. In some embodiments, if no exogenous promoter is provided, gene expression may be driven by the 5' LTR, which is a weak promoter and requires the presence of Tat to activate expression. In many embodiments, structural genes can be provided in separate vectors for manufacture of the lentivirus, rendering the produced virions replication-defective. Specifically, with respect to lentivirus, the packaging system may comprise a single packaging vector encoding the Gag, PO1, Rev, and Tat genes, and a third, separate vector encoding the envelope protein Env (usually VSV-G due to its wide infectivity). To improve the safety of the packaging system, the packaging vector can be split, expressing Rev from one vector, Gag and POI from another vector. Tat can also be eliminated from the packaging system by using a retroviral vector comprising a chimeric 5' LTR, wherein the U3 region of the 5' LTR is replaced with a heterologous regulatory element.

[0190] Nucleic acids (e.g., genes) to be packaged into a retrovirus (e.g., a lentivirus) can be incorporated into the proviral backbone in several general ways. The most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the LTR. Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.

[0191] Accordingly, nucleic acids (e.g., genes) to be packaged into a retrovirus are flanked by 5 ' and 3 ' LTRs, which serve to promote transcription and polyadenylation of the virion RNAs, respectively. The term "long terminal repeat" or "LTR" refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R and U5 regions. LTRs generally provide functions fundamental to the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals, and sequences needed for replication and integration of the viral genome. The U3 region contains the enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. In certain embodiments, the R region comprises a trans-activation response (TAR) genetic element, which interacts with the trans-activator (tat) genetic element to enhance viral replication. This element is not required in embodiments wherein the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0192] In some embodiments, a retroviral vector comprises a modified 5' LTR and/or 3 ' LTR. Modifications of the 3 ' LTR are often made to improve the safety of lenti viral or retroviral systems by rendering viruses replication-defective. In some embodiments, a retroviral vector is a selfinactivating (SIN) vector. As used herein, a SIN retroviral vector refers to a replication defective retroviral vector in which the 3 ' LTR U3 region has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the 3 ' LTR U3 region is used as a template for the 5' LTR U3 region during viral replication and, thus, the viral transcript cannot be made without the U3 enhancer-promoter. In some embodiments, a 3' LTR is modified such that the U5 region is replaced, for example, with an ideal polyadenylation sequence. It should be noted that modifications to the LTRs such as modifications to the 3' LTR, the 5' LTR, or both 3 ' and 5' LTRs, are also included in some embodiments of the present disclosure.

[0193] In some embodiments, the U3 region of the 5' LTR is replaced with a heterologous promoter to drive transcription of the viral genome during production of viral particles. [0194] Examples of heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) [0195] (thymidine kinase) promoters. Typical promoters are able to drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination to generate replication-competent virus, because there is no complete U3 sequence in the virus production system.

[0196] Adjacent to a 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site). As used herein, the term "packaging signal" or "packaging sequence" refers to sequences located within the retroviral genome which are required for encapsidation of retroviral RNA strands during viral particle formation (see e.g., Clever et al., 1995 J. Virology, 69(4):2101-09). The packaging signal may be a minimal packaging signal (also referred to as the psi [yr] sequence) needed for encapsidation of the viral genome.

[0197] In some embodiments, a retroviral vector (e.g., lentiviral vector) further comprises a FLAP As used herein, the term "FLAP" refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (CPP T and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and in Zennou el al. (2000) Cell 101 : 173. During reverse transcription, central initiation of the plus-strand DNA at the cPPT and central termination at the CTS lead to the formation of a three-stranded DNA structure: a central DNA flap. While not wishing to be bound by any theory, the DNA flap may act as a cis-active determinant of lentiviral genome nuclear import and/or may increase the titer of the virus. In some embodiments, retroviral vector backbones comprise one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors. For example, in some embodiments, a transfer plasmid includes a FLAP element. In some embodiments, a vector of the present disclosure comprises a FLAP element isolated from HIV-1.

[0198] In some embodiments, a retroviral vector (e.g., lentiviral vector) further comprises an export element. In some embodiments, retroviral vectors comprise one or more export elements. The term "export element" refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. [0199] Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) RRE (see e.g., Cullen el al., (1991) J. Virol. 65: 1053; and Cullen et al., (1991) Cell 58: 423) and the hepatitis B virus post-transcriptional regulatory element (HPRE). Generally, the

[0200] RNA export element is placed within the 3' UTR of a gene, and can be inserted as one or multiple copies.

[0201] In some embodiments, a retroviral vector (e.g., lentiviral vector) further comprises a posttranscriptional regulatory element. A variety of posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) (see Zufferey et al., (1999) J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); an optimized posttranscriptional regulatory element (oPRE; see Schambach et al., (2006) Gene Therapy 13, 641-45); and the like (Liu et al., (1995), Genes Dev., 9: 1766). The posttranscriptional regulatory element is generally positioned at the 3' end the heterologous nucleic acid sequence. This configuration results in synthesis of an mRNA transcript whose 5' portion comprises the heterologous nucleic acid coding sequences and whose 3' portion comprises the posttranscriptional regulatory element sequence. In some embodiments, vectors of the present disclosure lack or do not comprise a posttranscriptional regulatory element such as a WPRE or HPRE, because in some instances these elements increase the risk of cellular transformation and/or do not substantially or significantly increase the amount of mRNA transcript or increase mRNA stability. Therefore, in certain embodiments, vectors of the present disclosure lack or do not comprise a WPRE or HPRE as an added safety measure.

[0202] Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. Accordingly, in some embodiments, a retroviral vector (e.g., lentiviral vector) further comprises a polyadenylation signal. The term "polyadenylation signal" or "polyadenylation sequence" as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase H. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a polyadenylation signal are unstable and are rapidly degraded. Illustrative examples of polyadenylation signals that can be used in a vector of the present disclosure, include an ideal polyadenylation sequence (e.g., AATAAA, ATTAAA AGTAAA), a bovine growth hormone polyadenylation sequence (BGHpA), a rabbit B-globin polyadenylation sequence (rBgpA), or another suitable heterologous or endogenous polyadenylation sequence known in the art.

[0203] In some embodiments, a retroviral vector further comprises an insulator element.

[0204] Insulator elements may contribute to protecting retrovirus-expressed sequences, e.g., therapeutic genes, from integration site effects, which may be mediated by cis-acting elements present in genomic DNA and lead to deregulated expression of transferred sequences (i.e., position effect; see, e.g., Burgess-Beusse et al., (2002) Proc. Natl. Acad. Sci., USA, 99: 16433; and Zhan et al., 2001, Hum. Genet., 109:471). In some embodiments, a retroviral vector comprises an insulator element in one or both LTRs, or elsewhere in the region of the vector that integrates into the cellular genome. Suitable insulators for use in the present disclosure include, but are not limited to, the chicken B-globin insulator (see Chung et al., (1993). Cell 74: 505; Chung et al., (1997) Proc. Natl. Acad, sci., USA 94:575; and Bell al., 1999. Cell 98:387). Examples of insulator elements include, but are not limited to, an insulator from a B-globin locus, such as chicken HS4.

[0205] Non-limiting examples of lentiviral vectors include pLVX-EF I alpha-AcGFPl-C I (Clontech Catalog 1984), pLVX-EF lalpha-IRES-mCherry (Clontech Catalog 1987), pLVX-Puro (Clontech Catalog #632159), pLVX-IRES-Puro (Clontech Catalog #632186), pLenti6/V5- DEST™ (Thermo Fisher), pLenti6.2/V5-DEST™ (Thermo Fisher), pLKO. l (Plasmid #10878 at Addgene), pLKO.3G (Plasmid #14748 at Addgene), pSico (Plasmid #11578 at Addgene), pLJMl- EGFP (Plasmid #19319 at Addgene), FUGW (Plasmid #14883 at Addgene), pLVTHM (Plasmid #12247 at Addgene), pLVUT-tTR-KRAB (Plasmid #11651 at Addgene), pLL3.7 (Plasmid #11795 at Addgene), pLB (Plasmid #11619 at Addgene), pWPXL (Plasmid #12257 at Addgene), pWPI (Plasmid #12254 at Addgene), EF.CMV.RFP (Plasmid #17619 at Addgene), pLenti CMV Puro DEST (Plasmid #17452 at Addgene), pLenti -puro (Plasmid #39481 at Addgene), pULTRA (Plasmid #24129 at Addgene), pLX301 (Plasmid #25895 at Addgene), pHIV-EGFP (Plasmid #21373 at Addgene), pLV-mCherry (Plasmid #36084 at Addgene), pLionll (Plasmid #1730 at Addgene), plnducerlO-mir-RUP-PheS (Plasmid #44011 at Addgene). These vectors can be modified to be suitable for therapeutic use. For example, a selection marker (e.g., puro, EGFP, or mCherry) can be deleted or replaced with a second exogenous gene of interest. Further examples of lentiviral vectors are disclosed in U. S. Patent Nos. 7,629,153, 7,198,950, 8,329,462, 6,863,884, 6,682,907, 7,745,179, 7,250,299, 5,994,136, 6,287,814, 6,013,516, 6,797,512, 6,544,771, 5,834,256, 6,958,226, 6,207,455, 6,531,123, 5,352,694, and PCT Publication No. WO 2017/091786.

[0206] In certain embodiments, a nucleic acid vector, such as a viral vector, encoding a heterologous protein disclosed herein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a multiplicity of infection (MOI) of between 0 and 24 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 4. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 5. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 6. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 7. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 8. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 9. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 10. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 12. In certain embodiments, a nucleic acid vector encoding a heterologous protein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 14.

Nucleic acid transfer vehicles [0207] Nucleic acid transfer vehicles are another advantageous method for the delivery of polynucleotides encoding a heterologous protein of the disclosure to target cells (e.g., a starting population of T cells). In certain embodiments, the nucleic acid transfer vehicle is a nanoparticle, such as a lipid nanoparticle (e.g., LNP), non-lipid polymeric core-shell nanoparticle, or a biodegradable nanoparticle. In certain embodiments, the LNP comprises one or more of ionizable lipids, PEGylated lipids, structural lipids (e.g., cholesterol), and/or helper lipids.

[0208] Without wishing to be bound by theory, it is thought that transfer vehicles described herein encapsulate nucleic acids encoding the heterologous protein from degradation and provide for effective delivery of the nucleic acid(s) to target cells in vivo and in vitro.

Cell culture conditions

[0209] In certain embodiments, contacting of the activated population of T cells with polynucleotides encoding a heterologous protein (e.g., a CAR) is performed in a culture medium containing no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum (e.g., human serum). In certain embodiments, contacting of the activated population of T cells with polynucleotides encoding a heterologous protein (e.g., a CAR) is performed in a culture medium containing 2% serum. In certain embodiments, during the transfection step, the activated population of T cells is incubated in a cultivation medium comprising a basal cultivation medium and serum (e.g., 2% serum).

[0210] In certain preferred aspects, the basal cultivation medium is a serum-free medium. In certain preferred methods, following contact with a polynucleotide encoding a heterologous protein (e.g., a CAR), the activated T cells are cultured in a serum-free cultivation medium for a period of between 24 hours and 60 hours. In certain methods of the disclosure, the cultivating step is for a period of between 29 hours and 59 hours. In preferred methods of the disclosure, the cultivating step is for a period of between 36 hours and 52 hours. In more preferred aspects, the cultivating step is for a period of between 46 hours and 50 hours. In certain aspects, the cultivating step is for a period of about 48 hours.

[0211] Methods of the disclosure may further include harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein. [0212] In certain embodiments, the cultivation medium does not further comprise any added cytokine or growth factor, other than the proteins from the serum. In certain embodiments, the cultivation medium does not further comprise any added protein, other than the proteins from the serum.

[0213] In certain embodiments, contacting of the activated population of T cells with polynucleotides encoding a heterologous protein (e.g., a CAR) is performed in a serum-free culture medium. In certain embodiments, contacting of the activated population of T cells with polynucleotides encoding a heterologous protein (e.g., a CAR) is performed in a basal cultivation medium not supplemented by serum. In certain embodiments, the basal cultivation medium does not comprise any cytokine or growth factor. In certain embodiments, the basal cultivation medium does not comprise any added protein. It is understood that during the cell culture (e g., during the transfection step, or in the previous activation step if there is no medium change between the activation step and transfection step), the cells in the cultivation medium may secrete proteins. It is also understood that the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule may naturally degrade overtime, releasing fragments into the cultivation medium. None of these proteins are considered as “added proteins”.

[0214] In certain embodiments, contacting of the activated population of T cells with polynucleotides encoding a heterologous protein (e.g., a CAR) is performed in a culture medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL- 15, and IL- 21. In certain embodiments, the one or more cytokines is IL-2. In certain embodiments, the one or more cytokines is IL-7 and IL-15. In certain embodiments, the one or more cytokines is IL-2, IL-

7, and IL-15. In certain embodiments, the one or more cytokines is IL-21. In certain embodiments, the one or more cytokines is IL-21, IL-7, and IL-15. In certain embodiments, the T cells are contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng/mL of IL-2, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 100 ng/mL of IL-2. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-7, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 10 ng/mL of IL-7. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-15, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 10 ng/mL of IL-15. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-21, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 20 ng/mL of IL-21, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are cultivated in the absence of any of the cytokines selected from IL-2, IL-7, IL- 15, and IL-21. Either a serum-free cultivation medium (e.g., a basal cultivation medium) or a serum supplemented cultivation medium can be provided in the transfection step, in the presence or absence of the cytokines and combinations thereof disclosed herein.

[0215] In certain embodiments, contacting of the activated population of T cells with polynucleotides encoding a heterologous protein (e.g., a CAR) is performed in a cytokine-free culture medium or without added cytokine. In other embodiments, cytokines were added to the cultivation medium at the initiation of the previous step (activation step). It is understood that at the beginning of the transfection step, the effective concentration of the cytokines may have decreased as a result of degradation or have increased as a result of secretion from the cells (e.g., T cells) in the culture. In certain embodiments, additional cytokines are added at the beginning of the transfection step (e.g., of the same kind and amount as added at the beginning of the activation step).

[0216] In certain embodiments, contacting of the activated population of T cells with a polynucleotide encoding a heterologous protein (e.g., a CAR) is performed simultaneously with contacting with the one or more cytokines.

[0217] In certain embodiments, contacting of the activated population of T cells with a polynucleotide encoding a heterologous protein (e.g., a CAR) is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 hours or more after contacting with the one or more cytokines.

[0218] In certain embodiments, contacting of the activated population of T cells with a polynucleotide encoding a heterologous protein (e.g., a CAR) is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 hours or more prior to contacting with the one or more cytokines.

[0219] In certain aspects, the culturing step produces between a 1.0- and a 4-fold expansion of the harvested T cells. In certain aspects, the culturing step produces between a 1.2- and a 4-fold expansion of the harvested T cells.

Cultivation duration

[0220] When the T cells are contacted with polynucleotides encoding a heterologous protein of the disclosure (e.g., a CAR), the cells may be cultivated in culture under conditions and for a time sufficient to facilitate integration of the nucleic acid into the genome of the T cells for stable expression of the heterologous protein in the T cells, and/or attainment of a desired T cell phenotype (e.g., TNSCM cell phenotype).

[0221] Surprisingly, the present Inventors have discovered that the two-to-three-day methods for manufacture of the disclosure obtain a very high CAR+ percentage after only a brief culture (generally about 48 hours). For example, the T cells contacted with polynucleotides encoding a heterologous protein of the disclosure (e.g., a CAR) are cultivated in culture between 24 and 72 hours (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours). In certain embodiments, the T cells contacted with polynucleotides encoding a heterologous protein of the disclosure (e.g., a CAR) are cultivated in culture for about 48 hours (e.g., about 44, 45, 46, 47, 48, 49, 50, 51, hours 52 hours). In certain embodiments, the T cells contacted with polynucleotides encoding a heterologous protein of the disclosure (e.g., a CAR) are cultivated in culture for 48 hours.

Harvest and storage

[0222] Following activation, introduction of a heterologous nucleic acid, and cultivation, the population of T cells may be harvested for storage and subsequent therapeutic use. In certain embodiments, the T cells are harvested for storage no later than 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, or 36 hours after initiation of the activation step.

[0223] In certain embodiments, the T cells are harvested for storage no later than about 64 (e.g., 62, 63, 64, 65, or 66) hours after initiation of the activation step. In certain embodiments, the T cells are harvested for storage no later than 64 hours after initiation of the activation step. In certain embodiments, the T cells are harvested for storage no later than about 72 (e.g., 70, 71, 72, 73, or 74) hours after initiation of the activation step. In certain embodiments, the T cells are harvested for storage no later than 72 hours after initiation of the activation step.

[0224] In certain embodiments, the T cells are harvested for storage no later than 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30 hours after completion of the activation step (i.e., the beginning of the transfection step). In certain embodiments, the T cells are harvested for storage about 48 (e.g., 46, 47, 48, 49, or 50) hours after contacting the T cells with a polynucleotide encoding a heterologous protein (e.g., a CAR).

[0225] In certain embodiments, the harvesting step is accompanied by one or more assays intended to test for one or more parameters of cell viability, cell count, purity (e.g., fraction of T cells in the total population of cells), fraction of cells expressing the heterologous protein, T cell phenotype, T cell activation/exhaustion status, amount of proliferation of T cells relative to the starting population, T cell cytotoxicity, cytokine release, among others.

[0226] The harvesting step may be followed by a storage step, whereby the T cells produced according to the disclosed method are maintained under conditions suitable to preserve the cells, including their viability, as well as functional and molecular profiles, until later therapeutic application or quality control testing. In certain embodiments, the storage step comprises one or more of: (1) reformulating the population of cells in a storage medium (e.g., a refrigeration medium, freezing medium, or cry opreservation medium); (2) transfer of the cells to a suitable container means for storage under appropriate storage conditions; and (3) maintenance of the cells under suitable conditions.

Properties of engineered T cells

[0227] Disclosed herein, in certain embodiments, are engineered T cells produced or obtainable according to the methods of the disclosure. In certain embodiments, the T cells are engineered (e.g., genetically manipulated) to express a heterologous protein, such as a CAR. In certain embodiments, an engineered T cell or a population of engineered T cells stably express a CAR, [0228] e g., by genomic integration of a heterologous nucleic acid sequence encoding the CAR in the T cell.

[0229] Protein/CAR expression

[0230] The brief two-to-three-day methods for manufacturing CAR T cells disclosed herein, including those starting from whole blood and/or PBMC samples, produce a population of engineered T cells expressing a heterologous protein (e.g., a CAR) from a starting population of T cells at a very high percentage such that at 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the cells of the starting population of T cells express the heterologous protein (e.g., CAR).

[0231] In certain embodiments, expression of the heterologous protein (e.g., CAR) is measured in the T cells 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step. In certain embodiments, expression of the heterologous protein (e.g., CAR) is measured in the T cells 3 days after the harvesting step. In certain embodiments, expression of the heterologous protein (e.g., CAR) is measured in the T cells at least 3, 4, 5, 6, 7, 8, 9, 10, or more days after initiation of the transfection step, such that transient expression from unintegrated vector is not significantly detected. Methods for quantifying expression of a heterologous protein at the genomic, transcriptomic, and proteomic levels in cells are well known in the art and include, without limitation, flow cytometry (e.g., fluorescence assisted cell sorting; FACS), quantitative (q)PCR, digital (d)PCR, fluorescence imaging, integration site analysis, RNA sequencing, in situ hybridization, immunoprecipitation, and Topanga assay, among others.

[0232] Surprisingly, the present Inventors have discovered that methods of the disclosure for manufacturing CAR T cells produce cells exhibiting an improved CAR+ percentage relative to other, existing methods. This improved CAR+ percentage, generally greater than at least 50-60%, was obtainable from methods of the disclosure using: (i) whole blood as a starting sample; (ii) isolation of T cells directly from whole blood; (iii) concurrent T cell isolation and activation; (iv) a transduction step approximately 10-25 hours (preferably about 18 hours) after T cells isolation; and (v) a brief culture step after transduction (generally about 40-60 hours and preferably around 48 hours). [0233] In certain embodiments, the methods disclosed herein produce a population of engineered T cells expressing a CAR that secretes increased amounts of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32) proteins selected from the group consisting of IFNg, Granzyme B, IL-113, IL -2, IL-4, IL-5, IL-6, IL-7, IL- 8, IL-9, IL- 10, IL- 12, IL-13, IL- 15, IL-17A, IL-17F, IL-21, IL-22, IP- 10, MCP1, MCP4, TNFa, TNF13, TGF13, GM-CSF, MIPla, MIPU3, CCL11, perform, RANTES, sCD137, and VEGF after being contacted with a target cell that expresses an antigen (e.g., CD 19) bound by the CAR, as compared to a T cell that has not been contacted with the target cell or has been contacted with a control target cell that does not express the antigen. In certain embodiments, secretion of the one or more proteins after contact with the target cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, or higher as compared to secretion of the one or more proteins in the absence of the target cell or in the presence of a control target cell that does not express the antigen. In certain embodiments, cytokine secretion is measured in the T cells 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step.

[0234] Methods for quantifying cytokine release from T cells are well known in the art and include, without limitation, ELISA, flow cytometry (e.g., cytometric bead array assay), and proteomic analysis (e.g., multiplexed single cell chip analysis), among others.

[0235] In certain preferred embodiments, the short manufacturing time afforded by the presently disclosed methods produce CAR T cells with an improved polyfunctional phenotype upon contact with a target (e.g., CD 19). In certain preferred embodiments, the methods disclosed herein produce a population of engineered T cells expressing a CAR of which at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, or more are a population of polyfunctional CAR T cells that secrete increased amounts of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32) proteins selected from the group consisting of IFNg, Granzyme B, IL-113, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL- 13, IL-15, IL-17A, IL-17F, IL-21, IL-22, IP-10, MCP1, MCP4, TNFa, TNF13, TGF13, GM-CSF, MIPla, MIP113, CCL11, perform, RANTES, sCD137, and VEGF after being contacted with a target cell that expresses an antigen (e.g., CD 19) bound by the CAR, as compared to a T cell that has not been contacted with the target cell or has been contacted with a control target cell that does not express the antigen. In certain embodiments, secretion of the one or more proteins after contact with the target cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, or higher as compared to secretion of the one or more proteins in the absence of the target cell or in the presence of a control target cell that does not express the antigen. In certain embodiments, cytokine secretion is measured in the T cells 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step.

[0236] In preferred methods of the disclosure, the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg. In preferred aspects, at least 1% of the harvested T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MIP-la and MIP-lb. In certain methods, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNg and Granzyme B.

[0237] In preferred methods of the disclosure, the polyfunctional T cells and/or a portion of the population thereof, comprise two or more of cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

[0238] In some methods of the disclosure, the polyfunctional T cells and/or a portion of the population of polyfunctional T cells comprise: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

[0239] In certain embodiments, the methods disclosed herein produce a population of engineered T cells expressing a CAR that exhibit increased expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD 137 after being contacted with a target cell that expresses an antigen (e.g., CD 19) bound by the CAR, as compared to expression of the one or more T cell activation markers in the absence of the target cell or in the presence of a control target cell that does not express the antigen.

[0240] In certain embodiments, expression of the one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, or more, as compared to expression of the one or more T cell activation markers in the absence of the target cell or in the presence of a control target cell that does not express the antigen. In certain embodiments, expression of the one or more T cell activation markers is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step. Methods for quantifying expression of T cell activation markers are well known in the art and include, without limitation ELISA, flow cytometry, quantitative (q)PCR, digital (d)PCR, fluorescence imaging, in situ hybridization, and proteomic analysis, among others.

[0241] In certain embodiments, the methods disclosed herein produce a population of engineered T cells expressing a CAR that exhibit increased cytotoxicity against a target cell expressing an antigen (e.g., CD 19) bound by the CAR, as compared to cytotoxicity against a cell that does not express the antigen. In certain embodiments, cytotoxicity is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, or more, as compared to cytotoxicity against a cell that does not express the antigen. In certain embodiments, cytotoxicity of the engineered T cells against a target cell expressing an antigen (e.g., CD19) is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step. Methods of assessing cytotoxicity of engineered T cells expressing a heterologous protein (e.g., a CAR) against a target cell are well known in the art and include, without limitation, chromium release assay, bioluminescence assay (e.g., luciferase-mediated bioluminescence imaging), real-time impedance-based analysis, flow cytometry (e.g., in combination with a viability dye, such as CTV), CFSE/PI assay, among others.

[0242] In certain embodiments, the methods disclosed herein produce a population of engineered T cells expressing a CAR that exhibit increased proliferation after being contacted with a target cell that expresses an antigen (e.g., CD 19) bound by the CAR, as compared to proliferation in the absence of the target cell or in the presence of a control target cell that does not express the antigen. In certain embodiments, proliferation is increased by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more, as compared to proliferation in the absence of the target cell or in the presence of a control target cell that does not express the antigen.

[0243] In certain embodiments, proliferation of the T cells following contact with a target cell expressing an antigen (e.g., CD19) is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step. Methods of assessing proliferation of engineered T cells expressing a heterologous protein (e.g., a CAR) are well-known in the art and include, without limitation, MTT assay, MTS assay, cell counting (e.g., via flow cytometry), CFSE/flow cytometric analysis, and [3H] thymidine incorporation, among others.

[0244] In certain embodiments, the methods disclosed herein produce a population of engineered T cells various phenotypes, such as naive T (TN) cells characterized as CD45RO- CCR7+, and CD95-, central memory T (TCM) cells characterized as CD45RO+ and CCR7+, effector memory T (TEM) cells characterized as CD45RO+ and CCR7-, stem memory T (TSCM) cells characterized as CD45RO-, CCR7+, and CD95+, and effector memory cells re-expressing CD45RA T (TEMRA) cells characterized as CD45RO- and CCR7-. Alternative characterizations of these T cell subsets include but are not limited to naive T (TN) cells characterized as CD45RA+, CCR7+, and CD95, central memory T (TCM) cells characterized as CD45RA- and CCR7+, effector memory T (TEM) cells characterized as CD45RA- and CCR7-, stem memory T (TSCM) cells characterized as CD45RA+, CCR7+, and CD95+, and effector memory cells re-expressing CD45RA T (TEMRA) cells characterized as CD45RA+ and CCR7-.

[0245] Surprisingly and advantageously, the methods disclosed herein are able to produce a population of engineered T cells expressing a heterologous protein (e.g., a CAR) that include an increased amount of naive and stem cell memory T (TNSC ) cells (identified by markers such as CD45RA+/CD45RO-/CCR7+/CD62L+; CD45RA+/CCR7+; or CD45RO-/CCR7+) cells as compared to the starting population of T cells. In certain embodiments, the T cells produced according to the disclosed methods comprise TNSCM cells in an amount higher by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 2-fold, 2.5-fold, 3-fold, 3.5- fold, 4-fold, or more), as compared to the number of TNSCM cells in the starting population of T cells. In certain embodiments, the T cells produced according to the disclosed methods comprise stem cell memory T (TSCM) cells (identified by markers such as CD45RA+/CD45RO- /CCR7+/CD62L+/CD95+; CD45RA+/CCR7+/CD95+; or CD45RO-/CCR7+/CD95+) in an amount higher by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, or more, as compared to the number of TSC cells in the starting population of T cells.

[0246] In certain preferred embodiments, the T cells produced according to the disclosed methods comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more of TNSC cells, out of all the T cells harvested. In certain embodiments, the T cells produced according to the disclosed methods comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more of TSCM cells, out of all the T cells harvested. In certain embodiments, the amount of TNSCM cells in the engineered population of T cells is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step. Methods for quantifying TNSCM cells are well known in the art and include, without limitation, flow cytometry (e.g., FACS) and fluorescence microscopy, among others.

[0247] In certain embodiments, the methods disclosed herein produce a population of engineered T cells expressing a heterologous protein (e g., a CAR) that include a decreased amount of effector memory (TEM) T cells (identified by markers such as D45RA-/CD45RO+/CCR7-/CD62L-; D45RA-/CCR7-; or CD45RO+/CCR7-) as compared to the staffing population of T cells. In certain embodiments, the T cells produced according to the disclosed methods include TEM cells in an amount lower by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, as compared to the number of TEM cells in the starting population of T cells. In certain embodiments, the amount of TEM cells in the engineered population of T cells is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step. Methods for quantifying TEM cells are well known in the art and include, without limitation, flow cytometry (e.g., FACS) and fluorescence microscopy, among others.

Chimeric Antigen Receptor

[0248] Disclosed herein, in certain embodiments, are T cells engineered (e.g., genetically modified) to express a heterologous protein. In certain embodiments, the heterologous protein is a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises: (1) an extracellular domain containing an antigen-binding site that specifically binds to a target antigen, (2) a transmembrane domain; (3) an intracellular signaling domain; and, optionally, (4) a costimulatory domain. In certain embodiments, a CAR disclosed herein further comprises a hinge region.

[0249] In certain embodiments, the CAR is a human CAR, comprising fully human sequences, e.g., naturally-occurring human sequences. The extracellular domain is, in certain embodiments, linked to one or more intracellular signaling domains that, in certain embodiments, can mediate cell activation through an antigen receptor complex. In certain embodiments, the transmembrane domain is linked to the extracellular domain. In certain embodiments, the transmembrane domain that is naturally associated with one of the domains in the CAR is used. In certain embodiments, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0250] A CAR disclosed herein can include any number of amino acids, provided that the CAR retains its biological activity, e.g., the ability to specifically bind to antigen, mediate cytotoxic activity, detect diseased cells in a mammal, or treat or prevent disease in a mammal, etc. In certain embodiments, the CAR includes 50 or more (e.g., 60 or more, 100 or more, or 500 or more) amino acids, but less than 1,000 (e.g., 900 or less, 800 or less, 700 or less, or 600 or less) amino acids. In certain embodiments, the CAR is about 50 to about 700 amino acids (e.g., about 300 to about 1,000 amino acids (e.g., about 300 to about 800, about 300 to about 600, or about 400 to about 600 amino acids), or a range defined by any two of the foregoing values.

[0251] In certain embodiments, a CAR contains additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the CAR, e.g., recognize target cells, mediate cytotoxic activity, treat or prevent a disease or disorder, etc. More desirably, the additional amino acids enhance the biological activity of the CAR, as compared to the biological activity of the parent CAR.

Extracellular domain

[0252] In certain embodiments, the CAR disclosed herein comprises an extracellular antigen binding domain comprising an antibody or an antigen-binding fragment thereof. Anticalins or other alternative scaffolds are also contemplated. The antigen binding domain of the CAR can be a whole antibody or an antibody fragment (e.g., scFv). A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2 and CH3) regions, and each light chain contains one N- terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. The VH and VL regions have similar general structures, with each region including three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for antigen recognition and binding. The three CDR regions are connected by four framework regions, whose sequences are relatively conserved.

[0253] The antigen-binding fragment of the antibody retains the ability of the antibody to specifically bind to its antigen. The antibody fragment desirably includes, for example, one or more CDRs or the variable region (or portions thereof). Examples of antibody fragments include, but are not limited to: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a Fab' fragment, which is monovalent fragment consisting of the VL, VH, CL, CHI domains, and a disulfide bridge thiol (iii) a F(ab')2 fragment, which is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain; (vi) a single domain antibody, such as a VHH or VNAR, which contain a single monomeric variable antibody domain; and (vii) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain includes a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule having two functional antigen binding sites. In certain embodiments, the antigen binding domain of the CAR includes a scFv that binds to a target antigen.

[0254] In certain embodiments, antibody or antigen-binding fragment thereof in the extracellular domain of the CAR can be obtained or derived from a mammal, including but not limited to, a mouse, a rat, or a human. In certain embodiments, the antigen binding domain includes a variable region of a mouse or human monoclonal antibody or antigen-binding fragment thereof that binds to an antigen. In this respect, the antigen binding domain includes a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of a mouse or human monoclonal antibody or antigen-binding fragment thereof that binds to an antigen.

[0255] In certain embodiments, an extracellular domain of a CAR disclosed herein includes a signal sequence. The signal sequence may be positioned at the amino terminus of the antigen recognition domain (e.g., the variable region of the antibody or antigen-binding fragment thereof). The signal sequence may include any suitable signal sequence. In one embodiment, the signal sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence or a CD8a signal sequence. For example, a CAR including a murine scFv can include a GM-CSF signal sequence, while a CAR including a human scFv can include a CD8a signal sequence. It is understood that N-terminal signal sequences are typically cleaved from the CAR protein after being expressed, but a nucleic acid encoding the CAR generally includes a sequence encoding the signal sequence.

[0256] In some embodiments, the antigen-binding domain binds to a target antigen (e.g., a polypeptide). In some embodiments, the antigen-binding domain binds specifically to a target antigen (e.g., a polypeptide).

Hinge domain [0257] In certain embodiments, the CAR further includes a hinge or spacer between the antigen binding domain and the transmembrane domain. The hinge or spacer may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., a CD8a hinge, an IgG4 hinge region, and/or a CH1/CL and/or FC region. In certain embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl In certain embodiments, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In certain embodiments, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 and including any integer between the endpoints of any of the listed ranges. In certain embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include CD8a hinge, IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. In certain embodiments, the CAR hinge comprises a CD8a, truncated CD8a, or CD28 hinge domain.

[0258] In some embodiments, the hinge region is a short sequence of amino acids that can facilitate structural flexibility between polypeptide domains, e.g., between an extracellular domain and a transmembrane domain (see, e.g., Woof et al., Nal. Rev. Immunol. 4(2):89-99 (2004)). In some embodiments, a hinge region may include all, or a portion of, an extracellular region of any suitable transmembrane protein (e.g., CD8a).

[0259] In some embodiments, the hinge region is derived from a CD8a protein or a CD28 protein. In some embodiments, a hinge region is derived from a CD8a protein. In some embodiments, the hinge region is derived from a CD28 protein. In some embodiments, a hinge region is or comprises a hinge region or functional fragment thereof from a CD28 protein. In some embodiments, the hinge region is or comprises a hinge region or functional fragment thereof from a CD8a protein. In some embodiments, the hinge region is derived from a human CD8a protein or a human CD28 protein. In some embodiments, the hinge region is derived from a human CD8a protein. In some embodiments, the hinge region is derived from a human CD28 protein. In some embodiments, the hinge region is or comprises a hinge region or functional fragment thereof from a human CD28 protein. In some embodiments, the hinge region is or comprises a hinge region or functional fragment thereof from a human CD8a protein.

[0260] In some embodiments, a hinge region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 28. In some embodiments, a hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 28. In some embodiments, a hinge region is derived from the same polypeptide as a transmembrane domain. In some embodiments, a hinge region and a transmembrane domain are derived from a CD8 polypeptide. In some embodiments, a hinge region and a transmembrane domain are derived from a CD8a polypeptide. In some embodiments, a hinge region and transmembrane domain comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 19. In some embodiments, a hinge region and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, a hinge region and transmembrane domain comprise an amino acid sequence as set forth in SEQ ID NO: 19.

[0261] In some embodiments, a hinge region and transmembrane domain are encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 20. In some embodiments, a hinge region and transmembrane domain are encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 20. In some embodiments, a hinge region and transmembrane domain are encoded by a nucleic acid sequence as set forth in SEQ ID NO: 20.

Transmembrane domain

[0262] Disclosed herein, in certain embodiments, are modified T cells expressing a CAR comprising transmembrane domain operably connected to an extracellular domain and an intracellular signaling domain of the CAR. The transmembrane domain can be any transmembrane domain derived or obtained from any molecule (e.g., type I transmembrane protein) known in the art.

[0263] In some embodiments, the transmembrane domain of the CAR is derived from a natural source (e.g., a natural or wild-type polypeptide). In some embodiments, the transmembrane domain, as used in accordance with the present disclosure, is derived from any suitable transmembrane protein or polypeptide known in the art. In some embodiments, a transmembrane domain is derived from a CD3 epsilon polypeptide, a CD4 polypeptide, a CD5 polypeptide, a CD8 polypeptide, a CD9 polypeptide, a CD 16 polypeptide, a CD22 polypeptide, a CD28 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD45 polypeptide, a CD64 polypeptide, a CD80 polypeptide, a CD86 polypeptide, a CD 134 polypeptide, a CD137 polypeptide, a CD154 polypeptide, a T cell receptor alpha chain polypeptide, a T cell receptor beta chain polypeptide, a T cell receptor zeta chain polypeptide, or any derivatives thereof and/or combination thereof. In some embodiments, a transmembrane is or comprises a transmembrane domain or functional fragment thereof from a CD3 epsilon polypeptide, a CD4 polypeptide, a CD5 polypeptide, a CD8 polypeptide, a CD9 polypeptide, a CD 16 polypeptide, a CD22 polypeptide, a CD28 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD45, polypeptide, a CD64 polypeptide, a CD80 polypeptide, a CD86 polypeptide, a CD 134 polypeptide, a CD 137 polypeptide, a CD 154 polypeptide, a T cell receptor alpha chain polypeptide, a T cell receptor beta chain polypeptide, a T cell receptor zeta chain polypeptide, or any combination thereof. In some embodiments, a transmembrane is synthetically derived, or engineered. In some embodiments, a synthetically derived or engineered transmembrane domain comprises predominantly hydrophobic residues (e.g., leucine, valine, etc.). In some embodiments, an engineered transmembrane domain is or comprises any engineered transmembrane domain known in the field.

[0264] Alternatively, in certain embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In certain embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s). [0265] The present disclosure appreciates that CD8 is a transmembrane glycoprotein that functions as a co-receptor for the T-cell receptor (TCR), and is expressed primarily on the surface of T cells, e g., cytotoxic T-cells. The most common form of CD8 exists as a dimer composed of a CD8a and CD813 chain. In some embodiments, a transmembrane domain is derived from a CD8a protein. In some embodiments, a transmembrane protein comprises an amino acid sequence having at 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 11. In some embodiments, a transmembrane protein comprises an amino acid sequence as set forth in SEQ LD NO: 11.

[0266] The present disclosure further appreciates that CD28 is expressed on T-cells and provides co- stimulatory signals required for T-cell activation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2). In some embodiments, a CAR of the present disclosure comprises a CD28 transmembrane domain. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least 770%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 12. In some embodiments, a transmembrane protein comprises an amino acid sequence as set forth in SEQ ID NO: 12.

Intracellular signaling domain

[0267] In certain embodiments, upon binding of the CAR to a target antigen, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune effector cell, e.g., T cell engineered to express the receptor. For example, in some contexts, the receptor induces a function of a T cell, such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In certain embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In certain embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.

[0268] In some aspects, the receptor includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM-containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, FceRl (e.g., an FceRl gamma chain polypeptide), FcyRI, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD8, CD22, CD79a, CD79b, LIGHT, NKG2C, 0X40, PD-1, CD66d, or any derivatives or any combination thereof.

[0269] In certain embodiments, the receptor includes an intracellular component of a TCR complex. It is understood that the most common intracellular signaling domain used in CAR therapies is an intracellular signaling domain of CD3 zeta (CD3Q. CD3 zeta associates with T cell receptors to produce a signal and contains ITAMs. In certain embodiments, intracellular signaling molecule(s) in the CAR contain(s) an intracellular signaling domain, portion thereof, or sequence derived from CD3 zeta. In certain embodiments, the intracellular signaling domain comprises a CD3(^ domain. In certain embodiments, the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain or functional fragment thereof, such as a 112AA cytoplasmic domain of isoform 3 of human CD3 zeta. (UniProt Accession No.: P20963.2).

[0270] In some embodiments, an intracellular signaling domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 23. In some embodiments, an intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 23.

[0271] In some embodiments, an intracellular signaling domain is encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 24. In some embodiments, an intracellular signaling domain is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 24. [0272] In some embodiments, the intracellular signaling domain comprises at least one intracellular signaling domain or functional fragment thereof from a 4-1BB polypeptide, a B7H3 polypeptide, a CD2 polypeptide, a CD3 gamma polypeptide, a CD3 delta polypeptide, a CD3 zeta polypeptide, a CD7 polypeptide, a CD27 polypeptide, a CD28 polypeptide, a CD30 polypeptide, a CD40 polypeptide, an FCERI polypeptide (e.g., an FceRI gamma chain polypeptide), an FcyRI polypeptide, LIGHT polypeptide, NKG2C polypeptide, 0X40 polypeptide, PD-1 polypeptide, or any derivatives thereof or any combination thereof. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof from a CD28 polypeptide. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof from a CD28 polypeptide and an intracellular signaling domain or functional fragment thereof from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus, an intracellular signaling domain or functional fragment thereof from a CD28 polypeptide and an intracellular signaling domain or functional fragment thereof from a CD3 zeta polypeptide.

[0273] In some embodiments, the intracellular signaling domain of the present disclosure comprises at least one signaling sequence from a 4-1BB polypeptide, a B7-H3 polypeptide, a CD2 polypeptide, a CD3 gamma polypeptide, a CD3 delta polypeptide, a CD3 zeta polypeptide, a CD7 polypeptide, a CD27 polypeptide, a CD28 polypeptide, a CD30 polypeptide, a CD40 polypeptide, an FceRI polypeptide (e.g., an FceRI gamma chain polypeptide), an FcyRI polypeptide, LIGHT polypeptide, NKG2C polypeptide, 0X40 polypeptide, PD-1 polypeptide, or any combination thereof. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence from a CD28 polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence from a CD28 polypeptide and at least one signaling sequence from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus, at least one signaling sequence from a CD28 polypeptide and at least one signaling sequence from a CD3 zeta polypeptide. Costimulatory domain

[0274] In certain embodiments, a CAR of the disclosure contains an intracellular domain of a T cell costimulatory molecule, e.g., positioned between the transmembrane domain and intracellular signaling domain. In certain embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of CD28 or 4-1BB, or a functional variant or portion thereof, such as a 41 -amino acid cytoplasmic domain of a human CD28 (UniProt Accession No. Pl 0747.1) or a 42-amino acid cytoplasmic domain of a human 4-1BB (UniProt Accession No. Q07011.1) or functional variant or portion thereof.

[0275] In certain embodiments, the receptor encompasses one or more, e.g., two or more, costimulatory domains in addition to an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary receptors include intracellular components of CD3-zeta and CD28, or intracellular components of CD3-zeta and 4-1BB.

[0276] In certain embodiments, the receptor includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40, DAP10, and ICOS. In some aspects, the same receptor includes both the activating and costimulatory components.

[0277] In certain embodiments, the intracellular signaling domain comprises a CD8a transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain, and further comprises a CD28 or 4- IBB (CD TNFRSF9) co-stimulatory domain, linked to a CD3 zeta intracellular domain.

[0278] In some embodiments, an intracellular signaling domain comprises a CD28 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises an intracellular signaling domain or a functional fragment thereof from a CD28 polypeptide. In some embodiments, a CD28 polypeptide intracellular signaling domain or functional fragment thereof comprises a co-stimulatory domain.

[0279] In some embodiments, an intracellular signaling domain disclosed herein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 21. In some embodiments, the intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 21. [0280] In some embodiments, an intracellular signaling domain is encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 22. In some embodiments, an intracellular signaling domain is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 22.

Chimeric antigen receptor — multiple domains

[0281] In some embodiments, a CAR of the present disclosure comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR of the present disclosure comprises a signal peptide sequence (also referred to as a targeting signal, localization signal, localization sequence, leader sequence, or leader peptide), an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR of the present disclosure comprises, from N- terminus to C-terminus, an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR of the present disclosure comprises, from N-terminus to C-terminus, a signal peptide sequence, an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the signal peptide sequence is cleaved from the CAR during or after its insertion into a membrane (e.g., ER membrane) during synthesis of the CAR protein. In some embodiments, domains or components (e.g., extracellular domains, hinge regions, transmembrane domains, intracellular signaling domains, etc.) of a CAR are directly linked, or are contiguous. In some embodiments, domains or components of a CAR are not-directly linked, or are non-contiguous.

[0282] In some embodiments, a CAR as described herein comprises an intracellular signaling domain, wherein the intracellular signaling domain comprises: (a) a CD3 zeta intracellular signaling domain or functional fragment thereof; and (b) at least one of a 4-1BB, an 0X40, or a CD28 intracellular signaling domain or functional fragment thereof. In some embodiments, a 4- 1BB intracellular signaling domain or functional fragment thereof, an 0X40 intracellular signaling domain, and/or a CD28 intracellular signaling domain or functional fragment thereof is or comprises a co-stimulatory domain. [0283] In some embodiments, a CAR of the present disclosure comprises: (a) a CD28 transmembrane domain; and (b) an intracellular signaling domain comprising: (i) a CD3^ intracellular signaling domain or functional fragment thereof; and (ii) a CD28 intracellular signaling domain or functional fragment thereof. In some embodiments, a CD28 intracellular signaling domain or functional fragment thereof is or comprises a CD28 co-stimulatory domain.

[0284] In some embodiments, a CAR of the present disclosure comprises: (a) a CD8a transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3^ intracellular signaling domain or functional fragment thereof; and (ii) a CD28, an FceRI gamma chain, and/or a 4-1BB intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises: (a) a CD8a transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3(^ intracellular signaling domain or functional fragment thereof; and (ii) a CD28, an FceRI gamma chain, and a 4- IBB intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises: (a) a CD8a transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3(^ intracellular signaling domain or functional fragment thereof; and (ii) an FceRI gamma chain intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises: (a) a CD8a transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3^ intracellular signaling domain or functional fragment thereof; and (ii) a 4- IBB intracellular signaling domain or functional fragment thereof. In some embodiments, a CD28 intracellular signaling domain or functional fragment thereof is or comprises a CD28 costimulatory domain. In some embodiments, a FceRI intracellular signaling domain or functional fragment thereof is or comprises a FceRI costimulatory domain. In some embodiments, a 4-1BB intracellular signaling domain or functional fragment thereof is or comprises a 4- IBB costimulatory domain.

[0285] In some embodiments, a CAR of the present disclosure comprises (a) a CD8a transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3^ intracellular signaling domain or functional fragment thereof, and (ii) a CD27 and/or a CD28 intracellular signaling domain or functional fragment thereof. In some embodiments, a CD27 intracellular signaling domain or functional fragment thereof is or comprises a CD27 costimulatory domain. In some embodiments, a CD28 intracellular signaling domain or functional fragment thereof is or comprises a CD28 co-stimulatory domain.

[0286] In some embodiments, a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3(^ intracellular signaling domain or functional fragment thereof; and (ii) a CD27, a 4- IBB, and/or an FceRI gamma chain intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3 intracellular signaling domain or functional fragment thereof; and (ii) a CD27, a 4- IBB, and an FceRI gamma chain intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3(^ intracellular signaling domain or functional fragment thereof; and (ii) a CD27 intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3^ intracellular signaling domain or functional fragment thereof; and (ii) a 4- IBB intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3^ intracellular signaling domain or functional fragment thereof; and (ii) an FceRI gamma chain intracellular signaling domain or functional fragment thereof. In some embodiments, a CD27 intracellular signaling domain or functional fragment thereof is or comprises a CD27 costimulatory domain. In some embodiments, an intracellular signaling domain or functional fragment thereof is or comprises a FceRI costimulatory domain. In some embodiments, a 4- IBB intracellular signaling domain or functional fragment thereof is or comprises a 4- IBB costimulatory domain.

[0287] The present disclosure further provides for CARs comprising an extracellular domain directed to any target molecule of interest (e.g., comprising any of known antigen-binding domain, e.g., antibody, scFv, etc.), and further comprising any transmembrane domain described herein (including any hinge domain described herein), any intracellular signaling domain described herein (including any signal sequences or motifs, any co-stimulatory domains, etc., described herein), present in any combination. [0288] In some embodiments, a CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8a polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3(^ intracellular signaling domain or fragment thereof; and (ii) a human CD28 intracellular signaling domain or fragment thereof, wherein the CD28 intracellular signaling domain or fragment thereof is or comprises a co-stimulatory domain. In some embodiments, a CAR comprises: (a) a hinge region derived from a human CD8a polypeptide, (b) a transmembrane domain derived from a human CD8a polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3 intracellular signaling domain; and (ii) a human CD28 intracellular signaling domain. In some embodiments, a CAR comprises a sequence as set forth in SEQ ID NO: 27.

[0289] In some embodiments, a CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8a polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3^ intracellular signaling domain or fragment thereof; and (ii) a CD27 and/or a CD28 intracellular signaling domain or fragment thereof, wherein the CD27 and/or CD28 intracellular signaling domain or fragment thereof is or comprises a co-stimulatory domain.

[0290] In some embodiments, a CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8a polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3^ intracellular signaling domain or fragment thereof; and (ii) a human CD28, a human CD27, and/or an FceRl gamma chain intracellular signaling domain or fragment thereof, wherein the human CD28, the human CD27, and/or the FceRl gamma chain intracellular signaling domain or fragment thereof are or comprise a co-stimulatory domain.

[0291] In some embodiments, a CAR can comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8a polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3^ intracellular signaling domain; and (ii) a human CD28 and/or an FceRl gamma chain intracellular signaling domain, wherein the CD28 and/or the FceRl gamma chain intracellular signaling domain or fragment thereof are or comprise a co-stimulatory domain. [0292] In some embodiments, a CAR as described herein, further comprises a signal peptide sequence. In some embodiments, a signal peptide is positioned at the amino terminus of an extracellular domain (e.g., at the N-terminus of an antigen-binding domain). A signal peptide as used in accordance with the present disclosure may comprise any suitable signal peptide sequence. In some embodiments, a signal peptide sequence is a human granulocyte-macrophage colonystimulating factor (GM-CSF) receptor signal peptide sequence or a CD8a signal peptide sequence. In some embodiments, a CAR provided herein comprises a human or humanized scFv comprising a CD8a signal peptide sequence. In some embodiments, a signal peptide sequence comprises an amino acid sequence as set forth in SEQ ID NO: 15.

[0293] In some embodiments, a provided CAR comprises: (a) a CD8a hinge region comprising SEQ ID NO: 28, (b) a CD8a transmembrane domain comprising SEQ ID NO: 11, (c) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (d) a CD3(^ intracellular signaling domain comprising SEQ ID NO: 23. In some embodiments, a provided CAR comprises, from N- terminus to C-terminus: (a) a CD8a hinge region comprising SEQ ID NO: 28, (b) a CD8a transmembrane domain comprising SEQ ID NO: 11, (c) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (d) a CD3^ intracellular signaling domain comprising SEQ ID NO: 23.

[0294] In some embodiments, a provided CAR comprises: (a) an antigen-binding domain comprising SEQ ID NO: 17, (b) a CD8a hinge region comprising SEQ ID NO: 28, (c) a CD8a transmembrane domain comprising SEQ ID NO: 11, (d) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (e) a CD3(^ intracellular signaling domain comprising SEQ ID NO: 23. In some embodiments, a provided CAR comprises, from N-terminus to C-terminus: (a) an antigen-binding domain comprising SEQ ID NO: 17, (b) a CD8a hinge region comprising SEQ ID NO: 28, (c) a CD8a transmembrane domain comprising SEQ ID NO: 11, (d) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (e) a CD3(^ intracellular signaling domain comprising SEQ ID NO: 23.

[0295] In some embodiments, a provided CAR comprises: (a) a CD8a signal peptide sequence comprising SEQ ID NO: 15, (b) an antigen-binding domain comprising SEQ ID NO: 17, (c) a CD8a hinge region as set forth in SEQ ID NO: 28, (d) a CD8a transmembrane domain as set forth in SEQ ID NO: 11, (e) a CD28 intracellular signaling domain as set forth in SEQ ID NO: 21, and (f) a CD3(^ intracellular signaling domain as set forth in SEQ ID NO: 23. In some embodiments, a provided CAR comprises, from N-terminus to C-terminus: (a) a CD8a signal peptide sequence comprising SEQ ID NO: 15, (b) an antigen-binding domain comprising SEQ ID NO: 17, (c) a CD8a hinge region as set forth in SEQ ID NO: 28, (d) a CD8a transmembrane domain as set forth in SEQ ID NO: 11, (e) a CD28 intracellular signaling domain as set forth in SEQ ID NO: 21, and (I) a CD3(^ intracellular signaling domain as set forth in SEQ ID NO: 23.

[0296] In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence as set forth in SEQ ID NO: 10.

[0297] In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, a least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence as set forth in SEQ ID NO: 13.

[0298] In some embodiments, a CAR of the present disclosure is encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 14 In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 98 ⁰0 sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 14.

Modifications

[0299] The CAR may comprise one or modified synthetic amino acids in place of one or more naturally-occurring amino acids. Exemplary modified amino acids include, but are not limited to, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, Sacetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4nitrophenyl alanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, (3-phenylserine (3- hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N -methyl-lysine,N',N'-dibenzyl-lysine, 6- hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norbomane)-carboxylic acid, a,y di aminobutyric acid, a,y -di ami nopropionic acid, homophenylalanine, and a-tertbutylglycine.

[0300] The CAR (including functional portions and functional variants thereof) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.

Pharmaceutical Compositions

[0301] The engineered T cells disclosed herein can be incorporated into a pharmaceutical composition. These compositions can comprise, in addition to the engineered T cells disclosed herein, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the intended route of administration, e g., intravenous, cutaneous, or subcutaneous, nasal, intramuscular, intraperitoneal routes. The pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and one of ordinary skill in the art is capable of selecting suitable pharmaceutical excipients.

[0302] Accordingly, the pharmaceutical excipients provided below are intended to be illustrative, and not limiting. Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), incorporated by reference in its entirety.

Kits

[0303] Disclosed herein, in certain embodiments, are kits that include one or more doses of a population of engineered T cells (e.g., CAR-T cells) produced or obtainable according to the methods disclosed herein in a suitable container means. [0304] In certain embodiments, the kit comprises a container means comprising the engineered T cells described herein. In certain embodiments, the container means is any suitable container which houses, e.g., a liquid or lyophilized composition including, but not limited to, a vial, syringe, bottle, and an intravenous (IV) bag or ampoule. A syringe holds any volume of liquid suitable for injection into a subject, including, but not limited to, 0.5 cc, 1 cc, 2 cc, 5 cc, 10 cc, or more. In certain embodiments, packages and kits include a label specifying information required by US FDA or similar regulatory authority, e.g., a product description, amount and mode of administration, and/or indication of treatment. In certain embodiments, packages provided herein include any of the compositions as described herein.

[0305] In certain embodiments, packages and kits additionally include a buffering agent, a preservative, and/or a stabilizing agent in a pharmaceutical formulation. In certain embodiments, each component of the kit is enclosed within an individual container and all of the various containers are within a single package. In certain embodiments, disclosure kits are designed for cold storage or room temperature storage.

[0306] Additionally, in certain embodiments, the preparations contain stabilizers to increase the shelf-life of the kits and include, e.g., bovine serum albumin (BSA). Where the compositions are lyophilized, the kit contains, in certain embodiments, further preparations of solutions to reconstitute the lyophilized preparations. Acceptable reconstitution solutions are well known in the art and include, e.g., pharmaceutically acceptable phosphate buffered saline (PBS).

[0307] In certain embodiments, a kit can further include instructions for performing any of the methods described herein. The term "packaging material" refers to a physical structure housing the components of the kit. In certain embodiments, the packaging material maintains the components sterile and is made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, etc.). In certain embodiments, the label or packaging insert includes appropriate written instructions (e.g., instructing the user of the kit to perform one or more methods disclosed herein). Kits, in certain embodiments, additionally include labels or instructions for using the kit components in any method of the disclosure. In certain embodiments, a kit includes a compound in a pack or dispenser together with instructions for administering the compound in a method described herein. [0308] In an aspect, the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and cultivating the T cells in a serum-free cultivation medium comprising an interleukin-2 (IL-2) protein for 29-71 hours; and (c) harvesting the T cells; optionally wherein step (b) is performed at least 1 hour after initiation of step (a); and wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein.

[0309] In an aspect, the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order:(a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and cultivating the T cells in a cultivation medium comprising interleukin 7 (IL-7) and interleukin- 15 (IL- 15) proteins for 31-71 hours; and (c) harvesting the T cells; wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein. [0310] In an aspect, the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and (c) cultivating the T cells in a serum-free cultivation medium comprising interleukin-7 (IL-7) and/or interleukin- 15 (IL-15) proteins for 30-60 hours; and (c) harvesting the T cells; wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein.

[0311] In an aspect, the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28 in the absence of a cytokine; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and cultivating the T cells in a cultivation medium comprising at least one cytokine for 24-72 hours; and (c) harvesting the T cells; wherein step (b) is performed about 18 hours after step (a); and wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein. In certain embodiments, the at least one cytokine in step (c) comprises one or more (e.g., 1, 2, 3, or 4) of IL-2, IL-7, IL-15, and/or IL- 21. In certain embodiments, the at least one cytokine in step (c) comprises IL-2. In certain embodiments, the at least one cytokine in step (c) comprises IL-21. In certain embodiments, the at least one cytokine in step (c) comprises IL-7 and IL-15. In certain embodiments, the at least one cytokine in step (c) comprises IL-2, IL-7, and IL-15. In certain embodiments, the at least one cytokine in step (c) comprises IL-21, IL-7, and IL-15.

[0312] In an aspect, the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and cultivating the T cells in a cultivation medium comprising interleukin-21 (IL-21) protein for 31-71 hours; and (c) harvesting the T cells; wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein. In certain embodiments, the cultivation medium further comprises IL-7 and/or IL- 15.

[0313] In an aspect, the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28;

(b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and cultivating the T cells in a cultivation medium for 24-72 hours; and

(c) harvesting the T cells; wherein the cultivation medium does not comprise IL-2, IL-7, IL- 15, or IL-21; and wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein. In certain embodiments, the cultivation medium is cytokine free. In certain embodiments, wherein step (a) does not comprise use of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, step (a) is performed in the absence of a cytokine. In certain embodiments, step (c) does not comprise use of IL-2, IL-7, IL-15, or IL-2L In certain embodiments, step (c) is performed in the absence of a cytokine. In certain embodiments, the method is performed in the absence of a cytokine. In some embodiments, the cultivation medium is a basal cultivation medium. [0314] In certain embodiments, the cultivation medium comprises serum. In some embodiments, the cultivation medium does not further comprise an added cytokine or growth factor. In certain embodiments, the cultivation medium is serum-free.

[0315] In certain embodiments, the starting population of T cells is seeded in culture at a concentration of about lx 10⁶ cells per mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of about 2xl0⁶ cells per mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of about 5xl0⁶ cells per mL. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about IxlO⁶ cells per mL; and (b) the cultivation medium comprises IL -2. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about IxlO⁶ cells per mL; and (b) the cultivation medium comprises IL-7 and IL-15. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about IxlO⁶ cells per mL; and (b) the cultivation medium comprises IL-7, IL- 15, and IL-21. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about IxlO⁶ cells per mL; and (b) the cultivation medium comprises IL-21. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about IxlO⁶ cells per mL; and (b) the cultivation medium does not comprise IL-2, IL-7, IL-15, or IL-21 (i.e., does not comprise any one of IL-2, IL-7, IL-15, and IL-21). In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about IxlO⁶ cells per mL; and (b) the cultivation medium comprises IL -2. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 2xl0⁶ cells per mL; and (b) the cultivation medium comprises IL-7 and IL-15. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 2xl0⁶ cells per mL; and (b) the cultivation medium comprises IL-7, IL- 15, and IL-21. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 2xl0⁶ cells per mL; and (b) the cultivation medium comprises IL-21. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 2xl0⁶ cells per mL; and (b) the cultivation medium does not comprise IL-2, IL-7, IL-15, or IL-21 (i.e., does not comprise any one of IL-2, IL-7, IL-15, and IL-21). In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 5xl0⁶ cells per mL; and (b) the cultivation medium comprises IL-2. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 5xl0⁶ cells per mb; and (b) the cultivation medium comprises IL-7 and IL-15. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 5xl0⁶ cells per mL; and (b) the cultivation medium comprises IL-7, IL-15, and IL-21. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 5xl0⁶ cells per mL; and (b) the cultivation medium comprises IL-21. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 5xl0⁶ cells per mL; and (b) the cultivation medium does not comprise IL-2, IL-7, IL-15, or IL-21 (i.e., does not comprise any one of IL-2, IL-7, IL-15, and IL -21).

[0316] In certain embodiments, the method further comprises (d) maintaining the T cells harvested in step (c) at a temperature of no greater than 38° C (e.g., no greater than 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9° C, or less), between 2-8° C (e.g., 2, 3, 4, 5, 6, 7, or 8° C), or no greater than -80° C (e.g., no greater than -80, - 81, -82, -83, -84, -85, -86, -87, -88, -89, -90, -91, -92, -93, -94, -95, -96, -97, -98, -99, -100, or less).

[0317] In certain embodiments, the one or more agents that activate CD3 and/or CD28 comprise an anti-CD3 antibody, anti-CD28 antibody, or both. In certain embodiments, the one or more agents that activate CD3 and/or CD28 comprise a first agent that binds CD3 and a second agent that binds CD28. In certain embodiments, the first agent and the second agent are the same agent (e.g., a bispecific antibody that specifically binds to CD3 and CD28).

[0318] In certain embodiments, the method comprises, prior to step (a), obtaining a sample comprising the starting population of T cells from a subject. In certain embodiments, the method comprises, prior to step (a), having obtained a sample comprising the starting population of T cells from a subject. In certain embodiments, the sample is a whole blood sample obtained from the subject.

[0319] In certain embodiments, the starting population of T cells comprises T helper (Th) cells, cytotoxic T (Tc) cells, memory T (LM) cells, regulatory T (Treg) cells, innate-like T cells. In certain embodiments, the Th cells comprise Thl cells, Th2 cells, Thl7 cells, Th9 cells, Tfh cells, and/or Th22 cells. In certain embodiments, the memory T cells comprise central memory T cells (TCM) cells, effector memory T (TEM) cells, tissue-resident memory T (TRM) cells, and virtual memory T (TVM) cells. In certain embodiments, the innate-like T cells are natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells, and y5 T cells.

[0320] In certain embodiments, the polynucleotide is comprised in a delivery vehicle. In certain embodiments, the delivery vehicle is a lipid nanoparticle. In certain embodiments, the delivery vehicle is a nucleic acid vector. In certain embodiments, the nucleic acid vector is a viral vector.

[0321] In certain embodiments, the viral vector is a lentiviral vector.

[0322] In certain embodiments, the method results in expansion of the starting population of T cells that is no greater than 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1.9- fold, 1.8-fold, 1.7-fold, 1.6-fold, 1.5-fold, 1.4-fold, 1.3-fold, 1.2-fold, 1.1-fold, or less, following step (c). In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, or at least 75%, of the cells of the starting population of T cells, following step (c), are engineered to express the heterologous protein.

[0323] In certain embodiments, the heterologous protein comprises a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises: (a) an antigen-binding fragment of an antiCD 19 antibody; (b) a transmembrane domain; (c) an intracellular T cell signaling domain from human CD3^, and (d) an intracellular T cell signaling domain from human CD28.

[0324] In certain embodiments, one or more of the T cells harvested in step (c) secretes increased amounts of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32) proteins selected from the group consisting of IFNg, Granzyme B, IL-113, IL -2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-21, IL-22, IP -IO, MCPI, MCP4, TNFa, TNF13, TGF13, GMCSF, MIPla, MIP113, CCL11, perforin, RANTES, sCD137, and VEGF after being contacted with a target cell that expresses CD 19, as compared to a T cell not contacted with the target cell In certain embodiments, secretion of the one or more proteins after contact with the target cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or, 500% higher, or more, as compared to secretion of the one or more proteins in the absence of the target cell.

[0325] In certain embodiments, one or more of the T cells harvested in step (c) exhibit increased expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD 137 after being contacted with a target cell that expresses a target antigen, as compared to expression of the one or more T cell activation markers in the absence of the target cell. In certain embodiments, expression of the one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 is increased by at least 55%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or, 500% higher, or more, as compared to expression of the one or more T cell activation markers in the absence of the target cell.

[0326] In certain embodiments, one or more of the T cells harvested in step (c) exhibit increased cytotoxicity against a target cell that expresses a target antigen (e.g., antigen related to a disease such as CD 19), as compared to cytotoxicity against a cell that does not express the antigen. In certain embodiments, cytotoxicity is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or, 500% higher, or more, as compared to cytotoxicity against a cell that does not express antigen.

[0327] In certain embodiments, one or more of the T cells harvested in step (c) exhibit increased proliferation after being contacted with a target cell that expresses a target, as compared to proliferation in the absence of the target cell. In certain embodiments, proliferation is increased by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15- fold, 20-fold, 30-fold, 40-fold, 50-fold, or more, as compared to proliferation in the absence of the target cell.

[0328] In certain embodiments, proliferation is measured between 0 and 240 hrs after contact with the target cell. In certain embodiments, proliferation is measured at 0 hr, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, 24 hrs, 30 hrs, 36 hrs, 42 hrs, 48 hrs, 72 hrs, 96 hrs, 120 hrs, 144 hrs, 168 hrs, 192 hrs, 216 hrs, and/or 240 hrs after contact with the target cell. In certain embodiments, proliferation is measured as the fold-change in the number of CD3-positive (CD3+) cells in the T cells of step (c) as compared to the number of CD3+ cells in the starting population of T cells. In certain embodiments, proliferation is measured as the fold-change in the number of T cells of step (c) expressing the heterologous protein after being contacted with a target cell that expresses a target antigen, as compared to the number of T cells of step (c) expressing the heterologous protein in the absence of the target cell. [0329] In certain embodiments, the T cells harvested in step (c) exhibit an increased amount of naive and stem cell memory T (collectively TNSCM) cells (e.g., TSCM cells) as compared to the starting population of T cells. In certain embodiments, the T cells harvested in step (c) comprise TNSCM cells (e.g., TSCM cells) in an amount of at least 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or, 500% higher, or more, as compared to the starting population of T cells. In certain embodiments, the T cells harvested in step (c) exhibit substantially the same percentage of naive and stem cell memory T (TNSCM) cells (e.g., TSCM cells) as compared to the starting population of T cells (e.g., the percentage of TNSCM cells in the T cells of step (c) is increased or decreased by no more than 4%, 3%, 2%, or 1%, or less (absolute difference between the percentages of TNSCM cells in the T cells), as compared to the number of TNSCM cells in the starting population of T cells). In certain embodiments, the T cells harvested in step (c) comprise a decreased amount of T effector memory (TEM) cells as compared to the starting population of T cells. In certain embodiments, the T cells harvested in step (c) comprise TEM cells in an amount that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower, or more, as compared to the starting population of T cells. In certain embodiments, the T cells harvested in step (c) exhibit substantially the same percentage of TEM cells as compared to the starting population of T cells (e.g., the percentage of TEM cells in the T cells of step (c) is increased or decreased by no more than 4% 3%, 2%, 1%, or less (absolute difference between the percentages of TEM cells in the T cells), as compared to the number of TEM cells in the starting population of T cells).

[0330] In certain embodiments, the method is performed ex vivo.

EXAMPLES

[0331] The following examples are put forth to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their invention. Example 1: Preparation of T cells engineered to express a chimeric antigen receptor using 3-day process

[0332] An initial set of experiments was undertaken to characterize the 2-3-day, 6-day, and 8-day methods for manufacturing CAR T cells from whole blood and/or PBMCs isolated from whole blood. As a comparison, for several experiments described below, a 3-Day process that requires leukapheresis and combines activation and transduction (“3 Day Alternative”) was included. As this example shows, the presently disclosed methods were shown to produce CAR T cells using PBMCs extracted from a small, whole-blood samples. This included successful CAR T cell production using the two-to-three-day processes of the disclosure. Furthermore, as explained below, the resulting CAR T cells exhibited favorable phenotypes, targeted cytotoxicity and proliferation, and yields showing viable commercial production and scaleup. This initial set of experiments also validated that, not only do the methods for manufacturing of the disclosure work, they are able to do so using PBMCs from whole blood sample without the necessity for using leukapheresis, including in the rapid two-to-three day processes.

Starting Samples

[0333] T-cells were extracted from the peripheral blood mononuclear cells (PBMCs) isolated from donor I (27 year old Asian male, with the BMI of 25.4, and a smoker), or using leukapheresis from donor 2 (31 year old Caucasian male, with the BMI of 42.0, and not a smoker), or donor 3 (52 year old male of mixed ethnicity, with the BMI of 78.1, and not a smoker). The T cells in the samples were isolated using Miltenyi StraightFrom CD3 Microbeads (Miltenyi Biotec: cat. no. 130-090- 874) and stored at 2-8 C until further processing.

Manufacturing Processes Investigated

[0334] The isolated T cells were then independently processed, using the various manufacturing methods outlined in FIG. 10, which includes the following protocols: A (“KYV 3-Day Alternative"), which relies on leukapheresis and combines activation and transduction; B ("KYV 3-Day vl), which may use PBMCs/whole blood; C ("KYV 3-Day v2"), which may use PBMCs/whole blood; D ("KYV 6 Day") which may use PBMCs/whole blood; E ("KYV 8 day") which may use PBMCs/whole blood; and F ("KYV 3-Day v3"), which may use PBMCs/whole blood. For protocols A-C and F the initial starting population cells was 6xl0⁷ cells (enriched T cell population containing 85%-95% T cells) and for protocols D and E IxlO⁷ cells (enriched T cell population containing 85%-95% T cells) in Conditions D and E. The cells were seeded at the density of 3xl0⁶/cm² (Conditions B, C, and F) or lxl0⁶/cm² (Conditions A, D and E) in a G-Rex bioreactor.

[0335] TransAct (T Cell TransAct, human; Miltenyi Biotec; cat. No. 130-111-160) activation reagent was added to the cells in Conditions A, B, C, D, and E. The cells in condition F were not activated. Cytokines were added together with the activation reagent in Conditions B (100 ng/mE human IL -2), condition C (10 ng/mL human IL-7 and 10 ng/mL human IL-15), condition D (10 ng/mL human IL-7 and 10 ng/mL human IL- 15), and condition E (10 ng/mL human IL-7 and 10 ng/mL human IL- 15). The cells were incubated in cultivation media (TexMACS Medium supplemented with CTS Immune Cell SR) in a CO2 incubator.

[0336] The T cells were transduced with KL-hl98a28z, a self-inactivating (SIN) vesicular stomatitis virus (VSV)-G pseudotyped 3rd generation lentiviral vector encoding a chimeric antigen receptor that binds CD19. This CAR construct, named Hul9-CD828Z, has the amino acid sequence set forth in SEQ ID NO: 13. The lentiviral vector contained an MSCV promoter and other regulatory factors, including a central polypurine tract/central termination sequence upstream of the promoter, and a post-transcriptional regulatory element (PRE) downstream of the CAR expression sequence. The lentiviral vector KL-hl98a28z was manufactured using a HEK 293T cell line transiently transfected with a state-of-the-art four-plasmid system. The envelope protein encoding plasmid (pLTG1292) expresses a heterologous spike protein, the VSV-G protein, under control of the cytomegalovirus (CMV) promoter. The transduction step was initiated simultaneously with the activation in condition A; at 18 hours after initiation of the activation step in Conditions B and C; at 18 hours after the seeding step in Condition F; and at 24 hours after initiation of the activation step in Conditions D and E.

[0337] As shown in FIG. 10, the cells were incubated with the lenti virus vector for various lengths of time: 30 hours in Condition A; 48 hours in Conditions B, C, and E; 5 days in Condition D; and 8 days in Condition E. Upon harvest, Conditions A-C and F yielded approximately 3xl0⁷ cells each, condition D yielded approximately IxlO⁸ cells, and condition E yielded approximately 3xl0⁸ cells. [0338] The transduced T cells were frozen using a standard protocol. The cells were thawed at a later time and characterized according to Example 2 below.

Example 2: Characterization of the lentivirus vector-transduced T cells

Viability

[0339] Viability and cell count were measured using a NucleoCounter NC-200™ (ChemoMetec A/S, Allerod, Denmark), an automated cell counter that utilizes fluorescence detection to distinguish between viable and non-viable cells. Each test article was loaded into a proprietary cassette which contains two separate dyes that stain for total nucleated cells and non-viable cells. The software then calculated the percent viability and cell count. The total cell count was performed at 0 hours and 72 hours post thaw.

[0340] As shown in FIG. 2, each batch manufactured using the activated KYV 3 -Day processes (“vl” and “v2”) showed better overall T cell expansion during the 72 hours post-thaw than the 8- day process, which was at least comparable to the KYV Alternative 3-day process, which requires leukapheresis. Donor 1 showed least expansion overall, possibly due to the low viability of the starting cell population.

CAR expression and T cell phenotype

[0341] The T cells were next assessed for CAR expression and memory T cell phenotype by flow cytometry using a Cytoflex LX (Beckman Coulter) cytometer, using fluorescent antibodies recognizing the CD19 CARs and cell-surface markers associated Tn, Tscm, Tcm, Tern, and Temra memory T cell subsets. Specifically, Tn cells were identified as CD45RO-/CCR7+/CD95-; Tscm cells were identified as CD45RO-/CCR7+/CD95+; Tcm cells were identified as CD45RO+/CCR7+; Tern cells were identified as CD45RO+/CCR7-; and Temra cells were identified as CD45RO-/CCR7-. Fluorescent signal associated with each marker was acquired using CytExpert software (Beckman Coulter) and the final data analysis was performed using FlowJo (BD Biosciences). As shown in FIG. 3, CAR expression for KYV 3-Day processes vl and v2 was extremely high, and generally exceeded 50% CAR+ cells. This CAR+ percentage also exceeded those of the KYV Alternative 3-Day protocol. KYV 3-Day processes vl and v2 also showed higher CAR expression levels within the CD4+ and CD8+ T cell subpopulations (data not shown). The KYV 6-Day and KYV 8-Day processes also showed slightly better transduction efficiency than the KYV Alternative 3 -Day.

[0342] With respect to memory T cell subsets, as shown in FIGs. 4A and 4B, the cells produced by the KYV Alternative 3-Day, which relies on leukapheresis, and KYV 3-v Day v3 methods maintained the highest percentage of Tnscm, and exceeded 15% Tnscm. Of note, it was observed that the Tnscm cells (CD45RO-/CCR7+) were almost exclusively CD95+ (Tscm). The percentages of CAR+ for KYV Alternative 3-Day and KYV 3-Day v3 cells were 0 upon thawing. [0343] Additional memory phenotypes of the CAR+ T cells were assessed. KYV Alternative 3- Day and KYV 3-Day v3 cells upon thawing were excluded from phenotyping due to low CAR expression, and KYV 3-Day v3 cells were excluded entirely from phenotyping in Run 2 due to lack of transduction.

[0344] As shown in FIGs. 5A-5D, upon full CAR expression 72 hours post-thawing, KYV Alternative 3-Day cells, and KYV 3-Day v3 cells displayed similar percentage of Tnscm as KYV 3-Day vl and KYV 3-Day v2 cells. The "Post Enrichment" sample was untransduced with lenti virus.

[0345] Overall, T cells processed using the 3-day processes showed comparable or greater CAR expression than T cells manufactured using the KYV Alternative 3 -Day process, which relies on leukapheresis. Additionally, taking donor-to-donor variability and CAR expression into account, the three 3-day processes showed similar percentage of Tnscm among each other and higher percentage of Tnscm than the longer KYV 8-day and KYV 6-Day processes.

Cytotoxicity

[0346] The activity of the CAR-T cells of killing CD 19+ target cells was measured by flow cytometry using a Cytoflex LX (Beckman Coulter) cytometer.

[0347] Briefly, as outlined in Fig. 5, CD19+ Raji or Nalm6 cells were labeled with cell trace violet (CTV) and co-cultured with effector CAR-T cells at several effector-to-target (E:T) cell ratios for 18-20 hours. Percent cell killing was determined by the ratio of live CTV positive cells cocultured with effector cells to live CTV positive cells cultured in the absence of effector cells, at each E: T ratio. Data analysis was performed using FlowJo (BD Biosciences). [0348] The percentage cytolysis was measured and plotted vs. E:T ratio, as shown in FIG. 6 for Nalm6 cells. As provided in Table 1 below, cytotoxic activity at E:T ratio of 1: 1 was comparable between cells manufactured using the Processes A-E.

Table 1. Cytotoxicity of CAR-T cells manufactured by various protocols as described herein.

Single cell cytokine secretome analysis

[0349] Single cell cytokine secretome was assessed using a human adaptive immune cytokine panel. The CAR-T cells from Condition C were compared to Condition E by overnight coincubation with NALM-6 cells (E:T=3 : 1).

[0350] The human adaptive immune panel included the following cytokines: CCL-11, GM-CSF, Granzyme B, IFNg, IL-113, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL- 17A, IL-17F, IL-21, IL-22, IP- 10, MCP-I, MCP-4, MIP-la, MIP-113, Perforin, RANTES, sCD137, TNF-a, TNF-13, TGF-13, and VEGF.

[0351] FIGS. 7A-7D illustrate percentage of cells secreting each of the 32 cytokines, and show higher levels of IFNg (IFNg) and TNF-13 observed in cells manufactured using the KYV 3-day v2 process as compared to the KYV 9-day process. [0352] Next assessed was polyfunctionality of the CAR-T cells. Polyfunctionality represented the percentage of subsets of highly polyfunctional cells that simultaneously secrete multiple sets of cytokines, following co-culture with CD19+ target cells.

[0353] As shown in FIG. 8, the CAR-T cells manufactured using the KYV 3 -day v2 process showed higher polyfunctionality than the CAR-T cells manufactured using the 9-day process. Further, the percentage of polyfunctional cells that simultaneously secreted multiple sets of cytokines was assessed with respect to each cytokine. FIG. 9 shows the top 15 cytokines, with the cells manufactured using the KYV 3-day v2 processes displaying highest percentage of polyfunctional cells that simultaneously secrete multiple sets of cytokines.

Example 3 - Assessing methods for isolating PBMC

[0354] Given the positive results from Examples 1 and 2 in which T cells were successfully isolated from PBMCs obtained from whole blood to produce CAR T cells, various protocols for PBMC isolation were assessed.

[0355] Donors provided whole blood samples from which PBMCs were isolated using either a Rotea Counterflow Centrifugation System (ThermoFisher Scientific) or the X-LAB System (Coming Life Sciences). The Rotea system applies a counterflow centrifugation method for a broad range of cell processing applications, including PBMC isolation. The Corning X-LAB System is a sedimentation-based process that isolates mononuclear cells (MNCs) in a closed, sterile, semi-automated fashion — without the need for density gradient media or manual transfer steps.

[0356] As shown in FIG. 11, same-donor studies validate that the X-LAB system provided superior PBMC enrichment from whole blood samples. The X-LAB system provided a higher average purity with less variability compared to the Rotea system.

Example 4 - Assessing whole blood sample stability

[0357] Given the desirability of obtaining T cells for CAR T cell manufacturing from whole blood samples, the stability of whole blood and its impact on T cell levels and viability was assessed. Briefly, as outlined in FIG. 12, whole blood samples were obtained from donors. [0358] The composition of whole blood samples was assessed over three days using samples from two donors. Over the course of those three days, whole blood samples from two donors were enriched using clinical grade Dynabeads and the composition of the enriched samples likewise assessed.

[0359] As shown in FIG. 12, the CD3+, CD4+, CD8+ cell percentage (T cells) in the whole blood samples was stable at 72 hours, while the percentage of CD45+ cells decreased over time. When considering PBMC enriched whole blood, samples obtained within 24 hours gave a better yield than those obtained within 48 hours of sample draw. Thus, as shown, whole blood contains a sufficient number of T cells for CAR T manufacture, with fresh whole blood providing the best starting product. Coupled with the shortened CAR T manufacturing methods of the disclosure, using fresh blood should produce a superior, polyfunctional end product.

Example 5 - Assessing whole blood sample stability

[0360] Based on the promising whole blood composition and stability results from Example 4, a set of experiments to assess the CAR T cell manufacturing processes of the disclosure in which the PBMC step is omitted in favor of isolating T cell directly from a whole blood sample. Further, this Example not only assesses the feasibility of using a direct-from-blood T cell isolation step, but pairing that step with a concurrent activation of the isolated T cells.

[0361] FIG. 13 outlines the protocols used to assess the direct-from-blood T cell isolation step. Briefly, whole blood samples were obtained from three donors. The samples were used either 24 hours or 48 post-sample draw. For the concurrent T cell isolation and activation protocol, whole blood samples were run through a ThermoFisher DynaCellect Cell Isolator/WasherSystem (ThermoFisher) using anti-CD3/anti-CD28 Dynabeads, which captured and activated T cells from whole blood. The bead-bound T cells were collected using a G-RexlOM cell sorter. As a comparison, PBMCs were extracted from whole blood samples using the X-LAB system or the Rotea System. T cells were extracted from the PBMCs using a CliniMACS Plus Cell Isolator (Miltenyi Biotec) and activated using TransACT. As a control, the DynaCellect Cell isolation/activation protocol was also used on PBMCs extracted from Rotea to assess the relative efficacy of the DynaCellect system’s and the CliniMACS system’s enrichment comparative enrichment capability and to determine whether concurrent activation/i solation impacts enrichment.

[0362] As shown in FIG. 14, the DynaCellect provides a higher yield of enriched and final product relative to CliniMACS. This indicates that concurrent activation and T cell isolation appears to provide a higher yield of isolated T cells from a sample and a resulting higher yield of CAR T cells produced using said T cells. As shown, DynaCellect gives an average fold expansion of 1.2 compared to 0.56 from CliniMACS.

[0363] As shown in FIG. 15 and FIG. 16, whether starting from whole blood or enriched PBMCs, the DynaCellect concurrent isolation/activation protocols produced a high level of CD3+ cells across all materials (whole blood, enriched PBMCs, and final product), a more consistent CD4+/CD8+ ratio across all donors from starting material to final product. Thus, the concurrent activation/i solation method appears superior to a split isolation and activation protocol. Furthermore, this set of experiments clearly validates methods of the disclosures, that omit not only a leukapheresis step, but also a PBMC enrichment step when obtaining T cells from a whole blood sample.

Example 6 - Phenotype of CAR T cells engineered from T cells isolated directly from whole blood.

[0364] The T cells isolated directly from whole blood were next assessed for memory T cell phenotype by flow cytometry using a Cytoflex LX (Beckman Coulter) cytometer, using fluorescent antibodies recognizing cell-surface markers associated Tn, Tscm, Tcm, Tern, and Temra memory T cell subsets. Specifically, Tn cells were identified as CD45RO-/CCR7+/CD95- ; Tscm cells were identified as CD45RO-/CCR7+/CD95+; Tcm cells were identified as CD45RO+/CCR7+; Tern cells were identified as CD45RO+/CCR7-; and Temra cells were identified as CD45RO-/CCR7-. Fluorescent signal associated with each marker was acquired using CytExpert software (Beckman Coulter) and the final data analysis was performed using FlowJo (BD Biosciences). As shown in FIG. 17, the cells produced using the KYV 3-v Day protocol and produced from T cells isolated directly from whole blood show less differentiated phenotype compared to mock (untransduced) control cells. Surprisingly, products made from directly -isolated T cells showed a dramatic increase in the desirable Tcm and Tscm T cell types, which comports with the results from the cells in Examples 1-2 made using T cells isolated from PBMCs.

Example 7 - Functional characteristics of CAR T cells engineered from T cells isolated directly from whole blood.

[0365] Anti-CD19 CAR T cells were produced using the KYV 3 -Day process as outlined in Examples 1 and 2, but by isolating T cells directly from whole blood from healthy donors rather than PBMCs and omitting the TransACT activation step in favor of concurrent activation and isolation using CD3/CD28 Dynabeads.

[0366] Briefly, the T cells were transduced with KL-hl98a28z, a self-inactivating (SIN) vesicular stomatitis virus (V SV)-G pseudotyped 3rd generation lentiviral vector encoding a chimeric antigen receptor that binds CD19. This CAR construct, named Hul9-CD828Z, has the amino acid sequence set forth in SEQ ID NO: 13. The lentiviral vector contained an MSCV promoter and other regulatory factors, including a central polypurine tract/central termination sequence upstream of the promoter, and a post-transcriptional regulatory element (PRE) downstream of the CAR expression sequence. The lentiviral vector KL-hl98a28z was manufactured using a HEK 293T cell line transiently transfected with a state-of-the-art four-plasmid system. The envelope protein encoding plasmid (pLTG1292) expresses a heterologous spike protein, the VSV-G protein, under control of the cytomegalovirus (CMV) promoter. The isolation and activation step was initiated simultaneously as set forth in FIG. 10 for the KYV 3-Day “vl” and “v2” processes. At 18 hours after the isolation/activation step the cells were incubated with the lentivirus vector for 48 hours and the transduced T cells harvested, frozen using a standard protocol, and thawed at a later time for characterization.

[0367] The short-term ability of the CAR-T cells to specifically kill CD 19+ target cells was measured by flow cytometry using a Cytoflex LX (Beckman Coulter) cytometer. Nalm6 cells were labeled with cell trace violet (CTV) and co-cultured with effector CAR-T cells at several effector- to-target (E:T) cell ratios for 24 hours. Percent cell killing was determined by the ratio of live CTV positive cells cocultured with effector cells to live CTV positive cells cultured in the absence of effector cells, at each E: T ratio. Data analysis was performed using FlowJo (BD Biosciences). [0368] As shown in FIG. 18, following 24h in vitro co-culture with CD 19+ NALM6 target cells at 0.1 : 1 E:T (effectortarget) ratio, the results show that the mock untransduced (UT) cells, which do not express a CAR, exhibit low nonspecific killing activity compared to the engineered anti- CD19 CAR T cells produced using the KYV 3-Day process using T cells that were concurrently isolated from whole blood and activated, which showed a clear target-dependent cytotoxicity.

[0369] The target-dependent cytokine release of the CAR T cells was also assessed. FIG. 19 provides the results for the target-dependent cytokine release by the CAR T cells derived from healthy donor (HD) whole blood, following 24h in vitro co-culture with CD 19+ NALM6 target cells at the indicated E:T (effectortarget) ratios. Supernatants were collected and analyzed by ELLA assay. Mock untransduced (UT) cells, which do not express a CAR, exhibited low background levels of cytokine release. In contrast, the CAR T cells exhibit a clear target-dependent cytokine release, indicative of their target-dependent cytotoxic behavior.

[0370] Based on the positive results of the short-term killing assay, the exhaustion resistance of the CAR T cells and the durability of their target-dependent cytotoxicity was assessed using a long-term, in vitro serial rechallenge assay.

[0371] As shown in FIG. 20, the CD19-targeted CAR-T cells and CD 19+ NALM6 target cells were co-cultured at the indicated E:T ratios (CAR-T effector:NALM6 target) and % killing of target cells was measured by flow cytometry at each indicated timepoint. At each timepoint, a fresh round of target cells was added to the co-culture to assess the serial, repeated cytotoxicity of the CAR-T over time. Mock transduced (“UT”) cells, which do not express a CAR, did not demonstrate any cytotoxic activity. In contrast, the CAR-T cells showed a durable immune response and provided recurring cytotoxic response upon rechallenge for at least 30-days post first exposure.

[0372] Accordingly, cells produced using the two-to-three-day methods of the disclosure, which include methods in which the T cells are isolated and activated directly from whole blood, provide an exhaustion-resistant phenotype that exhibits the desired target-specific cytotoxic activity.

[0373] Advantageously, not only were the resulting CAR T cells effective, they were shown to expand upon contact with target cells at a higher rate than CAR T cells made using the "KYV 8 day" or “KYV 6 Day” process, outlined in FIG. 10. As explained the 8-day process: (i) uses T cells obtained from an enriched population (e g., from a leukapheresis sample or isolated PBMCs); (2) a separated T cell isolation and activation step; and (3) longer periods of time for culture and expansion.

[0374] Briefly, the anti-CD19 CAR-T cells were generated from the conventional 8-day manufacturing process (“Conv”) or the KYV 3 -day process outlined in this example. Both sets of CAR T cells were derived from healthy donor (HD) whole blood starting material. CAR-T cells were stimulated by co-culturing with mitomycin C-treated CD 19+ NALM6 target cells added at a 1 : 1 ratio every 3-4 days. Expansion of viable T cells was calculated at each indicated timepoint using Vicell cell count analysis.

[0375] The results are provided in FIG. 21. As shown, the cells made using the 3-day process showed a greater rate of target-specific expansion, which indicates a desirable hi vivo immune profile. Thus, the short CAR T manufacturing methods of the disclosure, which include those using direct-from-whole-blood T cell isolation, concurrent activation and T cell isolation, and brief periods of culture and cultivation produces cells with favorable phenotypes, target-specific cytotoxicity, durable immune responses, and a high rate of expansion upon target stimulus.

[0376] As shown, the CAR T cells manufactured using the methods of the disclosure provide a high CAR expression, including a higher CAR expression within CD4 and CD8 subsets relative to other existing methods. Moreover, the cells exhibited a high proportion of the desired Tcm and Tscm cell subsets. Single cell cytokine analyses revealed cell produced using the methods of the disclosure have higher levels of IFNg and TNF-0 co pared to traditional processes, including other shortened processes such as the KYV Alternative 3 day, which relies on leukapheresis. Furthermore, the cells produced using the methods of the disclosure exhibited a higher level of polyfunctionality relative to CAR T cells made using alternative methods, which is likely due to the brief period of time between isolating the T cells from whole blood and harvesting the finale CAR T product. Further, the resulting CAR T product was shown to have an increased, relative to other methods as described, level of antigen-induced proliferation.

[0377] Accordingly, as shown, the methods of the disclosure provide the shortest path from sample to CAR T product, while providing cells of higher quality than existing CAR T manufacturing processes.

Example 8 - T cell subpopulations from whole blood samples [0378] To assess the subpopulations of CAR T cells obtained from whole blood, three sets of CAR T cells were produced using a whole blood starting sample: “AR037” which used isolation of T cells from whole blood (1 week old at 2-8 °C) and in which cells were processed downstream in the classic 9-day process from Examples 1-2; “AR039” did not use any downstream processing of the fresh whole blood material; and “AR050” which applied the 3-day v2 (CPD-23-007) process to isolate and engineer T cells from fresh whole blood.

[0379] The TBNK/memory phenotype of Pre- and post-enrichment material and TBNK/memory phenotype of the final CAR T cell product (e.g., after expansion) for AR037 is provided in FIGS. 22-23. The TBNK/memory phenotype of Pre- and post-enrichment material for AR037 is provided in FIG. 24. The TBNK/memory phenotype of Pre- and post-enrichment material for AR050 is provided in FIG. 25.

[0380] As shown, using whole blood as a starting material produces viable CAR T cells of which a high percentage are Tnscm. As shown in FIG. 23, the number of Tnscm is shown to increase upon expansion of the cells. Surprisingly, using the shortened 3-day process (FIG. 25), the initial number of Tnscm surpasses that of the 8-day process. Given the increase shown in the final product of the 8-day process, the 3-day process should produce a final product with an extraordinarily high percentage of Tnscm cells.

Example 9 - Ingenui-T platform

[0381] Traditionally, apheresis has been the source of cellular starting material for T-cell therapy products due to the large numbers of T-cells required to go through the conventional manufacturing process and generate sufficient modified T-cells for a therapeutic dose to treat oncology patients. The associated burden on patients due to the length and invasiveness of the apheresis cell collection procedure and the logistical constraints of transporting the apheresis product to the manufacturing location are challenges currently associated with CAR T-cell products that limit accessibility and necessitate a new approach. The challenges encountered in obtaining an optimal leukapheresis product for CAR T-cell manufacturing encompass operational hurdles like access to specialized apheresis centers that meet guidelines along with resourced and trained staff,¹ and technical issues such as vascular access, contamination from other cell types, and effectively managing adverse events during the collection process.² [0382] Furthermore, conventional CAR T-cell manufacturing involves a protracted culture of patient apheresis material over 7—10 days designed to maximize expansion, resulting in a final product with a highly differentiated T-cell phenotype. However, studies in oncology showing that T cells with younger, more stem-like phenotype are correlated with improved clinical benefit compared with those with differentiated memory, effector function, or exhaustion phenotypes, which are signatures of more differentiated T-cell types. Shorter manufacturing processes can alleviate these issues and lead to improved CAR T-cell products³.

[0383] Starting from a whole blood draw instead of apheresis, together with shortening the duration of CAR T-cell manufacturing, holds the potential to revolutionize the patient experience of CAR T-cell therapy by addressing key challenges associated with conventional methods. This optimization could decrease production costs⁴, increase treatment accessibility, and increase the overall feasibility of CAR T-cell therapies.

[0384] Ingenui-T is a next-generation CAR T-cell manufacturing platform initially being developed for autoimmune disease, utilizing the same fully human anti-CD19 CAR construct as KYV-101. KYV-101 is an investigational autologous anti-CD19 CAR T-cell therapy (manufactured using conventional methods) under investigation for patients with B-cell-driven autoimmune diseases, including lupus nephritis, systemic sclerosis, myasthenia gravis, multiple sclerosis, and other diseases with a strong rationale for B-cell involvement in the disease pathology. This example provides an exemplary use of the the novel Ingenui-T manufacturing platform of the disclosure, which highlights the platform’s ability to generate high-purity and functional CAR T cells.

[0385] Importantly, the Ingenui-T platform yields CAR T cells with a potent functional profile and a less differentiated phenotype compared to CAR T cells generated in a conventional manufacturing process that uses apheresis-derived cellular starting material. By circumventing the challenges associated with apheresis, the Ingenui-T platform offers a promising avenue for enhancing the efficiency and accessibility of CAR T-cell therapy, lowering costs, and ultimately advancing its application in the realm of autoimmune diseases.

Methods

[0386] Patient Whole Blood and Leukapheresis Collection [0387] Peripheral whole blood (up to 200 mb) from healthy donors (n=9, AllCells or Bloodworks Northwest, USA) was collected and transported fresh for immediate processing. Cry opreservation was intentionally avoided to maximize cell viability. A cell count was performed to quantify the incoming blood cell population. Donor-matched cryopreserved leukapheresis material (n=4, AllCells, USA) was also obtained to generate CAR T cells according to a conventional manufacturing process.

[0388] Ingenui-T Manufacturing Platform

[0389] Fig. 26 provides an overview of the exemplary use of the presently disclosed Ingenui-T manufacturing platform to produce engineered immune cells starting from whole blood.

[0390] As shown in Fig. 26, briefly, up to 200 mb of collected whole blood was added directly to the Gibco™ CTS™ DynaCellect™ Magnetic Separation System (Thermo Fisher Scientific, Waltham, MA), and anti-CD3/CD28 Dynabeads (CTS™ Detachable Dynabeads™ CD3/CD28 Kit; Thermo Fisher Scientific, Waltham, MA) were used at a defined ratio for enrichment and activation of T cells. Isolated and activated T cells were counted, analyzed by flow cytometry for CD3⁺ T-cell purity, and seeded into vessels containing culture media enriched with cytokines (human interleukin 2 [IL-2], IL-21, IL- 15, IL-7, or a combination thereof). Less than 24 hours post-seeding, T-cells were transduced using a lentiviral vector encoding the Hul9-CD828Z anti- CD19 CAR construct at a fixed multiplicity of infection (MOI). This is the same construct used in KYV-101-a first-in-class, fully human autologous anti-CD19 CAR T-cell therapy (Kyverna Therapeutics, Emeryville, C A). After a targeted in vitro cell culture period of less than 72 hours post-seeding, beads were removed from the culture, and cells were harvested, formulated into final product containers, and cryopreserved. In parallel, untransduced cells were also generated through the same manufacturing process in the absence of lentiviral transduction for use as control cells.

[0391] Conventional Research-Grade CAR T-Cell Manufacturing

[0392] For conventional Research Use Only (RUO) manufacturing of anti -CD 19 CAR T cells, the cell product was manufactured to represent the KYV-101 product. Cryopreserved leukapheresis material was thawed, washed, and subjected to antibody-driven T-cell isolation using magnetic beads (Miltenyi Biotec). The isolated T cells were counted, analyzed by flow cytometry for CD3⁺ T cell purity, and activated using T Cell TransAct (Miltenyi Biotec) in the presence of supporting cytokines (human IL-2, IL-21, IL-15, IL-7, or a combination thereof). As with the Ingenui-T platform, T cells were transduced at a fixed MOI using the same lentiviral vector incorporating the Hul9-CD828Z anti-CD19 CAR construct (Kyvema Therapeutics, Emeryville, CA). Cells were then cultured for 8- 10 days before harvesting, formulation, and cry opreservation of the final product.

[0393] In Vitro CAR T-Cell Phenotyping

[0394] Flow cytometry was used to analyze CD3⁺ T-cell purity, CD4⁺ and CD8⁺ T-cell populations, and anti-CD19 CAR expression. CD4 and CD8 T-cell memory phenotypes of the Ingenui-T final product were compared to the T-cell memory populations in whole blood starting material based on the expression of CD45RO, CCR7, and CD95 surface markers. Similarly, the T-cell memory phenotypes in conventional CAR T cells were compared to the T-cell memory populations in the apheresis starting material (donor-matched with whole blood Ingenui-T cells). CAR expression was analyzed at 0 hour and 72 hours post-thaw of the final drug product, to ensure an accurate determination of stably integrated expression.

[0395] In Vitro CAR T-Cell Functional Activity

[0396] To evaluate the functional activity of CAR T cells in a short-term, single-challenge cytotoxicity assay, donor-matched Ingenui-T cells or conventional CAR T cells were co-cultured with CD 19+ NALM6 target cells expressing an mCherry fluorescent reporter protein at the indicated effector to target (E:T) ratios for 120 hours. Target-specific cytotoxic activity was assessed by imaging co-cultures using an Incucyte Sx5 (Sartorius) instrument and calculating the survival or outgrowth of fluorescent target cells over time, normalized to the signal intensity at the start of co-culture.

[0397] To evaluate the long-term functionality of CAR T cells, donor-matched Ingenui-T cells or conventional CAR T cells were serially rechallenged every 2-3 days with CD19+ NALM6 target cells at the indicated E:T ratios. At each time point, samples were split in half to assess the percent cytotoxicity and to re-plate with fresh target cells. Percent cytotoxicity was calculated at each timepoint by measuring the target cell survival using flow cytometry and was normalized to the survival of target cells in the absence of CAR T effector cells.

[0398] To evaluate cytolytic activity of CAR T cells against autologous primary B cells, Ingenui- T cells, or control untransduced T cells were co-cultured with peripheral blood mononuclear cells (PBMCs) obtained from donor-matched leukapheresis material. Effector-to-target (E:T) ratios were defined according to the number of CAR⁺ Ingenui-T cells (effector) to total PBMCs (targets). After 48 hours, target-specific cytolytic activity against B cells was measured by flow cytometry. B cells were defined by surface expression of either CD19 or CD20, within gated CD3' cells, to ensure proper detection of B cells even in the presence of interactions with anti-CD19 CAR T cells. Percent cytolysis against B cells was calculated by normalizing to the survival of B cells in PBMC-only cultures.

Results

[0399] Ingenui-T Cell Manufacturing Platform

[0400] The objective was to demonstrate the technical feasibility of generating anti-CD19 CAR T cells starting from fresh whole blood material in a shortened manufacturing process. As shown in Fig. 26, fresh whole blood from healthy donors was loaded onto the DynaCellect platform for simultaneous isolation and activation of T cells using anti-CD3/CD28 Dynabeads at a defined bead: cell ratio. The isolated/activated T cells were sampled to confirm the isolation purity (>95% CD3+) by flow cytometry and subsequently seeded into culture with media containing the cytokines IL-2, IL-7, IL- 15, IL-21, or a combination thereof. Transduction using a lentiviral vector encoding the anti-CD19 CAR construct occurred at a fixed MOI within the first 24 hours of culture, followed by a brief period in culture to allow for cell recovery and integration of the transgene (<72 hours post-seeding). Following this brief culture period, CAR T cells were collected for bead removal and subsequent formulation in cryopreservation media. T-cell purity analysis of the final Ingenui-T cell product showed a T-cell percentage of 93.9±1.6%, obtained from a starting T-cell frequency of 42.3±6.8% in whole blood. These results were comparable to the T-cell enrichment obtained via a conventional CAR T-cell manufacturing process using donor- matched cells (final T-cell purity of 94.0±3.3% from a starting T-cell frequency of 46.3±7.2% in conventional apheresis; Table 1).

[0401] Given the short culture time, Ingenui-T cells demonstrated minimal expansion during the manufacturing process, resulting in a 0.68±0.09-fold change of the total T-cell number from the time of culture seeding to final formulation (including any losses due to washing and bead removal procedures). Nevertheless, the final yield of Ingenui-T cell product was 38.5±6.6>< 10⁶ T cells per 100 mL of starting whole blood. Product attributes were tested upon harvest and after 72 hours of post-thaw culture to simulate product performance in the patient. At 72 hours post-thaw of the drug product, CAR+ expression ranged between 45.1% 54.5% in Ingenui-T cells, which was statistically similar to the 37.4%-56.3% CAR+ expression obtained from the conventional CAR T-cell manufacturing process derived from apheresis (Table 1). This demonstrates that using the Ingenui-T platform, anti-CD19 CART cells can be successfully manufactured directly from whole blood, in a shortened manufacturing process, at a scale sufficient for therapeutic dosing of B-cell- driven autoimmune disease patients.

[0402] Phenotypic and Functional Comparison of Ingenui-T Cells and Conventional CAR T Cells [0403] To demonstrate the pharmacologic activity of anti-CD19 CAR T cells generated in the Ingenui-T platform, we performed a set of phenotypic and functional in vitro characterization experiments, comparing Ingenui-T cells with CAR T cells expressing the same anti-CD19 CAR construct generated with a conventional manufacturing process. Whole blood for the Ingenui-T process and apheresis for the conventional process were sourced from the same donors as part of the same collection. As expected, due to the shortened culture period in the Ingenui-T platform, Ingenui-T cells comprised a less differentiated T cell memory phenotype than CAR T cells generated in a conventional manufacturing process. Whole blood-derived Ingenui-T cells preserved a T-cell memory phenotype that closely resembled the phenotype observed in the starting material, with slight increases in the overall effector/memory compartment (combined TCM, TEM, and TE populations).

[0404] From starting material to the Ingenui-T final product, the effector/memory compartment shifted from a mean of 48.8±5.6% to 69.4±4.8% within the CD4+ T cell fraction, and from 42.9±3.9% to 46.6±5.5% within the CD8+ T cell fraction, while maintaining a substantial proportion of cells within the TN+TSCM compartment (Fig. 27A).

[0405] In contrast, CAR T cells obtained from the traditional manufacturing process (~9 days in culture) had mostly converted to the effector/memory compartment, shifting from a mean of 58.4±3.5% to 94.0±2.8% and from 40.1±5.2% to 86.2±4.9% within the CD4+ and CD8+ T cell fractions, respectively.

[0406] Importantly, minimizing the differentiation of Ingenui-T cells in vitro is anticipated to preserve their activation potential for in vivo expansion and activity in the patient. This in turn enables the administration of a significantly lower dose for equivalent therapeutic efficacy in clinical application, which is a critical attribute for Ingenui-T considering the inherently lower starting number of T cells that can be obtained from a whole blood draw relative to apheresis.

[0407] The functional activity of Ingenui-T cells was assessed in both short-term and long-term in vitro preclinical assays in order to demonstrate target-specific cytotoxicity against CD 19- expressing cells. The cytotoxic activity of Ingenui-T cells and donor-matched, apheresis-derived conventional CAR T cells (from a traditional 9-day culture expressing the same CAR construct) were compared in a short-term cytotoxicity assay against CD19+ NALM6 tumor cells, as a representative target cell line.

[0408] As shown in Fig. 27B, in an Incucyte imaging-based assay, Ingenui-T cells controlled the outgrowth of NALM6 target cells over a period of 120 hours, at lower E:T ratios than conventional CAR T cells. This reflects the expected increase in CAR T-cell potency and target-mediated CAR T-cell proliferation due to the less differentiated memory phenotype of Ingenui-T cells. Minimal cytotoxicity was observed against a control CD19-negative target cell line (CEM/C1), nor was any cytotoxic activity observed in untransduced Ingenui-T cells (i.e., without CAR expression; against CD 19-positive targets data not shown). These results confirmed the anti-CD19 target-specific activity of Ingenui-T cells and their increased functional potency relative to conventional CAR T cells that had been generated in a conventional manufacturing process.

[0409] To further evaluate the functional activity of Ingenui-T cells, we performed a long-term serial rechallenge assay in vitro. In this assay, CAR T cells and CD 19+ NALM6 target cells were co-cultured at the indicated E:T ratios starting at day 0, followed by the serial addition of the same number of NALM6 target cells every 2 or 3 days, to evaluate the potency and durability of targetspecific serial cytotoxicity over an extended time.

[0410] As shown in Fig. 27C, Ingenui-T cells continued to kill target cells for a significantly longer period at a given E:T ratio, and required a >4-fold lower E:T ratio than donor-matched CAR T cells generated from a 9-day culture process to maintain the same duration of killing. This finding again matched our expectation that the younger differentiation phenotype of Ingenui-T cells results in higher functional potency, greater proliferation (data not shown), and more prolonged cytolytic activity compared to conventional CAR T cells, which are more prone to exhaustion and loss of functionality over time. [0411] Finally, the in vitro cytolytic activity of Ingenui-T cells was assessed against autologous primary B cells, which are the cells targeted for depletion in the treatment of patients with B-cell- driven autoimmune diseases. When Ingenui-T cells were co-cultured for 48 hours with autologous total peripheral blood mononuclear cells (PBMCs), B cells were eliminated in a specific and dosedependent manner (Fig. 27D). As expected, Ingenui-T cells demonstrated greater potency of B cell killing than conventional CAR T cells when tested at dose-limiting E:T ratios (e.g., at 0.011 :1; Fig. 27D and data not shown).

[0412] These preclinical assays provide proof-of-concept data that highly functional anti -CD 19 CAR T cells could be generated using our Ingenui-T platform starting from fresh autologous whole blood material. These results have paved the way for the clinical development of Ingenui-T cells as a therapy for autoimmune disease, which will improve the patient experience, increase treatment accessibility, and reduce costs, by eliminating the need for patients to undergo apheresis.

Example 10 - Ingenui-T platform - Leukapheresis & Whole Blood (WB) Starting Material

SM [0413] This example provides further data related to cells manufactured from leukapheresis or WB SM using the Ingenui-T platform using methods as disclosed herein. The objective was to characterize anti-CD19 CAR T cells starting from fresh whole blood material in a shortened manufacturing process as compared with those made using a longer, 9-day process and/or starting from a leukapheresis SM.

[0414] Briefly, manufactured CAR T cells were prepared using the method outlined in Fig. 26, in which the starting material obtained from the subject in Fig. 26 was from a leukapheresis sample. [0415] Peripheral whole blood and leukapheresis SM was obtained from healthy donors, which was collected and transported fresh for immediate processing.

[0416] As shown in Fig. 26, briefly, a 100 mL whole blood or leukapheresis sample was collected and an aliquot was added directly to the Gibco™ CTS™ DynaCellect™ Magnetic Separation System (Thermo Fisher Scientific, Waltham, MA), and anti-CD3/CD28 Dynabeads (CTS™ Detachable Dynabeads™ CD3/CD28 Kit; Thermo Fisher Scientific, Waltham, MA) were used at a defined ratio for enrichment and activation of T cells. Isolated and activated T cells were counted, analyzed by flow cytometry for CD3⁺ T-cell purity, and seeded into vessels containing culture media enriched with cytokines (human interleukin 2 [IL-2], IL-21, IL- 15, IL-7, or a combination thereof). Less than 24 hours post-seeding, T-cells were transduced using a lentiviral vector encoding the Hul9-CD828Z anti-CD19 CAR construct at a fixed multiplicity of infection (MOI). This is the same construct used in KYV-101, which was described above. After a targeted in vitro cell culture, beads were removed from the culture, and cells were harvested, formulated into final product containers, and cryopreserved. In parallel, untransduced cells were also generated through the same manufacturing process in the absence of lentiviral transduction for use as control cells.

[0417] As shown in Fig. 28A, flow cytometry was used to analyze the overall T cell expansion, T cell viability, and T cell purity of CAR-T cells manufactured using the 3-day process of the Ingenui-T platform using a starting leukapheresis sample. Across a variety of different cytokine cultures, the 3-day process consistently produced T cells with over a 1-fold expansion, with over 90% viability, and over 95% purity. Importantly, these results remained consistent whether or not the T-cells were transduced with an exogenous immune receptor. [0418] Similarly, as shown in Fig. 28B, when starting with a whole blood sample, a high final concentrations of expanded T cells were produced, with a very high T-cell purity, using the 3-day process of the disclosure.

[0419] These results were comparable to the T-cell enrichment obtained via a conventional CAR T-cell manufacturing process using donor-matched cells. For cells made from WB SM in particular, and despite the short culture time, the Ingenui-T cells demonstrated minimal expansion during the manufacturing process, yet concurrently produced a high final yield. Thus, not only was a sufficient amount of final product produced, it was produced with limited expansion, which should produce T-cells with favorable phenotypes as described above.

[0420] Figure 29A shows the % CAR+ expression analyzed by flow cytometry in CAR-T cells manufactured in the KYV 3-Day process starting from leukapheresis material. CAR expression was analyzed within total CD3+ T cells or within CD4+ or CD8+ T cells, at the time of harvest. KYV 3-Day Conditions “A”, “B”, “C”, “D” indicate different culture cytokine(s) used. N=4 healthy donors per condition. As explained in Example 8, at 72 hours post-thaw of the drug product, CAR+ expression in Ingenui-T cells (leukapheresis SM) was statistically similar to the CAR+ expression obtained from the conventional CAR T-cell manufacturing process.

[0421] Similarly, as shown in Fig. 29B, when starting with a whole blood starting sample, the % CAR+ expression surprisingly appeared to surpass the results achieved when using the 3-day process with a leukapheresis starting sample.

[0422] CD4 and CD8 T-cell memory phenotypes of the Ingenui-T final product, either produced using a leukapheresis starting material (SM) or a whole blood (WB) SM, were compared to the T- cell memory populations in whole blood/ leukapheresis starting material based on the expression of CD45RO, CCR7, and CD95 surface markers. Similarly, the T-cell memory phenotypes in conventional CAR T cells were compared to the T-cell memory populations in the leukapheresis starting material (donor-matched with whole blood Ingenui-T cells). CAR expression was analyzed at 0 hour and 72 hours post-thaw of the final drug product, to ensure an accurate determination of stably integrated expression.

[0423] Figure 30A shows the CD4+ to CD8+ ratio for CAR-T cells using a leukapheresis SM and the 3-day process compared to similar cells produced using a traditional 9-day process. As shown, the 3-day process produces CAR-T cells with a CD4:CD8 ratio that is similar to the far-longer 9- day process.

[0424] Figure 30B shows similar results for cells manufactured using the 3-day process, but starting from whole blood (WB).

[0425] To demonstrate the pharmacologic activity of anti-CD19 CAR T cells generated in the Ingenui-T platform, a set of phenotypic and functional in vitro characterization assays were performed to compare Ingenui-T cells (using WB or leukapheresis SM) with CAR T cells expressing the same anti -CD 19 CAR construct generated with a conventional (9-day) manufacturing process.

[0426] As shown in Figs. 31A-31B, when compared to the 9-day process, the 3-day processes of the disclosure using a leukapheresis SM produced T cells with fewer effector T cells and a larger proportion of TNSCM cells, particularly, Tscm cells. Surprisingly, as shown in Fig. 31C, when using a WB SM, the 3-day method of the disclosure produced T-cells with an even higher proportion of TNSCM cells. Unexpectedly, these TNSCM cells included a much larger proportion of naive T cells relative to methods starting with a leukapheresis SM and a 9-day method starting with WB SM.

[0427] Whole blood for the Ingenui-T process and leukapheresis SM for the 3-day and 9-day processes were sourced from the same donors as part of the same collection. As expected, due to the shortened culture period in the Ingenui-T platform, Ingenui-T cells comprised a less differentiated T cell memory phenotype than CAR T cells generated in a conventional manufacturing process. Whole blood-derived Ingenui-T cells preserved a T-cell memory phenotype that closely resembled the phenotype observed in the starting material, with slight increases in the overall effector/memory compartment (combined TCM, TEM, and TE populations). These were a dramatic improvement over the existing 9-day process. Thus, although the cells were transduced with the same exogenous CARs, they nevertheless are fundamentally different final products.

[0428] The functional activity of Ingenui-T cells was assessed using both WB SM and leukapheresis SM in order to demonstrate target-specific cytotoxicity against CD19-expressing cells. The cytotoxic activity of Ingenui-T cells and donor-matched, apheresis-derived conventional CAR T cells (from a traditional 9-day culture expressing the same CAR construct) were compared in a short-term cytotoxicity assay against CD 19+ NALM6 tumor cells, as a representative target cell line.

[0429] Fig. 32 shows % cytolysis of CD19+ NALM6 target cells or CD19- CEM/C1 control cells after co-culture with anti-CD19 CAR-T cells manufactured from KYV 3 -Day process, at the indicated E:T (Effector: Target) ratios. N=2 donors shown. Cytolytic activity was measured by luminescence assay and normalized to target cells alone (0: 1). As shown, the 3-day process cells produce a clear, target-dependent cytotoxic response against CD 19+ expressing cells.

[0430] Fig. 33A shows the results of killing or outgrowth of CD19+ NALM6 target cells during 120h co-culture with anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from leukapheresis starting material, at the indicated E:T (Effector:Target) ratios of 0.3: 1 or 1 : 1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized to time = 0. KYV 3-Day Conditions “Cl”, “C2”, “C3”, “C4” indicate different culture cytokine(s) used. “NT” = non-transduced control T cells. One representative donor shown from n=4. Similarly, Fig. 33B provides analogous results of killing or outgrowth of CD19+ NALM6 target cells during 120h co-culture with anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from freshly collected whole blood, at the indicated E:T (Effector:Target) ratios of 0.3: 1 or 1 :1. NALM6 growth was measured by fluorescence in an Incucyte-based imaging assay and normalized to time = 0. “NT” = non-transduced control T cells. “Conv 9-day” refers to donor- matched CAR-T cells manufactured in a conventional 9-day culture process, from leukapheresis starting material. One representative donor shown from n=4.

[0431] As shown in Fig. 33A, in an Incucyte imaging-based assay, Ingenui-T cells (leukapheresis SM) controlled the outgrowth of NALM6 target cells over a period of 120 hours, at lower E:T ratios than conventional CAR T cells. Similar results were found for cells produced using WB SM (Fig. 33B). T-cells produced using the three-day process, whether starting from WB SM or a leukapheresis sample produced a durable, target-specific cytotoxic response that surpassed the efficacy of comparable cells produced using a longer (e.g., 9-day) process. Cells produced using a WB SM produce a more effective and durable cytotoxic response even when compared to cells produced using a leukapheresis sample.

[0432] This reflects the expected CAR T-cell potency and target-mediated CAR T-cell proliferation of the cells. Furthermore, minimal cytotoxicity was observed against a control CD19- negative target cell line (CEM/C1), nor was any cytotoxic activity observed in untransduced Ingenui-T cells. These results confirmed the anti-CD19 target-specific activity of Ingenui-T cells made from WB SM.

[0433] The in vitro cytolytic activity of Ingenui-T cells was assessed against autologous primary B cells, which are the cells targeted for depletion in the treatment of patients with B-cell-driven autoimmune diseases. To evaluate cytolytic activity of CAR T cells against autologous primary B cells, Ingenui-T cells, or control untransduced T cells were co-cultured with peripheral blood mononuclear cells (PBMCs) obtained from donor-matched leukapheresis material. Effector-to- target (E:T) ratios were defined according to the number of CAR⁺ Ingenui-T cells (effector) to total PBMCs (targets). After 48 hours, target-specific cytolytic activity against B cells was measured by flow cytometry. B cells were defined by surface expression of either CD 19 or CD20, within gated CD3' cells, to ensure proper detection of B cells even in the presence of interactions with anti-CD19 CAR T cells. Percent cytolysis against B cells was calculated by normalizing to the survival of B cells in PBMC-only cultures.

[0434] When Ingenui-T cells made from WB SM were co-cultured for 48 hours with autologous total peripheral blood mononuclear cells (PBMCs), B cells were eliminated in a specific and dosedependent manner (Fig. 34). Ingenui-T cells demonstrated greater potency of B cell killing than conventional CAR T cells when tested at dose-limiting E:T ratios.

[0435] Fig. 35 shows IFN-gamma production by anti-CD19 CAR-T cells manufactured in the KYV 3 -Day process from freshly collected whole blood, in co-culture with CD 19+ NALM6 target cells or CD19- CEMC1 control cells at the indicated E:T (Effector: Target) ratios. Culture supernatants were collected and analyzed by ELLA. N=2 donors shown. These results show targetdependent cytokine release by anti-CD19 CAR-T cells manufactured from KYV 3-day process, in response to CD 19+ expressing target cells.

[0436] Fig 36A shows cytokine release by anti-CD19 CAR-T cells manufactured in the KYV 3- Day process from leukapheresis starting material, in co-culture with CD19+ NALM6 target cells at the indicated E:T (Effector: Target) ratios of 0.3 : 1 or 1 : 1. Figs. 36B-36C shows cytokine release by anti -CD 19 CAR-T cells manufactured in the KYV 3-Day process from freshly collected whole blood, in co-culture with CD19+ NALM6 target cells at the indicated E:T (Effector: Target) ratios of 0.3 : 1 or 1 : 1. Culture supernatants were collected and the indicated cytokines were analyzed by MSD. KYV 3-Day Conditions “Cl”, “C2”, “C3”, “C4” indicate different culture cytokine(s) used in the manufacturing process. “Conv 9-day” refers to donor-matched CAR-T cells manufactured in a conventional 9-day culture process, derived from leukapheresis starting material. “NT” = nontransduced control T cells. N=4 healthy donors per condition. As shown, cells produced using the 3-day methods of the disclosure provide an effector dose-dependent, CAR-mediated cytokine release in response to CD19+ expressing target cells.

[0437] The long-term serial cytotoxic activity by anti-CD19 CAR-T cells manufactured from KYV 3-day process was compared to a conventional 9-day process, in response to CD 19+ expressing target cells. Fig. 37 shows the duration of in vitro cytotoxicity by KYV 3-Day or Conv 9-Day anti-CD19 CAR T cells, derived from a healthy donor, in a serial rechallenge assay against CD19+ NALM6 tumor cells. KYV 3-Day CAR T cells were derived from leukapheresis starting material (“APH”, Top panel) or freshly collected whole blood (“WB”, Bottom panel). CAR T cells were co-cultured in triplicate with NALM6 target cells at the indicated Effector: Target (E:T) ratios, and the survival of NALM6 cells was analyzed every 2-3 days by flow cytometry. The time (days) to loss of CAR-mediated cytotoxic activity, defined as the assay timepoint at which >95% survival of target cells was detected, was measured for each individual replicate. Data representative of n=4 donors. As shown, the WB cells produced a more durable a long-lasting cytotoxic response.

[0438] Fig. 38 shows data comparing in vitro expansion by anti-CD19 CAR-T cells manufactured from KYV 3-day process compared to a conventional 9-day process, in response to CD19+ expressing target cells. The data provides the responses of KYV 3-Day or conventional (“Conv”) 9-Day anti-CD19 CAR T cells to repeated stimulation in co-culture with CD 19+ expressing REH target cells. KYV 3-Day CAR T cells were derived from leukapheresis starting material (“APH”, n=4) or other freshly collected starting material (“WB”, n=3), and compared to donor-matched Conv 9-Day CAR T cells derived from leukapheresis material. CAR T cells were co-cultured with mitomycin C-treated REH target cells at a 1 : 1 ratio, and cells were re-plated every 3-4 days with new target cells. Total fold expansion of CAR+ T cells (gated by flow cytometry analysis) was measured at day 16. As shown, the cells show the potential to expand upon contact with an appropriate target.

[0439] Based upon these encouraging results, the in vivo activity of the 3-day cells using freshly collected starting material was assessed in mice.

Ill [0440] Fig. 39A-39B show data pertaining to the in vivo activity of anti-CD19 CAR-T cells manufactured from KYV 3 -day process compared to conventional 9-day process, in CD19+ NALM6 tumor-bearing NSG mice.

[0441] Fig. 39A shows mean NALM6 tumor growth in NSG mice treated with the indicated doses of donor-matched anti-CD19 CAR T cells manufactured from the KYV 3-day process or a conventional (“Conv”) 9-day process, both derived from leukapheresis (“APH”) starting material. NALM6-luciferase tumor cells were injected intravenously into mice at day -7 prior to T cell transfer. On day 0, mice were given a single intravenous injection of the indicated doses of CAR T cells. Tumor burden in each animal was measured twice per week using IVIS bioluminescent imaging and shown as total flux (photons/sec). Data is shown as mean ± SEM of all animals per group. Data is representative of 2 studies using n=2 independent donors.

[0442] Fig. 39B shows individual NALM6 tumor growth curves in NSG mice treated with a le6 CAR+ T cell dose of donor-matched anti-CD19 CAR T cells. CAR T cells were manufactured from the KYV 3-day process, derived from freshly collected whole blood (“WB”), or a conventional (“Conv”) 9-day process, derived from leukapheresis starting material. Tumor cells were inoculated and mice were treated and analyzed as described for FIG. 39A. N=5 animals per group.

[0443] To further assess the success of the presently disclosed methods to produce CAR T cells using starting material derived from freshly collected whole blood, 9-day processes were developed based on the KYV 3-day process and Alternative 3-day process (Fig. 10). Fig. 40 outlines these whole-blood 9-day processes.

[0444] Briefly, anti-CD19 CAR-T drug products (DP) were engineered from T cells isolated directly from whole blood (WB) obtained from healthy donors (HD), using one of the two 9-day culture process. As shown in Fig. 40, T cells were isolated from whole blood either using anti- CD3 microbeads followed by TransAct activation (n=l), or concurrently isolated and activated using anti-CD3/CD28 Dynabeads (n=2). Less than 24 hours post-seeding, T-cells were transduced using a lentiviral vector encoding the Hul9-CD828Z anti-CD19 CAR construct at a fixed multiplicity of infection (MOI). Transduced cells were then cultured for a total of 9 days (from time of seeding). Figs. 41 A-41E provide data characterizing the products made usings these 9-day processes based on the 3-day whole blood processes of the disclosure. As shown in Fig. 41 A, for both whole-blood 9-day methods, whether T cells were isolated from WB using anti-CD3 microbeads followed by TransAct activation (solid triangle, n=l), or concurrently isolated and activated using anti-CD3/CD28 Dynabeads (solid circles, n=2), the final yield of drug product was high, and appeared to continue the trend set by the 3-day whole blood methods described herein (e.g., Fig. 28B). Fig. 4 IB provides flow cytometry data showing the % CAR+ cells within the DP. Fig. 41 C provides flow cytometry data showing % T cell purity within the DP compared to the WB starting material (SM). As shown, both variations of the 9-day whole blood method resulted in a successful CAR expression and >90% T cell purity within the final product. This is comparable to the 3-day whole blood methods (e.g., Figs. 29A-29B).

[0445] Fig. 4 ID provides flow cytometry data showing the CD4/8 fractions in the drug product while Fig. 4 IE provides flow cytometry data showing the T cell memory phenotype within the DP compared to the WB SM at the end of CAR T-cell production (n=3 combined from both isolation methods described for Figs. 41A-41C). The CD4/8 fraction is similar to the 3-day methods. However, the T-cell memory phenotype shows a different final drug product relative to both the 3-day whole blood methods and the conventional 9-day processes (not starting from whole blood) described above. Compared to the “conventional” 9-day process, these 9-day methods based on the whole-blood 3-day methods, produce a final drug product in which the memory or effector phenotype of the T cells (e.g., predominantly Tcm cells) is similar to that seen in the “conventional” 9-day process that starts with apheresis material (e.g., Fig. 31C). Accordingly, the 9-day whole blood processes also produced a lower TNSCM component relative to the 3-day processes (e.g., Fig. 31C), which had higher proportions of Tscm cells and naive T cells. The presently disclosed 9-day whole-blood methods offer an alternative to methods that, while requiring 9-days at the manufacturing step, must still rely on obtaining apheresis starting material. Thus, the presently disclosed methods are able to ease the bottlenecks in production associated with obtaining apheresis samples, while delivering an equivalent or better therapeutically effective cellular product.

[0446] To assess the therapeutic potential of T cells produced using these 9-day processes, the CD19-dependent, CAR-mediated cytotoxic activity of the cells was evaluated by co-culturing the CAR T-cells derived from WB starting material, with CD19+ NALM6 target cells or CD19- CEMC1 control cells, or co-culturing non-transduced control T cells with CD 19+ NALM6 target cells. The % cytolysis of target cells was evaluated by luminescence after 24h co-culture. Fig. 42 provides the % cytolysis results. Fig. 43 provides corresponding cytokine secretion data. As shown, cells produced from whole blood using the 9-day methods described herein provide a target-dependent cytotoxic response.

[0447] As shown, using whole blood starting material for the 3-day and 9-day Ingenui-T processes produces a final product with therapeutic potential. Confirming certain aspects of the Ingenui-T processes, drug product produced from the 3-day processes, as expected, due to the shortened culture period, comprised T cells with a higher effector/memory compartment relative to the whole-blood 9-day process, which itself had a comparable effector/memory compartment relative to a “conventional” 9-day process. Whole blood-derived Ingenui-T cells preserve a T-cell memory phenotype that more closely resembles the phenotypes observed in the starting materials, with slight increases in the overall effector/memory compartment (combined TCM, TEM, and TE populations) as the culture process lengthens. Both the 3-day and 9-day processes improved over the existing, conventional 9-day process. Thus, although the cells were transduced with the same exogenous CARs, they nevertheless are fundamentally different final products.

[0448] These preclinical assays provide proof-of-concept data, including in vivo data for the three- day processes, that highly functional anti-CD19 CAR T cells can be generated using the Ingenui- T platform starting from fresh autologous whole blood material. These results have paved the way for the clinical development of Ingenui-T cells as a therapy for autoimmune disease, which will improve the patient experience, increase treatment accessibility, and reduce costs, by eliminating the need for patients to undergo apheresis.

Discussion

[0449] The Ingenui-T platform is focused on enhancing patient experience and reducing the cost of manufacturing CAR T-cell therapies. The next-generation manufacturing process starts from autologous whole blood and uses a rapid (<3 day) manufacturing process, resulting in a potent CAR T-cell product with demonstrated target-specific killing activity. This manufacturing process marks a significant departure from traditional methods that necessitate apheresis, a laborious and resource-intensive process, and extended cell culture.

[0450] The use of whole blood eliminates the need for apheresis, alleviating the burden on the patient as it reduces the need for a lengthy and invasive collection process, ultimately enhancing the overall patient experience. Further, there is a fixed quantity of beds available for apheresis which limits the number of patients who can be treated, and which is becoming an even bigger concern as CAR T cells as CAR T therapies extend to non-Oncology indications such as autoimmunity where patient numbers are significantly higher (millions of patients compared to tens of thousands). The Ingenui-T platform reduces the differentiation of the cells in vitro by minimizing the culture time, resulting in both a shorter process and a more potent product that can potentially provide equivalent therapeutic benefit with a lower dose, while enabling the feasibility of using a limited volume of whole blood rather than apheresis as starting material. Streamlining the process through a combination of collecting up to 300 mb of whole blood, and minimizing the culture time, not only reduces and optimizes resource utilization, but also reduces the time spent in specialized facilities and reduces involvement of highly skilled personnel, enhancing the costefficiency of CAR T-cell therapy. This reduction in the overall cost of goods to manufacture and decreased burden on patients holds promise for broader accessibility and affordability This optimization also aligns with the goal of scalability of CAR T-cell therapies, addressing a critical need in the field.

[0451] The results of Ingenui-T cell manufacturing demonstrate the ability to enrich T cells from blood and successfully generate potent anti-CD19 CAR T cells, comparable or superior in activity to anti-CD19 CAR T cells derived from apheresis material in a conventional manufacturing process. Phenotypic characterization indicates a less differentiated phenotype in Ingenui-T cells compared to conventionally manufactured CAR T cells. This characteristic may have clinical implications, as less differentiated T cells are associated with enhanced in vivo expansion and efficacy.

[0452] The Ingenui-T anti-CD19 CAR T-cell product, currently in development for the treatment of B-cell-driven autoimmune diseases, introduces a novel treatment paradigm, addressing key challenges associated with traditional apheresis-based manufacturing methods. The reduced burden on patients, cost-efficiency, and the platform's unique approach highlights its potential to significantly impact the feasibility and accessibility of CAR T-cell therapy for the treatment of autoimmune diseases.

OTHER EMBODIMENTS [0453] Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure. Other embodiments are in the claims.

[0454] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

INCORPORATION BY REFERENCE

[0455] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

REFERENCES

[0001] (1) Mikhael, J., Fowler, J. & Shah, N. Chimeric Antigen Receptor T-Cell Therapies: Barriers and Solutions to Access. JCO Oncology Practice 18, 800-807 (2022).

[0002] (2) Qayed, M. et al. Leukapheresis guidance and best practices for optimal chimeric antigen receptor T-cell manufacturing. Cytotherapy 24, 869-878 (2022).

[0003] (3) Bulliard, Y., Andersson, B. S., Baysal, M. A., Damiano, J. & Tsimberidou, A. M. Reprogramming T cell differentiation and exhaustion in CAR-T cell therapy. J Hematol Oncol 16, 108 (2023).

[0004] (4) Ghassemi, S. et al. Rapid manufacturing of non-activated potent CAR T cells. Nat Biomed Eng 6, 118-128 (2022).

SEQUENCE APPENDIX

Claims

1. A method for producing a population of engineered T cells expressing a heterologous protein, the method comprising: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample; between 10 hours and 25 hours after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium for a period of between 24 hours and 60 hours; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.

2. The method of claim 1, wherein the T cells are isolated from the whole blood sample directly without an intervening T cell isolation step and/or leukapheresis.

3. The method of claim 1, wherein prior to the binding step, the method comprises isolating peripheral blood mononuclear cells (PBMC) comprising the T cells from the whole blood sample without a leukapheresis step.

4. The method of any one of claims 1-3, wherein the harvested T cells comprise between about 10% and 60% CD45RO-/CCR7+ T cells (Tnscm).

5. The method of claim 4, wherein the harvested T cells comprise at least 18% Tnscm.

6. The method of claim 4, wherein the harvested T cells comprise at least 22% Tnscm.

7. The method of claim 4, wherein the harvested T cells comprise at least 25% Tnscm.

8. The method of any one of claims 4-7, wherein the Tnscm comprise naive T cells (Tn) and stem cell memory T cells (Tscm).

9. The method of claim 8, wherein the Tnscm comprise more Tscm than Tn.

10. The method of claim 9, wherein the Tnscm comprise at least 1.5x more Tscm than Tn.

11. The method of claim 10, wherein the Tnscm comprise at least twice as many Tscm as Tn.

12. The method of claim 11, wherein the Tnscm comprise at least 3x more Tscm than Tn.

13. The method of claim 12, wherein the Tnscm comprise at least 5x more Tscm than Tn.

14. The method of claim 13, wherein the Tnscm comprise at least l Ox more Tscm than Tn.

15. The method of claim 14, wherein the Tnscm comprise at least 50x more Tscm than Tn.

16. The method of any one of claims 1-15, wherein the contacting step occurs between 15 hours and 19 hours after the binding step.

17. The method of any one of claims 1-16, wherein the contacting step occurs between 17 hours and 19 hours after the binding step.

18. The method of any one of claims 1-17, wherein the contacting step occurs about 18 hours after the binding step.

19. The method of any one of claims 1-18, wherein the cultivating step is for a period of between 36 hours and 52 hours.

20. The method of any one of claims 1-19, wherein the cultivating step is for a period of between 46 hours and 50 hours.

21. The method of any one of claims 1-20, wherein the cultivating step is for a period of about 48 hours.

22. The method of any one of claim 1-21, wherein the one or more anti-CD3 antibodies and the one or more anti-CD28 antibodies attached to the same support.

23. The method of any one of claims 1-22, wherein the one or more anti-CD3 antibodies and/or the one or more anti-CD28 antibodies are attached to the support via a cleavable linker.

24. The method of claim 22, wherein the linker is enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker.

25. The method of claim 23 or claim 24, further comprising contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface.

26. The method of any one of claims 1-25, wherein the surface is a solid surface.

27. The method of claim 26, wherein the solid surface is a bead, well, chip, or microfluidic channel.

28. The method of claim 28, wherein the solid surface is a bead.

29. The method of any one of claims 1-25, wherein the surface comprises a polymer.

30. The method of claim 29, wherein the polymer is a hydrogel.

31. The method of claim 29, wherein the surface comprises a polymer scaffold.

32. The method of any one of claims 1-31, wherein the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation.

33. The method of claim 32, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb.

34. The method of claim 32, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.

35. The method of claim 34, wherein at least 1% of the harvested T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.

36. The method of claim 32, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MIP-la and MIP-lb.

37. The method of claim 32, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNg and Granzyme B.

38. The method of claim 32, wherein the polyfunctional T cells and/or a portion of the population thereof, comprise two or more of: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP-la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

39. The method of claim 38, wherein the polyfunctional T cells and/or a portion of the population of polyfunctional T cells comprise: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP-la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

40. The method of any one of claims 1-39, wherein the serum free cultivation medium comprises at least one cytokine.

41. The method of claim 40, wherein the at least one cytokine comprises one or more of IL- 2, IL-21, IL-7, and IL-15.

42. The method of claim 40, wherein the at least one cytokine comprises one or more of IL- 21, IL-7, and IL-15 and does not comprise IL-2.

43. The method of any one of claims 1-42, wherein the number of isolated T cells from the whole blood sample is between about IxlO⁶ and about IxlO⁸ total T cells and the number of harvested T cells is between about IxlO⁸ and about 5xl0⁸.

44. The method of claim 43, wherein the number of isolated T cells from the whole blood sample is between about 5xl0⁷ and about 7.5xl0⁷ total T cells and the number of harvested T cells is between about 7.5xl0⁷ and about 1.2xl0⁸.

45. The method of claim 43 or claim 44, wherein the culturing step produces between a 1.0- and a 4-fold expansion of the harvested T cells.

46. A method for producing a population of engineered T cells expressing a heterologous protein, the method comprising: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample without an intervening PBMC isolation step or a leukapheresis step; contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.

47. The method of claim 46, wherein the contacting step occurs between 10 hours and 25 hours after binding the T cells.

48. The method of claim 46 or claim 47, wherein cultivating the T cells contacted with said nucleic acid occurs for a period of between 28 hours and 60 hours.

49. The method of any one of claims 46-48, wherein the harvested T cells comprise between about 10% and 60% CD45RO-/CCR7+ T cells (Tnscm).

50. The method of claim 49, wherein the harvested T cells comprise at least 18% Tnscm.

51. The method of claim 49, wherein the harvested T cells comprise at least 22% Tnscm.

52. The method of claim 49, wherein the harvested T cells comprise at least 25% Tnscm.

53. The method of any one of claims 49-52, wherein the Tnscm comprise naive T cells (Tn) and stem cell memory T cells (Tscm).

54. The method of claim 53, wherein the Tnscm comprise more Tscm than Tn.

55. The method of claim 54, wherein the Tnscm comprise at least 1.5x more Tscm than Tn.

56. The method of claim 55, wherein the Tnscm comprise at least twice as many Tscm as Tn.

57. The method of claim 56, wherein the Tnscm comprise at least 3x more Tscm than Tn.

58. The method of claim 57, wherein the Tnscm comprise at least 5x more Tscm than Tn.

59. The method of claim 58, wherein the Tnscm comprise at least lOx more Tscm than Tn.

60. The method of claim 59, wherein the Tnscm comprise at least 50x more Tscm than Tn.

61. The method of any one of claims 46-60, wherein the contacting step occurs between 15 hours and 19 hours after the binding step.

62. The method of any one of claims 46-61, wherein the contacting step occurs between 17 hours and 19 hours after the binding step.

63. The method of any one of claims 46-62, wherein the contacting step occurs about 18 hours after the binding step.

64. The method of any one of claims 46-63, wherein the cultivating step is for a period of between 36 hours and 52 hours.

65. The method of any one of claims 46-64, wherein the cultivating step is for a period of between 46 hours and 50 hours.

66. The method of any one of claims 46-65, wherein the cultivating step is for a period of about 48 hours.

67. The method of any one of claim 46-66, wherein the one or more anti-CD3 antibodies and the one or more anti-CD28 antibodies attached to the same support.

68. The method of any one of claims 46-67, wherein the one or more anti-CD3 antibodies and/or the one or more anti-CD28 antibodies are attached to the support via a cleavable linker.

69. The method of claim 68, wherein the linker is enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker.

70. The method of claim 68 or claim 69, further comprising contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface.

71. The method of any one of claims 46-70, wherein the surface is a solid surface.

72. The method of claim 71, wherein the solid surface is a bead, well, chip, or microfluidic channel.

73. The method of claim 72, wherein the solid surface is a bead.

74. The method of any one of claims 46-70, wherein the surface comprises a polymer.

75. The method of claim 74, wherein the polymer is a hydrogel.

76. The method of claim 74, wherein the surface comprises a polymer.

77. The method of claim 74, wherein the polymer forms a polymer scaffold.

78. The method of any one of claims 46-77, wherein the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation.

79. The method of claim 78, wherein the polyfunctional T cells and/or a portion of the population thereof that simultaneously secrete Granzyme B and TNFb.

80. The method of claim 78, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.

81. The method of claim 80, wherein at least 1% of the harvested T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.

82. The method of claim 78, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MTP-la and MIP-lb.

83. The method of claim 78, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNg and Granzyme B.

84. The method of claim 78, wherein the polyfunctional T cells and/or a portion of the population of polyfunctional T cells comprise two or more of: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP-la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

85. The method of claim 78, wherein the polyfunctional T cells comprise: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP-la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.

86. The method of any one of claims 46-85, wherein the serum free cultivation medium comprises at least one cytokine.

87. The method of claim 86, wherein the at least one cytokine comprises one or more of IL- 2, IL-21, IL-7, and IL-15.

88. The method of claim 86, wherein the at least one cytokine comprises one or more of IL- 21, IL-7, and IL- 15 and does not comprise IL-2.

89. The method of any one of claims 46-88, wherein the number of isolated T cells from the whole blood sample is between about IxlO⁶ and about IxlO⁸ total T cells and the number of harvested T cells is between about IxlO⁶ and about 1.5xl0⁸.

90. The method of claim 89, wherein the number of isolated T cells from the whole blood sample is between about 5xl0⁷ and about 7.5xl0⁷ total T cells and the number of harvested T cells is between about 7.5xl0⁷ and about 1.2xl0⁸.

91. The method of claim 89 or claim 90, wherein the culturing step produces between a 1.0- and a 4.0-fold expansion of the harvested T cells.

92. A method for producing a population of engineered T cells expressing a heterologous protein, the method comprising: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD4 antibodies and one or more anti-CD8 antibodies attached to a support, thereby isolating the T cells from the whole blood sample; between 10 hours and 25 hours after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium for a period of between 24 hours and 60 hours; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.

93. The method of claim 92, wherein the T cells are isolated from the whole blood sample directly without an intervening T cell isolation step and/or leukapheresis.

94. The method of claim 92, wherein prior to the binding step, the method comprises isolating peripheral blood mononuclear cells (PBMC) comprising the T cells from the whole blood sample without a leukapheresis step.

95. The method of any one of claims 92-94, further comprising a step of activating the isolated T cells.

96. The method of any one of claims 92-95, wherein the harvested T cells comprise between about 10% and 60% CD45RO-/CCR7+ T cells (Tnscm).

97. The method of claim 96, wherein the Tnscm comprise naive T cells (Tn) and stem cell memory T cells (Tscm).

98. The method of claim 97, wherein the Tnscm comprise more Tscm than Tn.

99. The method of any one of claims 92-98, wherein the contacting step occurs between 15 hours and 19 hours after the binding step.

100. The method of any one of claims 92-99, wherein the cultivating step is for a period of between 36 hours and 52 hours.

101. The method of any one of claims 92-100, wherein the one or more anti-CD4 antibodies and the one or more anti-CD8 antibodies attached to the same support.

102. The method of any one of claims 92-100, wherein the one or more anti-CD4 antibodies and/or the one or more anti-CD8 antibodies are attached to the support via a cleavable linker.

103. The method of claim 102, wherein the linker is enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker.

104. The method of claim 102 or claim 103, further comprising contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface.

105. The method of any one of claims 92-104, wherein the surface is a solid surface.

106. The method of claim 105, wherein the solid surface is a bead, well, chip, or microfluidic channel.

107. The method of any one of claims 92-104, wherein the surface comprises a polymer.

108. The method of claim 107, wherein the polymer is a hydrogel.

109. The method of claim 107, wherein the surface comprises a polymer scaffold.

110. The method of any one of claims 92-109, wherein the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation.

111. The method of claim 110, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb.

112. The method of claim 110, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.

113. The method of claim 110, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MIP-la and MIP-lb.

114. The method of claim 110, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNg and Granzyme B.

115. The method of any one of claims 92-114, wherein the serum free cultivation medium comprises at least one cytokine.

116. The method of claim 115, wherein the at least one cytokine comprises one or more of IL- 2, IL-21, IL-7, and IL-15.

117. The method of claim 115, wherein the at least one cytokine comprises one or more of IL- 21, IL-7, and IL-15 and does not comprise IL-2.

118. The method of any one of claims 92-117, wherein the number of isolated T cells from the whole blood sample is between about IxlO⁶ and about IxlO⁸ total T cells and the number of harvested T cells is between about IxlO⁸ and about 5xl0⁸.

119. The method of claim 118, wherein the number of isolated T cells from the whole blood sample is between about 5xl0⁷ and about 7.5xl0⁷ total T cells and the number of harvested T cells is between about 7.5xl0⁷ and about 1.2xl0⁸.

120. The method of claim 118 or claim 119, wherein the culturing step produces between a 1.0- and a 4-fold expansion of the harvested T cells.

121. A method for producing a population of engineered T cells expressing a heterologous protein, comprising: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample, wherein the T cells are isolated from the whole blood sample directly without an intervening T cell isolation step and/or leukapheresis; between 20 hours and 28 hours after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium for a period of between 4 days and 9 days; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.

122. A method for producing a population of engineered T cells expressing a heterologous protein, comprising: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD4 antibodies and one or more anti-CD8 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample, wherein the T cells are isolated from the whole blood sample directly without an intervening T cell isolation step and/or leukapheresis; between 20 hours and 28 hours after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium for a period of between 4 days and 9 days; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.

123. The method of claim 122, further comprising a step of activating the isolated T cells.

124. The method of any one of claims 121 to 123, wherein the contacting step occurs between about 22 and 26 hours after the binding step.

125. The method of claim 124, wherein the contacting step occurs between about 23 and 25 hours after the binding step.

126. The method of claim 124, wherein the contacting step occurs about 24 hours after the binding step.

127. The method of anyone of claims 121 to 126, wherein, the cultivating step is for a period of about 5 to about 7 days.

128. The method of anyone of claims 121 to 126, wherein, the cultivating step is for a period of about 6 days.

129. The method of anyone of claims 121 to 126, wherein, the cultivating step is for a period of about 8 days. 130. The method of any one of claims 121-129, wherein the harvested T cells comprise between about 10% and 60% CD45RO-/CCR7+ T cells (Tnscm).

130. The method of claim 129, wherein the Tnscm comprise naive T cells (Tn) and stem cell memory T cells (Tscm).

131. The method of claim 130, wherein the Tnscm comprise more Tscm than Tn.

132. The method of any one of claims 121-131, wherein the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation.

133. The method of claim 132, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb.

134. The method of claim 132, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.

135. The method of claim 132, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MIP-la and MIP-lb.

136. The method of claim 132, wherein the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNg and Granzyme B.

137. The method of any one of claims 121-136, wherein the serum free cultivation medium comprises at least one cytokine.

138. The method of claim 137, wherein the at least one cytokine comprises one or more of IL- 2, IL-21, IL-7, and IL-15.

139. The method of claim 137, wherein the at least one cytokine comprises one or more of IL- 21, IL-7, and IL-15 and does not comprise IL-2.