Methods for producing genetically engineered T cells

Abstract

The present disclosure provides methods for stimulating, culturing, expanding, and / or genetically manipulating cell populations enriched for CD57- T cells or depleted for CD57- positive cells, as well as cell populations enriched for CD57- T cells or depleted for CD57+ T cells. Methods for generating, isolating, enriching, or selecting CD57- T cells or depleting CD57+ cells, e.g., by negative selection, are also included. TIFF2022512913000026.tif109128

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 756,571, filed November 6, 2018, and entitled "PROCESS FOR PRODUCING GENETICALLY ENGINEERED T CELLS"; and U.S. Provisional Application No. 62 / 798,457, filed January 29, 2019, and entitled "PROCESS FOR PRODUCING GENETICALLY ENGINEERED T CELLS," the contents of which are incorporated by reference in their entirety for all purposes.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is filed with an electronic Sequence Listing, which is provided as a file entitled 735042017640SeqList.TXT, created on November 5, 2019, and is 74 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure, in some aspects, relates to cell populations enriched for CD57-negative T cells or depleted for CD57-positive cells, and methods for stimulating, culturing, expanding, and / or genetically manipulating cell populations enriched for CD57-negative T cells or depleted for CD57-positive T cells, including methods for generating, isolating, enriching, or selecting CD57-negative T cells or depleting CD57-positive cells, such as by negative selection. [Background technology]

[0004] background A variety of cell therapies are available for treating diseases and conditions. Cell therapies include methods involving immune cells, such as T cells, genetically engineered with recombinant receptors, such as chimeric antigen receptors. However, in some cases, some existing methods may result in populations with low consistency, efficacy, or persistence in vivo. Improved methods for manufacturing and / or operating such cell therapies are needed, including providing improved cell therapy products with high consistency, efficacy, and persistence in vivo. Summary of the Invention

[0005] overview Provided herein are methods for enriching T cells, comprising: performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample containing primary human T cells, thereby generating a depleted population, the depleted population containing fewer CD57+ T cells than the biological sample; and performing a second selection on cells from the depleted population, the second selection comprising enriching CD3+ T cells from the depleted population, whereby enrichment generates an enriched population of CD57-CD3+ T cells.

[0006] Also provided herein are methods for enriching T cells, comprising: performing a first selection, the first selection comprising enriching CD3+ T cells from a biological sample containing primary human T cells, thereby generating an enriched T cell population; and performing a second selection on cells from the enriched T cell population, the second selection comprising removing CD57+ T cells from the enriched sample cell population, thereby generating a depleted population, the depleted population containing fewer CD57+ T cells than the biological sample and / or enriched T cell population, and being enriched in CD3+ T cells.

[0007] Also provided herein is a method for enriching T cells, comprising removing CD57+ T cells from an apheresis product or leukapheresis product containing primary human T cells, thereby producing a depleted population of T cells, wherein the depleted population contains fewer CD57+ T cells than the biological sample, the depleted population containing at least one of the following: (i) less than or about 5% CD57+ T cells; (ii) a frequency of CD57+ T cells that is less than or about 35% of the frequency of CD57+ T cells present in the biological sample; and (iii) CD3+ T cells, wherein at least 95% or at least about 95% of the CD3+ T cells are CD57-.

[0008] In some of any such embodiments, the depleted population contains at least one of the following: (i) less than 5% or less than about 5% CD57+ T cells; (ii) a frequency of CD57+ T cells that is less than 35% or less than about 35% of the frequency of CD57+ T cells present in the biological sample; (iii) CD4+ T cells, of which at least 95% or at least about 95% are CD57- CD4+ T cells; and (iv) CD8+ T cells, of which at least 95% or at least about 95% are CD57- CD8+ T cells. In some of any such embodiments, at least 95% or at least about 95% of the CD3+ T cells of the depleted population contain CD57- CD3+ T cells. In some of any such embodiments, the depleted population is a first depleted population, and the method further comprises enriching CD3+ T cells from the first depleted population, thereby generating an enriched population of CD57- CD3+ T cells.

[0009] In some of any such embodiment, the biological sample comprises an apheresis product or a leukapheresis product.

[0010] Also provided herein are methods for enriching T cells, comprising: (a) performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample containing an apheresis product or leukapheresis product comprising primary human T cells, thereby generating a first depleted population, the first depleted population containing fewer CD57+ T cells than the biological sample; and (b) performing a second selection on cells from the first depleted population, the second selection comprising enriching CD3+ T cells from the first depleted population, whereby enrichment generates an enriched population of CD57-CD3+ T cells.

[0011] Also provided herein is a method for enriching T cells, comprising the steps of: (a) performing a first selection, where the first selection comprises enriching CD3+ T cells from a biological sample containing an apheresis product or a leukapheresis product containing primary human T cells, thereby generating an enriched biological sample; and (b) performing a second selection on cells from the enriched biological sample, where the second selection comprises removing CD57+ T cells from the enriched biological sample, thereby generating a depleted population that is an enriched population of CD57-CD3+ T cells.

[0012] Also provided herein is a method for enriching T cells, comprising removing CD57+ T cells from a biological sample containing an apheresis product or leukapheresis product containing primary human T cells, thereby producing a depleted population of T cells, wherein the depleted population contains fewer CD57+ T cells than the biological sample, the depleted population containing (i) less than or about 5% CD57+ T cells; (ii) a frequency of CD57+ T cells that is less than or about 35% of the frequency of CD57+ T cells present in the biological sample; (iii) CD4+ T cells, of which at least 95% or at least about 95% are CD57−; and / or (iv) CD8+ T cells, of which at least 95% or at least about 95% are CD57−.

[0013] In some of any such embodiments, the depleted population contains CD57-CD4+ T cells. In some of any such embodiments, the depleted population contains CD57-CD8+ T cells. In some of any such embodiments, the depleted population contains CD57-CD4+ T cells and CD57-CD8+ T cells.

[0014] In some of any such embodiments, the depleted population is a first depleted population, and the method further comprises selecting CD4+ T cells from the first depleted population, thereby generating a second depleted population enriched in CD57-CD4+ T cells and an unselected population. In some of any such embodiments, the method further comprises selecting CD8+ T cells from the unselected population, thereby generating a third depleted population that is an enriched population of CD57-CD8+ T cells and a second unselected population.

[0015] In some of any such embodiments, the depleted population is a first depleted population, and the method further comprises selecting CD8+ T cells from the first depleted population, thereby generating a second depleted population that is an enriched population of CD57-CD8+ T cells and an unselected population. In some of any such embodiments, the method further comprises selecting CD4+ T cells from the unselected population, thereby generating a third depleted population that is an enriched population of CD57-CD4+ T cells and the second unselected population.

[0016] In some of any of such embodiments, the depleted population is a first depleted population, and the method further comprises selecting CD3+ T cells from the first depleted population, thereby generating a second depleted population that is an enriched population of CD57-CD3+ T cells, and an unselected population.

[0017] (a) performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample containing an apheresis product or a leukapheresis product containing primary human T cells, thereby generating a first depleted population, the first depleted population containing fewer CD57+ T cells than the biological sample; (b) performing a second selection on cells from the first depleted population, the second selection comprising enriching one of (i) CD4+ T cells and (ii) CD8+ T cells from the first depleted population, whereby enrichment generates a second depleted population enriched in one of (i) CD4+ T cells and (ii) CD8+ T cells, and an unselected population; and (c) performing a third selection, the third selection enriching the other of (i) CD4+ cells and (ii) CD8+ cells from the unselected population, whereby enrichment generates a second depleted population enriched in one of (i) CD4+ T cells and (ii) CD8+ T cells. Also provided are methods for enriching T cells, comprising a step comprising generating a third depleted population enriched in another of the T cells. In some of any such embodiments, the frequency of CD57+ cells in the depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) is less than or less than about 35%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the frequency of CD57+ T cells in the biological sample. In some of any such embodiments, the frequency of CD57+ T cells in the depleted population (optionally the first, second, or third depleted population) is less than or about 35%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the frequency of CD57+ T cells in the biological sample. In some of any such embodiments, the depleted population (optionally the first, second, or third depleted population) comprises less than or about 3%, less than or about 2%, less than or about 1%, less than or about 0.1%, or less than or about 0.1%, or less than or about 0.01% CD57+ T cells.In some of any such embodiments, the depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) is free of or essentially free of CD57+ T cells. In some embodiments, a depleted population that is essentially free of CD57+ T cells comprises less than or about 3%, less than or about 2%, less than or about 1%, less than or about 0.1%, or less than or about 0.01% CD57+ T cells.

[0018] In some of any such embodiments, at least 95% or at least about 95% of the CD4+ T cells of the depleted population (optionally the first, second, or third depleted population) comprise CD57-CD4+ T cells. In some of any such embodiments, at least 95% or at least about 95% of the CD8+ T cells of the depleted population (optionally the first, second, or third depleted population) comprise CD57-CD8+ T cells. In some of any such embodiments, at least 95% or at least about 95% of the CD4+ T cells and 95% of the CD8+ T cells of the depleted population (optionally the first, second, or third depleted population) comprise CD57-CD4+ T cells and CD57-CD8+ T cells, respectively.

[0019] In some of any such embodiments, the frequency of naive-like cells in a depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) is at least 10%, 20%, 30%, 40%, or 50% greater than, or at least about 10%, 20%, 30%, 40%, or 50% greater than, the frequency or approximately the frequency of naive-like cells in the biological sample. In some of any such embodiments, the frequency of one or more of CD25+ T cells, CD27+ T cells, CD28+ T cells, CCR7+ T cells, or CD45RA+ T cells in a depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) is at least 10%, 20%, 30%, 40%, or 50% greater than or about the frequency of the respective cells in the biological sample.

[0020] In some of any such embodiments, the depleted population (optionally the first, second, or third depleted population) comprises at least, or at least about, 15%, 20%, 25%, 30%, 35%, or 40% CD27+ T cells. In some of any such embodiments, the depleted population (optionally the first, second, or third depleted population) comprises at least, or at least about, 10%, 15%, 20%, 25%, 25%, 30%, 35%, or 40% CD28+ T cells. In some of any such embodiments, the depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) comprises at least or at least about 10%, 15%, 20%, 25%, 25%, 30%, 35%, or 40% CD27+CD28+ T cells. In some of any such embodiments, the depleted population (optionally the first, second, or third depleted population) comprises at least, or at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, or 80% CD27+CD28+ T cells. In some of any such embodiments, the depleted population (optionally the first, second, or third depleted population) comprises at least, or at least about, 70% or 80% CD27+CD28+ T cells. In some of any such embodiments, the depleted population (optionally the first depleted population, the second depleted population or the third depleted population) comprises at least or at least about 10%, 15%, 20%, or 25% CCR7+ T cells.

[0021] In some of any of the provided embodiments, the depleted population (optionally the first, second, or third depleted population) is used as an input composition for a subsequent step in the process for genetically engineering T cells. In some embodiments, the input composition comprises a CD57-depleted population enriched in CD4+ cells (CD57-CD4+) and a CD57-depleted population enriched in CD8+ cells (CD57-CD8+). In some embodiments, the input composition comprises a CD57-depleted population enriched in CD4+ T cells (CD57-CD4+) and a CD57-depleted population enriched in CD8+ T cells (CD57-CD8+). In some embodiments of any of the methods, the method includes combining cells of the second and third depleted populations, thereby generating an input composition. In some of any of the provided embodiments, the method further includes combining the second and third depleted populations. In some of any of the provided embodiments, the second depleted population and the third depleted population are combined in a ratio of at or about 1:3 to at or about 3:1, thereby generating a depleted population comprising the second depleted population and the third depleted population. In some of any of the provided embodiments, the second depleted population and the third depleted population are combined in a ratio of at or about 1:1, thereby generating a depleted population comprising the second depleted population and the third depleted population.

[0022] In some of the provided methods, the methods produce T cell compositions containing at least 90%, at least 95%, at least 97%, at least 97%, at least 99%, or at least 99.9% or at least 99.9% CD57-CD4+ T cells. In some of the provided methods, the methods produce T cell compositions containing at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.9% or at least 99.9% CD57-CD8+ T cells. In some of any of the provided methods, the method produces a T cell composition that contains at least 90%, or at least about 90%, at least 95%, or at least about 95%, at least 97%, or at least about 97%, at least 99%, or at least about 99%, or at least 99.9%, or at least about 99.9% CD57-CD3+ T cells.

[0023] In some of the provided embodiments, the ratio of CD4+ to CD8+ T cells in the composition is 3:1 to 1:3, inclusive. In some of the provided embodiments, the ratio of CD4+ to CD8+ T cells in the composition is 2:1 to 1:2, inclusive. In some of the provided embodiments, the ratio of CD4+ to CD8+ T cells in the composition is 1.5:1 to 1:1.5, inclusive. In some of the provided embodiments, the ratio of CD4+ to CD8+ T cells in the composition is 1.2:1 to 1:1.2, inclusive. In some of the provided embodiments, the ratio of CD4+ T cells to CD8+ T cells in the composition is 1:1 or about 1:1 CD4+. In some of the provided embodiments, the primary T cells are derived from a human subject. In some of the provided embodiments, the primary T cells are derived from a subject having a disease or condition. In some of the provided embodiments, the disease or condition is cancer. In some of any of the provided embodiments, the primary T cells are derived from a healthy subject.

[0024] In some of any of the provided embodiments, depleting CD57+ T cells comprises immunoaffinity-based selection. In some of the provided embodiments, immunoaffinity-based selection comprises contacting the cells with an antibody capable of specifically binding to CD57 and recovering cells that do not bind to the antibody, thereby inducing negative selection, wherein the recovered cells are depleted of CD57+ cells. In some of the provided embodiments, enriching the cells comprises immunoaffinity-based selection. In some of the provided embodiments, the first, second, and / or third selection enriches for CD4 or CD8 T cells, and the immunoaffinity-based selection is caused by contacting the cells with an antibody capable of specifically binding to CD4 or CD8, respectively, and recovering cells that bind to the antibody, thereby inducing positive selection, wherein the recovered cells are enriched for CD4+ cells or CD8+ cells. In some of the provided embodiments, the selection enriches for CD3 T cells, and the immunoaffinity-based selection is caused by contacting the cells with an antibody capable of specifically binding to CD3 and recovering the antibody-bound cells, thereby causing positive selection, wherein the recovered cells are enriched for CD3+ cells. In some of the provided embodiments, the antibody is immobilized on a solid surface. In some of the provided embodiments, the solid surface is a magnetic particle. In some of the provided embodiments, the antibody is immobilized or attached to an affinity chromatography matrix.

[0025] In some of any of the provided embodiments, the antibody further comprises one or more binding partners capable of forming a reversible bond with a binding reagent immobilized on the matrix, wherein the antibody is reversibly bound to the chromatography matrix during contact. In some of the provided embodiments, the binding reagent is a streptavidin mutein that reversibly binds to the binding partner. In some of the provided embodiments, the streptavidin mutein contains the amino acid sequence Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44-47 in streptavidin of the amino acid sequence set forth in SEQ ID NO:66; or the streptavidin mutein contains the amino acid sequence Va144-Thr45-Ala46-Arg47 at sequence positions corresponding to positions 44-47 in streptavidin of the amino acid sequence set forth in SEQ ID NO:66. In some of the provided embodiments, the binding partner is a streptavidin-binding peptide. In some of any of the provided embodiments, the streptavidin-binding peptide is In some of the provided embodiments, the streptavidin-binding peptide is selected from the group consisting of the sequence TIFF0007604366000002.tif4128. In some of the provided embodiments, the method further comprises contacting cells in the sample with the affinity chromatography matrix, followed by applying a competing reagent to disrupt the binding between the binding partner and the binding reagent, thereby recovering the cells bound to the antibody. In some of the provided embodiments, the competing reagent is biotin or a biotin analog. In some of the provided embodiments, the chromatography matrix is ​​packed into a separation tube that is a column. In some of the provided embodiments, the biological sample comprising primary T cells is a sample formulated with a cryoprotectant. In some of the provided embodiments, the apheresis or leukapheresis sample is a sample formulated with a cryoprotectant.

[0026] Also provided is a method for stimulating T cells, comprising incubating T cells of an input composition under stimulatory conditions, thereby generating a stimulated population of T cells, wherein the input composition contains a depleted population (optionally a first depleted population, a second depleted population, or a third depleted population) generated by any of the methods described herein.

[0027] Also provided are methods for stimulating T cells, comprising incubating T cells of an input composition under stimulatory conditions, wherein the percentage of CD57+ T cells in the input composition is less than a threshold percentage of CD57+ T cells, the threshold being 30% or less, or about 30% or less, of CD57+ T cells. In some of any such embodiments, the threshold percentage is 25%, 20%, 15%, 10%, 5%, or 1% or less, or about 25%, 20%, 15%, 10%, 5%, or 1% or less of CD57+ T cells.

[0028] In some of any such embodiments, the input composition contains: (i) less than 5% or less than about 5% CD57+ T cells; (ii) at least 95% or at least about 95% CD57- T cells; (iii) at least 90% or at least about 90% CD57-CD4+ T cells; (iv) at least 90% or at least about 90% CD57-CD8+ T cells; and (v) at least 90% or at least about 90% CD57-CD3+ cells.

[0029] Also provided herein is a method for stimulating T cells, comprising incubating T cells of an input composition under stimulatory conditions, thereby generating a stimulated population of T cells, wherein the input composition contains at least one of the following: (i) less than or about 5% CD57+ T cells; (ii) at least 95% or at least about 95% CD57- T cells; (iii) at least 90% or at least about 90% CD57-CD4+ T cells; (iv) at least 90% or at least about 90% CD57-CD8+ T cells; and (v) at least about 90% or at least about 90% CD57-CD3+ T cells.

[0030] In some of any such embodiments, the input composition comprises cells obtained from a biological sample containing an apheresis product or a leukapheresis product containing primary human T cells.

[0031] In some of any such embodiments, the method further comprises removing CD57+ T cells from the biological sample, thereby generating an input composition containing a depleted population of T cells.

[0032] (a) performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample containing primary human T cells, thereby generating a first depleted population, the first depleted population containing fewer CD57+ T cells than the biological sample; (b) performing a second selection on cells from the first depleted population, the second selection comprising enriching one of (i) CD4+ T cells and (ii) CD8+ T cells from the first depleted population, whereby enrichment generates a second depleted population enriched in one of (i) CD57-CD4+ T cells and (ii) CD57-CD8+ T cells, and an unselected population; (c) performing a third selection on cells from the unselected population, whereby the third selection comprises enriching the other of (i) CD4+ cells and (ii) CD8+ cells from the unselected population, whereby enrichment generates a second depleted population enriched in one of (i) CD57-CD4+ T cells and (ii) CD8+ T cells. Also provided is a method for stimulating T cells, comprising: (i) generating a third depleted population enriched in the other of T cells and CD57-CD8+ T cells; and (d) incubating one or more input compositions under stimulatory conditions, wherein the one or more input compositions comprise T cells from the second depleted population or the third depleted population.

[0033] In some of any such embodiments, step (d) comprises incubating an input composition comprising CD4+ T cells and CD8+ T cells from the second and third depleted populations. In some of any such embodiments, step (d) comprises separately incubating a first input composition and a second input composition, wherein the first input composition contains T cells from the second depleted population and the second input composition contains T cells from the third depleted population.

[0034] In some of any such embodiments, the second selection comprises (i) enriching CD4+ T cells, thereby producing (i) a first enriched population enriched in CD57-CD4+ T cells, wherein the first input composition contains enriched CD57-CD4+ T cells; and the third selection comprises (ii) enriching CD8+ T cells, thereby producing (ii) a second enriched population enriched in CD57-CD8+ T cells, wherein the second input composition contains enriched CD57-CD8+ T cells. In some of any such embodiments, the second selection comprises (ii) enriching for CD8+ T cells, thereby producing a (ii) first enriched population enriched in CD57-CD8+ T cells, wherein the first input composition contains enriched CD57-CD8+ T cells; and the third selection comprises (i) enriching for CD4+ T cells, thereby producing a (i) second enriched population enriched in CD57-CD4+ T cells, wherein the second input composition contains enriched CD57-CD4+ T cells. Also provided herein is a method for stimulating T cells, comprising: (a) performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample containing primary human T cells, thereby generating a first depleted population, the depleted population containing fewer CD57+ T cells than the biological sample; (b) performing a second selection on cells from the first depleted population, the second selection comprising enriching CD3+ T cells from the first depleted population, whereby enrichment produces a second depleted population enriched in CD3+ T cells; and (c) incubating one or more input compositions under stimulatory conditions, the one or more input compositions containing T cells from the second depleted population.

[0035] Also provided herein is a method for stimulating T cells, comprising: (a) performing a first selection on cells from a biological sample, the first selection comprising enriching CD3+ T cells from the biological sample, whereby enrichment produces an enriched biological sample enriched in CD3+ T cells; (b) performing a second selection, whereby the second selection comprises removing CD57+ T cells from the enriched biological sample containing primary human T cells, whereby producing a depleted population, whereby the depleted population contains fewer CD57+ cells than the enriched biological sample; and (c) incubating one or more input compositions under stimulatory conditions, whereby the one or more input compositions contain T cells from the depleted population.

[0036] In some of any such embodiments, the input composition comprises less than or about 5% CD57+ T cells. In some of any such embodiments, the input composition contains less than or about 3%, less than or about 2%, less than or about 1%, less than or about 0.1%, or less than or about 0.1%, or less than or about 0.01% CD57+ T cells. In some of any such embodiments, the frequency of CD57+ cells in the input composition is less than or about 35%, 30%, 25%, 20%, 10%, 5%, 1%, or 0.1% of the frequency of CD57+ T cells present in the biological sample. In some of any such embodiments, the frequency of CD57+ T cells in the input composition is less than or about 35%, 30%, 25%, 20%, 10%, 5%, 1%, or 0.1% of the frequency of CD57+ T cells present in the biological sample. In some of any such embodiments, the input composition is free of or essentially free of CD57+ T cells. In some embodiments, a depleted population that is essentially free of CD57+ T cells comprises less than about or about 3%, less than about or about 2%, less than about or about 1%, less than about or about 0.1%, or less than about or about 0.01% CD57+ T cells.

[0037] In some of any such embodiments, the input composition contains at least 95% or at least about 95% CD57- T cells. In some of any such embodiments, the input composition contains at least 90% or at least about 90%, at least 95% or at least about 95%, at least 97% or at least about 97%, at least 99% or at least about 99%, or at least 99.9% or about 99.9% CD57-CD4+ T cells. In some of any such embodiments, the input composition contains at least 90% or at least about 90%, at least 95% or at least about 95%, at least 97% or at least about 97%, at least 99% or at least about 99%, or at least 99.9% or about 99.9% CD57-CD8+ T cells. In some of any such embodiments, the CD57- T cells include CD57-CD4+ T cells and CD57-CD8+ T cells. In some of any such embodiments, the input composition contains a ratio of CD4+ T cells to CD8+ T cells of 3:1 to 1:3, 2:1 to 1:2, 1.5:1 to 1:1.5, or 1.2:1 to 1:1.2 (inclusive). In some of any such embodiments, the input composition contains a ratio of CD4+ T cells to CD8+ T cells of 1:1 or about 1:1.

[0038] In some of any such embodiments, the frequency of naive-like cells in the input composition is at least, or at least about, 10%, 20%, 30%, 40%, or 50% greater than the frequency or approximately the frequency of naive-like cells in the biological sample. In some of any such embodiments, the frequency of one or more of CD25+ T cells, CD27+ T cells, CD28+ T cells, CCR7+ T cells, or CD45RA+ T cells in the input composition is at least, or at least about, 10%, 20%, 30%, 40%, or 50% greater than the frequency or approximately the frequency of the respective cells in the biological sample.

[0039] In some of any such embodiments, the input composition contains at least, or at least about, 15%, 20%, 25%, 30%, 35%, or 40% CD27+ T cells. In some of any such embodiments, the input composition contains at least, or at least about, 10%, 15%, 20%, 25%, 25%, 30%, 35%, or 40% CD28+ T cells. In some of any such embodiments, the input composition contains at least, or at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, or 80% CD27+CD28+ T cells. In some of any such embodiments, the input composition contains at least, or at least about, 70% or 80% CD27+CD28+ T cells. In some of any such embodiments, the input composition contains at least, or at least about, 10%, 15%, 20%, or 25% CCR7+ T cells.

[0040] In some of any such embodiments, removing CD57+ T cells and / or enriching cells from the biological sample in the first and / or second selection comprises immunoaffinity-based selection. In some of any such embodiments, immunoaffinity-based selection is caused by contacting cells with an antibody capable of specifically binding to CD57, CD4, or CD8, and recovering cells that do not bind to the antibody, thereby causing negative selection, or by recovering cells that bind to the antibody, thereby causing positive selection, wherein the recovered cells are depleted of CD57+ cells and / or enriched for CD4+ or CD8+ cells, and the antibody is immobilized on a magnetic particle. In some of any such embodiments, immunoaffinity-based selection is caused by contacting cells with an antibody immobilized or attached to an affinity chromatography matrix, wherein the antibody can specifically bind to CD57, CD4, or CD8, causing negative selection of CD57+ cells or positive selection of CD4+ or CD8+ cells. In some of any such embodiments, immunoaffinity-based selection is effected by contacting cells with an antibody capable of specifically binding to CD57, CD4, or CD8, and recovering cells that do not bind to the antibody, thereby causing negative selection, or by recovering cells that bind to the antibody, thereby causing positive selection, wherein the recovered cells are depleted of CD57+ T cells and / or enriched for CD4+ or CD8+ T cells, and the antibody is immobilized on a magnetic particle. In some of any such embodiments, immunoaffinity-based selection is effected by contacting cells with an antibody that is immobilized or attached to an affinity chromatography matrix, wherein the antibody can specifically bind to CD57, CD4, or CD8, causing negative selection of CD57+ T cells or positive selection of CD4+ or CD8+ T cells.

[0041] In some of any such embodiments, the stimulatory conditions include the presence of a stimulatory reagent, which can activate one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules. In some of any such embodiments, the stimulatory reagent contains (i) a primary agent that specifically binds to a member of the TCR complex, optionally, that specifically binds CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, optionally, the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS.

[0042] In some of any such embodiments, one or both of the primary agent and the secondary agent comprise an antibody or antigen-binding fragment thereof. In some of any such embodiments, the primary agent and the secondary agent comprise an antibody, and optionally, the stimulating reagent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody, or antigen-binding fragment thereof. In some of any such embodiments, the primary agent is an m-anti-CD3 antibody or antigen-binding fragment thereof, and the secondary agent is an m-anti-CD28 antibody or antigen-binding fragment thereof. In some of any such embodiments, the antigen-binding fragment is a monovalent antibody fragment selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In some of any such embodiments, the primary agent is an anti-CD3 Fab, and the secondary agent comprises an anti-CD28 Fab. In some of any such embodiments, the primary agent and the secondary agent are present on or attached to the surface of a solid support. In some of any such embodiments, the solid support is or comprises beads, optionally paramagnetic beads. In some of any such embodiments, the solid support is a paramagnetic bead having anti-CD3 and anti-CD28 antibodies attached to its surface, and the stimulatory reagent is present at a bead to cell ratio of less than about 3:1 or about 3:1.

[0043] In some of any such embodiments, the stimulating conditions include the presence of a stimulating reagent containing paramagnetic beads having anti-CD3 and anti-CD28 antibodies attached to their surfaces, and the stimulating reagent is present at a bead to cell ratio of less than or less than about 3: 1. In some of any such embodiments, the stimulating reagent is present at a bead to cell ratio of 1: 1 or about 1: 1.

[0044] In some of any such embodiments, the method further comprises separating the stimulatory reagent from the cells, wherein the separating comprises exposing the cells to a magnetic field.

[0045] In some of any such embodiments, the primary agent and secondary agent are reversibly bound to the surface of an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules. In some of any such embodiments, the streptavidin or streptavidin mutein molecules bind or are capable of binding to biotin, avidin, biotin analogs or muteins, avidin analogs or muteins, and / or biologically active fragments thereof. In some of any such embodiments, the streptavidin mutein molecule comprises the amino acid sequence Val at sequence positions corresponding to positions 44-47 in streptavidin of the amino acid sequence set forth in SEQ ID NO:66. 44 -Thr 45 -Ala 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47In some of any such embodiments, the streptavidin mutein molecule comprises: a) an amino acid sequence set forth in any of SEQ ID NOs: 66-68, 73, 80-82, or 85-88; b) at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 70-73, 78, and 85-89, and 44 -Thr 45 -Ala 46 -Arg 47 Or Ile 44 -Gly 45 -Ala 46 -Arg 47 and / or an amino acid sequence that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide; or c) a functional fragment of a) or b) that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide.

[0046] In some of any such embodiments, the streptavidin mutein molecule contains the amino acid sequence set forth in SEQ ID NO:73 or 78. In some of any such embodiments, the streptavidin mutein molecule contains the amino acid sequence set forth in SEQ ID NO:73. In some of any such embodiments, the streptavidin mutein molecule contains the amino acid sequence set forth in SEQ ID NO:78. In some of any such embodiments, the primary agent and the secondary agent each contain a streptavidin-binding peptide. In some of any such embodiments, the streptavidin-binding peptide is In some of any such embodiments, the streptavidin-binding peptide is selected from the group consisting of: TIFF0007604366000004.tif34162. In some of any such embodiments, the primary agent comprises an anti-CD3 Fab, wherein the secondary agent contains an anti-CD28 Fab.

[0047] In some of any such embodiments, the oligomeric particle reagent contains a radius of greater than or about 60 nm, greater than or about 70 nm, greater than or about 80 nm, or greater than or about 90 nm. In some of any such embodiments, the oligomeric particle reagent contains a radius of at least 5×10 7 g / mol or at least about 5 × 10 7 g / mol, or at least 1 × 10 8 g / mol or at least about 1 × 10 8 g / mol; and / or 5 × 10 7 g / mol ~ 5 × 10 8 g / mol, 1×10 8 g / mol ~ 5 × 10 8 g / mol, or 1 × 10 8 g / mol~2×10 8 In some of any such embodiments, the oligomeric particle reagent contains at least 500 or at least about 500 streptavidin or streptavidin mutein tetramers, at least 1,000 or at least about 1,000 streptavidin or streptavidin mutein tetramers, at least 1,500 or at least about 1,500 streptavidin or streptavidin mutein tetramers, or at least and / or 1,000 to 20,000 streptavidin or streptavidin mutein tetramers, 1,000 to 10,000 streptavidin or streptavidin mutein tetramers, or 2,000 to 5,000 streptavidin or streptavidin mutein tetramers.

[0048] In some of any such embodiments, the method further includes a step of separating the stimulatory reagent from the cells, the separating step including contacting the cells with a substance capable of reversing the binding between the primary and secondary agents and the oligomeric particle reagent.

[0049] In some of any such embodiments, the substance is a free binding partner and / or a competitive agent. In some of any such embodiments, the presence of the substance terminates or reduces the signal induced or modulated by the primary and secondary agents in the T cell. In some of any such embodiments, the substance is or comprises a streptavidin-binding peptide, biotin or a biologically active fragment, or a biotin analog or a biologically active fragment. In some of any such embodiments, the substance is or comprises a biotin analog. In some of any such embodiments, the substance is or comprises a streptavidin-binding peptide, wherein the streptavidin-binding peptide is TIFF0007604366000005.tif41170. In some of any such embodiments, the agent is or comprises biotin or a biologically active fragment, or a biotin analog or biologically active fragment.

[0050] In some of any such embodiments, the stimulatory conditions include the presence of one or more recombinant cytokines. In some of any such embodiments, the stimulatory conditions include the presence of one or more of recombinant IL-2, IL-7, and IL-15.

[0051] In some of any such embodiments, the method further comprises introducing a heterologous polynucleotide encoding a recombinant receptor into a T cell population obtained from the depleted population or input composition, or the stimulated population, thereby generating an engineered population of T cells.

[0052] Also provided are methods for genetically engineering T cells, comprising introducing a heterologous polynucleotide encoding a recombinant receptor into a stimulated population of T cells generated by incubating the T cells of the input composition under stimulatory conditions if the percentage of CD57+ T cells in the input composition is below a threshold percentage of CD57+ T cells, where the threshold is at or below about 30%. In some of any such embodiments, the threshold is at or below about 25%, 20%, 15%, 10%, 5%, or 1%.

[0053] Also provided is a method for genetically engineering T cells, comprising introducing a heterologous polynucleotide encoding a recombinant receptor into a population of T cells from a T cell composition produced by any of the methods described herein, thereby generating an engineered population of T cells. Also provided is a method for genetically engineering T cells, comprising introducing a heterologous polynucleotide encoding a recombinant receptor into a population of T cells from any of the stimulated T cell populations provided herein, thereby generating an engineered population of T cells.

[0054] Also provided is a method for genetically engineering T cells, comprising introducing a heterologous polynucleotide encoding a recombinant receptor into T cells from an input composition containing one or more of: (i) less than or about 5% CD57+ T cells; (ii) at least 95% or at least about 95% CD57- T cells; (iii) at least 90% or at least about 90% CD57-CD4+ T cells; or (iv) at least 90% or at least about 90% CD57-CD8+ T cells.

[0055] (a) performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample containing primary human T cells, thereby generating a depleted population, the depleted population containing fewer CD57+ T cells than the biological sample; (b) performing a second selection on cells from the depleted population, the second selection comprising enriching one of (i) CD4+ T cells and (ii) CD8+ T cells from the biological sample containing primary human T cells, whereby the enrichment produces a first enriched population enriched in one of (i) CD57-CD4+ T cells and (ii) CD57-CD8+ T cells, and an unselected population; and (c) performing a third selection on cells from the unselected population, whereby the third selection comprises enriching the other of (i) CD4+ cells and (ii) CD8+ cells from the unselected population, whereby the enrichment produces a first enriched population enriched in one of (i) CD57-CD4+ T cells and (ii) CD57-CD8+ Also provided is a method for genetically engineering T cells, comprising the steps of (a) generating a second enriched population of T cells that is enriched for the other of the T cells; and (d) introducing a heterologous polynucleotide encoding a recombinant receptor into T cells from one or both of the first and second enriched populations, thereby generating first and second engineered populations of T cells.

[0056] Also provided are methods for genetically engineering T cells, comprising: performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample containing primary human T cells, thereby generating a depleted population, the depleted population containing fewer CD57+ T cells than the biological sample; performing a second selection on cells from the depleted population, the second selection comprising enriching CD3+ T cells from the biological sample containing primary human T cells, whereby enrichment generates a first enriched population enriched in CD57-CD3+ T cells; and introducing a heterologous polynucleotide encoding a recombinant receptor into T cells from the first enriched population, thereby generating a first engineered population of T cells. In some of any such embodiments, prior to the introducing step, the first enriched population is incubated under stimulatory conditions.

[0057] Also provided herein are methods for genetically engineering T cells, comprising: performing a first selection, the first selection comprising enriching CD3+ T cells from a biological sample containing primary human T cells, thereby generating an enriched biological sample enriched in CD3+ T cells; performing a second selection on cells from the enriched biological sample, the second selection comprising removing CD57+ T cells from the enriched biological sample containing primary human T cells, thereby generating a depleted population, the depleted population containing fewer CD57+ T cells than the enriched biological sample; and introducing a heterologous polynucleotide encoding a recombinant receptor into T cells from the depleted population, thereby generating a first engineered population of T cells. In some of any such embodiments, the depleted population is incubated under stimulatory conditions prior to the introducing step.

[0058] In some of any such embodiments, the input composition or enriched population is incubated under stimulatory conditions prior to the introducing step.

[0059] In some of any such embodiments, step (d) comprises introducing the heterologous polynucleotide into a population of cells containing CD4+ T cells and CD8+ T cells from the first and second enriched populations. In some of any such embodiments, step (d) comprises separately introducing the heterologous polynucleotide into cells of the first enriched population and cells of the second enriched population.

[0060] In some of any such embodiments, the second selection comprises (i) enriching for CD4+ T cells, thereby generating a first enriched population enriched in (i) CD57-CD4+ T cells, wherein the first engineered population comprises CD57-CD4+ T cells; and the third selection comprises (ii) enriching for CD8+ T cells, thereby generating a second enriched population enriched in (ii) CD57-CD8+ T cells, wherein the second engineered population comprises CD57-CD8+ T cells. In some of any such embodiments, the second selection comprises (ii) enriching for CD8+ T cells, thereby generating a (ii) first enriched population enriched in CD57-CD8+ T cells, wherein the first engineered population comprises CD57-CD8+ T cells; and the third selection comprises (i) enriching for CD4+ T cells, thereby generating a (i) second engineered population enriched in CD57-CD4+ T cells, wherein the second input population comprises CD57-CD4+ T cells.

[0061] In some of such embodiments, one or both of the cells of the first and second enriched populations are incubated under stimulatory conditions before the introducing step.In some of such embodiments, the introducing step comprises transduction with a viral vector comprising a heterologous polynucleotide.In some of such embodiments, the viral vector is a gammaretroviral vector or a lentiviral vector.In some of such embodiments, the viral vector is a lentiviral vector.

[0062] In some of any such embodiments, the method further comprises incubating the composition containing the transduced cells for up to 96 hours after the introducing step. In some of any such embodiments, the incubating step is at a temperature of or about 37°±2°C. In some of any such embodiments, the incubating step is performed for up to 72 hours after the introducing step. In some of any such embodiments, the incubating step is performed for up to 48 hours after the introducing step. In some of any such embodiments, the incubating step is performed for up to 24 hours after the introducing step. In some of any such embodiments, the incubating step results in integration of the viral vector into the genome of the T cell.

[0063] In some of any such embodiments, the method further includes culturing the cells of the transduced population under conditions that promote the growth or expansion of the engineered cells, thereby producing an expanded population of cells. In some of any such embodiments, the culturing is performed in the presence of one or more recombinant cytokines. In some embodiments, the one or more recombinant cytokines are one or more of IL-2, IL-7, and IL-15. In some of any such embodiments, the growth or expansion results in a 2-fold, 3-fold, 4-fold, 5-fold, or about 2-fold, 3-fold, 4-fold, 5-fold, or at least about 2-fold, 3-fold, 4-fold, 5-fold, or more than or more than about 5-fold increase in the number of viable T cells comprising the heterologous polynucleotide.

[0064] Also provided is a method of harvesting or collecting cells, comprising harvesting or collecting a population of cells produced by any of the methods described herein.

[0065] Also provided are methods for genetically engineering T cells, comprising: (a) incubating T cells from a depleted population generated by any of the methods described herein under stimulatory conditions, wherein the stimulatory conditions comprise the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules, thereby generating stimulated T cells; and (b) introducing a heterologous polynucleotide into the stimulated T cells, comprising transducing the stimulated T cells with a viral vector comprising a heterologous polynucleotide encoding a recombinant receptor, thereby generating an engineered population of T cells.

[0066] (a) performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample comprising primary human T cells, thereby generating a depleted population, the depleted population containing fewer CD57+ T cells than the biological sample; (b) performing a second selection on cells from the depleted population, the second selection comprising enriching one of (i) CD4+ T cells and (ii) CD8+ T cells from the biological sample comprising primary human T cells, whereby the enrichment produces a first enriched population enriched in one of (i) CD57-CD4+ T cells and (ii) CD57-CD8+ T cells, and an unselected population; and (c) performing a third selection on cells from the unselected population, whereby the third selection comprises enriching the other of (i) CD4+ cells and (ii) CD8+ cells from the unselected population, whereby the enrichment produces a first enriched population enriched in one of (i) CD57-CD4+ T cells and (ii) CD57-CD8+ Also provided are methods for genetically engineering T cells, comprising: (a) generating a second enriched population of T cells that is enriched for the other of the T cells; (d) incubating T cells from the first enriched population and the second enriched population under stimulatory conditions, wherein the stimulatory conditions comprise the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules, thereby generating the first and second enriched populations of stimulated T cells; and (e) introducing a heterologous polynucleotide into the stimulated T cells of the first and second enriched populations, comprising transducing the stimulated T cells with a viral vector containing the heterologous polynucleotide, thereby generating engineered populations of T cells from the first and second enriched populations.

[0067] performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample containing primary human T cells, thereby generating a depleted population, the depleted population containing fewer CD57+ T cells than the biological sample; and performing a second selection on cells from the depleted population, the second selection comprising enriching CD3+ T cells from the biological sample containing primary human T cells, whereby the enrichment is for CD57-CD3+ T cells. Also provided herein are methods of genetically engineering T cells, comprising: generating an enriched population enriched for T cells; incubating T cells from the enriched population under stimulatory conditions, wherein the stimulatory conditions comprise the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules, thereby generating an enriched population of stimulated T cells; and introducing a heterologous polynucleotide encoding a recombinant receptor into the stimulated T cells of the enriched population, comprising transducing the stimulated T cells with a viral vector containing the heterologous polynucleotide, thereby generating a population of engineered T cells from the enriched population.

[0068] performing a first selection, the first selection comprising enriching CD3+ T cells from a biological sample containing primary human T cells, thereby producing an enriched biological sample enriched for CD3+ T cells; and performing a second selection on cells from the enriched biological sample, the second selection comprising removing CD57+ T cells from the enriched biological sample containing primary human T cells, thereby producing a depleted population, the depleted population having fewer CD57+ T cells than the enriched biological sample. Also provided herein are methods of genetically engineering T cells, comprising: (a) incubating T cells from the depleted population under stimulatory conditions, wherein the stimulatory conditions comprise the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules, thereby generating stimulated T cells of the depleted population; and (d) introducing a heterologous polynucleotide encoding a recombinant receptor into the stimulated T cells of the depleted population, comprising transducing the stimulated T cells with a viral vector containing the heterologous polynucleotide, thereby generating an engineered population of T cells from the depleted population.

[0069] In some of any such embodiments, the method further comprises: (i) culturing the population of engineered T cells under conditions that promote growth or expansion of the engineered population, thereby producing an expanded population of T cells; and (ii) harvesting or collecting the expanded population.

[0070] In some of any such embodiments, prior to the incubating step, the first enriched population and the second enriched population contain one or more of: (i) less than or about 5% CD57+ T cells; (ii) a frequency of CD57+ T cells that is less than or about 35% of the frequency of CD57+ T cells present in the biological sample; or (iii) at least 95% or at least about 95% CD57- T cells. In some of any such embodiments, prior to the incubating step, the first enriched population and the second enriched population contain less than or about 5% CD57+ T cells. In some of any such embodiments, prior to the incubating step, the first enriched population and the second enriched population each contain less than or about 3%, less than or about 2%, less than or about 1%, less than or about 0.1%, or less than or about 0.01% CD57+ T cells. In some of any such embodiments, prior to the incubating step, the frequency of CD57+ cells in the first enriched population and the second enriched population contains less than or about 35%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the frequency of CD57+ T cells in the biological sample. In some of any such embodiments, prior to the introducing step, the first enriched population and the second enriched population are free of or essentially free of CD57+ T cells. In some embodiments, a depleted population that is essentially free of CD57+ T cells contains less than or about 3%, less than or about 2%, less than or about 1%, less than or about 0.1%, or less than or about 0.01% CD57+ T cells.

[0071] In some of any such embodiments, the cells of the first enriched composition and the second composition are incubated and / or cultured together as a single mixed population. In some of any such embodiments, prior to the introducing step, the single mixed population comprises a CD4+ T cell to CD8+ T cell ratio of 3:1 to 1:3, 2:1 to 1:2, 1.5:1 to 1:1.5, or 1.2:1 to 1:1.2 (inclusive). In some of any such embodiments, prior to the introducing step, the single mixed population comprises a CD4+ T cell to CD8+ T cell ratio of 1:1 or about 1:1. In some of any such embodiments, the cells of the first enriched population and the second enriched population are incubated and / or cultured separately.

[0072] In some of any such embodiments, the stimulation reagent comprises paramagnetic beads having anti-CD3 and anti-CD28 antibodies attached to their surfaces. In some of any such embodiments, the stimulation reagent comprises an oligomeric particle reagent containing multiple streptavidin or streptavidin mutein molecules having reversibly bound anti-CD3 and anti-CD28 Fabs.

[0073] In some of any such embodiments, removing CD57+ T cells from a biological sample and / or enriching cells in the first and / or second selection comprises immunoaffinity-based selection. In some of any such embodiments, immunoaffinity-based selection is caused by contacting cells with an antibody capable of specifically binding to CD57, CD4, or CD8, and recovering cells that do not bind to the antibody, thereby causing negative selection, or by recovering cells that bind to the antibody, thereby causing positive selection, wherein the recovered cells are depleted of CD57+ cells and / or enriched for CD4+ or CD8+ cells, and the antibody is immobilized on a magnetic particle. In some of any such embodiments, immunoaffinity-based selection is caused by contacting cells with an antibody immobilized or attached to an affinity chromatography matrix, wherein the antibody specifically binds to CD57, CD4, or CD8, causing negative selection of CD57+ cells or positive selection of CD4+ or CD8+ cells. In some of any such embodiments, immunoaffinity-based selection is effected by contacting cells with an antibody capable of specifically binding to CD57, CD4, or CD8, and recovering cells that do not bind to the antibody, thereby causing negative selection, or by recovering cells that bind to the antibody, thereby causing positive selection, wherein the recovered cells are depleted of CD57+ T cells and / or enriched for CD4+ or CD8+ T cells, and the antibody is immobilized on a magnetic particle. In some of any such embodiments, immunoaffinity-based selection is effected by contacting cells with an antibody that is immobilized or attached to an affinity chromatography matrix, wherein the antibody can specifically bind to CD57, CD4, or CD8, causing negative selection of CD57+ T cells or positive selection of CD4+ or CD8+ T cells.

[0074] In some of any such embodiments, the harvesting step occurs at or after the engineered or expanded population of T cells comprises a threshold number of T cells, viable T cells, engineered T cells, or viable engineered T cells, or a threshold concentration of T cells, viable T cells, engineered T cells, or viable engineered T cells. In some of any such embodiments, the threshold number or concentration of T cells, viable T cells, engineered T cells, or viable engineered T cells is reached within, or within about, 4, 5, 6, or 7 days after the start of stimulation.

[0075] In some of any such embodiments, a threshold number or concentration of T cells, viable T cells, engineered T cells, or viable engineered T cells among a plurality of populations of engineered T cells or a plurality of populations of expanded T cells is reached within 5 or 6 days, or within about 5 or 6 days, after initiation of stimulation in at least 70%, 80%, 90%, or 95% of the plurality. In some of any such embodiments, the threshold number or concentration of T cells, viable T cells, engineered T cells, or viable engineered T cells is reached within 2, 3, 4, or 5, or within about 2, 3, 4, or 5 population doublings after initiation of stimulation.

[0076] In some of any such embodiments, the engineered or expanded population contains less than or about 3%, less than or about 2%, less than or about 1%, less than or about 0.1%, or less than or about 0.1%, or less than or about 0.01% CD57+ T cells. In some of any such embodiments, the engineered or expanded population is free of or essentially free of CD57+ T cells. In some embodiments, a depleted population that is essentially free of CD57+ T cells contains less than or about 3%, less than or about 2%, less than or about 1%, less than or about 0.1%, or less than or about 0.01% CD57+ T cells.

[0077] In some of any such embodiments, the frequency of naive-like cells in the engineered or expanded population is at least, or at least about, 10%, 20%, 30%, 40%, or 50% of the cells in the population. In some of any such embodiments, the frequency of one or more of CD25+ T cells, CD27+ T cells, CD28+ T cells, CCR7+ T cells, or CD45RA+ T cells in the engineered or expanded population is at least, or at least about, 10%, 20%, 30%, 40%, or 50% greater than the frequency or about the frequency of the respective cells in the population. In some of any such embodiments, the engineered or expanded population contains at least, or at least about, 15%, 20%, 25%, 30%, 35%, or 40% CD27+ T cells. In some of any such embodiments, the engineered or expanded population contains at least, or at least about, 10%, 15%, 20%, 25%, 25%, 30%, 35%, or 40% CD28+ T cells. In some of any such embodiments, the engineered or expanded population contains at least or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, or 80% CD27+CD28+ T cells.

[0078] In some of any such embodiments, the engineered or expanded population contains at least 70% or 80%, or at least about 70% or 80% CD27+CD28+ T cells. In some of any such embodiments, the engineered or expanded population contains at least 10%, 15%, 20%, or 25%, or at least about 10%, 15%, 20%, or 25% CCR7+ T cells.

[0079] In some of any such embodiments, the step of harvesting the cells includes rinsing or washing the cells to remove cellular debris. In some of any such embodiments, the step of harvesting or collecting further includes formulating the cells for cryopreservation or administration to a subject. In some of any such embodiments, the harvested or collected cells are formulated in the presence of a pharmaceutically acceptable excipient. In some of any such embodiments, the harvested or collected cells are formulated for cryopreservation in the presence of a cryoprotectant. In some of any such embodiments, the cryoprotectant comprises DMSO. In some of any such embodiments, the harvested or collected cells are formulated in a container, optionally a vial or bag.

[0080] In some of any such embodiments, the separation occurs before the harvesting step. In some of any such embodiments, the separation occurs before or during culturing. In some of any such embodiments, the separation occurs after the introducing step.

[0081] In some of any such embodiments, the recombinant receptor is capable of binding to a target antigen associated with, specific for, and / or expressed by cells or tissues of a disease, disorder, or condition. In some of any such embodiments, the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In some of any such embodiments, the target antigen is a tumor antigen. In some of any such embodiments, the target antigen is selected from the group consisting of αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (also known as CA9, CAIX, or G250), cancer-testis antigen, cancer / testis antigen 1B (also known as CTAG, NY-ESO-1, and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), epidermal growth factor receptor type III mutant (EGFR), and cytotoxic T cell proliferation and differentiation (CTL) domains.vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing 8 family member A (LRRC8A), LewisY, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG), 5T4 and / or a biotinylated molecule and / or a molecule expressed by HIV, HCV, HBV, or other pathogens.

[0082] In some of any such embodiments, the recombinant protein is or comprises a functional non-TCR antigen receptor or TCR, or an antigen-binding fragment thereof. In some of any such embodiments, the recombinant protein is a chimeric antigen receptor (CAR). In some of any such embodiments, the recombinant receptor is an anti-BCMA CAR. In some of any such embodiments, the recombinant protein is an anti-CD19 CAR. In some of any such embodiments, the chimeric antigen receptor comprises an antigen-binding domain, an extracellular domain containing a spacer and / or hinge region, a transmembrane domain, and an intracellular signaling domain containing a costimulatory signaling region. In some of any such embodiments, the extracellular domain containing the antigen-binding domain comprises an scFv.

[0083] In some of any such embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some of any such embodiments, the intracellular signaling domain is or comprises the intracellular signaling domain of a CD3 chain, optionally the CD3-zeta (CD3ζ) chain, or a signaling portion thereof. In some of any such embodiments, the costimulatory signaling region comprises the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof.

[0084] Also provided is a method for identifying a population of cells capable of expansion, comprising measuring the frequency of CD57+ cells in the population, wherein the population of cells is identified as capable of expansion if the frequency of CD57+ cells is below a threshold frequency.Also provided is a method for identifying a population of cells capable of expansion, comprising measuring the frequency of CD57+ T cells in the population, wherein the population of cells is identified as capable of expansion if the frequency of CD57+ T cells is below a threshold frequency.

[0085] In some of any such embodiments, the threshold frequency is a percentage that is less than, or less than about, 35%, 30%, 20%, 10%, 5%, 1%, or 0.1%. In some of any such embodiments, the population capable of expansion expands at least 2-fold, 4-fold, 8-fold, or 16-fold within 4, 5, 6, 7, or 8 days of culture under conditions that promote growth or expansion.

[0086] Also provided is a method for determining the expansion potential of a population of T cells, comprising measuring a value of a trait associated with CD57 expression in the T cell population, wherein the population of T cells is determined to be capable of expansion if the value of the trait is below or below approximately a threshold value for the trait.

[0087] In some of any such embodiments, the threshold value is: i) 25%, 20%, 15%, 10%, or 5% less than, about 25%, 20%, 15%, 10%, or 5% less than, or within 25%, 20%, 15%, 10%, or 5% less than the mean or median of measurements of a trait associated with CD57 expression in a plurality of reference T cell populations, and less than one standard deviation below or about the mean or median of the measurements; ii) less than the lowest measurement of a trait associated with CD57 expression in a population from among the plurality of reference T cell populations, and optionally less than the lowest measurement iii) is less than the mean or median of a measure of a trait related to CD57 expression calculated among more than 65%, 75%, 80%, 85% of samples from a plurality of reference T cell compositions; wherein the plurality of reference T cell populations are populations that did not expand when cultured under conditions that promote T cell proliferation or expansion, and optionally, the cells did not expand at least 2-fold, 4-fold, 8-fold, or 16-fold within 4, 5, 6, 7, or 8 days of culture.

[0088] In some of any such embodiments, the trait is the level or amount of CD57 polypeptide expressed in total T cells, CD4+ T cells, or CD8+ T cells. In some of any such embodiments, the trait is the frequency, percentage, or amount of CD57+ T cells, CD57+CD4+ T cells, or CD57+CD8+ T cells present in the cell population. In some of any such embodiments, the trait is the level or amount of CD57 mRNA present in T cells in the cell population. In some of any such embodiments, the trait is the level or amount of chromatin accessibility of the gene encoding CD57 (B3GAT1).

[0089] In some of any such embodiments, the method further comprises measuring a second value of a second trait associated with expression of one or more second gene products in the T cell population, wherein expansion is permitted if the trait value is less than or less than about a trait threshold value and if the second value of the second trait is greater than or greater than about a second threshold value for the second trait.

[0090] In some of any such embodiments, the second gene product is a marker associated with naive-like T cells. In some of any such embodiments, the one or more second gene products are selected from CD27, CD28, CCR7, or CD45RA. In some of any such embodiments, the one or more second gene products are CD27 and CD28.

[0091] In some of any such embodiments, the second threshold is: i) 25%, 20%, 15%, 10%, or 5%, or about 25%, 20%, 15%, 10%, or 5%, or within 25%, 20%, 15%, 10%, or 5% greater than the mean or median of measurements of a trait associated with expression of a second gene in a plurality of second reference T cell compositions, and / or greater than one standard deviation above the mean or median or about the mean or median of measurements; ii) greater than the highest measurement of a second trait associated with expression of a second gene in a composition from the plurality of reference T cell compositions, optionally within 50%, 25%, 20%, 15%, 10%, or 5% greater than the highest measurement; or iii) less than the mean or median of measurements of a trait associated with expression of a second gene calculated from more than 65%, 75%, 80%, 85% of samples from the plurality of reference T cell compositions.

[0092] In some of any such embodiments, the second trait is: (i) the level or amount of polypeptide encoded by the second gene present in total T cells, CD4+ T cells, or CD8+ T cells; (ii) the level or amount of polypeptide encoded by the second gene present on the surface of total T cells, CD4+ T cells, or CD8+ T cells; (iii) a trait that is the frequency, percentage, or amount of T cells, CD4+ T cells, or CD8+ T cells that are positive for expression of the second gene; (iv) the level or amount of mRNA of the second gene present in T cells; or (v) the level or amount of accessibility of the second gene.

[0093] Also provided are methods for genetically engineering T cells, comprising: (a) incubating T cells from a population of T cells identified or determined by any of the methods described herein under stimulatory conditions, wherein the stimulatory conditions comprise the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules, thereby generating stimulated T cells; and (b) introducing a heterologous polynucleotide into the stimulated T cells, wherein the stimulated T cells are transduced with a viral vector comprising a heterologous polynucleotide encoding a recombinant receptor, thereby generating an engineered population of T cells.

[0094] Also provided herein are compositions of cells, including any of those produced by the methods described herein. Also provided herein are compositions of cells comprising CD57-CD4+ cells. Also provided herein are compositions of cells comprising CD57-CD8+ cells. Also provided herein are compositions of cells comprising CD57-CD3+ cells.

[0095] Also provided are compositions of cells comprising a depleted population of CD57- T cells produced by any of the methods described herein.

[0096] Also provided is a composition of enriched CD57-CD4+ T cells, comprising CD57-CD4+ T cells of an enriched population of CD57-CD4+ T cells produced by any of the methods described herein.

[0097] Also provided is a composition of enriched CD57-CD8+ T cells, comprising CD57-CD8+ T cells of an enriched population of CD57-CD8+ T cells produced by any of the methods described herein.

[0098] Also provided are compositions of enriched CD57-CD3+ T cells, including CD57-CD3+ T cells of an enriched population of CD57-CD3+ T cells produced by any of the methods described herein.

[0099] Also provided are compositions of cells comprising a stimulated population of T cells produced by any of the methods described herein.

[0100] Also provided are compositions comprising cells of the engineered population of T cells produced by any of the methods described herein.

[0101] Also provided are compositions comprising cells of the expanded population of T cells produced by any of the methods described herein.

[0102] Also provided are compositions comprising a population of T cells identified or determined to be capable of expansion by any of the methods described herein.

[0103] Also provided are methods of treating a subject having or suspected of having a disease, disorder, or condition, comprising administering to the subject a dose of T cells from a population of engineered T cells produced by any of the methods described herein or an expanded population of T cells produced by any of the methods described herein.

[0104] Also provided is the use of a population of engineered T cells produced by any of the methods described herein or an expanded population of T cells produced by any of the methods described herein for the treatment of a disease, disorder, or condition in a subject.

[0105] Also provided is the use of a population of engineered T cells produced by any of the methods described herein or an expanded population of T cells produced by any of the methods described herein for the manufacture of a medicament for the treatment of a disease, disorder, or condition in a subject.

[0106] Also provided are articles of manufacture that include: (i) one or more reagents for immunoaffinity-based selection of cells specific for CD57, CD4, and / or CD8; and (ii) instructions for use of the one or more reagents to perform any of the methods described herein.

[0107] Also provided are articles of manufacture that include: (i) one or more reagents for immune affinity-based selection of cells specific for CD57, CD4, and / or CD8; (ii) one or more stimulatory reagents capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; and (iii) instructions for use of the one or more reagents to perform any of the methods described herein.

[0108] In some of any such embodiments, the reagent for immunoaffinity-based selection is or includes an antibody capable of specifically binding to CD57, CD4, or CD8. In some of any such embodiments, the reagent for immunoaffinity-based selection is or includes an antibody capable of specifically binding to CD57. In some of any such embodiments, the antibody is immobilized on a magnetic particle or immobilized or attached to an affinity chromatography matrix.

[0109] In some of any such embodiments, the stimulatory reagent comprises (i) a primary agent that specifically binds to a member of the TCR complex, optionally specifically binding to CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, optionally the costimulatory molecule being selected from CD28, CD137 (4-1-BB), OX40, or ICOS. In some of any such embodiments, one or both of the primary and secondary agents comprise an antibody or antigen-binding fragment thereof. In some of any such embodiments, the primary and secondary agents comprise antibodies, and optionally the stimulatory reagent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody, or antigen-binding fragments thereof. In some of any such embodiments, the primary and secondary agents are present on or attached to the surface of a solid support. In some of any such embodiments, the solid support is or comprises beads, optionally paramagnetic beads. In some of any such embodiments, the primary and secondary agents are reversibly bound to the surface of an oligomeric particle reagent that includes a plurality of streptavidin or streptavidin mutein molecules.

[0110] Also provided is an article of manufacture comprising: (i) any composition described herein; and (ii) instructions for administering the composition to a subject.

[0111] Also provided herein are therapeutic T cell compositions comprising recombinant receptor-expressing CD4+ T cells and recombinant receptor-expressing CD8+ T cells, wherein the total receptors in the composition are + / CD8 + At least 80% of the cells are CD57- and the total receptor in the composition + / CD4 + At least 80% of the cells are CD57-.

[0112] In some of any such embodiments, at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, at least 95% or at least about 95%, at least 96% or at least about 96%, at least 97% or at least about 97%, at least 98% or at least about 98%, at least 99% or at least about 99%, about 100%, or 100% of the cells in the composition are CD4+ T cells and CD8+ T cells. + / CD3 + At least 80% of the cells are CD57-. In some of any such embodiments, at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, at least 95% or at least about 95%, at least 96% or at least about 96%, at least 97% or at least about 97%, at least 98% or at least about 98%, at least 99% or at least about 99%, about 100%, or 100% of the cells in the composition are CD3+ T cells. In some of any such embodiments, the ratio of Receptor+ / CD4+ T cells to Receptor+ / CD8+ T cells in the composition is about 1:3 to about 3:1. In some of any such embodiments, the ratio of Receptor+ / CD4+ T cells to Receptor+ / CD8+ T cells in the composition is 1:1 or about 1:1.

[0113] In some of any such embodiments, the recombinant protein is or contains a recombinant receptor capable of binding to a target protein associated with, specific for, and / or expressed in cells or tissues of a disease, disorder, or condition. In some of any such embodiments, the recombinant protein is a chimeric antigen receptor (CAR). In some of any such embodiments, the number of viable T cells in the composition is 10 x 10 6 pieces or approximately 10 x 10 6 From 200 x 10 6 or approximately 200 x 10 6and optionally, the number of viable T cells in the composition is 10 x 10 6 pieces or approximately 10 x 10 6 From 100 x 10 6 pieces or approximately 100 x 10 6 pieces, 10×10 6 pieces or approximately 10 x 10 6 From 70 x 10 6 or approximately 70 x 10 6 pieces, 10×10 6 pieces or approximately 10 x 10 6 From 50 x 10 6 or approximately 50 x 10 6 pieces, 50×10 6 or approximately 50 x 10 6 From 200 x 10 6 pieces or approximately 200 x 10 6 pieces, 50×10 6 or approximately 50 x 10 6 From 100 x 10 6 pieces or approximately 100 x 10 6 pieces, 50×10 6 or approximately 50 x 10 6 From 70 x 10 6 or approximately 70 x 10 6 pieces, 70×10 6 or approximately 70 x 10 6 From 200 x 10 6 pieces or approximately 200 x 10 6 pieces, 70×10 6 or approximately 70 x 10 6 From 100 x 10 6 pieces or approximately 100 x 10 6 pieces, or 100 x 10 6 pieces or approximately 100 x 10 6 From 200 x 10 6 pieces or approximately 200 x 10 6In some of any such embodiments, the volume of the composition is between 1.0 mL and 10 mL inclusive, optionally 2 mL or about 2 mL, 3 mL or about 3 mL, 4 mL or about 4 mL, 5 mL or about 5 mL, 6 mL or about 6 mL, 7 mL or about 7 mL, 8 mL or about 8 mL, 9 mL or about 9 mL, or 10 mL or about 10 mL, or any value between any of the foregoing. [The present invention 1001] (a) performing a first selection step, the first selection step comprising enriching either CD57- or CD3+ T cells from a biological sample containing primary human T cells, thereby generating an enriched T cell population; and (b) subjecting cells from the enriched T cell population to a second selection 1. A method for enriching T cells, comprising: the first selection comprises enriching for CD57- T cells and the second selection comprises enriching for CD3+ T cells from the enriched population; or the first selection comprises enriching for CD3+ T cells, and the second selection comprises depleting CD57+ T cells from the enriched T cell population; The method produces a depleted population that contains fewer CD57+ T cells than the biological sample and is enriched for CD3+ T cells. [The present invention 1002] (a) performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample comprising primary human T cells, thereby generating a depleted population, the depleted population comprising fewer CD57+ T cells than the biological sample; and (b) performing a second selection on cells from the depleted population, the second selection comprising enriching for CD3+ T cells from the depleted population, whereby enrichment produces an enriched population of CD57-CD3+ T cells. 1. A method for enriching T cells, comprising: [The present invention 1003] (a) performing a first selection, the first selection comprising enriching CD3+ T cells from a biological sample containing primary human T cells, thereby generating an enriched T cell population; and (b) subjecting cells from the enriched T cell population to a second selection, the second selection comprising removing CD57+ T cells from the enriched sample cell population, thereby generating a depleted population, the depleted population containing fewer CD57+ T cells than the biological sample and / or the enriched T cell population, and being enriched for CD3+ T cells. 1. A method for enriching T cells, comprising: [The present invention 1004] (a) performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample comprising primary human T cells, thereby generating a first depleted population, the first depleted population comprising fewer CD57+ T cells than the biological sample; (b) subjecting cells from the first depleted population to a second selection, the second selection comprising enriching one of (i) CD4+ T cells and (ii) CD8+ T cells from the first depleted population, whereby the enrichment produces a second depleted population enriched in one of (i) CD4+ T cells and (ii) CD8+ T cells and an unselected population; and (c) performing a third selection, the third selection comprising enriching the other of (i) CD4+ cells and (ii) CD8+ cells from the unselected population, whereby enrichment produces a third depleted population enriched in the other of (i) CD4+ T cells and (ii) CD8+ T cells. 1. A method for enriching T cells, comprising: [The present invention 1005] The depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) comprises one of the following: (i) less than or about 5% CD57+ T cells; (ii) a frequency of CD57+ T cells that is less than or less than about 35% of the frequency of CD57+ T cells present in the biological sample; (iii) CD4+ T cells, wherein at least 95% or at least about 95% of the CD4+ T cells are CD57−; and (iv) CD8+ T cells, wherein at least 95% or at least about 95% of the CD8+ T cells are CD57-. The method according to any one of 1001 to 1004 of the present invention, which includes at least one of the following. [The present invention 1006] The method of any of claims 1001 to 1005, wherein the biological sample comprises an apheresis product or a leukapheresis product. [The present invention 1007] 1. A method for enriching T cells, comprising removing CD57+ T cells from a biological sample comprising an apheresis product or leukapheresis product containing primary human T cells, thereby generating a depleted population of T cells, comprising: the depleted population contains fewer CD57+ T cells than the biological sample; The depleted populations are: (i) less than or about 5% CD57+ T cells; (ii) a frequency of CD57+ T cells that is less than or less than about 35% of the frequency of CD57+ T cells present in the biological sample; (iii) CD4+ T cells, wherein at least 95% or at least about 95% of the CD4+ T cells are CD57−; and (iv) CD8+ T cells, wherein at least 95% or at least about 95% of the CD8+ T cells are CD57-. The method includes at least one of the following: [The present invention 1008] 8. The method of any of claims 1001 to 1007, wherein at least 95% or at least about 95% of the CD4+ T cells of the depleted population comprise CD57-CD4+ T cells. [The present invention 1009] 8. The method of any of claims 1001 to 1007, wherein at least 95% or at least about 95% of the CD8+ T cells of the depleted population comprise CD57-CD8+ T cells. [The present invention 1010] 1009. The method of any of claims 1001-1009, wherein at least 95%, or at least about 95%, of the CD4+ T cells and at least 95%, or at least about 95% of the CD8+ T cells of the depleted population comprise CD57-CD4+ T cells and CD57-CD8+ T cells, respectively. [The present invention 1011] 1010. The method of any of claims 1001 to 1010, wherein at least 95% or at least about 95% of the CD3+ T cells of the depleted population comprise CD57-CD3+ T cells. [The present invention 1012] the depleted population is a first depleted population; the method further comprising selecting CD4+ T cells from the first depleted population, thereby generating a second depleted population that is an enriched population of CD57- CD4+ T cells and an unselected population. Any of the methods 1007 to 1011 of the present invention. [The present invention 1013] selecting CD8+ T cells from the unselected population, thereby generating a third depleted population that is an enriched population of CD57-CD8+ T cells, and a second unselected population. The method of the present invention 1012 further comprises: [The present invention 1014] the depleted population is a first depleted population; the method further comprising selecting CD8+ T cells from the first depleted population, thereby generating a second depleted population that is an enriched population of CD57-CD8+ T cells and an unselected population. Any of the methods 1007 to 1011 of the present invention. [The present invention 1015] selecting CD4+ T cells from the unselected population, thereby generating a third depleted population that is an enriched population of CD57-CD4+ T cells, and a second unselected population. The method of the present invention 1014 further comprises: [The present invention 1016] the depleted population is a first depleted population; the method further comprising selecting CD3+ T cells from the first depleted population, thereby generating a second depleted population that is an enriched population of CD57-CD3+ T cells and an unselected population. Any of the methods 1007 to 1011 of the present invention. [The present invention 1017] Any of the methods of claims 1001 to 1016, wherein the frequency of CD57+ T cells in the depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) is less than about 35%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the frequency of CD57+ T cells in the biological sample. [The present invention 1018] Any of the methods of claims 1001-1017, wherein the depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) comprises less than or about 3%, less than or about 2%, less than or about 1%, less than or about 0.1%, or less than or about 0.01% CD57+ T cells. [The present invention 1019] The method of any of claims 1001 to 1018, wherein the depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) does not include or is essentially free of CD57+ T cells. [The present invention 1020] Any of the methods of claims 1001 to 1019, wherein the frequency of naive-like T cells in the depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) is at least about 10%, 20%, 30%, 40%, or 50% greater than the frequency of naive-like T cells in the biological sample, or is at least about 10%, 20%, 30%, 40%, or 50% greater. [The present invention 1021] The method of claim 1020, wherein the naive-like T cells are surface positive for one or more of the markers selected from CD45RA, CD27, CD28, and CCR7. [The present invention 1022] 10. The method of any of claims 1001 to 1021, wherein the frequency of CD27+ T cells in the depleted populations (optionally the first depleted population, the second depleted population, or the third depleted population) is at least about 10%, 20%, 30%, 40%, or 50% greater than the frequency of the respective cells in the biological sample. [The present invention 1023] 10. The method of any of claims 1001 to 1022, wherein the frequency of CD28+ T cells in the depleted populations (optionally the first depleted population, the second depleted population, or the third depleted population) is at least about 10%, 20%, 30%, 40%, or 50% greater than the frequency of the respective cells in the biological sample. [The present invention 1024] Any of the methods of claims 1001 to 1023, wherein the frequency of CD27+ / CD28+ T cells in the depleted population (optionally the first depleted population, the second depleted population, or the third depleted population) is at least about 10%, 20%, 30%, 40%, or 50% greater than the frequency of the respective cells in the biological sample, or is at least about 10%, 20%, 30%, 40%, or 50% greater. [The present invention 1025] combining the second depleted population with the third depleted population, optionally in a ratio of between at or about 1:3 and at or about 3:1, optionally at a ratio of 1:1 or about 1:1, thereby generating a depleted population comprising the second depleted population and the third depleted population. Any of the methods 1004 to 1006, 1013, 1015 and 1017 to 1024 of the present invention further comprises: [The present invention 1026] Any of the methods of claims 1001 to 1025, producing a T cell composition comprising at least 90%, at least 95%, at least 95%, at least 97%, at least 97%, at least 99%, or at least 99.9%, or at least 99.9% CD57-CD4+ T cells. [The present invention 1027] Any of the methods of claims 1001 to 1025, producing a T cell composition comprising at least 90%, or at least about 90%, at least 95%, or at least about 95%, at least 97%, or at least about 97%, at least 99%, or at least about 99%, or at least 99.9%, or at least about 99.9% CD57-CD8+ T cells. [The present invention 1028] Any of the methods of claims 1001 to 1025, producing a T cell composition comprising at least 90%, at least 95%, at least 95%, at least 97%, at least 97%, at least 99%, or at least 99.9%, or at least 99.9% CD57-CD3+ T cells. [The present invention 1029] 1028. The method of claim 1028, wherein the ratio of CD4+ to CD8+ T cells in said composition is 3:1 to 1:3, 2:1 to 1:2, 1.5:1 to 1:1.5, or 1.2:1 to 1:1.2 (each inclusive). [The present invention 1030] 1029. The method of claim 1028 or claim 1029, wherein the ratio of CD4+ to CD8+ T cells in said composition is 1:1 or about 1:1. [The present invention 1031] The method of any of claims 1001 to 1030, wherein the primary T cells are derived from a human subject. [The present invention 1032] The method of any of claims 1001 to 1031, wherein the primary T cells are derived from a subject having a disease or condition. [The present invention 1033] The method of claim 1032, wherein the disease or condition is cancer. [The present invention 1034] The method of any of claims 1001 to 1030, wherein the primary T cells are derived from a healthy subject. [This invention 1035] The method of any of claims 1001 to 1034, wherein the biological sample comprises CD4+ and CD8+ cells. [The present invention 1036] The method of any of claims 1001 to 1035, wherein depleting CD57+ T cells comprises immunoaffinity-based selection. [This invention 1037] immunoaffinity-based selection includes contacting the cells with an antibody capable of specifically binding to CD57 and recovering cells that do not bind to the antibody, thereby resulting in negative selection; The recovered cells are depleted of CD57+ cells. The method of the present invention 1036. [The present invention 1038] Optionally, in the first, second, and / or third selection, enriching for cells comprises immunoaffinity-based selection. [This invention 1039] The first, second, and / or third selections enrich for CD4 or CD8 T cells, Immunoaffinity-based selection is performed by contacting the cells with an antibody capable of specifically binding to CD4 or CD8, respectively, and recovering the cells that bind to the antibody, thereby resulting in positive selection; The recovered cells are enriched for CD4+ or CD8+ cells. The method of the present invention 1038. [The present invention 1040] The selection enriches for CD3 T cells, Immunoaffinity-based selection is performed by contacting the cells with an antibody capable of specifically binding to CD3 and recovering the cells that bind to the antibody, thereby resulting in positive selection; The recovered cells are enriched for CD3+ cells. The method of the present invention 1039. [The present invention 1041] 1040. The method of any of claims 1037 to 1040, wherein the antibody is immobilized on a solid surface, and optionally the solid surface is a magnetic particle. [The present invention 1042] 1040. The method of any of claims 1037 to 1040, wherein the antibody is immobilized or attached to an affinity chromatography matrix. [This invention 1043] The method of claim 1042, wherein the antibody further comprises one or more binding partners capable of forming a reversible bond with a binding reagent immobilized on the matrix, whereby the antibody is reversibly bound to the chromatography matrix during contact. [This invention 1044] The method of claim 1043, wherein the binding reagent is a streptavidin mutein that reversibly binds to the binding partner. [This invention 1045] The streptavidin mutein has the amino acid sequence Ile at positions corresponding to positions 44-47 in streptavidin in the amino acid sequence shown in SEQ ID NO:66. 44 -Gly 45 -Ala 46 -Arg 47 Contains, or The streptavidin mutein has the amino acid sequence Va1 at positions corresponding to positions 44-47 in streptavidin of the amino acid sequence shown in SEQ ID NO:66. 44 -Thr 45 -Ala 46 -Arg 47 Including, The method of the present invention 1044. [The present invention 1046] 1046. The method of any one of claims 1043 to 1045, wherein the binding partner is a streptavidin-binding peptide. [This invention 1047] Streptavidin-binding peptides TIFF0007604366000006.tif48169 The method of claim 1046, wherein the compound is selected from the group consisting of: [This invention 1048] The streptavidin-binding peptide has the sequence TIFF0007604366000007.tif4128 The method of the present invention 1046 or 1047, [This invention 1049] contacting the cells in the sample with the affinity chromatography matrix and then applying a competing reagent to disrupt the binding between the binding partner and the binding reagent, thereby recovering the cells bound to the antibody. Any of the methods of claims 1043 to 1048, further comprising: [The present invention 1050] 1049. The method of claim 1049, wherein the competitive reagent is biotin or a biotin analogue. [This invention 1051] The method of any one of claims 1042 to 1050, wherein the chromatography matrix is ​​packed in a separation tube that is a column. [This invention 1052] The method of any one of claims 1001 to 1051, wherein the biological sample comprising primary T cells is a sample formulated with a cryoprotectant. [This invention 1053] 1053. The method of any of claims 1006 to 1052, wherein the apheresis or leukapheresis sample is a sample formulated with a cryoprotectant. [This invention 1054] 1. A method for stimulating T cells, comprising incubating T cells of an input composition under stimulatory conditions, thereby generating a stimulated population of T cells, The input composition is produced by any one of the methods of the present inventions 1001 to 1053. method. [This invention 1055] The input composition is: (i) less than or about 5% CD57+ T cells; (ii) at least 95% or at least about 95% CD57 − T cells; (iii) at least 90% or at least about 90% CD57 − CD4 + T cells; (iv) at least 90% or at least about 90% CD57-CD8+ T cells; and (v) at least about 90% or about 90% CD57-CD3+ T cells The method of the present invention 1054, comprising at least one of the following: [This invention 1056] 1. A method for stimulating T cells, comprising incubating T cells of an input composition under stimulatory conditions, thereby generating a stimulated population of T cells, The input composition is: (i) less than or about 5% CD57+ T cells (ii) at least 95% or at least about 95% CD57 − T cells; (iii) at least 90% or at least about 90% CD57 − CD4 + T cells; (iv) at least 90% or at least about 90% CD57-CD8+ T cells; and (v) at least 90% or at least about 90% CD57-CD3+ T cells The method includes at least one of the following: [This invention 1057] The method of the present invention 1056, wherein the stimulatory conditions include the presence of a stimulatory reagent, which is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules. [This invention 1058] The stimulating agent is (i) a primary agent that specifically binds to a member of the TCR complex, optionally specifically binding to CD3; and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, optionally wherein the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS. The method of the present invention 1057, comprising: [This invention 1059] The method of claim 1058, wherein at least one of the primary agent and the secondary agent comprises an antibody or an antigen-binding fragment thereof. [The present invention 1060] The method of claim 1058 or claim 1059, wherein the primary agent is an anti-CD3 antibody or an antigen-binding fragment thereof and the secondary agent is an anti-CD28 antibody or an antigen-binding fragment thereof. [This invention 1061] The method of claim 1059 or claim 1060, wherein the antigen-binding fragment is a monovalent antibody fragment selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). [This invention 1062] The method of any of claims 1058 to 1061, wherein the primary agent is an anti-CD3 Fab and the secondary agent comprises an anti-CD28 Fab. [This invention 1063] The method of any of claims 1058 to 1062, wherein the primary agent and the secondary agent are each present on or attached to the surface of a solid support. [This invention 1064] 1063. A method according to claim 1063, wherein the solid support is or comprises beads, optionally paramagnetic beads. [This invention 1065] 1064. The method of claim 1064, wherein the solid support is a paramagnetic bead having anti-CD3 and anti-CD28 antibodies attached to its surface, and the stimulating reagent is present at a bead to cell ratio of less than about 3:1 or about 3:1. [The present invention 1066] The method of claim 1065, wherein the stimulatory reagent is present at a bead to cell ratio of 1:1 or about 1:1. [This invention 1067] The method of any of claims 1058-1062, wherein the primary agent and secondary agent are reversibly bound to the surface of an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules. [The present invention 1068] 1067. The method of claim 1067, wherein the streptavidin or streptavidin mutein molecule binds or is capable of binding to biotin, avidin, a biotin analogue or biotin mutein, an avidin analogue or avidin mutein, and / or biologically active fragments thereof. [The present invention 1069] The streptavidin mutein molecule has the amino acid sequence Val at amino acid residues corresponding to residues 44-47 with respect to the amino acid sequence set forth in SEQ ID NO:66. 44 -Thr 45 -Ala 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47 The method of the present invention 1067 or 1068, comprising: [The present invention 1070] The streptavidin mutein molecule (a) an amino acid sequence shown in any one of SEQ ID NOs: 70 to 73, 78, and 85 to 89; (b) exhibiting at least about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 70-73, 78, 85-89, or about 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and 44 -Thr 45 -Ala 46 -Arg 47 Or Ile 44 -Gly 45 -Ala 46 -Arg 47 and / or an amino acid sequence that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide; or (c) a functional fragment of (a) or (b) that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide. Any of the methods of claims 1067 to 1069, comprising: [This invention 1071] 1070. The method of any one of claims 1067 to 1070, wherein the streptavidin mutein molecule comprises the amino acid sequence shown in SEQ ID NO:73. [This invention 1072] The method of any of claims 1067 to 1070, wherein the streptavidin mutein molecule comprises the amino acid sequence shown in SEQ ID NO:78. [This invention 1073] 13. The method of any of claims 1067 to 1072, wherein the primary agent and the secondary agent each comprise a streptavidin-binding peptide. [This invention 1074] Streptavidin-binding peptides TIFF0007604366000008.tif34168 The method of the present invention 1073, selected from the group consisting of: [This invention 1075] The method of any of claims 1067 to 1074, wherein the primary agent comprises an anti-CD3 Fab and the secondary agent comprises an anti-CD28 Fab. [This invention 1076] 1076. The method of any of claims 1067-1075, wherein the oligomeric particle reagent comprises a radius of greater than or about 60 nm, greater than or about 70 nm, greater than or about 80 nm, or greater than or about 90 nm. [This invention 1077] The oligomeric particle reagent comprises at least about 5×10 7 g / mol or approximately 5 × 10 7 g / mol or at least about 1 × 10 8 g / mol or approximately 1 × 10 8 g / mol; or 5 × 10 7 g / mol ~ 5 × 10 8 g / mol, 1×10 8 g / mol ~ 5 × 10 8 g / mol, or 1 × 10 8 g / mol~2×10 8 1067. The method of any one of claims 1067 to 1076, comprising a molecular weight of 1067 g / mol (inclusive). [This invention 1078] The oligomeric particle reagent may comprise at least about 500 streptavidin or streptavidin mutein tetramers, at least about 1,000 streptavidin or streptavidin mutein tetramers, at least about 1,500 streptavidin or streptavidin mutein tetramers, or at least about 2,000 streptavidin or streptavidin mutein tetramers. streptavidin or streptavidin mutein tetramers; or 1,000 to 20,000 streptavidin or streptavidin mutein tetramers, 1,000 to 10,000 streptavidin or streptavidin mutein tetramers, or 2,000 to 5,000 streptavidin or streptavidin mutein tetramers. [This invention 1079] The method further comprises the step of separating the stimulatory reagent from the cells, the separating step comprises contacting the cells with a substance capable of reversing the binding between the primary and secondary agents and the oligomeric particle reagent; Any of the methods of the present invention 1061 to 1078. [The present invention 1080] 1079. The method of claim 1079, wherein said agent is a free binding partner and / or a competitive agent. [This invention 1081] 1079. The method of claim 1080, wherein said substance is or comprises a streptavidin-binding peptide, biotin or a biologically active fragment thereof, or a biotin analogue or a biologically active fragment thereof. [This invention 1082] 1081. The method of claim 1081, wherein said substance is or comprises biotin or a biotin analogue. [This invention 1083] 1083. The method of any of claims 1056 to 1082, wherein the stimulatory conditions comprise the presence of one or more recombinant cytokines. [This invention 1084] The method of any of claims 1056 to 1083, wherein the stimulatory conditions include the presence of one or more of recombinant IL-2, IL-7, and IL-15. [This invention 1085] introducing a heterologous polynucleotide encoding a recombinant receptor into a population of T cells from a T cell composition produced by any of the methods of claims 1001 to 1053, thereby generating an engineered population of T cells. 1. A method for genetically engineering T cells, comprising: [The present invention 1086] introducing a heterologous polynucleotide encoding a recombinant receptor into a population of T cells from a stimulated T cell population of any of claims 1054 to 1084, thereby generating an engineered population of T cells. 1. A method for genetically engineering T cells, comprising: [This invention 1087] The method of claim 1085 or claim 1086, wherein the introducing step comprises transduction with a viral vector comprising the heterologous polynucleotide. [This invention 1088] 1087. The method of claim 1087, wherein the viral vector is a gammaretroviral vector or a lentiviral vector. [This invention 1089] The method of claim 1087 or claim 1088, wherein the viral vector is a lentiviral vector. [The present invention 1090] after the introducing step, incubating the composition comprising the transduced cells for up to 96 hours, optionally at a temperature of at or about 37°±2°C. Any of the methods of claims 1085 to 1089, further comprising: [This invention 1091] The method of claim 1090, wherein after the introducing step, the incubating step is carried out for up to 72 hours. [This invention 1092] The method of claim 1090, wherein after the introducing step, the incubating step is carried out for up to 48 hours. [This invention 1093] The method of claim 1090, wherein after the introducing step, the incubating step is carried out for up to 24 hours. [This invention 1094] The method of any of claims 1090 to 1093, wherein the incubating step results in integration of the viral vector into the genome of the T cell. [This invention 1095] Culturing the cells of the engineered population under conditions that promote the growth or expansion of the engineered cells, thereby producing an expanded population of cells. Any of the methods of claims 1085 to 1094, further comprising: [This invention 1096] 1095. The method of claim 1095, wherein the culturing step is optionally carried out in the presence of one or more recombinant cytokines, including one or more of IL-2, IL-7, and IL-15. [This invention 1097] The method of claim 1095 or claim 1096, wherein the proliferation or expansion results in an increase of about 2-fold, 3-fold, 4-fold, 5-fold, or at least 2-fold, 3-fold, 4-fold, 5-fold, or at least about 2-fold, 3-fold, 4-fold, 5-fold, or more than 5-fold or more than about 5-fold in the number of viable T cells comprising the heterologous polynucleotide compared to the initiation of the culture. [This invention 1098] The method of any of claims 1001 to 1097, further comprising the step of harvesting or collecting the population of cells produced by said method. [This invention 1099] The method of claim 1098, wherein the harvesting or collecting step further comprises formulating the cells for cryoprotection in the presence of a cryoprotectant. [The present invention 1100] The method of claim 1098 or claim 1099, wherein the harvested or collected cells are formulated in the presence of a pharmaceutically acceptable excipient. [The present invention 1101] The recombinant receptor Target antigens associated with, specific to, and / or expressed on cells or tissues of a disease, disorder, or condition Any of the methods of 1085 to 1100 of the present invention, which can be bound to [The present invention 1102] The method of claim 1101, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. [The present invention 1103] The method of claim 1101 or 1102, wherein the target antigen is a tumor antigen. [The present invention 1104] Target antigens include αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9; also known as CAIX or G250), cancer-testis antigen, cancer / testis antigen 1B (CTAG; also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), and type III epidermal growth factor receptor mutant (EGFR). vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5;Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate-binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), and Her4 (erb-B4). , erbB dimer, human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein G (TPBG; also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2;The method of any of claims 1101 to 1103, wherein the antigen is selected from among dopachrome tautomerase, also known as dopachrome delta-isomerase or DCT, vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens; [This invention 1105] 1104. The method of any of claims 1101 to 1103, wherein the recombinant receptor is or comprises a functional non-TCR antigen receptor or TCR or an antigen-binding fragment thereof. [The present invention 1106] The method of any of claims 1101 to 1104, wherein the recombinant receptor is a chimeric antigen receptor (CAR). [This invention 1107] The recombinant receptor an extracellular domain, including an antigen-binding domain, a spacer, and / or a hinge region; a transmembrane domain, and Intracellular signaling domains containing costimulatory signaling regions Any of the methods 1101 to 1106 of the present invention, comprising: [This invention 1108] 1107. The method of claim 1107, wherein the extracellular domain comprises an antigen-binding domain comprising an scFv. [This invention 1109] The method of claim 1107 or claim 1108, wherein the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). [The present invention 1110] 1109. The method of any of claims 1107 to 1109, wherein the intracellular signalling domain is or comprises the intracellular signalling domain of a CD3 chain, optionally the CD3-zeta (CD3ζ) chain, or a signalling portion thereof. [The present invention 1111] The method of any of claims 1107 to 1110, wherein the costimulatory signaling region comprises the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. [The present invention 1112] A cellular composition produced by any of the methods of present inventions 1001 to 1053. [The present invention 1113] The composition of claim 1112, wherein the cells comprise CD57-CD4+ T cells. [This invention 1114] 1112. The composition of claim 1112, wherein the cells comprise CD57-CD8+ T cells. [This invention 1115] 1112. The composition of claim 1112, wherein the cells comprise CD57-CD3+ T cells. [The present invention 1116] A composition comprising an engineered population of T cells produced by the method of any of claims 1085 to 1111. [This invention 1117] A therapeutic T cell composition comprising CD4+ T cells expressing a recombinant receptor and CD8+ T cells expressing a recombinant receptor, wherein the total receptors in the composition are + / CD8 + At least 80% of the cells are CD57- and the total receptor in the composition + / CD4 + A therapeutic T cell composition, wherein at least 80% of the cells are CD57-. [This invention 1118] 1117. A therapeutic T cell composition of the present invention, wherein at least 80%, or at least about 80%, at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, at least 98%, or at least about 98%, at least 99%, or at least about 99%, about 100%, or 100% of the cells in the composition are CD4+ T cells and CD8+ T cells. [This invention 1119] the total receptors in the composition + / CD3 + A therapeutic T cell composition comprising CD3+ T cells expressing a recombinant receptor, wherein at least 80% of the cells are CD57-. [The present invention 1120] A therapeutic T cell composition of the present invention 1119, wherein at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, at least 95% or at least about 95%, at least 96% or at least about 96%, at least 97% or at least about 97%, at least 98% or at least about 98%, at least 99% or at least about 99%, about 100%, or 100% of the cells in the composition are CD3+ T cells. [This invention 1121] 1120. The therapeutic T cell composition of any of claims 1117 to 1120, wherein the ratio of receptor+ / CD4+ T cells to receptor+ / CD8+ T cells in said composition is about 1:3 to about 3:1. [This invention 1122] 1122. The therapeutic T cell composition of any of claims 1117 to 1121, wherein the ratio of receptor+ / CD4+ T cells to receptor+ / CD8+ T cells in said composition is 1:1 or about 1:1. [This invention 1123] The recombinant protein Target proteins associated with, specific to, and / or expressed in cells or tissues of a disease, disorder, or condition The therapeutic composition of any one of claims 1117 to 1122, which is or comprises a recombinant receptor capable of binding to [This invention 1124] The therapeutic composition of any of claims 1117 to 1123, wherein the recombinant protein is a chimeric antigen receptor (CAR). [This invention 1125] The number of viable T cells in the composition is 10×106 pieces or approximately 10 x 10 6 From 200 x 10 6 pieces or approximately 200 x 10 6 and optionally, the number of viable T cells in the composition is 10 x 10 6 pieces or approximately 10 x 10 6 From 100 x 10 6 pieces or approximately 100 x 10 6 pieces, 10×10 6 pieces or approximately 10 x 10 6 From 70 x 10 6 or approximately 70 x 10 6 pieces, 10×10 6 pieces or approximately 10 x 10 6 From 50 x 10 6 or approximately 50 x 10 6 pieces, 50×10 6 or approximately 50 x 10 6 From 200 x 10 6 pieces or approximately 200 x 10 6 pieces, 50×10 6 or approximately 50 x 10 6 From 100 x 10 6 pieces or approximately 100 x 10 6 pieces, 50×10 6 or approximately 50 x 10 6 From 70 x 10 6 or approximately 70 x 10 6 pieces, 70×10 6 or approximately 70 x 10 6 From 200 x 10 6 pieces or approximately 200 x 10 6 pieces, 70×10 6 or approximately 70 x 10 6 From 100 x 10 6 pieces or approximately 100 x 10 6 pieces, or 100 x 10 6 pieces or approximately 100 x 10 6 From 200 x 10 6 pieces or approximately 200 x 10 6 The therapeutic composition of any one of 1117 to 1124 of the present invention, wherein the number of the compounds is 1 (each including both end values). [The present invention 1126] The therapeutic composition of any of claims 1117 to 1125, wherein the volume of the composition is 1.0 mL to 10 mL (inclusive), optionally 2 mL or about 2 mL, 3 mL or about 3 mL, 4 mL or about 4 mL, 5 mL or about 5 mL, 6 mL or about 6 mL, 7 mL or about 7 mL, 8 mL or about 8 mL, 9 mL or about 9 mL, or 10 mL or about 10 mL, or any value between any of the foregoing. [This invention 1127] A method of treating a subject having or suspected of having a disease, disorder, or condition, comprising administering to the subject a dose of T cells from an engineered population of T cells produced by the method of any of claims 1085 to 1111. [This invention 1128] A method of treating a subject having or suspected of having a disease, disorder, or condition, comprising administering to the subject a dose of T cells from any of the compositions of inventions 1112-1126. [This invention 1129] The method of claim 1127 or claim 1128, wherein the recombinant receptor, optionally a CAR, specifically recognizes or specifically binds to an antigen associated with the disease or condition or expressed or present on cells of the disease or condition. [The present invention 1130] The T cell dose was 5 × 10 6 pieces or approximately 5 x 10 6 From 1.5 x 10 8 pieces or approximately 1.5 x 10 8 5 × 10 recombinant receptor-expressing T cells 6 pieces or approximately 5 x 10 6 From 1 x 10 8 pieces or approximately 1 x 10 8 5 × 10 recombinant receptor-expressing T cells 6 pieces or approximately 5 x 10 6 From 50 x 10 6 pieces or approximately 50 x 10 6 5 × 10 recombinant receptor-expressing T cells 6 pieces or approximately 5 x 10 6 From 25 x 10 6 pieces or approximately 25 x 10 6 5 × 10 recombinant receptor-expressing T cells 6 pieces or approximately 5 x 10 6 From 10 pieces to 10 pieces 6 pieces or approximately 10 x 10 6 10 x 10 recombinant receptor-expressing T cells 6 pieces or approximately 10 x 10 6 From 1.5 x 10 8 pieces or approximately 1.5 x 10 8 10 x 10 recombinant receptor-expressing T cells 6 pieces or approximately 10 x 10 6 From 1 x 10 8 pieces or approximately 1 x 108 10 x 10 recombinant receptor-expressing T cells 6 pieces or approximately 10 x 10 6 From 50 x 10 6 pieces or approximately 50 x 10 6 10 x 10 recombinant receptor-expressing T cells 6 pieces or approximately 10 x 10 6 From 25 x 10 6 pieces or approximately 25 x 10 6 25 x 10 recombinant receptor-expressing T cells 6 pieces or approximately 25 x 10 6 From 1.5 x 10 8 pieces or approximately 1.5 x 10 8 25 x 10 recombinant receptor-expressing T cells 6 pieces or approximately 25 x 10 6 From 1 x 10 8 pieces or approximately 1 x 10 8 25 x 10 recombinant receptor-expressing T cells 6 pieces or approximately 25 x 10 6 From 50 x 10 6 pieces or approximately 50 x 10 6 50 x 10 recombinant receptor-expressing T cells 6 pieces or approximately 50 x 10 6 From 1.5 x 10 8 pieces or approximately 1.5 x 10 8 50 x 10 recombinant receptor-expressing T cells 6 pieces or approximately 50 x 10 6 From 1 x 10 8 pieces or approximately 1 x 10 8 1 x 10 recombinant receptor-expressing T cells 8 pieces or approximately 1 x 10 8 From 1.5 x 10 8 pieces or approximately 1.5 x 10 8 The method of any one of 1127 to 1129, comprising recombinant receptor-expressing T cells (both inclusive). [This invention 1131] 1130. The method of any of claims 1127 to 1130, wherein the T cells in the dose of T cells are total T cells, total viable T cells, total viable recombinant receptor-expressing T cells, total viable recombinant receptor-expressing CD4+ T cells, or total viable recombinant receptor-expressing CD8+ T cells. [This invention 1132] 1132. The method of any of claims 1127 to 1131, wherein the dose of T cells is allogeneic to the subject being treated. [This invention 1133] The method of any of claims 1127 to 1131, wherein the dose of T cells is autologous to the subject being treated. [This invention 1134] The method of any one of claims 1127 to 1133, wherein the disease or condition is cancer. [This invention 1135] Use of a population of engineered T cells produced by the method of any of inventions 1085 to 1111 or a composition of any of inventions 1112 to 1126 for the treatment of a disease, disorder, or condition in a subject. [This invention 1136] 11. Use of a population of engineered T cells produced by any of the methods of claims 1085 to 1111 for the manufacture of a medicament for the treatment of a disease, disorder, or condition in a subject. [This invention 1137] Any of the compositions of claims 1112 to 1126 for use in treating a disease, disorder, or condition in a subject. [This invention 1138] 8. The use or composition for use of any of claims 1135 to 1137, wherein the recombinant receptor, optionally a CAR, specifically recognizes or specifically binds to an antigen associated with the disease or condition or expressed or present on cells of the disease or condition. [This invention 1139] 9. The use or composition for use of any of claims 1135 to 1138, wherein the disease or condition is cancer. [This invention 1140] 1139. A use or composition for use of any of claims 1135 to 1139 which is autologous to the subject. [This invention 1141] A use or composition for use of any of claims 1135 to 1140 which is allogeneic to the subject. [This invention 1142] (i) one or more reagents for immunoaffinity-based selection of cells specific for CD57 and one or more of CD3, CD4, and / or CD8; and (ii) Instructions for the use of one or more reagents for carrying out any of the methods of inventions 1001 to 1141. Manufactured articles, including: [This invention 1143] (i) one or more reagents for immunoaffinity-based selection of cells specific for CD57 and one or more of CD3, CD4, and / or CD8; (ii) one or more stimulatory reagents capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; and (iii) Instructions for the use of one or more reagents for carrying out any of the methods of inventions 1001 to 1141. Manufactured articles, including: [This invention 1144] 1144. The article of manufacture of claim 1142 or 1143, wherein the reagent for immunoaffinity-based selection is or comprises an antibody capable of specifically binding to CD57, CD3, CD4, or CD8. [Invention 1145] 114. The article of manufacture of any of claims 1142 to 1144, wherein the reagent for immunoaffinity-based selection is or comprises an antibody capable of specifically binding to CD57. [Invention 1146] 1145. The article of manufacture of claim 1145, wherein the antibody is immobilized on magnetic particles. [This invention 1147] 1145. An article of manufacture of the invention wherein the antibody is immobilized or attached to an affinity chromatography matrix. [This invention 1148] The stimulating agent is (i) a primary agent that specifically binds to a member of the TCR complex, optionally specifically binding to CD3; and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, optionally wherein the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS. Any one of the manufactured articles of inventions 1143 to 1147, comprising: [This invention 1149] 1148. An article of manufacture of the present invention, wherein one or both of the primary agent and the secondary agent comprises an antibody or antigen-binding fragment thereof. [This invention 1150] The article of manufacture of claim 1148 or claim 1149, wherein the primary agent and the secondary agent comprise antibodies, and optionally the stimulating reagent comprises incubation with an anti-CD3 antibody or antigen-binding fragment thereof and an anti-CD28 antibody or antigen-binding fragment thereof. [This invention 1151] The article of manufacture of any of claims 1148-1150, wherein the primary agent and secondary agent are present on or attached to the surface of a solid support. [This invention 1152] 1151. An article of manufacture of the invention 1151, wherein the solid support is or comprises a bead, optionally a paramagnetic bead. [This invention 1153] The article of manufacture of any of claims 1148-1152, wherein the primary agent and secondary agent are reversibly bound to the surface of an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules. [This invention 1154] (i) any one of the compositions 1112 to 1126 of the present invention; and (ii) instructions for administering the composition to a subject; Manufactured articles, including: [Brief explanation of the drawings]

[0114] [Figure 1] Figures 1A-1D show the total cell count (Figure 1A), fold expansion (Figure 1B), viability (Figure 1C), and percentage of Ki-67+ cells (a marker associated with cell cycle entry; Figure 1D) among CD8+ T cells obtained from seven different donors (donors A-G) over approximately 240 hours after the start of stimulation in an exemplary manufacturing process for primary human T cells, or a similar process without cell transduction. [Figure 2]Figure 2A shows flow cytometry analysis of CD57 and Ki-67 expression at various time points over approximately 216 hours after the initiation of stimulation in an exemplary process for primary human T cells. Figure 2B shows the percentage of CD57+ cells in the cell population during stimulation and culture in three different donors (donors A-C). Figure 2C shows the percentage of Ki-67+ cells among CD57+ or CD57- cells in the cell population during stimulation and culture. Figure 2D shows the percentage of CD57+ cells in the CD4+ cell population during stimulation and culture in three different donors (donors A-C). Figure 2E shows the expression of CD57 and Ki67 on cells from donor cell compositions over time after stimulation. Cells from one donor cell composition took 8 days to reach harvest criteria ("8d to harvest"), while cells from the other donor cell composition took only 7 days to reach harvest criteria ("7d to harvest"). FIG. 2F shows the expression of CD45RA and CD27 on cells from the donor cell composition described in FIG. 2E. [Figure 3] Figure 3A shows flow cytometry analysis of CD69 and CD25 expression (activation-associated markers) among CD57+ and CD57- cells in cell populations during stimulation and culture in an exemplary process for primary human T cells. Figures 3B and 3C show hierarchical clustering analysis of the expression of various surface markers associated with T cell differentiation phenotypes (e.g., naive-like T cells, effector T (TEFF) cells, memory T cells, central memory T cells (TCM), effector memory T (TEM) cells, and effector memory RA T (TEMRA) cells), as well as CD57 (Figure 3B) or Ki-67 (Figure 3C). Each row represents a cell population from an individual donor, and each column represents a marker or combination of markers associated with a T cell differentiation phenotype. [Figure 4]Figure 4A shows the total cell count and percentage of viable cells over approximately 240 hours after the start of stimulation in an exemplary manufacturing process for a titrated cell composition containing a mixture of CD57+ and CD57- cells at the following frequencies: (1) 100% CD57+ cells; (2) 75% CD57+ cells; (3) 25% CD57+ cells; and (4) 0% CD57+ cells. Exemplary harvest criteria are displayed. Figure 4B shows an image of a cell culture well 48 hours after the start of stimulation, demonstrating cell clustering in the presence of a stimulating reagent, during stimulation and culture of the titrated cell composition. Figure 4C shows the amount of IL-2 (μg / mL) present in the culture medium 12 and 48 hours after the start of stimulation during stimulation and culture of the titrated cell composition. [Figure 5] Figures 5A and 5B show the percentages of CD57+ T cells (Figure 5A) and CD27+CD28+ T cells (Figure 5B). Figure 5C shows CD27 expression for four donor cell compositions in which CD57+ cells were not depleted (non-depleted) or CD57+ cells were depleted (depleted). Figure 5D shows Ki67 expression in depleted and non-depleted CD3+ cell compositions. Figure 5E shows the culture duration (from the start of stimulation to the time when the harvest criteria were reached) for primary CD8+ cells obtained from subjects in which CD57+ cells were depleted (depleted) or CD57+ cells were not depleted (non-depleted). Figure 5F shows the total number of T cells over time in depleted and non-depleted cell compositions after stimulation. Figure 5G shows the percentage of cells expressing chimeric antigen receptors (CARs) after transduction in depleted and non-depleted cell compositions. [Figure 6]Figure 6A shows a boxplot depicting the percentage of CD57+ T cells among CD4+ and CD8+ T cells obtained from a non-Hodgkin's lymphoma (NHL) patient for engineering cells to express a chimeric antigen receptor (CAR) using an exemplary manufacturing process. The box represents the interquartile range, and the whiskers represent the full range of the data set. Figure 6B shows the relationship between the percentage of CD57+ CD8+ cells and the percentage of Ki-67+ cells in various cell populations obtained from an NHL patient. Figure 6C shows a hierarchical clustering analysis of the expression of various surface markers associated with T cell differentiation phenotypes (e.g., naive-like T cells, effector T (TEFF) cells, memory T cells, central memory T cells (TCM), terminal effector cells), and CD57 expression in cell populations obtained from an NHL patient. Each row represents an individual patient, and each column represents a marker or combination of markers associated with a T cell differentiation phenotype. Figure 6D shows the percentage of live CD57+ cells expressing Ki67 in subsets of cell populations grouped by CD27 and CD45RA expression status. Figure 6E shows the percentage of live CD8+CD57+ cells in subsets of cell populations sorted by CD27 and CD45RA expression (upper panel) and the percentage of live CD4+CD57+ cells in subsets of cell populations sorted by CD27 and CD45RA expression (lower panel). [Figure 7] Figure 1 shows the relationship between the percentage of central memory / naive-like CD4+ T cells in a therapeutic output T cell composition (e.g., pharmaceutical product) and the number of population doublings to achieve harvest criteria (Spearman ρ: -0.54; p-value: <0.001). Similar results were observed for CD8+ T cells in a therapeutic output T cell composition (e.g., pharmaceutical product). [Figure 8]Figure 8A shows the number of population doublings to reach the harvest criteria for high (0.35 x 10^6 cells / mL) and low (0.05 x 10^6 cells / mL) seeding densities during the expansion step of the production process. Figure 8B shows the percentage of CD27+CAR+CD8+ T cells in the output therapeutic composition as a function of seeding density during the expansion step of the production process. Figure 8C shows the effect of process duration on the amount of central memory T cells in the output T cell composition. [Figure 9] Figures 9A-9D show Kaplan-Meier survival curves for subjects receiving CAR+ T cell compositions divided into groups receiving compositions containing percentages of CCR7+CD27+ CAR+ T cells among CD4+ CAR+ T cells (Figure 9A for progression-free survival, Figure 9C for duration of response) and among CD8+ CAR+ T cells (Figure 9B for progression-free survival, Figure 9D for duration of response) above or below certain threshold levels. [Figure 10] PFS curves based on the optimal-split log-rank test are shown for patients with "high" or "low" population doubling numbers (PDL) in CD8+ / CAR+ T cells. A low PDL refers to a PDL of <6, and a PDL of >6 refers to a high PDL. [Figure 11] Shown are the percentages of CD27+CD28+ T cells in enriched CD4+ (left column) and CD8+ (right column) input compositions from non-Hodgkin's lymphoma patients. [Figure 12] Figure 1 shows the relationship between the percentage of effector memory T cells in the enriched CD4+ input composition and the number of population doublings required to achieve harvest criteria (Spearman ρ: 0.43; p-value: <0.001). Similar results were observed for the enriched CD8+ input composition. [Figure 13] 13A-D show the total viable cells (FIG. 13A), viability (FIG. 13B), and phenotype (FIGS. 13C and 13D) during and after a manufacturing run involving various non-expansion and expansion manipulation processes. [Figure 14] Shown are the percentages of CD57+ cells in the CD4+ and CD8+ cell populations during and after manufacturing runs involving various non-expansion and expansion manipulation processes. DETAILED DESCRIPTION OF THE INVENTION

[0115] Detailed Description Provided herein are methods and compositions that are particularly useful for selecting, isolating, enriching, stimulating, activating, genetically manipulating, or expanding samples, populations, or compositions of cells that have a decreased CD57+ T cell frequency, e.g., compared to the starting cell material or biological sample. In some aspects, provided herein are methods for enriching T cells that consist of or include selecting, isolating, or removing CD57+ T cells from a biological sample, e.g., to generate an enriched population of CD57- T cells or a population depleted of CD57+ T cells. In some aspects, provided herein are methods for enriching T cells that consist of or include selecting, isolating, or removing CD57+ T cells from a biological sample, e.g., to generate an enriched population of CD57- T cells or a population depleted of CD57+ T cells.

[0116] In some aspects, provided herein are methods for stimulating a population of CD57- T cells by incubating cells of the population under stimulatory conditions. Also provided herein are methods for genetically engineering T cells by introducing a heterologous polypeptide into cells, e.g., cells of or derived from a population of enriched CD57- T cells.

[0117] In certain embodiments, existing methods for generating engineered T cells, e.g., engineered T cells expressing a chimeric antigen receptor (CAR), may include a step, stage, or phase in which a population or composition of T cells grows or expands. However, in some cases, a portion of the population or composition may not exhibit any growth or expansion, or in some cases, may expand slowly, thus requiring extra days to complete the engineering process.

[0118] In some aspects, existing methods or processes for generating or manufacturing engineered cell compositions can result in heterogeneity in the manufacturing process. In some aspects, the phenotype associated with memory T cells can affect clinical outcomes (see, e.g., Fraietta et al., Nat Med. 2018; 24(5):563-571; and Larson et al., Cancer Res. 2018; 78(13 Suppl):Abstract nr 960). In some cases, enriching cells exhibiting early memory T cell phenotypes can improve the manufacturing of engineered cell compositions (see, e.g., Singh et al., Sci Trans Med. 2016; 8(320):320ra3).

[0119] The provided methods and compositions address these problems. The provided methods and compositions relate, at least in part, to a population of cells, e.g., an enriched population of CD57- T cells, that undergo improved or more rapid proliferation and expansion, e.g., during a process for stimulating or manipulating T cells. CD57 (also known as HNK1 and LEU7) is a beta-1,3-glucuronyltransferase that may be expressed on the surface of T and NK lymphocytes. In some aspects, CD57 expression, e.g., surface expression, is associated with mature, effector-differentiated subpopulations of T and NK cells. In some aspects, CD57 expression corresponds to T cells or T cell populations that lack expression of the costimulatory receptors CD28 and CD27, which, in certain aspects, can affect sustained proliferation and cell survival. In certain aspects, CD57 expression may also identify cells with reduced or impaired proliferative capacity. In some aspects, CD57+CD28- cell populations may exhibit shorter telomere lengths and reduced proliferative capacity compared to CD57- cell populations (reviewed in Strioga, Pasukoniene, & Characiejus, Immunol. (2011)).

[0120] Certain embodiments contemplate that a reduction in the frequency of CD57+ T cells from the starting cell material used in the genetic engineering process enriches for cells with greater proliferative potential. In some aspects, negative selection of CD57+ T cells improves manufacturing success and pharmaceutical consistency by pre-enriching cells that are more poised for expansion. Furthermore, in some embodiments, enriching a cell population or composition with cells exhibiting increased proliferative potential may reduce the degree of effector cell differentiation, which should help improve the consistency of the target product profile (CD27+, CCR7+ T cells) between subjects in an autologous setting or between batches in an allogeneic setting.

[0121] In some embodiments, the method is used in connection with the process of generating or producing genetically engineered cells suitable for cell therapy in a manner that may be faster and more efficient than alternative processes. In certain embodiments, the methods provided herein have a high success rate for generating or manufacturing compositions of engineered cells from a wider range of subjects than may be possible with alternative processes. Thus, in some aspects, the speed and efficiency of the provided methods for generating engineered cells for cell therapy allow for easier planning and adjustment of cell therapy treatments, such as autologous therapy, to a wider range of subjects than may be possible with some alternative methods. In some aspects, depletion of CD57+ cells (e.g., CD57+ T cells) is believed to be advantageous, such as by improving the consistency of cell populations in downstream processes. For example, it has been observed herein that depleting CD57+ cells may deplete cells with lower or reduced proliferation capacity, and as a result, the depleted compositions exhibit improved consistency in cell proliferation rate. In this regard, improving the consistency in cell proliferation rate may improve the consistency in the duration required for a cell population to reach harvest criteria. It has additionally been observed herein that depleting CD57+ cells prior to transducing a cell population with a vector encoding a chimeric antigen receptor (CAR) can improve the consistency of CAR expression in transduced cells.

[0122] In some aspects, preselecting recipient donor cells with improved proliferation potential, for example, by depleting CD57+ T cells or screening for low amounts of CD57+ T cells, can provide improved process control for the number of cells used in the process to generate a cell therapy. In certain embodiments, CD57 expression may serve as a biomarker for cells exhibiting delayed or poor growth. Accordingly, some of the provided embodiments relate to methods that utilize one or more selection reagents or process steps to selectively remove CD57+ cells prior to the process of stimulating, genetically engineering, or expanding the cells. In some aspects, such reagents and process steps may be used in conjunction with CD8+ and CD4+ selection strategies. For example, in some embodiments, selection of CD57+ cells is employed to remove or deplete CD57+ cells from a sample, composition, or population of cells prior to any steps for CD8+ or CD4+ selection. For example, in some embodiments, selection of CD57+ T cells is employed to remove or deplete CD57+ T cells from a sample, composition, or population of cells prior to any step for CD8+ or CD4+ selection, thereby generating a CD57-depleted population. In some aspects, it may be advantageous to deplete CD57+ cells by negative selection (as opposed to positive selection), e.g., prior to any step for CD8+ or CD4+ selection. In some aspects, depleting CD57+ cells by negative selection, e.g., prior to any step for CD8+ or CD4+ selection, reduces the likelihood that the CD57-depleted population is contaminated with one or more reagents or solutions used in the CD57 selection step.

[0123] In some embodiments, CD57+ T cell selection is employed to remove or deplete CD57+ T cells from a cell sample, composition, or population after any step for CD8+ or CD4+ selection. In some aspects, such reagents and process steps may be used in conjunction with a CD3+ selection strategy. For example, in some embodiments, CD57+ T cell selection is employed to remove or deplete CD57+ T cells from a cell sample, composition, or population before any step for CD3+ selection. In some embodiments, CD57+ T cell selection is employed to remove or deplete CD57+ T cells from a cell sample, composition, or population after any step for CD3+ selection. In some embodiments, CD57+ cells are selected or removed using magnetic beads against CD57, such as in a column, which bind to CD57+ cells, and the column flow-through (unbound fraction) then contains a CD57+-depleted cell source, e.g., a population of cells enriched for CD57- T cells. In some embodiments, CD57+ T cells are selected or removed using magnetic beads against CD57, such as in a column, which bind to CD57+ T cells, and the column flow-through (unbound fraction) then contains a CD57+-depleted cell source, e.g., a population of cells enriched for CD57- T cells.

[0124] Because CD57+ T cells are less likely to proliferate, certain embodiments contemplate that some existing ex vivo T cell activation or expansion protocols may reduce the presence of CD57+ T cells after a period of time, e.g., after the first 48 hours of the process. In some aspects, CD57+ cells (e.g., CD57+ T cells) either die during such processes or their frequency decreases due to a subset of cells capable of robust expansion. However, while natural attrition of CD57+ T cells can occur during such processes, natural attrition does not control the amount of cells entering the process that exhibit high proliferation potential (e.g., CD57- cells). Thus, in some aspects, CD57+ cells (e.g., CD57+ T cells) are viable but do not proliferate significantly, so they contribute to the overall starting cell number input to the process. Thus, in some embodiments, an advantage of depleting CD57+ T cells or demonstrating a low CD57+ T cell content in a sample, composition, or population is to ensure that CD57- T cells, e.g., cells with the capacity to proliferate, do not constitute a minority population in the incoming material. Thus, in some embodiments, ensuring a low frequency of CD57+ T cells or a reduced proportion of proliferating cells can also reduce the incidence of extended processing times during the manipulation process, increased cell differentiation, and / or non-compliance with collection criteria.

[0125] In some aspects, provided embodiments are based on the observation that during a process for manipulating cells, such as a manufacturing process for generating a composition containing recombinant receptor-expressing T cells for cell therapy, a large number of cells express or upregulate markers associated with T cell stimulation or activation, including CD25 and CD69, following stimulation, such as incubation of the cells in the presence of anti-CD3 / anti-CD8 antibodies. In some aspects, only a subset of cells has been observed to enter the cell cycle, as indicated by expression of Ki-67. In some cases, Ki-67+ cells primarily comprise cells expressing CD27 and CD28, while the Ki67- population has been observed to be enriched for CD57+ cells, exhibiting a CD27-CD28- phenotype. In some aspects, CD57+ cells have been observed herein to exhibit a phenotype associated with stimulation or activation and persist throughout the early stages of the manufacturing process. In some aspects, the frequency of CD57+ T cells decreased approximately 48 hours after stimulation, which typically coincided with T cell expansion and increased survival. In some aspects, certain types of cells, e.g., CD57+ cells (e.g., CD57+ T cells), exhibit low or no expansion during stimulation or culture, while continued use of growth factors and / or activation reagents results in process and product heterogeneity.

[0126] In some aspects, when CD57+ cells (e.g., CD57+ T cells) were selected and combined in various ratios with CD57- cells (e.g., CD57+ T cells) prior to stimulation, the frequency of CD57+ cells in the cell composition (e.g., input composition) prior to stimulation was associated with a longer process duration. In some aspects, it was also observed that cell compositions, e.g., compositions containing CAR+ T cells, did not expand or proliferate when 95% or more, e.g., 100%, of the T cells in the composition were CD57+ T cells.

[0127] In some aspects, it has been observed that CD27+ T cells can contribute to cell expansion during the manufacturing process to generate engineered T cells, whereas CD57+ cells, e.g., CD57+ T cells, generally do not expand and contribute minimally to the cells in an engineered cell composition (e.g., a cell composition for administration). Thus, the presence of CD57+ T cells can affect the manufacturing process, e.g., during culture for expansion, and has also been observed to contribute to variability in the process and other cell composition attributes. In some aspects, as provided herein, selective depletion of CD57+ T cells at the beginning of a manufacturing method, e.g., prior to stimulation of the cells, can improve the consistency, quality, and potency of engineered cell compositions.

[0128] Thus, in some embodiments, the provided methods can reduce process duration for producing cell therapies, thus improving the consistency of manufacturing schedules in certain aspects. Furthermore, in some aspects, the speed and efficiency of the provided methods for producing engineered cells for cell therapy allows for easier planning and tailoring of cell therapy treatments, such as autologous therapy, to a wider subject population than may be possible with some alternative methods.

[0129] In certain embodiments, the provided methods successfully remove at least a portion of non-expanding cells at the beginning of a process to generate a population or composition of cells useful for cell therapy, e.g., engineered T cells. In some aspects, this is achieved through selecting for CD57+ cells (e.g., CD57+ T cells) before the start of such a process, or by screening to ensure that only cell compositions or populations free of or low in CD57+ T cells are used for such a process, improving, for example, the speed or frequency of successfully generating a cell population suitable for use in cell therapy. In some embodiments, the provided methods reduce the duration and number of cell doublings required, for example, during growth, culture, or expansion prior to harvest to provide the required number of T cells for use as cell therapy. Thus, without wishing to be bound by theory, some embodiments contemplate that the provided methods increase or demonstrate a sufficient number of recipient donor cells that exhibit improved expansion capabilities.

[0130] In some embodiments, cell therapies produced from enriched populations of CD57- T cells contain T cells with a lower degree of differentiation than cell therapies produced from alternative processes. In certain embodiments, reduced cell differentiation of a cell therapy improves consistency among cell therapies produced by a provided process (e.g., compared to alternative processes, e.g., cell therapies produced from populations of cells containing varying amounts of CD57+ T cells). In certain embodiments, reduced cell differentiation of a cell therapy improves the product quality profile of a cell therapy.

[0131] Certain aspects contemplate that CD57 is expressed by NK and NKT cells in addition to T cells (all of which may be present in a biological sample, e.g., leukapheresis material). Thus, in some embodiments, negative selection for CD57+ cells (e.g., CD57+ T cells) reduces residual non-T cells and improves T cell purity. Thus, the provided methods increase the purity of the T cell population being treated, such as by stimulation, transduction, or expansion, as well as the purity of the resulting cell therapy.

[0132] All publications, including patent documents, scientific literature, and databases, referenced in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall control over the definition incorporated herein by reference.

[0133] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0134] I. Enriched CD57- T cell population In certain embodiments, provided herein are populations of enriched CD57- T cells (also referred to herein as CD57- T cell populations, compositions of enriched CD57- T cells, or CD57- T cell compositions). In certain embodiments, the provided populations of enriched CD57- T cells are used in connection with methods for stimulating, activating, manipulating, transducing, culturing, or expanding T cells, e.g., T cells of an enriched population of CD57- T cells or T cells derived therefrom. In some embodiments, the enriched population of CD57- T cells results from, or is a product of, e.g., the isolation, selection, or enrichment of a biological sample, e.g., a biological sample containing one or more immune cells. In certain embodiments, the enriched population of CD57- T cells is or comprises viable T cells, CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In certain embodiments, the cells of the enriched population of CD57- T cells are or comprise viable T cells, CD3+ T cells, CD4+ T cells, and / or CD8+ T cells, or any combination thereof. In various embodiments, the cells of the enriched population of CD57- T cells are or comprise viable CD57- T cells, CD57- CD3+ T cells, CD57- CD4+ T cells, CD57- CD8+ T cells, or any combination thereof.

[0135] Certain embodiments contemplate that the amount of CD57 expression, e.g., the amount of CD57+ T cells, in a cell-containing sample, population, or composition can be measured by any suitable known means. In some embodiments, CD57 expression is measured in a sample, population, or composition to measure, assess, or determine the amount, frequency, or percentage of CD57+ cells, e.g., CD57+ T cells, in the sample, population, or composition. In certain embodiments, CD57 expression is measured in a sample, population, or composition to measure, assess, or determine the amount, frequency, or percentage of CD57+ cells, e.g., CD57+ T cells, in the sample, population, or composition.

[0136] In some embodiments, cell compositions having a higher percentage of CD57+ cells can result in a lower percentage of cells capable of reproductive expansion. In some cases, engineered cell compositions having a high percentage of CD57+ cells are associated with reduced proliferative capacity, which may result in longer process times, more doublings to achieve threshold cell numbers, increased cell differentiation, and / or failure to meet harvest criteria in manufacturing processes for producing engineered T cell compositions for cell therapy.

[0137] Also provided in some aspects are methods for identifying a population of cells capable of expansion, comprising measuring the frequency of CD57+ cells in the population, wherein the population of cells is identified as capable of expansion if the frequency of CD57+ cells is below a threshold frequency. In some of any such embodiments, the threshold frequency is a percentage that is less than or less than about 35%, 30%, 20%, 10%, 5%, 1%, or 0.1%. In some of any such embodiments, the population capable of expansion expands at least 2-fold, 4-fold, 8-fold, or 16-fold, or at least about 2-fold, 4-fold, 8-fold, or 16-fold within 4, 5, 6, 7, or 8 days of culture under conditions that promote growth or expansion.

[0138] In some aspects, methods are also provided for determining the expansion potential of a T cell population, comprising measuring a value of a trait associated with CD57 expression in the T cell population, wherein the population of T cells is determined to be capable of expansion if the value of the trait is less than or less than about a threshold value for the trait.

[0139] In some embodiments, the threshold value is: i) 25%, 20%, 15%, 10%, or 5% less than, about 25%, 20%, 15%, 10%, or 5% less than, or within 25%, 20%, 15%, 10%, or 5% less than the mean or median of measurements of a trait associated with CD57 expression across a plurality of reference T cell populations, and / or less than one standard deviation below or about the mean or median of the measurements; ii) less than the lowest measurement of a trait associated with CD57 expression across a population from a plurality of reference T cell populations, and optionally, more than 50% less than the lowest measurement. within, within 25%, within 20%, within 15%, within 10%, or within 5% less; iii) less than the mean or median value of a measure of a trait related to CD57 expression calculated from more than 65%, 75%, 80%, 85% of samples from a plurality of reference T cell compositions; wherein the plurality of reference T cell populations are populations that did not expand when cultured under conditions that promote T cell proliferation or expansion, optionally wherein the cells did not expand at least 2-fold, 4-fold, 8-fold, or 16-fold, or at least about 2-fold, 4-fold, 8-fold, or 16-fold within 4, 5, 6, 7, or 8 days of culture.

[0140] In some embodiments, the trait is the level or amount of CD57 polypeptide expressed in total T cells, CD4+ T cells, or CD8+ T cells. In some embodiments, the trait is the frequency, percentage, or amount of CD57+ T cells, CD57+CD4+ T cells, or CD57+CD8+ T cells present in a cell population. In some embodiments, the trait is the level or amount of CD57 polypeptide expressed in CD3+ T cells. In some embodiments, the trait is the frequency, percentage, or amount of CD57+CD3+ T cells present in a cell population. In some embodiments, the trait is the level or amount of CD57 mRNA present in T cells in a cell population. In some embodiments, the trait is the level or amount of chromatin accessibility of the gene encoding CD57 (B3GAT1).

[0141] In some embodiments, the method further comprises measuring a second value of a second trait associated with expression of one or more second gene products in the T cell population, wherein the population is capable of expansion if the trait value is less than or less than about a trait threshold value and the second value of the second trait is greater than or greater than about a second threshold value for the second trait.

[0142] In some embodiments, the second gene product is a marker associated with naive-like T cells. In some embodiments, the one or more second gene products are selected from CD27, CD28, CCR7, or CD45RA. In some embodiments, the one or more second gene products are CD27 and CD28.

[0143] In certain embodiments, negative expression, e.g., negative expression of CD57 or CD57-, is expression equal to or below the level of background expression, e.g., when detected using standard techniques, e.g., techniques involving antibody staining. In certain embodiments, negative expression is equal to or below the level of background expression, when detected by techniques suitable for assessing protein or gene expression, e.g., but not limited to, immunohistochemistry, immunofluorescence, or flow cytometry-based techniques. In some embodiments, for example, positive expression of a particular protein is or includes surface expression of the protein at an amount, level, or concentration above background. In certain embodiments, for example, negative expression of a particular protein is or includes surface expression of the protein at an amount, level, or concentration that is at or below background.

[0144] In certain embodiments, the methods provided herein include one or more steps of assessing, measuring, determining, and / or quantifying the expression of one or more proteins or genes (e.g., CD57) in a sample, population, or composition, e.g., to quantify cells in the sample, composition, or population that have positive or negative expression of the protein or gene (e.g., CD57). Such steps may include assessing, measuring, determining, and / or quantifying any suitable trait associated with expression, e.g., measuring the level of a protein, surface protein, mRNA, or gene accessibility, e.g., epigenetic gene accessibility.

[0145] In some embodiments, protein (e.g., CD57) expression is or includes assessing, measuring, determining, and / or quantifying the level, amount, or concentration of a protein or a gene-encoded protein expressed on the surface of a cell. In certain embodiments, protein (e.g., CD57) expression is assessed by assessing, measuring, determining, and / or quantifying the surface expression of the protein, e.g., the level, amount, or concentration of the protein on the cell surface. In certain embodiments, the amount, frequency, or percentage of cells positive for surface expression of the protein is measured, e.g., cells having a surface with a greater amount, concentration, or density of the protein on their surface that is greater than the background signal of the technique used to measure the surface protein. In certain embodiments, surface expression of a protein (e.g., CD57) is measured by immunohistochemistry, immunofluorescence, or flow cytometry-based techniques. In some embodiments, the amount, frequency, or percentage of cells positive for surface expression of the protein is determined by an appropriate known technique, such as immunohistochemistry, immunofluorescence, or flow cytometry-based techniques.

[0146] In certain embodiments, the amount, frequency, or percentage of cells that are negative or positive for protein expression, e.g., surface expression, in a sample, composition, or population is determined by flow cytometry. In some embodiments, the protein is CD3, CD4, CD8, CD25, CD27, CD28, CD57, CCR7, or CD45RA. In certain embodiments, the protein is CD57.

[0147] In certain embodiments, expression of a protein (e.g., CD57) in a sample, population, or composition is or includes any suitable method for assessing, measuring, determining, and / or quantifying the level, amount, or concentration of a protein. Such methods include, but are not limited to, detection by immunoassays, nucleic acid- or protein-based aptamer technology, HPLC (high performance liquid chromatography), peptide sequencing (e.g., Edman degradation sequencing or mass spectrometry (MS / MS), etc., optionally coupled with HPLC), and microarray adaptations of any of the foregoing (including nucleic acid, antibody, or protein-protein (i.e., non-antibody) arrays). In some embodiments, an immunoassay is or includes a method or assay that detects a protein based on an immunological reaction, for example, by detecting binding of an antibody or antigen-binding antibody fragment to a gene product. Immunoassays include, but are not limited to, quantitative immunocytochemistry or immunohistochemistry, ELISA (including direct, indirect, sandwich, competitive, multiple and portable ELISA (see, e.g., U.S. Pat. No. 7,510,687)), Western blot (including one-, two- or higher-dimensional blotting or other chromatographic means, optionally including peptide sequencing), enzyme immunoassay (EIA), RIA (radioimmunoassay), and SPR (surface plasmon resonance).

[0148] In certain embodiments, expression of a protein or its corresponding gene is measured, assessed, or quantified by measuring the mRNA (or cDNA product derived from the mRNA) encoding the protein (e.g., CD57). In certain embodiments, the amount or level of mRNA (or corresponding cDNA) is measured, assessed, or quantified by any suitable means, including reverse transcriptase (rt) PCR, droplet digital PCR, real-time, and quantitative PCR methods (PCR) (e.g., TAQMAN®, molecular beacons, LIGHTUP™, SCORPION™, SIMPLEPROBES®; see, e.g., U.S. Pat. Nos. 5,538,848; 5,925,517; 6,174,670; 6,329,144). see US Pat. Nos. 6,326,145 and 6,635,427); Northern blotting; Southern blotting, e.g., of reverse transcription products and derivatives; array-based methods, including blotted arrays, microarrays, or in situ synthesized arrays; and sequencing, e.g., sequencing-by-synthesis, pyrosequencing, dideoxy sequencing, or sequencing by ligation, or any other assay method, e.g., Scatterdure. et al., Nat. Rev. Genet. 5:335-44 (2004) or Nowrousian, Euk. Cell 9(9): 1300-1310 (2010) (including specific platforms such as HELICOS®, ROCHE® 454, ILLUMINA® / SOLEXA®, ABI SOLiD®, and POLONATOR® sequencing). In some embodiments, mRNA expression is determined by next-generation sequencing methods, such as RNA sequencing (RNA-Seq).RNA sequencing methods have been adapted to the most common DNA sequencing platforms (HiSeq system (Illumina), 454 Genome Sequencer FLX System (Roche), Applied Biosystems SOLiD (Life Technologies), IonTorrent (Life Technologies)). These platforms require the initial reverse transcription of RNA into cDNA. Conversely, the single-molecule sequencer HeliScope (Helicos BioSciences) can use RNA as a template for sequencing.

[0149] In some embodiments, the expression of protein or its corresponding gene is or comprises epigenetic analysis of protein.In some embodiments, a population of cells is evaluated for gene accessibility, for example, the accessibility of B3GAT1, which encodes CD57.Epigenetic analysis can be carried out by any suitable known means, including but not limited to, assay for transposase-accessible chromatin (ATAC-seq), which uses sequencing to examine chromatin accessibility.

[0150] In some embodiments, the enriched population of CD57- cells contains 25%, 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001% CD57+ T cells, or about 25%, 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001% CD57+ T cells. or less than or about 25%, 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%. In certain embodiments, the enriched population of CD57- cells is essentially free of CD57+ cells. In certain embodiments, the enriched population of CD57- cells is essentially free of CD57+ cells. In certain embodiments, the enriched population of CD57- cells contains less than or less than about 20% CD57+ cells. In certain embodiments, the enriched population of CD57- cells contains less than or about 10% CD57+ cells. In some embodiments, the enriched population of CD57- cells contains less than or about 5% CD57+ cells. In various embodiments, the enriched population of CD57- cells contains less than 1%, 0.1%, or 0.01%, or less than about 1%, 0.1%, or 0.01% CD57+ cells.

[0151] In certain embodiments, the enriched population of CD57- cells contains at or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% CD57- T cells. The enriched population of CD57- T cells contains at least, or at least about, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100% CD57- T cells. In certain embodiments, the enriched population of CD57- T cells contains at least, or at least about, 80% CD57- T cells. In certain embodiments, the enriched population of CD57- T cells contains at least, or at least about, 95% CD57- T cells. In various embodiments, the enriched population of CD57- T cells contains at least, or at least about, 99%, 99.9%, or 99.99% CD57- T cells. In certain embodiments, all or essentially all of the cells in the enriched population of CD57- T cells are CD57- T cells.In some embodiments, the enriched population of CD57- cells contains 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100% CD57-CD3+ T cells, or contains about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100% CD57-CD3+ T cells; At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100%. In certain embodiments, the enriched population of CD57− T cells contains at least 80% or at least about 80% CD57− CD3+ T cells. In certain embodiments, the enriched population of CD57− T cells contains at least 95% or at least about 95% CD57− CD3+ T cells. In various embodiments, the enriched population of CD57- T cells contains at least, or at least about, 99%, 99.9%, or 99.99% CD57- CD3+ T cells. In certain embodiments, all or essentially all of the cells in the enriched population of CD57- T cells are CD57- CD3+ T cells.

[0152] In certain embodiments, cells of the enriched population of CD57- T cells are or comprise viable cells. In some embodiments, cell viability is assessed using assays that may include, but are not limited to, dye uptake assays (e.g., calcein AM assay), XTT cell viability assays, and dye exclusion assays (e.g., trypan blue, eosin, or propidium dye exclusion assays). In certain embodiments, viable cells have negative expression of one or more apoptotic markers, e.g., annexin V or active caspase 3. In some embodiments, viable cells are negative for expression of one or more apoptotic markers, which may include, but are not limited to, caspases or active caspases, e.g., caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, or caspase 10, Bcl-2 family members, e.g., Bax, Bad, and Bid, annexin V, or TUNEL staining. In certain embodiments, viable cells are negative for active caspase 3. In certain embodiments, viable cells are negative for Annexin V. In certain embodiments, at least 60% or at least about 60%, at least 65% or at least about 65%, at least 70% or at least about 70%, at least 75% or at least about 75%, at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, at least 95% or at least about 95%, at least 97% or at least about 97%, at least 99% or at least about 99%, at least 99.5% or at least about 99.5%, at least 99.9% or at least about 99.9%, or at least about 100% of the cells in the enriched population of CD57− T cells are viable cells. In some embodiments, the viable cells are or comprise viable CD3+, viable CD4+, viable CD8+, viable CD57-, viable CD57- CD3+, viable CD57- CD4+, or viable CD57- CD8+ T cells, or any combination thereof. In some embodiments, the viable cells are negative for active caspase 3.In certain embodiments, viable cells are Annexin V negative.

[0153] In certain embodiments, the enriched population of CD57− cells contains 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100% CD57− CD4+ T cells, or contains about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100% CD57− CD4+ T cells; At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100%. In certain embodiments, the enriched population of CD57− T cells contains at least 80% or at least about 80% CD57− CD4+ T cells. In certain embodiments, the enriched population of CD57− T cells contains at least 90% or at least about 90% CD57− T cells. In various embodiments, the enriched population of CD57- T cells contains at least 95% or at least about 95% CD57- CD4+ T cells. In particular embodiments, all or essentially all of the cells in the enriched population of CD57- T cells are CD57- CD4+ T cells.In some embodiments, the enriched population of CD57- cells contains 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100% CD57-CD8+ T cells, or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100% CD57-CD8+ T cells; At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100%. In certain embodiments, the enriched population of CD57− T cells contains at least 80% or at least about 80% CD57− CD8+ T cells. In certain embodiments, the enriched population of CD57− T cells contains at least 90% or at least about 90% CD57− CD8+ T cells. In various embodiments, the enriched population of CD57- T cells contains at least 95% or at least about 95% CD57- CD8+ T cells. In particular embodiments, all or essentially all of the cells in the enriched population of CD57- T cells are CD57- CD8+ T cells.In some embodiments, the enriched population of CD57- cells contains 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100% CD57-CD3+ T cells, or contains about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100% CD57-CD3+ T cells; At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% or about 100%. In certain embodiments, the enriched population of CD57− T cells contains at least 80% or at least about 80% CD57− CD3+ T cells. In certain embodiments, the enriched population of CD57− T cells contains at least 90% or at least about 90% CD57− CD3+ T cells. In various embodiments, the enriched population of CD57- T cells contains at least 95% or at least about 95% CD57- CD3+ T cells. In particular embodiments, all or essentially all of the cells in the enriched population of CD57- T cells are CD57- CD3+ T cells.

[0154] In certain embodiments, the frequency of cells in the population of enriched CD57- cells are naive-like cells. In some embodiments, naive-like T cells are T cells that are positive for the expression of one or more markers that indicate that the cells are naive and / or naive-like cells. In certain embodiments, naive-like T cells are cells that are positive for the expression of markers associated with a naive or naive-like state in T cells. In certain embodiments, naive-like T cells are T cells that are negative for the expression of one or more markers that indicate that the cells are not naive and / or naive-like cells. In certain embodiments, a non-naive or non-naive-like state in T cells is characterized by, for example, an effector T (T EFF ) cells, memory T cells, central memory T cells (T CM ), Effector Memory T(T EM ) cells, and combinations thereof.

[0155] In some embodiments, the naive-like T cells are positive for the expression of at least one or more markers that indicate that the cells are naive and / or naive-like cells and / or associated with a naive or naive-like state in T cells. In some embodiments, the markers are expressed on the cell surface. In certain embodiments, the naive-like T cells are negative for the expression of at least one or more markers that indicate that the cells are non-naive and / or non-naive-like cells and / or associated with a non-naive or non-naive-like state in T cells.

[0156] Markers indicating that a T cell is naive and / or a naive-like T cell and / or associated with a naive or naive-like state in T cells include, but are not limited to, CD27, CD28, CD45RA, CD62L, and / or CCR7. In some embodiments, naive-like T cells, e.g., naive-like CD4+ and / or CD8+ T cells, are positive for the expression of CD27, CD28, CD45RA, and / or CCR7. In certain embodiments, naive-like T cells are positive for the surface expression of one or more of CD27, CD28, CD45RA, and / or CCR7. In some embodiments, naive-like T cells, e.g., naive-like CD4+ and / or CD8+ T cells, are negative for the expression of CD62L. In some embodiments, naive-like T cells, e.g., naive-like CD3+ T cells, are negative for the expression of CD62L. In some embodiments, the naive-like T cells, eg, naive-like CD3+, CD4+, and / or CD8+ T cells, are negative for expression of CD62L.

[0157] Markers that indicate that a cell is non-naive and / or a non-naive-like T cell and / or associated with a non-naive or non-naive-like state in T cells include, but are not limited to, CD25, CD45RO, CD56, KLRG1, and / or CD95. In some embodiments, naive-like T cells, e.g., naive-like CD4+ and / or CD8+ T cells, are negative for expression of CD25, CD45RO, CD56, and / or KLRG1. In certain embodiments, naive-like T cells, e.g., naive-like CD4+ and / or CD8+ T cells, have low expression of markers associated with non-naive or non-naive-like cells. In some embodiments, naive-like T cells, e.g., naive-like CD3+ T cells, are negative for expression of CD25, CD45RO, CD56, and / or KLRG1. In certain embodiments, naive-like T cells, e.g., naive-like CD3+ T cells, have low expression of markers associated with non-naive or non-naive-like cells. In certain embodiments, naive-like T cells have low expression of CD95. In certain embodiments, naive-like T cells are negative for surface expression of one or more of CD25, CD45RO, CD56, and / or KLRG1.

[0158] In some embodiments, low expression of a marker associated with non-naive or non-naive-like cells is or includes at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% lower than expression of the marker in a cell that is a non-naive-like cell and / or a non-naive-like T cell and / or positive for one or more markers indicative of the cell being non-naive and / or a non-naive-like T cell and / or associated with a non-naive or non-naive-like state in T cells. In certain embodiments, low expression of a marker associated with non-naive or non-naive-like cells is or includes expression of a marker that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% lower than expression of the marker in a cell that is a non-naive-like cell and / or a non-naive-like T cell and / or positive for one or more markers indicative of the cell being associated with a non-naive or non-naive-like state in T cells. In certain embodiments, low expression of a marker associated with non-naive or non-naive-like cells is or includes expression of a marker that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% lower than expression of the marker in a cell that is a non-naive-like cell. EFF ) cells, memory T cells, central memory T cells (T CM), and / or Effector Memory T(T EM ) is or comprises expression that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% lower than expression of the marker in the cell.

[0159] In some embodiments, markers indicating that a cell is non-naive and / or a non-naive-like T cell and / or associated with a non-naive or non-naive-like state in T cells include one or more cytokines. For example, in certain embodiments, non-naive or non-naive-like T cells are negative for expression and / or production of one or more of IL-2, IFN-γ, IL-4, and IL-10. In some embodiments, one or more cytokines are secreted. In certain embodiments, one or more cytokines are expressed internally by non-naive-like T cells, e.g., during or after treatment with an agent that prevents, inhibits, or reduces secretion.

[0160] In certain embodiments, naive-like T cells, e.g., naive-like CD57- T cells, are positive for expression, e.g., surface expression, of CD45RA and CCR7. In certain embodiments, naive-like CD4+ T cells are positive for expression, e.g., surface expression, of CD45RA and CCR7. In some embodiments, naive-like CD8+ T cells are positive for expression, e.g., surface expression, of CD45RA and CCR7. In some embodiments, naive-like CD3+ T cells are positive for expression, e.g., surface expression, of CD45RA and CCR7. In certain embodiments, naive-like T cells are positive for expression, e.g., surface expression, of CD45RA, CD27, and CCR7, and negative for expression, e.g., surface expression, of CD45RO. In certain embodiments, naive-like CD4+ T cells are positive for expression, e.g., surface expression, of CD45RA, CD27, and CCR7, and negative for expression, e.g., surface expression, of CD45RO. In some embodiments, naive-like CD8+ T cells are positive for expression, e.g., surface expression, of CD45RA, CD27, and CCR7, and negative for expression, e.g., surface expression, of CD45RO. In some embodiments, naive-like CD3+ T cells are positive for expression, e.g., surface expression, of CD45RA, CD27, and CCR7, and negative for expression, e.g., surface expression, of CD45RO.

[0161] In certain embodiments, the enriched population of CD57− T cells contains 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD25 expression, or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD25 expression, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD25 expression. In various embodiments, the enriched population of CD57- T cells contains 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD25+ T cells, or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD25+ T cells, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD25+ T cells. In various embodiments, the enriched population of CD57- cells contains 10% to 60%, 20% to 50%, or 25% to 40% or about 10% to 60%, 20% to 50%, or about 25% to 40% CD25+ T cells (inclusive). In some embodiments, the enriched population of CD57- cells contains 10% to 60%, 20% to 50%, or 25% to 40%, or about 10% to 60%, 20% to 50%, or about 25% to 40% CD57- CD25+ T cells (inclusive).

[0162] In certain embodiments, the enriched population of CD57− T cells contains 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD27 expression, or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD27 expression, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD27 expression. In various embodiments, the enriched population of CD57- T cells contains 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD27+ T cells, or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD27+ T cells, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD27+ T cells. In various embodiments, the enriched population of CD57- cells contains 10% to 60%, 20% to 50%, or 25% to 40%, or about 10% to 60%, about 20% to 50%, or about 25% to 40% CD27+ T cells (inclusive). In some embodiments, the enriched population of CD57- cells contains 10% to 60%, 20% to 50%, or 25% to 40%, or about 10% to 60%, about 20% to 50%, or about 25% to 40% CD57- CD27+ T cells (inclusive). In certain embodiments, the enriched population of CD57- T cells contains at least 25% or at least about 25% CD27+ T cells.

[0163] In certain embodiments, the enriched population of CD57− T cells contains 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD28 expression, or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD28 expression, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD28 expression. In various embodiments, the enriched population of CD57- T cells contains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD28+ T cells, or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD28+ T cells, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD28+ T cells. In certain embodiments, the enriched population of CD57- cells contains from 5% or about 5%, to 50% or about 50%, to 5% or about 5%, to 35% or about 35%, or to 10% or about 10%, to 25% or about 25% CD27+ T cells (inclusive). In some embodiments, the enriched population of CD57- cells contains 5% to 50%, 5% to 35%, or 10% to 25%, or about 5% to 50%, about 5% to 35%, or about 10% to 25% CD57- CD27+ T cells (inclusive). In certain embodiments, the enriched population of CD57- T cells contains at least 25% or at least about 25% CD27+ T cells.

[0164] In some embodiments, the enriched population of CD57- T cells contains 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CCR7 expression, or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CCR7 expression, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CCR7 expression. In certain embodiments, the enriched population of CD57- T cells contains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CCR7+ T cells, or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CCR7+ T cells, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CCR7+ T cells. In some embodiments, the enriched population of CD57- cells contains from 5% or about 5% to 50% or about 50%, 5% or about 5% to 35% or about 35%, or 10% or about 10% to 25% or about 25% CCR7+ T cells (inclusive). In specific embodiments, the enriched population of CD57- cells contains from 5% or about 5% to 50% or about 50%, 5% or about 5% to 35% or about 35%, or 10% or about 10% to 25% or about 25% CD57- CCR7+ T cells (inclusive). In some embodiments, the enriched population of CD57- T cells contains at least 25% or at least about 25% CCR7+ T cells.

[0165] In some embodiments, the enriched population of CD57− T cells contains 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD45RA expression, or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD45RA expression, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% T cells that are positive for CD45RA expression. In certain embodiments, the enriched population of CD57- T cells contains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD45RA+ T cells, or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD45RA+ T cells, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD57- CD45RA+ T cells. In some embodiments, the enriched population of CD57- cells contains from 5% or about 5% to 50% or about 50%, 5% or about 5% to 35% or about 35%, or 10% or about 10% to 25% or about 25% CD45RA+ T cells (inclusive). In specific embodiments, the enriched population of CD57- cells contains from 5% or about 5% to 50% or about 50%, 5% or about 5% to 35% or about 35%, or 10% or about 10% to 25% or about 25% CD57- CD45RA+ T cells (inclusive). In some embodiments, the enriched population of CD57- T cells contains at least 25% or at least about 25% CD45RA+ T cells.

[0166] In some embodiments, the frequency of naive-like cells in the depleted population is at least, or at least about, 10%, 20%, 30%, 40%, or 50% greater than the frequency or approximately the frequency of naive-like cells in the biological sample. In some embodiments, the frequency of one or more of CD25+ T cells, CD27+ T cells, CD28+ T cells, CCR7+ T cells, or CD45RA+ T cells in the depleted population is at least, or at least about, 10%, 20%, 30%, 40%, or 50% greater than the frequency or approximately the frequency of the respective cells in the biological sample. In some embodiments, the depleted population comprises at least, or at least about, 15%, 20%, 25%, 30%, 35%, or 40% CD27+ T cells. In some embodiments, the depleted population comprises at least, or at least about, 10%, 15%, 20%, 25%, 25%, 30%, 35%, or 40% CD28+ T cells. In some embodiments, the depleted population comprises at least, or at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, or 80% CD27+CD28+ T cells. In some embodiments, the depleted population comprises at least, or at least about, 70% or 80% CD27+CD28+ T cells. In some embodiments, the depleted population comprises at least, or at least about, 10%, 15%, 20%, or 25% CCR7+ T cells.

[0167] II. Selection of CD57- T cells and / or depletion of CD57+ T cells In some embodiments, the enriched population of CD57- T cells is obtained from a biological sample. In certain embodiments, the enriched population of CD57- T cells is selected, isolated, or enriched from a biological sample. In certain embodiments, CD57+ T cells are removed, separated, or depleted from a biological sample. In certain embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% of the CD57+ T cells are removed, separated, or depleted from the biological sample.

[0168] In certain embodiments, a subset of cells, e.g., a subset of T cells, is selected, isolated, or enriched from the biological sample prior to selecting, isolating, or enriching CD57- T cells from the biological sample. In some embodiments, a subset of cells, e.g., T cells, is selected, isolated, or enriched from an enriched population of CD57- T cells.

[0169] In some embodiments, the enriched population of CD57− T cells contains, or about, or less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.1%, 0.01%, or 0.001% of the CD57+ T cells of the biological sample, e.g., prior to selection, isolation, or enrichment. In certain embodiments, the enriched population of CD57- T cells contains 20%, about 20%, or less than 20% of the CD57+ T cells of the biological sample. In certain embodiments, the enriched population of CD57- T cells contains 5%, about 5%, or less than 5% of the CD57+ T cells of the biological sample. In some embodiments, the enriched population of CD57- T cells contains 1%, about 1%, or less than 1% of the CD57+ T cells of the biological sample, e.g., prior to selection, isolation, or enrichment. In various embodiments, the enriched population of CD57- T cells contains 0.1%, about 0.1%, or less than 0.1% of the CD57+ T cells of the biological sample. In certain embodiments, the enriched population of CD57- T cells contains 0.01%, about 0.01%, or less than 0.01% of the CD57+ T cells of the biological sample. In some embodiments, the frequency of CD57+ T cells in the depleted population is less than or about 35%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the frequency of CD57+ T cells in the biological sample. In some embodiments, the depleted population contains less than or about 3%, less than or about 2%, less than or about 1%, less than or about 0.1%, or less than or about 0.1%, or less than or about 0.01% CD57+ T cells. In some embodiments, the depleted population is free of or essentially free of CD57+ T cells.

[0170] In certain embodiments, the cells of the enriched population of CD57- T cells are less differentiated than the cells of the biological sample, e.g., prior to selection, isolation, or enrichment. In certain embodiments, the enriched population of CD57- T cells contains a higher frequency of naive-like cells than the biological sample. In certain embodiments, the enriched population of CD57+ T cells contains a higher frequency of naive-like cells than the biological sample. A population of T cells may be, for example, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, or 10 fold more than the biological sample prior to selection, isolation, or enrichment. fold more, or at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1, 2, 3, 4, 5, or 10 fold more, or at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1, 2, 3, 4, 5, or 10 fold more naive-like cells.

[0171] In some embodiments, naive-like cells include naive T cells or central memory T cells. In some embodiments, naive-like cells can include cells that are positive for or express high levels of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells. In some aspects, the cells are CD27+. In some aspects, the cells are CD28+. In some aspects, the cells are CCR7+. In certain aspects, CCR7 is expressed by naive or naive-like T cells (e.g., CCR7+CD45RA+ or CCR7+CD27+) and central memory T cells (CCR7+CD45RA-). In certain embodiments, naive-like T cells or T cells that are surface-positive for markers expressed on naive-like T cells are CCR7+CD45RA+, wherein the cells are CD27+ or CD27-. In certain embodiments, naive-like T cells or T cells that are surface-positive for markers expressed on naive-like T cells are CD27+CCR7+, wherein the cells are CD45RA+ or CD45RA-. In certain embodiments, naive-like T cells or T cells that are surface-positive for markers expressed on naive-like T cells are CD62L-CCR7+. In certain embodiments, naive-like cells include cells at an early stage of differentiation (e.g., cells that are CCR7+CD27+).

[0172] In certain embodiments, central memory T cells may include cells of various differentiation states and may be characterized by positive or high expression (e.g., surface expression) of certain cell markers and / or negative or low expression (e.g., surface expression) of other cell markers. In some aspects, less differentiated cells, e.g., central memory cells, are longer-lived and less rapidly depleted, thereby increasing persistence and durability. In some aspects, responders to cell therapy, such as CAR-T cell therapy, have increased expression of central memory genes. See, e.g., Fraietta et al. (2018) Nat Med. 24(5):563-571. In some aspects, central memory T cells are characterized by positive or high expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127. In some aspects, central memory T cells are characterized by negative or low expression of CD45RA and / or granzyme B. In certain embodiments, central memory T cells or T cells that are surface positive for markers expressed on central memory T cells are CCR7+CD45RA-.

[0173] In certain embodiments, the enriched population of CD57- T cells is about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold more abundant than the biological sample prior to selection, isolation, or enrichment, e.g., or at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1, 2, 3, 4, 5, or 10 times more CD27+ T cells. A population of T cells may be, for example, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold more abundant than the biological sample prior to selection, isolation, or enrichment. or at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, or 10 fold more CD28+ T cells.In various embodiments, the enriched population of CD57- T cells is, for example, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold more abundant than the biological sample prior to selection, isolation, or enrichment. or at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1, 2, 3, 4, 5, or 10 times more CD25+ T cells. A population of T cells may be, for example, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold more abundant than the biological sample prior to selection, isolation, or enrichment. or at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, or 10 fold more CCR7+ T cells.In certain embodiments, the enriched population of CD57- T cells is, e.g., about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold more abundant than the biological sample prior to selection, isolation, or enrichment. or at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold more CD45RA+ cells.

[0174] In certain embodiments, T cells, e.g., CD3+ T cells, are selected, isolated, or enriched from a biological sample prior to selecting, isolating, or enriching CD57− T cells from the biological sample. In some embodiments, T cells, e.g., CD3+ T cells, are selected, isolated, or enriched from an enriched population of CD57− T cells. In certain embodiments, selecting, isolating, or enriching T cells, e.g., CD3+ T cells, involves positive selection of cells from the sample.

[0175] In some embodiments, CD57+ cells are selected, isolated, or enriched from a sample, cell composition, or cell population, thereby producing isolated or selected CD57+ cells and a population of enriched CD57- cells, e.g., T cells. In certain embodiments, CD57+ cells are selected, isolated, or enriched from a biological sample, thereby producing isolated or selected CD57+ cells and a population of enriched CD57- cells, e.g., T cells. In certain embodiments, CD3+ T cells are enriched, selected, or isolated from a population of enriched CD57- cells, thereby producing a population of enriched CD57- CD3+ T cells and an unselected population of enriched CD57- cells. In certain embodiments, CD3+ T cells are enriched, selected, or isolated from an unselected population of enriched CD57- cells, thereby producing an enriched population of CD57- CD3+ T cells. In various embodiments, CD3+ T cells are enriched, selected, or isolated from the enriched population of CD57− cells, thereby generating an enriched population of CD57− CD3+ T cells and an unselected population enriched for CD57− cells, and then CD4+ or CD8+ T cells are enriched, selected, or isolated from the enriched unselected population of CD57− cells, thereby generating an enriched population of CD57− CD4+ T cells or CD57− CD8+ T cells.

[0176] In certain embodiments, a subset of T cells, e.g., CD4+ or CD8+ T cells, is selected, isolated, or enriched from a biological sample prior to selecting, isolating, or enriching CD57− T cells from the biological sample. In some embodiments, a subset of T cells, e.g., CD4+ or CD8+ T cells, is selected, isolated, or enriched from an enriched population of CD57− T cells. In certain embodiments, selecting, isolating, or enriching a subset of T cells, e.g., CD4+ or CD8+ T cells, involves positive selection of cells from the sample.

[0177] In some embodiments, CD57+ cells are selected, isolated, or enriched from a sample, cell composition, or cell population, thereby producing isolated or selected CD57+ cells and a population of enriched CD57- cells, e.g., T cells. In certain embodiments, CD57+ cells are selected, isolated, or enriched from a biological sample, thereby producing isolated or selected CD57+ cells and a population of enriched CD57- cells, e.g., T cells. In certain embodiments, CD4+ T cells are enriched, selected, or isolated from the population of enriched CD57- cells, thereby producing an enriched population of CD57- CD4+ T cells and an unselected population enriched in CD57- cells. In certain embodiments, CD8+ T cells are enriched, selected, or isolated from the population of enriched CD57- cells, thereby producing an enriched population of CD57- CD8+ T cells and an enriched population of CD57- cells. In certain embodiments, CD8+ T cells are enriched, selected, or isolated from an unselected population of enriched CD57- cells, thereby generating an enriched population of CD57-CD8+ T cells. In certain embodiments, CD4+ T cells are enriched, selected, or isolated from an unselected population of enriched CD57- cells, thereby generating an enriched population of CD57-CD4+ T cells.

[0178] In certain embodiments, CD4+ T cells are enriched, selected, or isolated from the population from the enriched CD57- cells, thereby generating an enriched population of CD57- CD4+ T cells and an unselected population enriched for CD57- cells, and then CD8+ T cells are enriched, selected, or isolated from the enriched unselected population of CD57- cells, thereby generating an enriched population of CD57-CD8+ T cells. In various embodiments, CD4+ T cells are enriched, selected, or isolated from the enriched population of CD57- cells, thereby generating an enriched population of CD57- CD4+ T cells and an unselected population enriched for CD57- cells, and then CD8+ T cells are enriched, selected, or isolated from the enriched unselected population of CD57- cells, thereby generating an enriched population of CD57-CD8+ T cells.

[0179] In certain embodiments, (1) CD4+ T cells are enriched, selected, or isolated from the biological sample, thereby generating an enriched population of CD4+ T cells and an enriched unselected population of CD4- cells; (2) CD8+ T cells are enriched, selected, or isolated from the enriched unselected population of CD4- cells, thereby generating an enriched population of CD8+ T cells; and (3) CD57+ T cells are depleted from the enriched CD4+ and CD8+ T cell populations, thereby generating enriched populations of CD57-CD4+ and CD57-CD8+ T cells. In certain embodiments, (1) CD8+ T cells are enriched, selected, or isolated from the biological sample, thereby generating an enriched population of CD8+ T cells and an unselected population enriched in CD8− cells; (2) CD4+ T cells are enriched, selected, or isolated from the enriched unselected population of CD4− cells, thereby generating an enriched population of CD4+ T cells; and (3) CD57+ T cells are depleted from the enriched CD4+ and CD8+ T cell populations, thereby generating enriched populations of CD57−CD4+ and CD57−CD8+ T cells.

[0180] In certain embodiments, CD4+ T cells are enriched, selected, or isolated from the biological sample, thereby generating an enriched population of CD4+ T cells, and then CD57+ cells are removed from the enriched population of CD4+ T cells, thereby generating an enriched population of CD57-CD4+ T cells. In certain embodiments, CD8+ T cells are enriched, selected, or isolated from the biological sample, thereby generating an enriched population of CD8+ T cells, and then CD57+ cells are removed from the enriched population of CD8+ T cells, thereby generating an enriched population of CD57-CD8+ T cells.

[0181] In some embodiments, one or more populations of enriched CD57- T cells are frozen, e.g., cryopreserved and / or cryoprotected, after isolation, selection, and / or enrichment. In certain embodiments, an enriched population of CD57- CD4+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, after isolation, selection, and / or enrichment. In certain embodiments, an enriched population of CD57- CD8+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, after isolation, selection, and / or enrichment. In certain embodiments, an enriched population of CD57- CD3+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, after isolation, selection, and / or enrichment. In some embodiments, one or more populations of enriched T cells are frozen, e.g., cryopreserved and / or cryoprotected, before any step of incubating, activating, stimulating, manipulating, transducing, transfecting, culturing, expanding, harvesting, and / or formulating the population of cells. In certain embodiments, the enriched population of CD57-CD4+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, prior to any step of incubating, activating, stimulating, manipulating, transducing, transfecting, culturing, expanding, harvesting, and / or formulating the population of cells. In some embodiments, the enriched population of CD57-CD8+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, prior to any step of incubating, activating, stimulating, manipulating, transducing, transfecting, culturing, expanding, harvesting, and / or formulating the population of cells. In some embodiments, the enriched population of CD57-CD3+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, prior to any step of incubating, activating, stimulating, manipulating, transducing, transfecting, culturing, expanding, harvesting, and / or formulating the population of cells. In certain embodiments, the one or more cryoprotected input compositions are stored, e.g., at or about -80°C, for 12 hours to 7 days, 24 hours to 120 hours, or 2 days to 5 days.In certain embodiments, the one or more cryoprotected input compositions are stored at or about −80° C. for a period of less than 10, 9, 8, 7, 6, or 5, 4, 3, 2, or 1 day at or about −80° C. In some embodiments, the one or more cryoprotected input compositions are stored at or about −70° C., or at or about −80° C. for less than 3 days, e.g., about 2 days.

[0182] In some embodiments, "depleting" or "removing," when referring to one or more particular cell types or cell populations, refers to reducing the number or percentage of a cell type or population, e.g., compared to the total number of cells in a composition or volume thereof, or compared to other cell types, e.g., by negative selection based on a marker expressed by the population or cells, or by positive selection based on a marker not present in the cell population or cells to be depleted. Generally, the terms depleting or removing do not require the complete removal of the cells, cell type, or population from the composition.

[0183] In some embodiments, "enriching," when referring to one or more particular cell types or cell populations, refers to increasing the number or percentage of a cell type or population, e.g., compared to the total number of cells in a composition or volume thereof, or compared to other cell types, for example, by positive selection based on a marker expressed by the population or cells, or by negative selection based on a marker not present in the cell population or cells to be depleted. Generally, the term enriching does not require the complete removal of other cells, cell types, or populations from the composition, nor does it require that the cells so enriched be present at 100% or even close to 100% in the enriched composition.

[0184] In some aspects, cell populations or cell compositions obtained from a subject, e.g., a human subject, for cell therapy, e.g., adoptive cell therapy, may exhibit low or slow growth, such that they do not reach a threshold (e.g., no growth) for harvesting cells to generate a therapeutic composition (e.g., harvest criteria), or do not reach a threshold (e.g., slow growth) for harvesting cells to generate a therapeutic composition within a certain period of time. In some aspects, some of such cell populations can contain a high frequency of CD57+ cells, e.g., a frequency of CD57+ cells greater than a threshold. In other aspects, cell populations or cell compositions obtained from a subject, e.g., a human subject, for cell therapy, e.g., adoptive cell therapy, can exhibit improved growth compared to populations exhibiting no growth or slow growth. In some aspects, such cell populations or cell compositions can contain a low frequency of CD57+ cells, e.g., a frequency of CD57+ cells below a threshold. In some aspects, cell populations or cell compositions exhibiting improved growth can exhibit phenotypes or express markers associated with naive-like or central memory-like phenotypes, such as CD27+, CD28+, and / or CCR7+. In some embodiments, provided methods are based on the observation that there is variability or heterogeneity in CD57+ T cell expression among T cells in biological samples (e.g., leukapheresis or apheresis samples) from human subjects, which, in some aspects, can result in variability in the phenotype and function of engineered T cell compositions produced for use in adoptive cell therapy from multiple different subjects, even using the same manufacturing process. In certain embodiments, provided methods control or reduce such variability by selecting, isolating, or enriching CD57− T cells from the biological sample, e.g., by removing, separating, or depleting CD57+ T cells from the biological sample. Such cells can then be used in processes to engineer or manufacture cells for cell therapy, minimizing variability between products while also improving certain attributes and characteristics of the product, such as its ability to expand and persist when administered to a subject.

[0185] A. Sample and Cell Preparation In certain embodiments, the provided methods are used in connection with isolating, selecting, or enriching cells from a biological sample to generate one or more populations of enriched cells, e.g., CD57- T cells. In some embodiments, the provided methods involve isolating cells or populations thereof from a biological sample, e.g., a biological sample obtained or derived from a subject, such as a subject with a particular disease or condition, in need of, or to whom cell therapy is administered. In some aspects, the subject is a human, e.g., a patient in need of a particular therapeutic intervention, such as adoptive cell therapy, for which cells are isolated, processed, and / or manipulated. Thus, the cells, in some embodiments, are primary cells, e.g., primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject. Biological samples can be samples obtained directly from a biological source or samples that are processed. Biological samples include, but are not limited to, body fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue, and organ samples, as well as processed samples derived therefrom.

[0186] In some aspects, the sample is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy. In some embodiments, the sample is or includes a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.

[0187] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some aspects, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects, contains cells other than red blood cells and platelets.

[0188] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the wash step is accomplished by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the wash step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are washed after washing, e.g., by filtration of Ca. ++ / Mg ++ In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0189] In some embodiments, the cell-containing sample (e.g., an apheresis product or leukapheresis product) is washed to remove one or more anticoagulants, such as heparin, that were added during apheresis or leukapheresis.

[0190] In some embodiments, a cell-containing sample (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product) is cryopreserved and / or cryoprotected (e.g., frozen), and then thawed and optionally washed prior to any steps for isolating, selecting, activating, stimulating, manipulating, transducing, transfecting, incubating, culturing, harvesting, formulating a population of cells, and / or administering the formulated cell population to a subject.

[0191] In some embodiments, a sample containing autologous peripheral blood mononuclear cells (PBMCs) from a subject is collected in a manner suitable for ensuring adequate manufacturing quality. In one aspect, the sample containing PBMCs is derived from fractionated whole blood. In some embodiments, whole blood from a subject is fractionated by leukapheresis using centrifugal force to exploit density differences between cell phenotypes, whereby autologous mononuclear cells (MNCs) are preferentially enriched while other cell phenotypes, such as red blood cells, are reduced in the collected cell composition. In some embodiments, autologous plasma is collected simultaneously during MNC collection, which in some aspects can enable extended stability of the leukapheresis product. In one aspect, autologous plasma is added to the leukapheresis product to improve the buffering capacity of the leukapheresis product matrix. In some aspects, the total volume of whole blood processed to generate the leukapheresis product is or is about 2 L, 4 L, 6 L, 8 L, 10 L, 12 L, 14 L, 16 L, 18 L, or 20 L, or any value between any of the foregoing. In some embodiments, the volume of collected autologous plasma is or is about 10 mL, 50 mL, 100 mL, 150 mL, 200 mL, 250 mL, or 300 mL or more, or any value between any of the foregoing. In some embodiments, the leukapheresis product is subjected to procedures, e.g., washing and formulation for in-process cryopreservation, within about 48 hours of completing the leukapheresis collection. In some embodiments, the leukapheresis product is subjected to one or more washing steps, e.g., within about 2, 6, 12, 18, 24, 36, or 48 hours of completing the leukapheresis collection. In some aspects, the one or more washing steps remove anticoagulant during the leukapheresis collection, cellular waste products that may accumulate in the leukapheresis product, residual platelets, and / or cellular debris. In some embodiments, one or more buffer exchanges are performed between the one or more washing steps.

[0192] In certain embodiments, the apheresis product or leukapheresis product is cryopreserved and / or cryoprotected (e.g., frozen), as described below, and then thawed before being subjected to a cell enrichment, selection, or isolation step (e.g., a T cell selection or isolation step). In some embodiments, after the cryopreserved and / or cryoprotected apheresis product or leukapheresis product is subjected to a T cell selection or isolation step, no additional cryopreservation and / or cryoprotection step is performed during or between any subsequent steps, such as activating, stimulating, manipulating, transducing, transfecting, incubating, culturing, harvesting, formulating, and / or administering the formulated cell population to a subject. For example, T cells selected from a thawed cryopreserved and / or cryoprotected apheresis product or leukapheresis product are not again cryopreserved and / or cryoprotected before being thawed and optionally washed for downstream processes, such as T cell activation / stimulation or transduction.

[0193] In certain embodiments, the apheresis product or leukapheresis product is frozen in a cryopreservation solution or buffer containing 5×10 6 cells / mL, 10 x 10 6 cells / mL, 20 x 10 6 cells / mL, 30 x 10 6 cells / mL, 40 x 10 6 cells / mL, 50 x 10 6 cells / mL, 60 x 10 6 cells / mL, 70 x 10 6 cells / mL, 80 x 10 6 cells / mL, 90 x 10 6 cells / mL, 100 x 10 6 cells / mL, 110 x 10 6 cells / mL, 120 x 10 6 cells / mL, 130 x 10 6 cells / mL, 140 x 10 6 cells / mL or 150 x 10 6cells / mL, approximately 5 x 10 6 cells / mL, 10 x 10 6 cells / mL, 20 x 10 6 cells / mL, 30 x 10 6 cells / mL, 40 x 10 6 cells / mL, 50 x 10 6 cells / mL, 60 x 10 6 cells / mL, 70 x 10 6 cells / mL, 80 x 10 6 cells / mL, 90 x 10 6 cells / mL, 100 x 10 6 cells / mL, 110 x 10 6 cells / mL, 120 x 10 6 cells / mL, 130 x 10 6 cells / mL, 140 x 10 6 cells / mL or 150 x 10 6 cells / mL, or at least 5 x 10 6 cells / mL, 10 x 10 6 cells / mL, 20 x 10 6 cells / mL, 30 x 10 6 cells / mL, 40 x 10 6 cells / mL, 50 x 10 6 cells / mL, 60 x 10 6 cells / mL, 70 x 10 6 cells / mL, 80 x 10 6 cells / mL, 90 x 10 6 cells / mL, 100 x 10 6 cells / mL, 110 x 10 6 cells / mL, 120 x 10 6 cells / mL, 130 x 10 6 cells / mL, 140 x 10 6 cells / mL or 150 x 10 6 The cells are cryopreserved and / or cryoprotected (e.g., frozen) at a density of 10 ...

[0194] In certain embodiments, the cryopreserved and / or cryoprotected apheresis or leukapheresis product is banked (e.g., without T cell selection prior to freezing the sample), which, in some aspects, can provide more flexibility in subsequent manufacturing steps. In some aspects, the cryopreserved and / or cryoprotected apheresis or leukapheresis product is divided into multiple cryopreservation containers (e.g., bags) that can each be used individually or in combination in processing the product. For example, when the total number of viable cells in the apheresis or leukapheresis product is 15×10 9 When the total number of viable cells in the apheresis product or leukapheresis product is less than 15-30 x 10, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product is divided into four cryopreservation containers (e.g., bags). In some embodiments, the total number of viable cells in the apheresis product or leukapheresis product is between 15-30 x 10. 9 When the total number of cells is eight, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product is divided into eight cryopreservation containers (e.g., bags).

[0195] In one aspect, banking cells prior to selection can increase cell yield for downstream processes, and banking cells earlier can mean that they are healthier and may be easier to meet manufacturing success criteria. In another aspect, once thawed, the cryopreserved and / or cryoprotected apheresis or leukapheresis product can be subjected to one or more different selection methods. The advantage of this approach is, among other things, enhancing the availability, efficacy, and / or other aspects of cells in cell therapy for the treatment of a disease or condition of interest, such as in the sample donor and / or another recipient.

[0196] In some embodiments, the sample (e.g., an apheresis or leukapheresis sample) is collected after the donor has been diagnosed with a disease or condition, either before cell selection or without prior cell selection (e.g., without prior T cell selection, such as chromatographic selection), and cryopreserved and / or cryoprotected. In some aspects, the time of cryopreservation is also before the donor receives one or more of any initial treatment for the disease or condition, any targeted therapy or any therapy designated for treatment of the disease or condition, or any therapy other than radiation and / or chemotherapy. In some embodiments, the sample is collected after the first recurrence of the disease after initial treatment for the disease and before the donor or subject receives subsequent treatment for the disease. The initial treatment and / or subsequent treatment can be a therapy other than cell therapy. In some embodiments, the collected cells may be used for cell therapy after the initial treatment and / or subsequent treatment. In one aspect, cryopreserved and / or cryoprotected samples without prior cell selection can help reduce initial costs, such as those associated with untreated patients in randomized clinical trials who may cross over and later require treatment.

[0197] In some embodiments, the sample (e.g., an apheresis or leukapheresis sample) is collected, cryopreserved, and / or cryoprotected, either before or without prior cell selection (e.g., without prior T cell selection, such as by chromatographic selection), at a time point after second-line treatment for the disease and after a second recurrence of the disease before the donor or subject receives subsequent treatment for the disease. In some embodiments, the patient is identified as likely to relapse after second-line treatment, for example, by assessing certain risk factors. In some embodiments, the risk factors are based on disease type and / or genetic traits, such as double-hit lymphoma, primary refractory cancer, or activated B-cell lymphoma. In some embodiments, the risk factors are based on clinical features, such as early recurrence after first-line treatment or other poor prognostic indicators after treatment (e.g., an IPI (International Prognostic Index) > 2).

[0198] In some embodiments, a sample (e.g., an apheresis or leukapheresis sample) is collected and cryopreserved and / or cryoprotected before cell selection or without prior cell selection (e.g., without prior T cell selection, such as chromatographic selection) at a time before the donor or subject is diagnosed with any disease. In some aspects, the donor or subject may be determined to be at risk of developing any disease. In some aspects, the donor or subject may be a healthy subject. In certain cases, the donor or subject may never be considered at risk of developing or being diagnosed with any disease, but may choose to bank or preserve the cells in case cell therapy is needed at a later stage in life. In some embodiments, the donor or subject may be considered at risk of developing any disease based on factors such as gene mutations, gene abnormalities, gene disruptions, family history, protein abnormalities (such as defects in protein production and / or processing), and lifestyle choices that may increase the risk of developing any disease. In some embodiments, the cells are collected as a prophylactic.

[0199] In some embodiments, cryopreserved and / or cryoprotected samples of cells (e.g., apheresis or leukapheresis samples), e.g., samples of cells that have not been subjected to prior cell selection (e.g., without prior T cell selection, such as chromatographic selection), are stored or banked for a period of more than 12 hours, more than 24 hours, more than 36 hours, or more than 48 hours, or a period equal to 12, 24, 36, or 48 hours, or more than 0.5, 1, 1.5, or 2 days, or a period equal to 0.5, 1, 1.5, or 2 days. In some embodiments, samples are stored or banked for a period of more than 1 week, more than 2 weeks, more than 3 weeks, or more than 4 weeks, or a period equal to 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, samples are stored or banked long-term. In some aspects, the sample is greater than 1 month, greater than 2 months, greater than 3 months, greater than 4 months, greater than 5 months, greater than 6 months, greater than 7 months, greater than 8 months, greater than 9 months, greater than 10 months, greater than 11 months, greater than 1 year, greater than 2 years, greater than 3 years, greater than 4 years, greater than 5 years, greater than 6 years, greater than 7 years, greater than 8 years, greater than 9 years, greater than 10 years, greater than 11 years, greater than 12 years, greater than 13 years, greater than 14 years, greater than 15 years, greater than 16 years, greater than 17 years, greater than 18 years, greater than 19 years, greater than 20 years, greater than 25 years, greater than 30 years, greater than 35 years, greater than 36 years, greater than 37 years, greater than 38 years, greater than 39 years, greater than 40 years, greater than 41 years, greater than 42 years, greater than 43 years, greater than 44 years, greater than 45 years, greater than 46 years, greater than 47 years, greater than 48 years, greater than 49 years, greater than 50 years, greater than 51 years, greater than 52 years, greater than 53 years, greater than 54 years, greater than 55 years, greater than 56 years, greater than 57 years, greater than 58 years, greater than 59 years, greater than 60 years, greater than 61 years, greater than 62 years, greater than 63 years, greater than 64 years, greater than 65 years, greater than 66 years, greater than 67 years, greater than 68 years, greater than 69 years, greater than 70 years, greater than 71 years, greater than 72 years, greater than 73 years, greater than 74 years, greater than 75 years, greater than 7 years, greater than 40 years or more, or a period equal to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years or more.

[0200] In some embodiments, apheresis or leukapheresis samples collected from donors are transported to a storage or processing facility in a refrigerated environment and / or cryogenically stored at a storage facility or processed at a processing facility. In some embodiments, the sample is processed prior to transport by, for example, selecting T cells, such as CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, such processing is performed after transport and before cryogenic storage of the sample. In some embodiments, processing is performed after thawing the sample following cryogenic storage.

[0201] By allowing a donor, and thus their cells, to store their cells at a stage when they have not received extensive treatment for a disease and / or before the onset or diagnosis of a disease or condition, such cells may have certain advantages for use in cell therapy compared to cells collected after one or more treatments. For example, cells collected before one or more treatments may be healthier, may exhibit higher levels of specific cellular activity, may proliferate more rapidly, and / or may be more amenable to genetic manipulation than cells that have undergone several treatments. Another example of an advantage of the embodiments described herein may include convenience. For example, by collecting, optionally processing, and storing a donor's cells before they are needed for cell therapy, the cells are readily available if a recipient later needs them. This increases the capacity of apheresis laboratories and provides technicians with greater flexibility in scheduling the apheresis collection process.

[0202] Exemplary methods and systems for cryogenically storing and processing cells from a sample, such as an apheresis sample, may include those described in WO2018170188. In some embodiments, the methods and systems involve collecting an apheresis sample before a patient requires cell therapy, then cryopreserving the apheresis sample for later use in a process to engineer cells (e.g., T cells) with a recombinant receptor (e.g., a CAR). In some cases, such a process may include a process described herein. In some embodiments, an apheresis sample is collected from a subject and cryopreserved prior to subsequent T cell selection, activation, stimulation, manipulation, transduction, transfection, incubation, culture, harvesting, formulation, and / or administration of a formulated cell population to the subject. In such examples, the cryopreserved apheresis sample is thawed before subjecting the sample to one or more selection steps, such as any described herein.

[0203] In some embodiments, a cryopreserved and / or cryoprotected sample of cells (e.g., an apheresis or leukapheresis sample), e.g., a sample of cells that has not been subjected to prior cell selection (e.g., without prior T cell selection, such as chromatographic selection), is thawed prior to use in downstream processes to manufacture a cell population for cell therapy, e.g., a T cell population containing CAR+ T cells. In some embodiments, such a cryopreserved and / or cryoprotected sample of cells (e.g., an apheresis or leukapheresis sample) is used in connection with the processes provided herein for engineered T cell therapy, such as CAR+ T cell therapy. In certain examples, no further step of cryopreservation is performed prior to or during the harvesting / formulation step.

[0204] In some embodiments, a cryopreserved and / or cryoprotected apheresis product or leukapheresis product is thawed. In some embodiments, the thawed cell composition is subjected to dilution (e.g., with serum-free medium) and / or washing (e.g., with serum-free medium), which can optionally remove or reduce unwanted or undesirable components. In some cases, the dilution and / or washing removes or reduces the presence of cryoprotectants, such as DMSO, contained in the thawed sample, which can otherwise negatively affect cell viability, yield, and recovery rate upon prolonged exposure to room temperature. In some embodiments, the dilution and / or washing allows for medium exchange of the thawed cryopreserved product with serum-free medium, as described in PCT / US2018 / 064627, which is incorporated herein by reference.

[0205] In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more supplements (e.g., OpTmizer™ T-Cell Expansion Basal Medium (ThermoFisher)). In some embodiments, the one or more supplements are serum-free. In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more additional components for the maintenance, expansion, and / or activation of cells (e.g., T cells), e.g., provided by additional supplements (e.g., OpTmizer™ T-Cell Expansion Supplement (ThermoFisher)). In some embodiments, the serum-free medium further comprises a serum replacement supplement, e.g., an immune cell serum replacement, e.g., CTS™ Immune Cell Serum Replacement (ThermoFisher, #A2596101), or an immune cell serum replacement as described in Smith et al. Clin Transl Immunology. 2015 Jan;4(1):e31. In some embodiments, the serum-free medium further comprises a free form of an amino acid, such as L-glutamine. In some embodiments, the serum-free medium further comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), e.g., the dipeptide in Glutamax™ (ThermoFisher). In some embodiments, the serum-free medium further comprises one or more recombinant cytokines, e.g., recombinant human IL-2, recombinant human IL-7, and / or recombinant human IL-15.

[0206] B. T cell selection In some embodiments, the selection, isolation, or enrichment of cells, e.g., CD57+ or CD57- cells (e.g., CD57+ T cells), involves one or more preparative and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove unwanted components, enrich for desired components, or lyse or remove cells sensitive to a particular reagent. In some examples, cells are separated based on one or more properties, e.g., density, adhesiveness, size, sensitivity to, and / or resistance to, a particular component. In some embodiments, the method involves density-based cell separation, e.g., preparation of white blood cells from peripheral blood by lysis of red blood cells and centrifugation through a Percoll or Ficoll gradient. In certain embodiments, methods, techniques, and reagents for selection, isolation, and enrichment are described, e.g., in PCT Publication Nos. WO 2013124474 and WO 2015164675, which are incorporated herein by reference in their entireties.

[0207] In certain embodiments, CD57- cells are isolated, enriched, or selected by a process or procedure that includes one or more selection steps. In some embodiments, the one or more selection steps are or include negative selection. In certain embodiments, CD57- cells are isolated, enriched, or selected by separating or removing CD57+ cells. In certain embodiments, a cell population enriched in CD57- cells is obtained as a result of negative selection of CD57+ cells from a population.

[0208] In certain embodiments, a bivalent antibody to bind CD57+ cells to large density cells or beads. This technology has been used most notably with red blood cells (e.g., RosetteSep™ STEMCELL Technologies) or any other similar or suitable technology to bind target cells, e.g., CD57+ T cells, to a density gradient for removal.

[0209] In some embodiments, at least a portion of the selection step involves incubation of cells with a selection reagent. The incubation with the selection reagent as part of a selection method can be performed using one or more selection reagents for the selection of one or more different cell types based on the expression or presence of one or more specific molecules, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, in or on the cells. In certain embodiments, such surface proteins can include CD57, CD4, or CD8. In certain embodiments, such surface proteins can include CD3. In some embodiments, any known method using a selection reagent for separation based on such markers can be used. In some embodiments, the selection reagent results in a separation that is an affinity or immunoaffinity-based separation. For example, in some aspects, the selection involves incubation with a reagent for separating cells and cell populations based on the cellular expression or expression level of one or more markers, usually cell surface markers, e.g., incubation with an antibody or binding partner that specifically binds to such marker, followed generally by a washing step and separation of cells that have the antibody or binding partner bound from cells that do not have the antibody or binding partner bound. In some embodiments, the reagent for cell separation is or comprises an antibody or antigen-binding fragment thereof that binds to or recognizes CD4, CD8, or CD57. In some embodiments, the reagent for cell separation is or comprises an antibody or antigen-binding fragment thereof that binds to or recognizes CD3.

[0210] In some aspects of such processes, a volume of cells is mixed with a certain amount of a desired affinity-based selection reagent. Immunoaffinity-based selection can be performed using any system or method that results in favorable energetic interactions between the cells to be separated and a marker on the cells, e.g., an antibody or other binding partner on a solid surface, e.g., a molecule that specifically binds to a particle. In some embodiments, the method is performed using particles such as beads, e.g., magnetic beads, coated with a selection agent (e.g., an antibody) specific to a marker on the cells. The particles (e.g., beads) can be incubated or mixed with the cells in a container, such as a tube or bag, with shaking or mixing, at a certain cell density to particle (e.g., bead) ratio to promote energetically favorable interactions. In other cases, the method involves cell selection, where all or part of the selection is performed within the internal cavity of a centrifuge chamber, e.g., under centrifugal rotation. In some embodiments, incubation of the cells with the selection reagent, e.g., an immunoaffinity-based selection reagent, is performed within the centrifuge chamber. In certain embodiments, the isolation or separation is performed using a system, device, or apparatus described in International Patent Application Publication No. WO2009 / 072003 or US20110003380A1. In one example, the system is the system described in International Publication No. WO2016 / 073602.

[0211] In some embodiments, performing such a selection step, or portions thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads), within the cavity of a centrifuge chamber allows the user to control certain parameters, such as the volume of various solutions, the addition of solutions during processing, and their timing, which can provide advantages over other available methods. For example, the ability to reduce the volume of liquid within the cavity during incubation allows the concentration of particles (e.g., bead reagents) used for selection, and therefore the chemical potential of the solution, to be increased without affecting the total number of cells within the cavity. This can enhance pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, performing an incubation step within the chamber, for example, when associated with the systems, circuits, and controls described herein, allows the user to agitate the solution at desired times during incubation, which can also improve interactions.

[0212] In some embodiments, at least a portion of the selection step, including incubation of cells with selection reagents, is performed in a centrifuge chamber. In some aspects of such processes, a volume of cells is mixed with a selection reagent based on the desired affinity in an amount that is significantly less than the amount typically used when performing a similar selection in a tube or container to select the same number and / or volume of cells according to the manufacturer's instructions. In some embodiments, an amount of one or more selection reagents is used that is 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 50% or less, 60% or less, 70% or less, or 80% or less of the amount of the same selection reagent used to select cells in a tube or container-based incubation according to the manufacturer's instructions for the same number and / or volume of cells.

[0213] In some embodiments, for selection, e.g., selection based on the immunoaffinity of cells, cells are incubated in the cavity of the chamber in a population that also contains a selection buffer containing a selection reagent, such as a molecule that specifically binds to a surface marker on the cells desired to be enriched and / or depleted but not to surface markers on other cells in the population, e.g., an antibody optionally bound to a scaffold such as a polymer or surface, e.g., beads, e.g., magnetic beads, e.g., magnetic beads bound to monoclonal antibodies specific for CD4 and CD8. In some embodiments, for selection of cells, e.g., immunoaffinity-based selection, cells are incubated in the cavity of the chamber in a population that also contains a selection buffer with a selection reagent, e.g., a molecule that specifically binds to a surface marker on the cells desired to be enriched and / or depleted but not to surface markers on other cells in the population, e.g., an antibody optionally bound to a scaffold such as a polymer or surface, e.g., beads, e.g., magnetic beads, e.g., magnetic beads bound to monoclonal antibodies specific for CD3. In some embodiments, the selection reagent is added to the cells in the cavity of the chamber in a substantially lesser amount (e.g., 5% or less, 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, or 80% or less) than would normally be used or required to achieve approximately the same or similar efficiency of selecting the same number of cells or the same volume of cells when selection is performed in a tube with shaking or rotation as described.In some embodiments, incubation is performed by adding a selection buffer to the cells and selection reagent, e.g., 10 mL to 200 mL, e.g., at least 10 mL, at least 20 mL, at least 30 mL, at least 40 mL, at least 50 mL, at least 60 mL, at least 70 mL, at least 80 mL, at least 90 mL, at least 100 mL, at least 150 mL, or at least 200 mL, or at least about 10 mL, at least about 20 mL, at least about 30 mL, at least about 40 mL, at least about 50 mL, or less. The target volume is achieved by incubation of at least about 60 mL, at least about 70 mL, at least about 80 mL, at least about 90 mL, at least about 100 mL, at least about 150 mL, or at least about 200 mL, or about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, about 100 mL, about 150 mL, or about 200 mL, or 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL of reagent. In some embodiments, the selection buffer and selection reagent are premixed before addition to the cells. In some embodiments, the selection buffer and selection reagent are added to the cells separately. In some embodiments, the selection incubation is performed under cyclic gentle mixing conditions that help promote energetically favorable interactions, which may allow for the use of relatively less overall selection reagent while achieving high selection efficiency.

[0214] In some embodiments, the total duration of incubation with the selection reagent is between 5 minutes and 6 hours, or about 5 minutes and 6 hours, e.g., between 30 minutes and 3 hours, e.g., at least 30 minutes, at least 60 minutes, at least 120 minutes, or at least 180 minutes, or at least about 30 minutes, at least about 60 minutes, at least about 120 minutes, or at least about 180 minutes.

[0215] In some embodiments, incubation is generally under mixing conditions, such as in the presence of a spin, generally at a relatively low force or speed, such as a speed slower than that used to pellet the cells, e.g., at or about 600 rpm to 1700 rpm (e.g., 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm, or about 600 rpm, about 1000 rpm, or about 1500 rpm or about 1700 rpm, or at least 60 80 g, at least 1000 rpm, or at least 1500 rpm or at least 1700 rpm), e.g., 80 g to 100 g or about 80 g to about 100 g (e.g., 80 g, 85 g, 90 g, 95 g, or 100 g, or about 80 g, about 85 g, about 90 g, about 95 g, or about 100 g, or at least 80 g, at least 85 g, at least 90 g, at least 95 g, or at least 100 g) of sample or RCF at the wall of a chamber or other container. In some embodiments, spinning is performed using repeated intermittents of spinning at such low speeds followed by rest periods, e.g., 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, or 10 seconds of spin and / or rest, e.g., about 1 second or 2 seconds of spin followed by about 5 seconds, about 6 seconds, about 7 seconds, or about 8 seconds of rest.

[0216] In some embodiments, such a process is performed within a completely closed system in which the chamber is integrated. In some embodiments, this process (and in some aspects also one or more additional steps, such as a preceding washing step to wash a cell-containing sample, such as an apheresis sample) is performed automatically using an automated program to draw, push, and centrifuge cells, reagents, and other components into the chamber at the appropriate times to complete the washing and binding steps in a single closed system.

[0217] In some embodiments, after incubation and / or mixing of the cells and the selection reagent(s), the incubated cells are separated to select cells based on the presence or absence of a particular reagent(s). In some embodiments, the separation is performed in the same closed system in which the cells were incubated with the selection reagent. In some embodiments, after incubation with the selection reagent, the incubated cells, including the cells to which the selection reagent has bound, are transferred to a system for immunoaffinity-based separation of the cells. In some embodiments, the system for immunoaffinity-based separation is or includes a magnetic separation column.

[0218] Such separation steps can be based on positive selection, in which cells that bind to a reagent, such as an antibody or binding partner, are retained for subsequent use, and / or negative selection, in which cells that do not bind to a reagent, such as an antibody or binding partner, are retained. In some instances, both fractions are retained for subsequent use. In some aspects, negative selection, which specifically identifies cell types within a heterogeneous population, can be particularly useful when antibodies are not available, such that separation is best performed based on markers expressed by cells other than the desired population.

[0219] In some embodiments, the process steps further comprise negative and / or positive selection of the incubated cells, e.g., using a system or device capable of affinity-based selection. In some embodiments, isolation is achieved by enrichment of a particular cell population by positive selection, or depletion of a particular cell population by negative selection. In some embodiments, positive or negative selection is achieved by detecting a specific or relatively high level of a marker (e.g., a marker) expressed in the positively or negatively selected cells, respectively. high This is achieved by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers expressed (marker+) on the surface of the cells.

[0220] Separation does not necessarily result in the enrichment or elimination of a particular cell population, or 100% of cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, may indicate an increase in the number or proportion of such cells, but need not result in the complete absence of cells that do not express the marker. Similarly, negative selection, elimination, or depletion of a particular type of cell, such as cells expressing a marker, may indicate a decrease in the number or proportion of such cells, but need not result in the complete elimination of any such cells.

[0221] In some examples, multiple separation steps are performed, in which a positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells that simultaneously express multiple markers, such as by incubating cells with multiple antibodies or binding partners, each specific for a marker targeted by negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on different cell types. In certain embodiments, the separation step is repeated and / or performed more than once, in which a positively or negatively selected fraction from one step is subjected to the same separation step, such as repeated positive or negative selection. In some examples, a single separation step is repeated and / or performed more than once, for example, to increase the purity of the selected cells and / or to further remove and / or deplete negatively selected cells from the negatively selected fraction. In certain embodiments, one or more separation steps are performed 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In certain embodiments, one or more selection steps are performed and / or repeated 1 to 10, 1 to 5, or 3 to 5 times.

[0222] For example, in some aspects, a specific subpopulation of T cells, such as cells that are positive for or express high levels of one or more surface markers, e.g., CD3+, CD4+, CD8, or CD57+ T cells, is isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more antibodies or binding partners that specifically bind to such markers. In some embodiments, the antibodies or binding partners can be bound, for example, directly or indirectly, to a solid support or matrix for selection, such as magnetic or paramagnetic beads. For example, in some embodiments, CD4+ T cells, CD8+ T cells, or CD57+ T cells can be selected, e.g., positively selected, using CD4 Microbeads, CD8 Microbeads, or CD57 Microbeads (Miltenyl Biotec). For example, in some embodiments, CD3+ T cells can be selected, e.g., positively selected, using CD3 Microbeads (Miltenyl Biotec).

[0223] In certain embodiments, CD57- cells are separated from a PBMC sample by negative selection of cells positive for CD57 expression. In various embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, such as CD14. In some aspects, a CD3+ selection step is used to separate T cells from non-T cells. Such CD3+ populations can be further separated into subpopulations by positive or negative selection of markers expressed on, or relatively highly expressed by, CD4+ or CD8+ and / or one or more naive-like, memory, and / or effector T cell subpopulations. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further separated into subpopulations by positive or negative selection of markers expressed on, or relatively highly expressed by, one or more naive-like, memory, and / or effector T cell subpopulations.

[0224] In some embodiments, CD8+ cells are further enriched or depleted for CD57- T cells, e.g., by positive or negative selection based on surface expression of CD57. In certain embodiments, CD4+ cells are further enriched or depleted for CD57- T cells, e.g., by positive or negative selection based on surface expression of CD57. In certain embodiments, CD3+ cells are further enriched or depleted for CD57- T cells, e.g., by positive or negative selection based on surface expression of CD57.

[0225] In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to enhance efficacy, e.g., to improve long-term survival, expansion, and / or engraftment after administration, which in some aspects are particularly robust in such subpopulations. See Terakura et al., (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells with CD4+ T cells further enhances efficacy.

[0226] In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, and both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more additional positive or negative selection steps. In some embodiments, the selection of the CD4+ cell population and the CD8+ cell population are performed simultaneously. In some embodiments, the selection of the CD4+ cell population and the CD8+ cell population are performed sequentially in either order. In some embodiments, the method of selecting cells can include the methods described in U.S. Patent Application Publication No. 20170037369. In some embodiments, the selected CD4+ cell population and the selected CD8+ cell population can be combined after selection. In some aspects, the selected CD4+ cell population and the selected CD8+ cell population can be combined in a bioreactor bag as described herein.

[0227] In various embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, is subjected to selection for CD57+ T cells, whereby the negative fraction containing enriched CD57- cells is retained. In some embodiments, the negative fraction enriched for CD57- cells is subjected to selection for CD3+ T cells, whereby the positive fraction is retained. In particular embodiments, CD8+ T cells are selected from the negative fraction enriched for CD57- cells. In some embodiments, the negative fraction enriched for CD57- cells is subjected to selection for CD8+ T cells, whereby both the negative and positive fractions are retained. In particular embodiments, CD4+ T cells are selected from the negative fraction. In particular embodiments, the negative fraction enriched for CD57- cells is subjected to selection for CD4+ T cells, whereby both the negative and positive fractions are retained. In particular embodiments, CD8+ T cells are selected from the negative fraction.

[0228] In some aspects, the incubated sample or population of cells to be separated is incubated with a selection reagent containing a small magnetizable or magnetically responsive substance, e.g., a magnetically responsive particle or microparticle, e.g., a paramagnetic bead (e.g., Dynalbeads or MACS® beads). The magnetically responsive substance, e.g., a particle, is generally attached, directly or indirectly, to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on the cells or cell population desired to be separated, e.g., negatively or positively selected. In some aspects, the selection agent is or comprises a paramagnetic bead and an attached antibody or antigen-binding fragment thereof that binds to or recognizes CD3, CD4, CD8, or CD57. In some embodiments, the selection agent is a CD3, CD4, CD8, or CD57 MACS® microbead.

[0229] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. Many well-known magnetically responsive materials are known for use in magnetic separation methods, such as the magnetic particles described in U.S. Patent No. 4,452,773 to Molday and European Patent Specification EP 452342 B, which are incorporated herein by reference. Colloidal-sized particles, such as those described in U.S. Patent No. 4,795,698 to Owen and U.S. Patent No. 5,200,084 to Liberti et al., may also be used.

[0230] Incubation is generally carried out under conditions in which the antibody or binding partner, or a molecule, e.g., a secondary antibody or other reagent that specifically binds to such an antibody or binding partner attached to a magnetic particle or bead, specifically binds to the cell surface molecule if present on cells in the sample.

[0231] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are attached to cells via coating with a primary antibody specific for one or more markers. In certain embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell-type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with a biotinylated primary antibody or biotinylated secondary antibody.

[0232] In some aspects, separation is achieved by placing a sample in a magnetic field, and cells with attached magnetically responsive or magnetizable particles are attracted to a magnet to separate them from unlabeled cells. In the case of positive selection, cells that are attracted to the magnet are retained. In the case of negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subjected to additional separation steps.

[0233] In some embodiments, affinity-based selection is by magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic-activated cell sorting (MACS), for example, the CliniMACS system, allows for high-purity selection of cells with magnetized particles attached to them. In certain embodiments, MACS operates in a mode in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells attached to magnetized particles are held in place while unattached species are eluted. Then, after this initial elution step is complete, species trapped in the magnetic field and prevented from elution are released in some manner so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from the heterogeneous cell population. In various embodiments, the selection agent is CD3, CD4, CD8, or CD57 MACS® microbeads.

[0234] In some embodiments, a suboptimal yield concentration of an affinity reagent is a concentration lower than that used or desired to achieve an optimal or maximum yield of bound cells in a given selection or enrichment process, which involves incubating cells with the reagent and recovering or separating the cells bound to the reagent (where "yield" is, for example, the number of such recovered or selected cells relative to the total number of cells in the incubation that are targeted by the reagent, or for which the reagent exhibits specificity, or that bear a marker to which the reagent exhibits specificity and can bind). A suboptimal yield concentration is generally a concentration or amount of reagent that, when recovered in such a process or step, achieves a yield of less than the total of bound cells, e.g., CD57+, CD4+, or CD8+ T cells, e.g., 70% or less. In some embodiments, a suboptimal yield concentration is generally a concentration or amount of reagent that, when recovered in such a process or step, achieves a yield of less than the total of bound cells, e.g., CD3+ T cells, e.g., 70% or less. In some embodiments, a yield of 50% or less, 45% or less, 40% or less, 30% or less, or 25% or less is achieved with a suboptimal concentration of affinity reagent. Concentration may be expressed in terms of number or mass of particles or surfaces per cell and / or number of mass molecules of agent (e.g., antibody, e.g., antibody fragment) per cell.

[0235] In some embodiments, e.g., when operating at suboptimal yield concentrations for each or one or more of two or more selection reagents having affinity for CD57+, CD4+, or CD8+ T cells, one or more of such reagents is used at a higher concentration than one or more of the other such reagents to bias the ratio of cell types recognized by that reagent relative to the cell types recognized by the other such reagents. In some embodiments, e.g., when operating at suboptimal yield concentrations for each or one or more of two or more selection reagents having affinity for CD57+, CD3+, CD4+, or CD8+ T cells, one or more of such reagents is used at a higher concentration than one or more of the other such reagents to bias the ratio of cell types recognized by that reagent relative to the cell types recognized by the other such reagents. For example, a reagent that specifically binds to a marker whose ratio is desired to be biased can be included at a concentration (e.g., agent or mass per cell) that is half, 1x, 2x, 3x, 4x, 5x, 10x, or more increased than the other, depending on how much of a ratio increase is desired. In some embodiments, when operating in a suboptimal range and / or using enough cells to achieve reagent saturation, the amount of immunoaffinity reagent is proportional to the approximate yield of enriched cells. In certain embodiments, the appropriate amount or concentration of immunoaffinity reagent, depending on the desired ratio of the resulting population containing enriched or selected cells, e.g., CD57-, CD4+, or CD8+ T cells, can be routinely determined. In certain embodiments, the appropriate amount or concentration of immunoaffinity reagent, depending on the desired ratio of the resulting population containing enriched or selected cells, e.g., CD3+ T cells, can be routinely determined.

[0236] In some embodiments, the separation and / or isolation step is carried out using magnetic beads, in which an immunoaffinity reagent is reversibly bound to a streptavidin mutein, for example, via a peptide-ligand interaction, as described in WO 2015 / 164675. An example of such magnetic beads is Streptamers®. In some embodiments, the separation and / or isolation step is carried out using magnetic beads, for example, magnetic beads commercially available from Miltenyi Biotec.

[0237] In some embodiments, the magnetically responsive particles are left attached to the cells, which are then incubated, cultured, and / or manipulated; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known, and include, for example, the use of competitive unlabeled antibodies, magnetizable particles, or antibodies bound to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0238] In some embodiments, the isolation and / or selection results in one or more populations of enriched T cells, e.g., CD57- T cells, CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, two or more separate populations of enriched T cells are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, the separate populations are isolated, selected, enriched, and / or obtained from separate biological samples collected, harvested, and / or obtained from the same subject.

[0239] In certain embodiments, the isolation and / or selection results in one or more populations of enriched T cells that comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD57-CD3+ T cells. In certain embodiments, the enriched population of T cells consists essentially of CD57-CD3+ T cells.

[0240] In certain embodiments, the isolation and / or enrichment results in an enriched population of CD4+ T cells that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD57-CD4+ T cells. In certain embodiments, the input composition of CD4+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or no CD8+ T cells, and / or is free or substantially free of CD8+ T cells. In some embodiments, the enriched population of T cells consists essentially of CD57-CD4+ T cells.

[0241] In certain embodiments, the isolation and / or enrichment results in an enriched population of CD57-CD8+ T cells that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD57-CD8+ T cells. In certain embodiments, the population of CD8+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or no CD4+ T cells, and / or is free or substantially free of CD4+ T cells. In some embodiments, the enriched population of T cells consists essentially of CD57-CD8+ T cells.

[0242] C. Chromatographic Cell Selection In aspects of the methods provided herein, cells of a sample, such as T cells, are selected by chromatographic isolation, for example, column chromatography, including affinity chromatography or gel permeation chromatography. In some embodiments, cells, such as CD57- T cells, are isolated, selected, or enriched by chromatographic isolation, for example, column chromatography, including affinity chromatography or gel permeation chromatography. In some embodiments, the method uses a receptor binding reagent that binds to a receptor molecule (e.g., CD57) located on the surface of target cells, such as cells to be isolated, selected, or enriched (e.g., CD57+ cells). Such methods may be referred to as (traceless) cell affinity chromatography technology (CATCH) and may include any of the methods or techniques described in PCT Application Publication Nos. WO 2013124474 and WO 2015164675, which are incorporated herein by reference in their entirety. In certain embodiments, CD57+ cells are negatively selected by chromatographic isolation.

[0243] In some embodiments, target cells (e.g., CD57+ cells) have or express a receptor molecule on the cell surface such that the cells to be isolated, selected, or enriched are determined by the presence of at least one common specific receptor molecule (e.g., CD57). In some embodiments, a sample containing target cells may also contain additional cells that lack receptor molecules. For example, in some embodiments, T cells are isolated, enriched, and / or selected from a sample containing multiple cell types, e.g., red blood cells or B cells. In certain embodiments, CD57+ cells are isolated, enriched, and / or selected from a sample containing multiple cell types, e.g., red blood cells or B cells, thereby providing a population of isolated CD57+ cells and unselected cells, e.g., an enriched population of CD57- T cells.

[0244] In some embodiments, the receptor binding reagent is directly or indirectly bound to, for example, a chromatography matrix (e.g., stationary phase) contained in a chromatography column. In some embodiments, the receptor binding reagent is present on the chromatography matrix (e.g., stationary phase) when a sample is added to the column. In some embodiments, the receptor binding reagent can be indirectly bound to the chromatography matrix (e.g., stationary phase) via a reagent, such as an affinity reagent described herein. In some embodiments, the affinity reagent is covalently or non-covalently bound to the stationary phase of the column. In some embodiments, the affinity reagent is reversibly immobilized to the chromatography matrix (e.g., stationary phase). In some cases, the affinity reagent is immobilized to the chromatography matrix (e.g., stationary phase) via a covalent bond. In some aspects, the affinity reagent is reversibly immobilized to the chromatography matrix (e.g., stationary phase) non-covalently.

[0245] In some embodiments, the receptor binding reagent can be present on the chromatography matrix (e.g., stationary phase) when the sample is added to the chromatography column (e.g., stationary phase), bound directly (e.g., covalently or non-covalently) or indirectly via an affinity reagent. Thus, when the sample is added, the target cells can bind to the receptor binding reagent and be immobilized on the chromatography matrix (e.g., stationary phase) of the column. Alternatively, in some embodiments, the receptor binding reagent can be added to the sample. In this manner, the receptor binding reagent binds to the target cells (e.g., T cells) in the sample, and the sample can then be added to the chromatography matrix (e.g., stationary phase) containing the affinity reagent, whereupon the receptor binding reagent already bound to the target cells binds to the affinity reagent, thereby immobilizing the target cells on the chromatography matrix (e.g., stationary phase). In some embodiments, the receptor binding reagent binds to an affinity reagent, as described herein, e.g., via a binding partner C, as described herein, contained in the receptor binding reagent, as described herein.

[0246] In some aspects, a receptor binding reagent is added to the sample. In certain embodiments, the receptor binding reagent has a binding site B that specifically binds to a receptor molecule on the surface of a cell, for example, a target cell. In some aspects, the receptor binding reagent also includes a binding partner C that can specifically and reversibly bind to the binding site Z of the affinity reagent. For example, in certain aspects, a receptor binding reagent that binds to or recognizes CD57 is added to the sample, and it binds to CD57 on the surface of cells that are positive for CD57 expression in binding site B.

[0247] In certain aspects, the affinity reagent may also contain two or more binding sites Z that can bind to the binding partner C, thereby providing multimerization of the receptor-binding reagent. Thus, the affinity reagent used herein may also be referred to as a multimerization reagent. The affinity reagent may be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. In some aspects, various chromatography matrices may be coupled to various affinity reagents and stacked into a column to form a multicomponent system for separation.

[0248] In some embodiments, two or more receptor binding reagents associate with, e.g., reversibly or irreversibly bind to, the affinity reagent, e.g., via one or more binding sites Z present on the affinity reagent. In some cases, this results in the receptor binding reagents being positioned close to each other such that an avidity effect can occur if a target molecule bearing (at least two copies of) a cell surface molecule (e.g., a selection marker) comes into contact with a receptor binding reagent that can bind to that particular molecule (e.g., a selection marker).

[0249] In some embodiments, two or more different receptor binding reagents that are the same, i.e., have the same selection marker binding specificity, can reversibly bind to the affinity reagent. In some embodiments, it is possible to use at least two different receptor binding reagents that bind to different selection markers, and in some cases three or four different receptor binding reagents. In some aspects, each of the at least two receptor binding reagents can bind to a different molecule (e.g., selection marker), such as a first molecule, a second molecule, etc. In some cases, different molecules (e.g., selection markers), e.g., cell surface molecules, can be present on the same target cell. In other cases, different molecules (e.g., selection markers), e.g., cell surface molecules, can be present on different target cells present in the same cell population. In some cases, a third, fourth, etc. receptor binding reagent, each containing an additional different binding site, can also reversibly associate with the same reagent.

[0250] In some embodiments, two or more different receptor-binding reagents contain the same binding partner C. In some embodiments, two or more different receptor-binding reagents contain different binding partners. In some aspects, a first receptor-binding reagent can have a binding partner C1 that can specifically bind to binding site Z1 present on the affinity reagent, and a second receptor-binding reagent can have a binding partner C2 that can specifically bind to binding site Z1 or binding site Z2 present on the affinity reagent. Thus, in some examples, the multiple binding sites Z contained by the affinity reagent include binding sites Z1 and Z2 that can reversibly bind to binding partners C1 and C2 contained by the receptor-binding reagent, respectively. In some embodiments, C1 and C2 are the same, and / or Z1 and Z2 are the same. In other aspects, one or more of the multiple binding sites Z can be different. In other examples, one or more of the multiple binding partners C can be different. It is within the skill level of a person skilled in the art to select any combination of different binding partners C that are compatible with an affinity reagent containing binding site Z, so long as each binding partner C is capable of interacting with, e.g., specifically binding to, one of the binding sites Z.

[0251] In certain embodiments, a sample, e.g., a sample containing cells and a receptor-binding reagent (e.g., an antibody), is loaded onto or contacted with a chromatography matrix containing an attached or immobilized affinity reagent (e.g., a binding reagent). In certain aspects, the affinity reagent has multiple binding sites Z that specifically bind to the binding partner C of the receptor-binding reagent. In certain aspects, the receptor-binding reagent binds to the affinity reagent through the interaction between the binding partner C and the binding site Z. Thus, in some embodiments, cells, e.g., target cells, are immobilized via a complex formed by one or more binding sites Z of the affinity reagent and the binding site Z of the receptor-binding reagent on the chromatography matrix. In further aspects, cells, e.g., target cells, can be depleted from the sample, e.g., by rinsing, releasing, or washing the remaining sample from the chromatography matrix. In certain aspects, the receptor-binding reagent may be included in the cell-containing sample and applied to or contacted with the chromatography matrix for binding to the attached affinity or multimerization reagent, e.g., before adding the sample to the chromatography matrix.

[0252] In some embodiments, chromatography matrix is ​​used to remove or separate target cells from sample, for example, by negative selection.For example, in certain embodiments, a sample containing CD57+ cells and CD57- cells is contacted with or incubated with a receptor binding reagent that binds to and / or recognizes CD57.In certain embodiments, the sample and receptor binding reagent are loaded onto the matrix, where, in some aspects, a complex is formed between the immobilized or attached affinity reagent, the receptor binding reagent, and CD57+ T cells.In some embodiments, unbound cells are removed or rinsed from the chromatography matrix, thereby removing bound CD57+ cells and providing a sample, for example, a population, enriched with CD57- cells.

[0253] In certain embodiments, a chromatography matrix is ​​used to isolate, select, or enrich target cells from a sample, for example, by positive selection. For example, in some embodiments, a sample containing CD4+ or CD8+ T cells and other cells, such as non-T cell immune cells, is contacted with or incubated with a receptor binding reagent that binds to and / or recognizes CD4 or CD8. In certain embodiments, the sample and receptor binding reagent are loaded onto the matrix, where, in some aspects, a complex is formed between the immobilized or attached affinity reagent, the receptor binding reagent, and the CD4+ or CD8+ T cells. In certain embodiments, unbound cells are removed or rinsed from the chromatography matrix. In certain embodiments, the immobilized CD4+ or CD8+ cells can be removed or released by adding a competing reagent, for example, to disrupt the complex. In some aspects, the separated, released, or eluted CD4+ or CD8+ T cells are a sample, composition, or population of cells enriched for CD4+ or CD8+ T cells.

[0254] In certain embodiments, a chromatography matrix is ​​used to isolate, select, or enrich target cells from a sample, for example, by positive selection. For example, in some embodiments, a sample containing CD3+ T cells and other cells, such as non-T cell immune cells, is contacted with or incubated with a receptor binding reagent that binds to and / or recognizes CD3. In certain embodiments, the sample and receptor binding reagent are loaded onto the matrix, where, in some aspects, a complex is formed between the immobilized or attached affinity reagent, the receptor binding reagent, and the CD3+ T cells. In certain embodiments, unbound cells are removed or rinsed from the chromatography matrix. In certain embodiments, the immobilized CD3+ cells can be removed or released by adding a competing reagent, for example, to disrupt the complex. In some aspects, the separated, released, or eluted CD3+ T cells are a sample, composition, or population of cells enriched in CD3+ T cells.

[0255] In some aspects, a competing reagent is loaded onto the chromatography column. In some embodiments, the reversible bond formed between binding partner C and binding site Z can be disrupted by the competing reagent. In certain embodiments, the competing reagent has a binding site capable of binding to binding site Z of the affinity reagent. In some embodiments, the competing reagent can be biotin, a biotin derivative or analog, or a streptavidin-binding peptide that can compete for binding with binding partner C for one or more binding sites Z. In certain embodiments, the competing reagent forms a complex with the affinity reagent and is thereby immobilized on the chromatography matrix. In some embodiments, unlike binding partner C and the competing reagent, the competing reagent exhibits a higher binding affinity for one or more binding sites Z compared to the affinity of the binding partner. In certain aspects of any of the methods provided herein, the addition of a competing reagent to the stationary phase of the chromatography column to disrupt binding of a selection agent (e.g., a receptor-binding agent) to the affinity reagent in order to separate target cells (e.g., T cells) from the chromatography matrix (e.g., stationary phase) is not required.

[0256] This competitive binding results in displacement of the binding between the receptor-binding reagent and the affinity reagent at binding partner C and binding site Z. In certain embodiments, adding or loading the competing reagent to a chromatography matrix with an attached complex containing the affinity reagent, receptor-binding reagent, and cells, e.g., target cells, results in the cells being eluted from the chromatography matrix. In some aspects, the receptor-binding reagent has low affinity for the cell's receptor molecule at binding site B, such that the receptor-binding reagent separates from the cells in the presence of the competing reagent. Thus, in some embodiments, cells, e.g., target cells, that are free or essentially free of bound receptor-binding molecules are eluted from the chromatography matrix.

[0257] In some embodiments, an elution sample containing cells, such as target cells, a competing reagent, and a receptor-binding reagent is collected from the eluate of the first chromatography column. In certain embodiments, the elution sample is loaded onto a second chromatography column having a suitable stationary phase that can serve as both an affinity chromatography matrix and a gel permeation matrix. In certain embodiments, the affinity chromatography matrix has an affinity reagent immobilized thereon. In some aspects, the receptor-binding reagent and the competing reagent bind to the binding site Z on the affinity reagent, thereby immobilizing it on the chromatography column. As a result, in certain aspects, the elution sample containing the isolated target cells is depleted of the receptor-binding reagent and the competing reagent. Thus, in some aspects, the target cells that do not contain any reaction are now ready for further use, for example, for processing by any of the methods described herein.

[0258] In some embodiments, cells of the sample, e.g., target cells, can be depleted from the sample, for example, by rinsing, releasing, or washing the remaining sample from the chromatography matrix (e.g., stationary phase). In some embodiments, one or more (e.g., two, three, four, five, six) washing steps are used to remove unbound cells and debris from the chromatography matrix (e.g., stationary phase). In some embodiments, at least two washing steps are performed. In some embodiments, the sample is allowed to permeate the matrix for at least or about 5, 10, 15, 16, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes before one or more washing steps are performed. In some embodiments, the wash step is performed for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes or for at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes after the sample is applied to the chromatography column (e.g., stationary phase). In some embodiments, the wash step is performed for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes or for at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes after the sample is applied to the chromatography column (e.g., stationary phase). In some embodiments, one or more wash steps are performed within or about 120, 100, 90, 80, 70, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 minutes after addition of the sample to the chromatography column (e.g., stationary phase).In some embodiments, one or more wash steps are performed within or about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 minutes after addition of the sample to the chromatography column (e.g., the stationary phase). In some embodiments, one or more wash steps are performed within or about 5 to 60 minutes after addition of the sample to the chromatography column (e.g., the stationary phase). In some embodiments, one or more wash steps are performed within or about 5 to 50 minutes after addition of the sample to the chromatography column (e.g., the stationary phase). In some embodiments, one or more wash steps are performed within or about 5 to 40 minutes after addition of the sample to the chromatography column (e.g., the stationary phase). In some embodiments, one or more wash steps are performed within or about 5-30 minutes after adding the sample to the chromatography column (e.g., the stationary phase). In some embodiments, one or more wash steps are performed within or about 5-20 minutes after adding the sample to the chromatography column (e.g., the stationary phase). In some embodiments, one or more wash steps are performed within or about 5-10 minutes after adding the sample to the chromatography column (e.g., the stationary phase). In some embodiments, one or more wash steps are performed within or about 10-60 minutes after adding the sample to the chromatography column (e.g., the stationary phase). In some embodiments, one or more wash steps are performed within or about 20-60 minutes after adding the sample to the chromatography column (e.g., the stationary phase). In some embodiments, one or more wash steps are performed within or about 30-60 minutes after adding the sample to the chromatography column (e.g., the stationary phase). In some embodiments, one or more wash steps are performed within or about 40-60 minutes after application of the sample to the chromatography column (eg, stationary phase).In some embodiments, one or more wash steps are performed within or about 50-60 minutes after application of the sample to the chromatography column (eg, stationary phase).

[0259] In some embodiments, multiple cell selection steps are performed, where a positively or negatively selected fraction from one step is subjected to another selection step, e.g., subsequent positive or negative selection. In certain embodiments, methods, techniques, and reagents for selection, isolation, and enrichment are described, for example, in PCT Application No. WO2015164675, which is incorporated herein by reference in its entirety.

[0260] In some embodiments, a single selection step can be used to isolate target cells (e.g., CD57- T cells) from a sample. In some embodiments, a single selection step can be performed on a single chromatography column. In some examples, a single selection step can simultaneously deplete cells expressing multiple markers. Similarly, multiple cell types can be simultaneously positively selected. In certain embodiments, a selection step is repeated and / or performed more than once, in which case the positively or negatively selected fraction from one step is subjected to the same selection step, e.g., repeated positive or negative selection. In some examples, a single selection step is repeated and / or performed more than once, e.g., to increase the purity of the selected cells and / or to further remove and / or deplete negatively selected cells from the negatively selected fraction. In certain embodiments, one or more selection steps are performed two, three, four, five, six, seven, eight, nine, ten, or more than ten times. In certain embodiments, one or more selection steps are performed and / or repeated 1 to 10 times, 1 to 5 times, or 3 to 5 times. In some embodiments, two selection steps are performed.

[0261] Cell selection can be performed using one or more chromatography columns. In some embodiments, the one or more chromatography columns are included in a closed system. In some embodiments, the closed system is an automated closed system, e.g., requiring minimal or no user (e.g., human) input. In some embodiments, cell selection is performed continuously (e.g., sequential selection techniques). In some embodiments, one or more chromatography columns are arranged in series. For example, a first column can be oriented so that the column output (e.g., eluate) can be fed to a second chromatography column, e.g., via a connecting tube. In some embodiments, multiple chromatography columns can be arranged in series. In some embodiments, cell selection can be achieved by performing sequential positive and negative selection steps, where subsequent steps subject negative and / or positive fractions from the previous step to further selection. In this case, the entire process is performed within the same tube or set of tubes. In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is accomplished to enrich for one of the CD4+ or CD8+ populations, and unselected cells from the first selection are used as the source of cells for a second selection to enrich for the other of the CD4+ or CD8+ populations. In some embodiments, one or more additional rounds of selection are performed to enrich for a subpopulation of one or both of the CD4+ or CD8+ populations, e.g., central memory T (T CM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some embodiments, a sample containing target cells is subjected to sequential selections, where a first selection is performed to enrich for a CD3+ population and the selected cells are used as a cell source for a second selection to enrich for a CD3+ population. In some embodiments, a sample containing target cells is subjected to sequential selections, where a first selection is performed on a first stationary phase (e.g., in a first chromatographic column) to enrich for a CD3+ population and the flow-through containing unbound cells is used as a cell source for a second selection to enrich for a CD3+ population on a second stationary phase (e.g., in a second chromatographic column), where the first and second stationary phases are arranged sequentially. In some embodiments, one or more additional rounds of selection are performed to select a subpopulation of the CD3+ population, such as a central memory T (T CM ) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is achieved to enrich for a CD3+ population, and the selected cells are used as a source of cells for a second selection to enrich for a CD4+ population. In some embodiments, one or more additional rounds of selection are performed to enrich for a subpopulation of the CD3+CD4+ population, e.g., central memory T (T CM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is achieved to enrich for a CD3+ population, and the selected cells are used as a source of cells for a second selection to enrich for a CD8+ population. In some embodiments, one or more additional rounds of selection are performed to enrich for a subpopulation of the CD3+CD8+ population, e.g., central memory T (T CM ) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some aspects, it is envisioned that specific subpopulations of T cells (e.g., CD3+ cells), such as cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells), are selected by sequential positive or negative selection techniques.

[0262] In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is performed to enrich for a CD57- population, and the selected cells are used as a source of cells for a second selection to enrich for a CD3+ population. In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is performed on a first stationary phase (e.g., in a first chromatographic column) to enrich for a CD57- population, and the flow-through containing unbound cells is used as a source of cells for a second selection to enrich for a CD3+ population on a second solid phase (e.g., in a second chromatographic column), where the first and second stationary phases are arranged sequentially. In some embodiments, a subpopulation of the CD57- population, such as central memory T (T CM Further selection can be performed to enrich for CD57-CD3+ cells, naive T cells, and / or cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is performed to enrich for a CD57- population, and the selected cells are used as a source of cells for a second selection to enrich for a CD3+ population. In some embodiments, a subpopulation of the CD57-CD3+ population, e.g., central memory T (T CMFurther selection can be performed to enrich for CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ cells, naive T cells, and / or cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some aspects, it is contemplated that specific subpopulations of T cells (e.g., CD57− cells), e.g., cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are selected by positive or negative sequential selection techniques.

[0263] In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is performed to enrich for a CD3+ population, and the selected cells are used as a source of cells for a second selection to enrich for a CD57- population. In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is performed on a first stationary phase (e.g., in a first chromatographic column) to enrich for a CD3+ population, and the flow-through containing unbound cells is used as a source of cells for a second selection to enrich for a CD57- population on a second solid phase (e.g., in a second chromatographic column), where the first and second stationary phases are arranged sequentially. In some embodiments, a subpopulation of the CD3+ population, such as central memory T (T CMFurther selection can be performed to enrich for CD3+CD57− cells, naive T cells, and / or cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is performed to enrich for a CD3+ population, and the selected cells are used as a source of cells for a second selection to enrich for a CD57− population. In some embodiments, a subpopulation of the CD3+CD57− population, e.g., central memory T (T CM Further selection can be performed to enrich for CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ cells, naive T cells, and / or cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some aspects, it is contemplated that specific subpopulations of T cells (e.g., CD57− cells), e.g., cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are selected by positive or negative sequential selection techniques.

[0264] In some embodiments, cell selection is performed in parallel (e.g., parallel selection techniques). In some embodiments, one or more chromatography columns are arranged in parallel. For example, two or more columns can be arranged so that a sample is loaded simultaneously onto two or more columns via tubing that allows the sample to be added to each column, e.g., without the sample crossing the first column. For example, when using parallel selection techniques, cell selection can be achieved by simultaneously performing positive and / or negative selection steps in a closed system, e.g., the entire process is performed in the same tube or set of tubes. In some embodiments, a sample containing target cells is subjected to parallel selection, in which the sample is loaded onto two or more chromatography columns, each performing selection of a cell population. In some embodiments, two or more chromatography columns individually perform selection of CD57-, CD3+, CD4+, or CD8+ populations. In some embodiments, two or more chromatography columns independently perform selection of the same cell population, including affinity chromatography or gel permeation chromatography. For example, two or more chromatography columns can perform selection of CD57- cells. In some embodiments, two or more chromatography columns independently select different cell populations, including affinity chromatography or gel permeation chromatography. For example, two or more chromatography columns may independently select CD57- cells, CD4+ cells, CD3+ and / or CD8+ cells. In some embodiments, further selections, e.g., using sequential selection techniques, can also be performed to enrich subpopulations of one or all of the cell populations selected by parallel selection. For example, the selected cells can be selected from central memory T (T CM) cells, naive T cells and / or cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to parallel selection, where parallel selection is performed to enrich for the CD57− population in two or more columns. In some embodiments, a subpopulation of the CD57− population, e.g., central memory T (T CM Further selection can be performed to enrich for CD57- and CD3+ populations, such as central memory T (T) cells, naive T cells, and / or cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to parallel selection, where selection is performed independently to enrich for CD57- and CD3+ populations in two or more columns. In some embodiments, subpopulations of the CD57- and CD3+ populations, such as central memory T (T CM Further selection can be performed to enrich for CD57- and CD4+ populations, such as central memory T (T) cells, naive T cells, and / or cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to parallel selection, where selection is performed independently to enrich for CD57- and CD4+ populations in two or more columns. In some embodiments, subpopulations of the CD57- and CD4+ populations, such as central memory T (T CMFurther selection can be performed to enrich for CD57- and CD8+ populations, such as central memory T (T), naive T cells, and / or cells that are positive for or express high levels of one or ...

Claims

1. 1. A method for enriching T cells, comprising: (a) performing a first selection, the first selection comprising enriching either CD57- or CD3+ T cells from a biological sample comprising primary human T cells, thereby generating an enriched T cell population; (b) subjecting cells from the enriched population of T cells to a second selection, the first selection comprises enriching for CD57- T cells and the second selection comprises enriching for CD3+ T cells from the enriched population; or the first selection comprises enriching for CD3+ T cells and the second selection comprises depleting CD57+ T cells from the enriched T cell population; The method produces a depleted population that contains fewer CD57+ T cells than the biological sample and is enriched for CD3+ T cells; (c) introducing a heterologous polynucleotide encoding a recombinant receptor into a population of T cells obtained from the depleted population, thereby generating an engineered population of T cells; (d) culturing cells of the engineered population of T cells under conditions that promote the proliferation or expansion of the engineered population of T cells, thereby producing an expanded engineered population of T cells; The method includes:

2. 2. The method of claim 1, wherein the first selection comprises enriching for CD57- T cells and the second selection comprises enriching for CD3+ T cells from the enriched population.

3. 2. The method of claim 1, wherein the first selection comprises enriching for CD3+ T cells and the second selection comprises depleting CD57+ T cells from the enriched T cell population.

4. (a) performing a first selection, the first selection comprising removing CD57+ T cells from a biological sample comprising primary human T cells, thereby generating a first depleted population, the first depleted population comprising fewer CD57+ T cells than the biological sample; (b) subjecting cells from the first depleted population to a second selection, the second selection comprising enriching one of (i) CD4+ T cells and (ii) CD8+ T cells from the first depleted population, whereby enrichment produces a second depleted population enriched in one of (i) CD4+ T cells and (ii) CD8+ T cells, and an unselected population; (c) performing a third selection, the third selection comprising enriching the other of (i) CD4+ cells and (ii) CD8+ cells from the unselected population, whereby enrichment produces a third depleted population enriched in the other of (i) CD4+ T cells and (ii) CD8+ T cells; (d) introducing a heterologous polynucleotide encoding a recombinant receptor into a population of T cells obtained from the depleted population, thereby generating an engineered population of T cells; and (e) culturing cells of the engineered population of T cells under conditions that promote proliferation or expansion of the engineered population of T cells, thereby producing an expanded engineered population of T cells.

23. A method for enriching T cells comprising:

5. The first depleted population, the second depleted population, or the third depleted population, (i) less than 5% CD57+ T cells; (ii) a frequency of CD57+ T cells that is less than 35% of the frequency of CD57+ T cells present in the biological sample; (iii) CD4+ T cells, wherein at least 95% of the CD4+ T cells are CD57-; and (iv) CD8+ T cells, wherein at least 95% of the CD8+ T cells are CD57-. The method according to any one of claims 1 to 4, comprising at least one of:

6. 6. The method of any one of claims 1 to 5, wherein the biological sample comprises an apheresis product or a leukapheresis product.

7. The method of any one of claims 1 to 6, wherein at least 95% of the CD3+ T cells of the depleted population comprise CD57-CD3+ T cells.

8. the frequency of CD57+ T cells in the first depleted population, the second depleted population, or the third depleted population is less than 35%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the frequency of CD57+ T cells in the biological sample; the first depleted population, the second depleted population, or the third depleted population comprises less than 3%, less than 2%, less than 1%, less than 0.1%, or less than 0.01% CD57+ T cells; and / or the first depleted population, the second depleted population, or the third depleted population is free or essentially free of CD57+ T cells; The method of any one of claims 1 to 7.

9. the frequency of naive-like T cells in the first depleted population, the second depleted population, or the third depleted population is at least 10%, 20%, 30%, 40%, or 50% greater than the frequency of naive-like T cells in the biological sample; the naive-like T cells are surface positive for one or more of the markers selected from CD45RA, CD27, CD28, and CCR7; and the frequency of CD27+ T cells in the first depleted population, the second depleted population, or the third depleted population is at least 10%, 20%, 30%, 40%, or 50% greater than the frequency of the respective cells in the biological sample; the frequency of CD28+ T cells in the first depleted population, the second depleted population, or the third depleted population is at least 10%, 20%, 30%, 40%, or 50% greater than the frequency of the respective cells in the biological sample; and / or the frequency of CD27+ / CD28+ T cells in the first depleted population, the second depleted population, or the third depleted population is at least 10%, 20%, 30%, 40%, or 50% greater than the frequency of the respective cells in the biological sample; The method according to any one of claims 1 to 8.

10. combining the second depleted population with the third depleted population in a ratio between 1:3 and 3:1, or in a 1:1 ratio, thereby generating a depleted population comprising the second depleted population and the third depleted population. The method of any one of claims 4 to 6 and 8 to 9, further comprising:

11. At least 90%, at least 95%, at least 97%, at least 99%, or at least 99.9% of CD57-CD4+ T cells, CD57-CD8+ T cells, or CD57-CD3+ T cells The method of any one of claims 1 to 10, comprising producing a T cell composition comprising:

12. the ratio of CD4+ to CD8+ T cells in the composition is between 3:1 and 1:3, between 2:1 and 1:2, between 1.5:1 and 1:1.5, or between 1.2:1 and 1:1.2, inclusive; or the ratio of CD4+ to CD8+ T cells in the composition is 1:1; 12. The method of claim 11.

13. the primary T cells are derived from a subject having a disease or condition, and the disease or condition is cancer; or The primary T cells are derived from a healthy subject. The method of any one of claims 1 to 12.

14. Deleting CD57+ T cells comprises immunoaffinity-based selection; Immunoaffinity-based selection includes contacting the cells with an antibody capable of specifically binding to CD57 and recovering cells that are not bound to the antibody, thereby resulting in negative selection; The harvested cells are depleted of CD57+ cells. The method of any one of claims 1 to 13.

15. 15. The method of any one of claims 1 to 14, wherein enriching the cells in the first, second and / or third selection comprises immunoaffinity-based selection.

16. The method of claim 4, wherein the selection to enrich for CD4 or CD8 T cells comprises an immunoaffinity based selection and is performed by contacting the cells with an antibody capable of specifically binding to CD4 or CD8, respectively, and recovering the cells that bind to the antibody, thereby resulting in a positive selection and the recovered cells are enriched for CD4+ cells or CD8+ cells.

17. 2. The method of claim 1, wherein the selection to enrich for CD3 T cells comprises immunoaffinity based selection and is performed by contacting the cells with an antibody capable of specifically binding to CD3 and recovering cells that bind to the antibody, thereby resulting in positive selection and the recovered cells being enriched for CD3+ cells.

18. The method of any one of claims 1 to 17, wherein prior to the introduction, the depleted population is incubated under stimulating conditions.

19. the step of introducing comprises transduction with a viral vector comprising the heterologous polynucleotide; The viral vector is a gamma retroviral vector or a lentiviral vector; 19. The method of any one of claims 1 to 18.

20. 20. The method of any one of claims 1 to 19, wherein the culturing step is carried out in the presence of one or more recombinant cytokines, including one or more of IL-2, IL-7, and IL-15.

21. The recombinant receptor Target antigens associated with, specific to, and / or expressed in cells or tissues of a disease, disorder, or condition can be bound to and / or the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer; The target antigen is a tumor antigen; 21. The method of any one of claims 1 to 20.

22. The method of any one of claims 1 to 21, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

23. The recombinant receptor an extracellular domain, comprising an antigen-binding domain, a single chain Fv fragment (scFv), a spacer, and / or a hinge region; A transmembrane domain, and Intracellular signaling domains containing costimulatory signaling regions 23. The method of claim 22, comprising:

24. and / or the intracellular signalling domain is or comprises the intracellular signalling domain of the CD3 chain, the intracellular signalling domain of the CD3-zeta (CD3ζ) chain, or a signalling portion thereof; the costimulatory signaling region comprises the intracellular signaling domain of CD28, 4-1BB, or ICOS or a signaling portion thereof; 24. The method of claim 23.

Images