Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy

Abstract

To provide: methods, cells, and compositions for preparing cells and compositions for genetic engineering and cell therapy; streamlined cell preparation methods, e.g., for isolation, processing, incubation, and genetic engineering of cells and populations of cells; and cells and compositions produced by the methods and methods of their use.SOLUTION: Provided is a method for enriching CD4+ or CD8+ cells in a closed system and producing an enriched composition including cells of a first selected population and cells of a second selected population. The method is capable of preparing of a plurality of different cell populations for adoptive therapy using fewer steps and / or resources and / or reduced handling compared with other methods.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 61 / 983,415, filed Apr. 23, 2014, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of sequence listings This application is filed with an electronic format sequence listing. The sequence listing is provided as a file named 735042000240seqlist.txt, size 13 kilobytes, created on Apr. 22, 2015. The information in the electronic format sequence listing is incorporated herein by reference in its entirety.

[0003] Field In some aspects, the present disclosure relates to methods for preparing cells, cells, and compositions, as well as compositions for genetic manipulation and cell therapy. In some embodiments, for example, rationalized cell preparation methods for the isolation, processing, incubation, and genetic manipulation of cells and cell populations are provided. Cells and compositions produced by the methods, as well as methods of using them, are also provided. The cells can include immune cells such as T cells, and generally include a plurality of isolated T cell populations or T cell subtypes. In some aspects, the method can prepare a plurality of different cell populations for adoptive therapy using fewer steps and / or resources and / or reduced handling compared to other methods.

Background Art

[0004] Background A variety of methods are available for preparing cells for therapeutic use. For example, multiple methods are available for isolating, processing, and manipulating cells, including T cells and other immune cells. Multiple methods are available for isolating such cells, as well as for expressing genetically engineered antigen receptors such as high-affinity T cell receptors (TCRs) and chimeric antigen receptors (CARs). Multiple methods are available for adoptively transferring such cells into a subject. Improved methods are needed for the preparation (e.g., isolation, processing, culturing, and manipulation) of cells for cell therapy applications. In particular, methods are needed for the preparation and manipulation of cells with improved efficiency, safety, variability, and conservation of resources, such as the preparation and manipulation of multiple isolated cell types or subtypes. Methods, cells, compositions, kits, and systems are provided for addressing such needs.

SUMMARY OF THE INVENTION

[0005] Summary Methods are provided for the preparation and manipulation of cells and cell populations, as well as cells and compositions produced by such methods. In some aspects, the cells can be used for immunotherapy, such as in adoptive immunotherapy methods. In some instances, the provided methods include the isolation, selection, or enrichment of CD4+ cells and CD8+ cells or subpopulations thereof from the same starting sample, such as a single apheresis sample, leukapheresis sample, or sample containing peripheral blood mononuclear cells (PBMCs). In some aspects, the method includes the selection or enrichment of at least two cell populations, such as a CD4+ cell population and a CD8+ population, in a single process flow, in which a sample of the negative fraction is not discarded from a first selection or enrichment of one of CD4+ or CD8+ in the process, and then a second selection is made for the other of the CD4+ cells or CD8+ cells. In some aspects, the first and second selections can occur simultaneously or sequentially.

[0006] In some aspects, the selection, enrichment, and / or isolation of both cell populations, such as CD4+ cells and CD8+ cells, is performed simultaneously, for example, within the same vessel or using the same apparatus, or sequentially as part of a system or apparatus in which the vessels, such as columns, chambers, used to make the first and second selections are operably connected. In some embodiments, the simultaneous and / or sequential enrichment or selection can occur as a single process stream without handling either the positive or negative fractions prepared as part of the first and / or second selection or enrichment. In some aspects, the isolation, culture, and / or manipulation of different populations is performed from the same starting composition or material, for example, from the same sample.

[0007] In some embodiments, the method comprises performing a first selection by enriching for either CD4+ cells or CD8+ cells from a sample containing primary human T cells to generate a first selected population and a non-selected population, and performing a second selection by enriching for the other of the CD4+ cells or CD8+ cells from the non-selected population, whereby the method produces a cell composition containing CD4+-enriched cells and CD8+-enriched cells.

[0008] In some embodiments, the second selection is performed by enriching the other T cell subtype from the unselected population generated by the first selection. Thus, in some embodiments, the provided method differs from other selection methods in that the negative fraction from the first selection is not discarded, but rather is used as a starting material for further selection to enrich for another cell type. Generally, if the T cell subset enriched by the first selection is the CD4+ subset (i.e., the first selection enriches for CD4+ cells), then of course the first selection is designed not to enrich for the other subtype of cells to be enriched by the second selection. For example, in some embodiments, the first selection enriches for CD4+ cells and not CD8+ cells, and the second selection enriches for CD8+ cells from the negative fraction recovered from the first selection. Similarly, generally, if the T cell subset enriched by the first selection is the CD8+ subset (i.e., the first selection enriches for CD8+ cells), then of course the first selection is designed not to enrich for the other subtype of cells to be enriched by the second selection. For example, in some embodiments, the first selection enriches for CD8+ cells and not CD4+ cells, and the second selection enriches for CD4+ cells from the negative fraction recovered from the first selection.

[0009] In some embodiments, the method further involves additional selections, such as a third, fourth, etc., that can enrich cells from the selected population and / or the unselected population from any previous selection step. For example, in some embodiments, cells from either the selected population (e.g., from the second selection step) or the unselected population are further enriched. For example, in some embodiments, CD8+ cell subtypes, such as resting cells or central memory cells, are further enriched from the selected CD8+ cells.

[0010] In some embodiments, (a) providing a starting culture composition, the composition being produced by performing a first selection in a closed system (the first selection being from a sample containing primary human T cells CD4 +(including generating a first selected population and a non - selected population by enriching one of cells and CD8+ cells), and produced by making a second selection in a closed system (the second selection includes generating a second selected population by enriching the other of CD4+ cells and CD8+ cells from the non - selected population); (b) generating stimulated cells by incubating a culture - starting composition containing cells of the first selected population and cells of the second selected population under stimulating conditions in a culture tank; and (d) introducing a genetically engineered antigen receptor into the stimulated cells generated in (b) A method for producing genetically engineered T cells is provided, wherein thereby the method generates an output composition comprising CD4 + T cells and CD8 + T cells that express the genetically engineered antigen receptor.

[0011] In some embodiments, methods are also provided that include the simultaneous enrichment or selection of first and second cell populations, such as CD4+ and CD8+ cell populations. In some embodiments, the method comprises contacting cells of a sample containing primary human T cells in an incubation composition with a first immunoaffinity reagent that specifically binds CD4 and a second immunoaffinity reagent that specifically binds CD8 under conditions in which the immunoaffinity reagents specifically bind to CD4 molecules and CD8 molecules on the cell surface in the sample, and generating an enriched composition comprising CD4+ and CD8+ cells by recovering the cells bound to the first and / or second immunoaffinity reagent. In some embodiments, the method is performed such that the enriched composition contains less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less thereof, of the total CD4+ cells in the incubation composition, or less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less thereof, of the CD8+ cells in the incubation composition, by including a concentration of the first and / or second immunoaffinity reagent that is suboptimal in the incubation composition.

[0012] In one aspect, there is provided a step of providing a composition enriched in CD4+ T cells and CD8+ T cells, wherein cells of a sample containing primary human T cells are contacted in an incubation composition with a first immunoaffinity reagent that specifically binds to CD4 and a second immunoaffinity reagent that specifically binds to CD8 under conditions in which the immunoaffinity reagents specifically bind to CD4 molecules and CD8 molecules on the cell surface in the sample, whereby an enriched composition is produced; and a step of generating an enriched composition containing CD4+ cells and CD8+ cells at a starting culture ratio by recovering cells bound to the first and / or second immunoaffinity reagent, wherein the first and / or second immunoaffinity reagent is present in the incubation composition at a sub-optimal yield concentration, and wherein the enriched composition contains less than 70% of all CD4+ cells in the incubation composition and / or less than 70% of CD8+ in the incubation composition, thereby producing a composition enriched in CD4+ and CD8+ T cells.

[0013] In some aspects of any aspect so provided, the method is performed by immunoaffinity-based selection, such as contacting the cells with an antibody that specifically binds to a cell surface marker, such as CD4, CD8, or other cell surface markers expressed on naive T cells, resting T cells, or central memory T cells. In some aspects, the solid support is a sphere, such as a bead, such as a microbead or a nanobead. In some aspects, the bead can be a magnetic bead. In some aspects, the solid support can be a column or other vessel for performing column chromatography.

[0014] In some embodiments, the antibody comprises one or more binding partners capable of forming a reversible bond with a binding reagent immobilized on a solid surface, such as a sphere or a chromatography matrix, wherein the antibody is reversibly immobilized on the solid surface. In some embodiments, cells expressing a cell surface marker bound by the antibody on the solid surface can be recovered from the matrix by disruption of the reversible bond between the binding reagent and the binding partner. In some embodiments, the binding reagent is streptavidin or a streptavidin analog or variant, such as streptavidin, an analog or variant shown in any of SEQ ID NOs: 11-16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence shown in any of SEQ ID NOs: 11-16 and retaining binding to a binding partner such as biotin or a peptide. In some embodiments, the binding partner is biotin, a biotin analog, or a peptide capable of binding to the binding reagent. In some embodiments, the binding partner is a peptide capable of binding to the binding reagent, such as a streptavidin-binding peptide, e.g., a peptide comprising a sequence shown in any of SEQ ID NOs: 1-10, or containing the same. In some embodiments, the method comprises, as part of a first and / or second selection, contacting cells in a sample with a solid support comprising an antibody immobilized thereon, and then adding a competing reagent to disrupt the bond between the binding partner and the binding reagent to recover selected cells from the solid surface. In some embodiments, the competing reagent is biotin or a biotin analog.

[0015] In some embodiments, the method produces a selected or enriched composition containing CD4+ cells that are selected or enriched relative to CD8+ cells present at the start of culture, or a subpopulation thereof. In some embodiments, the starting ratio of CD4+ cells to CD8+ cells is between 10:1 or about 10:1 and 1:10 or about 1:10, between 5:1 or about 5:1 and 1:5 or about 1:5, or between 2:1 or about 2:1 and 1:2 or about 1:2, such as 1:1 or about 1:1. Achieving or producing the starting ratio in the generated composition, such as the starting composition for culture, containing cells enriched or selected for CD4, CD8, or a subpopulation thereof, by choosing or selecting a sufficient amount or relative amount of an immunoaffinity-based reagent, such as antibody-coated beads (e.g., magnetic beads) or one or more affinity chromatography matrices, is within the level of skill of a person skilled in the art. Examples of such methods are described in the subsections below.

[0016] In some embodiments, the cells include immune system cells such as lymphocytes, e.g., T cells (e.g., CD4+ T cells and CD8+ T cells and isolated subpopulations thereof), and NK cells. In some embodiments, the cells are present in combination with multiple cell populations or cell types, which in some aspects are included in the composition in a specific ratio or number of cells or cell types. Also provided are methods for optimizing the method by selecting or determining appropriate ratios and numbers, such as the starting ratio of cell types and populations and the desired output ratio and dosage, for use in connection with the method.

[0017] Also provided are cells, cell populations, and compositions thereof for use in and produced by the method. Also provided are systems, devices, apparatuses, reagents, compounds, and kits for performing the method. Also provided are therapies and uses for the cells and compositions produced by the method, such as methods for adoptive cell therapy.

[0018] In some embodiments, methods for producing cells for adoptive cell therapy, methods for producing cells for genetic engineering, and methods for producing genetically engineered cells are provided. In some aspects, the cells are T cells such as CD4 + T cells and CD8 + T cells and / or their subtypes. In some aspects, the cell therapy is T cell therapy.

[0019] In some embodiments, the method is performed by: (a) isolating a cell population from a sample; and (b) incubating a culture initiation composition in a culture vessel containing the isolated population of cells. In some aspects, the method further comprises, for example, (c) genetically engineering the incubated cells or the cells in the culture vessel by introducing a genetically engineered antigen receptor into the cells in the culture vessel.

[0020] In some embodiments, the method is performed by: (a) incubating a culture initiation composition in a culture vessel containing a plurality of cell populations at a specific starting ratio or a specific number of cells; and (b) genetically engineering the cells, for example, by introducing a genetically engineered antigen receptor into the cells in the culture vessel.

[0021] In some embodiments, the genetically engineered antigen receptor is introduced into cells in the culture vessel, such as different cell types or subpopulations in the culture vessel, for example, CD4 + cells and CD8 + cells in the culture vessel.

[0022] In some aspects, the method produces an output composition for genetic engineering or adoptive cell therapy, or comprising cells expressing a genetically engineered antigen receptor.

[0023] In some embodiments, the isolation comprises or is effected by isolating a population of primary human CD4 + T cells and / or a population of primary human CD8 + T cells from the sample. In some aspects, it is CD4+ A step of depleting or enriching a subpopulation of cells, and / or CD8 + includes a step of depleting or enriching a subpopulation of cells. Thus, in some aspects, the starting composition for culturing is isolated CD4 + primary human T cells and CD8 + includes primary human T cells.

[0024] In some aspects, the step of enriching or depleting is performed by an immunoaffinity-based selection, such as binding with an antibody or other binding molecule that recognizes a surface marker on the cells. In some aspects, the antibody or other molecule is coupled to a magnetic response particle or magnetic particle, such as a bead. In some aspects, the selection includes a positive and / or negative selection step.

[0025] In some embodiments, CD8 + isolation of the primary human T cell population includes depleting or enriching a subpopulation of CD8 + cells. In some embodiments, CD4 + isolation of the primary human T cell population includes depleting or enriching a subpopulation of CD4 + cells. In some aspects, isolation of the T cell population includes enriching for T CM cells. In some aspects, CD8 + isolation of the primary human T cell population and / or CD4 + isolation of the primary human T cell population includes enriching for central memory T (T CM ) cells. In some aspects, enrichment of central memory T (T CM ) cells is by negative selection of cells expressing a surface marker present on naive T cells, such as CD45RA, or positive selection for cells expressing a surface marker present on central memory T cells and not present on naive T cells, such as CD45RO; and / or for central memory T (T CMIncluding positive selection of cells that express surface markers that are present in the cells and not present on another memory T cell subset.

[0026] In some embodiments, the isolation comprises (i) subjecting a sample to positive selection based on surface expression of CD4 to yield a positive fraction and a first negative fraction that are an isolated CD4 + population; and (ii) generating a second negative fraction by subjecting the first negative fraction to negative selection based on surface expression of non-T cell markers and surface markers present on naive T cells; and (iii) subjecting the second negative fraction to positive selection based on surface expression of a marker that is present on the surface of central memory T (T CM ) cells and not present on the surface of another memory T cell subset.

[0027] In some aspects, the marker present on naive T cells includes CD45RA. In some aspects, the surface marker that is present on central memory T (T CM ) cells and not present on another memory T cell subset includes CD62L, CCR7, CD27, CD127, and / or CD44.

[0028] In some aspects, the isolation or selection is performed in the same separation chamber. In some aspects, the isolation comprises (i) generating a CD4 + primary human T cell population and a sample that was not selected with one of a CD4 + primary human T cell population by subjecting the sample to a first selection; and (ii) generating the other of the CD4 + primary human T cell population and a CD8 + primary human T cell population by subjecting the unselected sample to a second selection. In some aspects, the CD4 + primary human T cell population is generated in the first selection and the CD8 + primary human T cell population is generated in the second selection. In some aspects, the first and / or second selection includes multiple positive selection steps or negative selection steps.

[0029] In some aspects, the isolation of one or more populations, such as a primary human T cell population, a primary human CD4+ T cell population, or a primary human CD8+ T cell population, includes positive selection based on the surface expression of CD62L, CCR7, CD44, or CD27. In some aspects, the primary human T cell population, the primary human CD4 + T cell population, or the primary human CD8 + The isolation of one or more populations, such as a T cell population, includes negative selection based on the surface expression of CD45RA or positive selection based on the surface expression of CD45RO.

[0030] In some embodiments, the isolation of multiple cell populations, such as CD4 + populations and CD8 + populations, is performed in the same separation vessel. In some aspects, the separation vessel is, or includes, a tube, a tubing set, a chamber, a unit, a well, a culture vessel, a bag, and / or a column. In some aspects, the separation vessel is maintained in an environment that confines the cells or in a sterile environment during separation.

[0031] In some embodiments, the incubating step is performed under stimulating conditions. In some aspects, the starting culture composition includes a primary human CD4 + T cell population and a primary human CD8 + T cell population at a starting culture ratio. In some aspects, the starting culture ratio is designed to obtain a desired output ratio (or a desired total number of T cells or subset numbers) of CD4 + cells to CD8 + cells after incubation, or at a somewhat later time, such as after manipulation, cryopreservation, or immediately prior to administration, such as bedside thawing.

[0032] In some embodiments, the desired output ratio is between 5:1 or about 5:1 and 1:5 or about 1:5 (or greater than about 1:5 and less than about 5:1), for example, between 1:3 or about 1:3 and 3:1 or about 3:1 (or greater than about 1:3 and less than about 3:1), for example, between 2:1 or about 2:1 and 1:5 or about 1:5 (or greater than about 1:5 and less than about 2:1), or is in the range of 2:1 or about 2:1 to 1:5 or about 1:5. In some aspects, the desired output ratio is 3:1, 2.9:1, 2.8:1, 2.7:1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5, or approximately those ratios.

[0033] In some aspects, the method results in a ratio of two different cell types or populations, such as the ratio of CD4 + cells to CD8 + cells in the output composition, and the ratio is between 5:1 or about 5:1 and 1:5 or about 1:5 (or greater than about 1:5 and less than about 5:1), for example, between 1:3 or about 1:3 and 3:1 or about 3:1 (or greater than about 1:3 and less than about 3:1), for example, between 2:1 or about 2:1 and 1:5 or about 1:5 (or greater than about 1:5 and less than about 2:1), or the ratio is 3:1, 2.9:1, 2.8:1, 2.7:1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5, or approximately those ratios.

[0034] In some aspects, the desired output ratio is 1:1 or about 1:1.

[0035] In some aspects, the method yields a desired output ratio or cell number in the output composition, yields a ratio or number in the output composition within a specific tolerance or error range of such desired output ratio or number, and / or yields such ratio or number at a specific percentage of the time the method is performed, such as at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% of the time.

[0036] In some aspects, the tolerance is within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio. In some aspects, the output ratio is within 20% of the desired ratio and / or falls within that ratio for at least 80% of the time the method is performed.

[0037] In some embodiments, the tolerance and / or the desired output ratio or number is determined or has been determined by administering different cell types at a plurality of test ratios or numbers to one or more subjects, for example CD4 + cells and CD8 + cells, and evaluating one or more parameters. In some aspects, the determination of the desired output ratio or number or tolerance includes evaluating one or more results after administration to the subject. In some aspects, the results include results selected from among remission of disease symptoms and results indicating safety and / or low toxicity or no toxicity.

[0038] In some embodiments, the starting culture ratio is CD4 + primary human T cell population and / or CD8 +Selected based on the growth rate or viability of isolated various cell types, such as a primary human T cell population. In some embodiments, the starting culture ratio is selected based on the origin of the sample, such as the subject from which the sample is derived. For example, in some aspects, the sample is derived from a subject, and the starting culture ratio is selected based on the disease or condition affecting the subject and / or the treatments the subject is receiving, has received, or will receive, such as co-treatments for administration using adoptive cell therapy. In some aspects, the starting culture ratio is selected based on the phenotype of one or more isolated or cultured cells or cell subtypes, such as the phenotype of a CD4+ cell population and / or a CD8+ cell population, such as the expression of cell surface markers or the synthesis or secretion of one or more factors such as cytokines or chemokines.

[0039] In some embodiments, the method includes the step of selecting a starting culture ratio prior to the incubation step. In some aspects, the selection is CD4 + Primary human T cells and / or isolated CD8 + Performed by measuring the growth rate or viability of one or more isolated cell populations or incubated cell populations, such as a primary human T cell population. In some aspects, the selection is isolated CD4 + Primary human T cells and / or isolated CD8 + Performed by evaluating the phenotype of primary human T cells. In some aspects, the phenotype is selected from the expression of surface markers and the secretion of cytokines or other factors. In some aspects, the step of selection is performed by evaluating the origin of the sample, such as when the starting culture ratio is selected based on the disease or condition affecting the subject from which the sample is derived.

[0040] In some embodiments, the method is CD4 present in the culture vessel at a time point following the start of incubation + Cells and CD8 +It further includes a step of determining an intermediate ratio or number of cells, such as an intermediate ratio between cells. In some aspects, the method further includes a step of adjusting one or more parameters based on the intermediate ratio, and / or increasing or decreasing the time for performing the incubation, and / or a step of operating. In one aspect, the adjusting step is CD4 in the culture tank + cells or CD8 + increasing or decreasing the number of one or more cell populations in the culture tank or enriching it, such as enriching CD4 cells or CD8 cells, adjusting the temperature, adding a stimulant to the culture tank, adjusting the concentration of one or more stimulants in the culture tank, and / or adding and / or removing a subpopulation of cells from the culture tank. In some aspects, the determining step and / or the adjusting step are performed while maintaining the composition incubated in a sterile environment or a contained environment. In some aspects, the determining and / or adjusting steps are performed in an automated manner, such as in a manner controlled by a computer attached to the device in which the steps are performed.

[0041] In some aspects, the steps of isolating, incubating, and / or operating are performed in an automated manner, such as in a sterile environment or a contained environment and / or in a manner controlled by a computer attached to the device in which the steps are performed.

[0042] In some aspects, the CD8 + population in the starting culture composition contains at least 50% central memory T (T CM ) cells or less than 20% naive T (T N ) cells.

[0043] In some embodiments, the sample is obtained from a subject. In some aspects, the subject is a subject to whom genetically engineered cells, such as T cells, or cells for adoptive cell therapy are administered, or a subject in need of such administration. In other aspects, the subject is a subject other than a subject to whom genetically engineered cells, such as T cells, or cells for adoptive therapy are administered, or a subject who does not require such treatment. Samples include blood and blood-derived samples such as leukocyte samples, apheresis samples, leukapheresis samples, peripheral blood mononuclear cell (PBMC) samples, and whole blood.

[0044] In some aspects, the stimulation conditions for incubation or manipulation include conditions under which the T cells of the starting culture composition proliferate or expand. For example, in some aspects, incubation is performed in the presence of an agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex, such as the CD3 zeta chain, or capable of activating activation signaling via such a complex or component. In some aspects, incubation is performed in the presence of an anti-CD3 antibody, and an anti-CD28 antibody, an anti-4-1BB antibody, such as an antibody coupled to a solid support such as beads or an antibody present on the surface of a solid support, and / or a cytokine such as IL-2, IL-15, IL-7, and / or IL-21.

[0045] In some embodiments, the genetically engineered antigen receptor is a T cell receptor (TCR), such as a high-affinity TCR, or a functional non-TCR antigen receptor, such as a chimeric antigen receptor (CAR), or includes the same. In some aspects, the receptor specifically binds to an antigen expressed by the cells of the disease or condition to be treated. In some aspects, the CAR includes an extracellular antigen recognition domain. In some aspects, it further includes an intracellular signaling domain that includes an ITAM-containing sequence and an intracellular signaling domain of a T cell costimulatory molecule.

[0046] Also provided are cells and compositions comprising a pharmaceutical composition produced by any method or manner, including genetically engineered cells and cells for adoptive cell therapy. Also provided are methods for administering such cells and compositions to a subject and the use of such cells and compositions in such methods. For example, provided are treatment methods implemented by producing cells according to a cell production method and administering to a subject a cell of the output composition or a composition derived therefrom. Provided is a treatment method comprising the step of administering to a subject the provided cells or composition. In some aspects, the sample from which the cells are isolated is derived from the subject to whom the cells are administered. In some aspects, the sample is derived from a different subject. Thus, the methods include autologous and allogeneic methods. In some embodiments, the method alleviates, treats, or prevents one or more symptoms of a disease or condition in a subject. In some aspects, the disease or condition is cancer or an associated symptom. In some embodiments, cancers include leukemia, lymphoma, such as chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, low-grade B-cell lymphoma, B-cell malignancies, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin (including melanoma) cancer, bone cancer, and brain cancer, ovarian cancer, epithelial cancer, renal cell cancer, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and / or mesothelioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] [[FIG. 1A]]Provide a schematic diagram of one aspect of a closed system for use in the aspects of the provided method. The exemplary system shown includes a cell sample 5, a wash buffer reservoir 6, an elution buffer reservoir 7, a first Fab reservoir 18, a second Fab reservoir 19, and a pump 8, which are connected via a series of tubing and valves 13 to a first chromatography column 1 containing a first matrix 3, and the first chromatography column is operably coupled via a series of tubing lines to a second chromatography column 2 containing a second matrix 4. The second chromatography column 2 is operably coupled to a removal chamber 9. The removal chamber 9 is operably coupled via a series of tubing lines to a valve 13 that directs cells and fluid to a waste container 10 or a culture tank 12. The system is enclosed within an enclosure 14. [[FIG. 1B]]Provide a schematic diagram of one embodiment of a closed system for use in the provided method embodiments. The exemplary system shown includes a cell sample 5, a wash buffer reservoir 6, an elution buffer reservoir 7, a first Fab reservoir 18, a second Fab reservoir 19, a third Fab reservoir 20, and a pump 8, which are connected via a series of tubing to a first chromatography column 1 containing a first matrix 3. A valve 13 operably connected to the series of tubing directs fluid via the series of tubing. The valve 13 operably connected to the first chromatography column 1 directs cells and fluid to a second chromatography column 2 containing a second matrix 4, and the second chromatography column 2 is operably connected to a removal chamber 9. The valve 13 operably connected to the first chromatography column 1 also directs cells and fluid to the removal chamber 9 operably connected to a third chromatography column 15 containing a third matrix 16. The third chromatography column 15 is operably connected to the removal chamber 9. The removal chamber 9 is operably connected to a first waste container 10 or a culture tank 12 via a series of tubing lines and valves 13. The system is enclosed within an enclosure 14.

Embodiments for Carrying Out the Invention

[0048] Detailed description Unless otherwise defined, all technical and scientific terms or terminology used herein, including all specialized terms, symbols, and other technical and scientific terms, are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, but the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0049] All publications, including patent documents, scientific literature, and databases, referred to in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were incorporated by reference separately. If the definitions set forth herein are contrary to or otherwise inconsistent with the definitions set forth in patents, applications, published applications, and other publications incorporated by reference herein, the definitions set forth herein shall control over the definitions incorporated by reference herein.

[0050] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0051] I. Methods and systems for isolating, culturing, and manipulating cells for adoptive therapy Methods for preparing cells, such as T cells, for use in therapies such as gene manipulation and adoptive cell therapy are provided. Specifically, in some embodiments, the method involves using or generating a composition comprising a plurality of different cell populations or cell types, such as isolated CD4 + and CD8 + T cell populations and subpopulations. In some embodiments, the method includes a step for isolating one or more cell populations, generally a plurality of cell populations. The cells are generally isolated from a sample derived from a subject.

[0052] In some aspects, the method is performed by using simultaneous or sequential selection or enrichment, in which a plurality of different cell populations, such as CD4+ cells or CD8+ cells, are selected, enriched, and / or isolated from a sample, such as a sample containing primary human T cells. In some embodiments, such a method is performed using a single processing stream in which a first population of cells, such as CD4+ cells or CD8+ cells, and a second population of cells, such as the other of CD4+ cells or CD8+ cells, are selected, enriched, and / or isolated, wherein no cells are discarded from the first population and thereafter the second population of cells is selected. In some aspects, the first and second selections can be performed simultaneously or sequentially. In some embodiments, the selection method is performed as a single process stream by performing a first selection to enrich a first population of cells, such as one of CD4+ and CD8+ cells, from a sample, such as a sample containing primary human T cells, and using the cells not selected from the first selection as a source of cells for a second selection, such as the other of CD4+ cells or CD8+ cells from the sample.

[0053] In some embodiments, one or more additional selections of the first or second selected cells can be performed. In some embodiments, the additional selection enriches for a subpopulation of CD4+ or CD8+ cells that express a marker on central memory T (T CM ) cells and / or enriches for a subpopulation of cells that express CD62L, CD45RA, CD45RO, CCR7, CD27, CD127, or CD44.

[0054] In some embodiments, the selection method is performed in a closed system or device. In some embodiments, within the closed system or device, a composition such as a culture initiation composition is produced that contains enriched or selected cell populations, such as both an enriched or selected CD4+ population and an enriched or selected CD8+ population, in the same composition.

[0055] For example, in some aspects, one or more steps are performed using multiple cell populations combined in the same composition, or multiple cell populations present in the same vessel, e.g., the same closed system or apparatus, or the same column, e.g., a magnetic separation column, tube, tubing set, culture or cultivation chamber, culture tank, processing unit, cell separation tank, centrifuge chamber, etc., or using the same separation matrix, medium, and / or reagent, e.g., the same magnetic or magnetoresponsive matrix, particles, or beads, the same solid support, e.g., an affinity-labeled solid support, and / or the same antibody and / or other binding partner, e.g., a fluorescently labeled antibody and binding partner, for multiple cell populations. In some embodiments, one or more vessels, units or chambers, such as columns, are operably connected in the same closed system or apparatus such that the method of isolation, selection and / or enrichment occurs in a single process flow where cell populations not selected from a first selection can be used as a source of cells for a second selection.

[0056] In some aspects, the isolation or enrichment of one or more specific populations or subpopulations of cells, e.g., CD4+ T cells and CD8+ T cells to be cultured or engineered for adoptive therapy, provides one or more advantages. For example, engineering cells enriched for multiple different cell populations or cell types, such as populations and subpopulations of isolated CD4 + T cells and CD8 + T cells, can improve efficacy or reduce or avoid unwanted effects. In some aspects, the isolation or enrichment increases the ability of the cells ultimately administered to the subject to persist, expand, activate, and / or engraft in vivo or when administered to the subject. In some aspects, it improves or increases one or more effector functions or activation phenotypes. For example, in some aspects, the central memory (T CM)Enriching cells can provide such advantages. In some aspects, one or more such advantages are provided by combining isolated populations or subpopulations of CD8+ T cells and CD4+ T cells, such as T CM enriched CD8 + populations and CD4 + populations, such as by combining two or more isolated populations or subtypes. For example, such advantages can, in some aspects, be achieved by administering a CD4+ population and a CD8+ population as compared to the CD8+ population alone.

[0057] The method, in some embodiments, includes processing the resulting composition containing a plurality of isolated or selected cell populations, such as a population of selected or enriched CD4+ cells and CD8+ cells. In one embodiment, processing the resulting composition includes incubating the cells under stimulating conditions to activate the cells, for example, in some aspects, for engineering or transduction, or for cell expansion. The method, in some embodiments, includes steps for engineering a plurality of cell types, such as CD4+ cells and CD8+ cells, present in an isolated, incubated composition such as a starting culture composition. In some aspects, engineering is performed to introduce into the cells a genetically engineered antigen receptor such as a TCR, e.g., a high-affinity TCR, or a functional non-TCR antigen receptor such as a chimeric antigen receptor (CAR). In some aspects, the method includes further processing, such as further incubation at, e.g., 37°C ± 2°C or about 37°C ± 2°C, and / or formulation of the cells and the composition containing them. In some embodiments, the processing produces a resulting output composition containing genetically engineered cells such as genetically engineered CD4+ cells and CD8+ cells. In some embodiments, the resulting, processed output composition can be used in methods for administering the cells and compositions prepared by the method to a patient, e.g., in the context of adoptive cell therapy.

[0058] In certain embodiments, isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method. In some aspects, the system is used to perform each of these steps in a closed or sterile environment to minimize, for example, errors, user handling, and / or contamination. In one example, the system is the system described in International Patent Application Publication No. WO2009 / 072003 or US20110003380A1. In some embodiments, the system is a closed device or system as shown in FIG. 1A or FIG. 1B. In some embodiments, the system or apparatus is automated and / or performs the selection step of enriching cells according to the method in an automated manner.

[0059] In some embodiments, the system or apparatus performs an isolation such as a selection step or an enrichment step. In some embodiments, a further system or apparatus such as a closed system or apparatus can be used to perform one or more other steps of the method, such as the cell preparation, processing, incubation, culture, and / or formulation steps. In some embodiments, the system or apparatus is an integrated or self-sufficient system and / or performs one or more, for example all, of the isolation, processing, manipulation, and formulation steps in an automated or programmable manner. In some aspects, the system or apparatus includes a computer and / or computer program that communicates with the system or apparatus and that enables a user to program, control, evaluate the results of, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0060] In some embodiments, enriched compositions such as the culture start composition are generated to contain different cell populations such as CD4+ and CD8+ by the provided method, so the generated compositions can be processed together and simultaneously under the same conditions and, in some aspects, in a closed system. Thus, in some aspects, the method provides steps for producing and processing a selected, enriched, or isolated population of cells containing different populations of cells such as CD4+ cells and CD8+ cells in a single process flow. In some aspects, this is different from prior art methods, which typically involve separate process flows, such as at least two process flows for each different cell population, for processing cells for adoptive cell therapy. For example, in some aspects of existing methods, CD4+ T cells are isolated, enriched, and / or selected separately and processed under stimulatory conditions for genetic manipulation, CD8+ T cells are isolated, enriched, and / or selected separately and processed under stimulatory conditions for genetic manipulation, the separately processed and manipulated CD4+ T cells and CD8+ T cells are recombined, and then administered to a subject.

[0061] In some embodiments, the method provides one or more advantages such as cost, time, and / or resource savings compared to other preparation, isolation, incubation, and manipulation methods. Such advantages can include being able to isolate, process, e.g., incubate, and / or manipulate multiple cell populations that are present at or near a desired ratio with increased efficiency and / or decreased complexity, time, cost, and / or resource use compared to other methods.

[0062] In some embodiments, such advantages are achieved by streamlining one or more method steps. For example, in some aspects, the isolation, culture, and / or manipulation of different populations are performed using the same apparatus or equipment and / or simultaneously. In some aspects, the isolation, culture, and / or manipulation of different populations are performed based on the same starting composition. In some aspects, such features of the method reduce the time, number of method steps, cost, amount of complexity, and / or number of resources compared to a method in which cell populations are isolated, incubated, and / or manipulated separately in separate vessels, using separate equipment, at separate times, and / or starting with different starting compositions.

[0063] In some aspects, a method of isolating, incubating, and manipulating cells results in an output composition in which different cell populations or cell types, such as CD4+ cells and CD8+ cells, are present at a desired ratio or within a certain tolerance of the desired ratio. Such ratios include ratios considered optimal for therapeutic use, e.g., output ratios considered appropriate or optimal for administration to a patient in the context of adoptive cell therapy. Also provided are methods for administering to a patient cells and compositions prepared by the method, e.g., in the context of adoptive cell therapy.

[0064] In some embodiments, a method of isolation or selection is performed to achieve selection of cells at a selected starting ratio of CD4+ cells to CD8+ cells or a subpopulation thereof. In some aspects, such a ratio is considered an optimal starting point for achieving an optimal ratio, such as a desired ratio in the output composition, at the completion of the method or one or more steps, such as after the culture, incubation, and / or manipulation steps. In some embodiments, providing cells at a starting ratio, such as a ratio of CD4+ cells to CD8+ cells, to achieve a desired ratio in the output composition accounts for differences in the expansion of CD4+ T cells and CD8+ T cells that may occur when stimulated or activated using different reagents.

[0065] In some embodiments, the method produces, or produces at least a certain percentage of the time, engineered cells or cells for engineering, within or at a certain percentage of a desired output ratio. The desired output ratio is typically the ratio considered optimal for administration to a patient via adoptive transfer. In some embodiments, CD8 + populations or subtypes of cells such as T cells and CD4 + T cells are administered within or at the tolerance of the desired dose of all cells such as the desired dose of T cells. In some aspects, the desired dose is the desired number of cells, or the desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at least the minimum number of cells or the minimum number of cells per unit of body weight. In some aspects, within the total cells administered at the desired dose, an individual population or subtype is present at or near the desired output ratio (e.g., the ratio of CD4 + to CD8 + ), e.g., within a certain tolerance or margin of error of such a ratio.

[0066] In some embodiments, the cells are administered within or at the tolerance of one or more individual cell populations or subtypes of the desired dose, such as the desired dose of CD4+ cells and / or the desired dose of CD8+ cells. In some aspects, the desired dose is the desired number of cells of a subtype or population, or the desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at least the minimum number of cells of a population or subtype, or the minimum number of cells of a population or subtype per unit of body weight.

[0067] Thus, in some embodiments, the dosage is based on the desired fixed dose of all cells and the desired ratio, and / or on one or more of the individual subtypes or subpopulations, e.g., their respective desired fixed doses. Thus, in some embodiments, the dosage is based on the desired fixed or minimum dose of T cells and the desired ratio of CD4 + to CD8 + cells, and / or on CD4+ cells and / or CD8 + based on a desired fixed dose or minimum dose of the cells.

[0068] In some embodiments, methods are provided for determining such optimal output ratios and / or desired doses, and / or acceptable levels of variance, such as tolerances, from a desired output ratio or desired dose. In some embodiments, the cell population is combined or incubated at a ratio (e.g., a starting culture ratio) designed to achieve a desired output ratio for adoptive transfer, in order to achieve the desired output ratio or dose (or to achieve such ratio within a particular percentage of time or within a particular tolerance).

[0069] Methods are also provided for determining a starting culture ratio designed to achieve a desired output ratio or dose within a particular tolerance and / or within a particular percentage of time. Methods are also provided for assessing a provisional ratio or number of a cell population over the course of various method steps, such as one or more periods during incubation. Based on such assessment, methods are also provided for adjusting various conditions, such as culture conditions. In some aspects, the adjustment is performed to confirm that a particular output ratio or dose is achieved or achieved within tolerance.

[0070] In certain embodiments, CD4 + T cell populations and CD8 + T cell populations (e.g., CD8 + populations enriched in subtypes of T cells, such as central memory T cells) at or near a desired output ratio, and / or T cells and / or CD4 + T cells and CD8 +A rationalized method is provided for preparing a composition having T cells at or near a desired dose (e.g., a number per unit of body weight or number). In this case, cell populations are combined, isolated, incubated, and / or manipulated, and the method is associated with increased efficiency and / or decreased complexity, time, cost, and / or resource use compared to methods in which the populations are isolated, incubated, and / or manipulated separately.

[0071] Also provided are cells and compositions, including pharmaceutical compositions and formulations, prepared by the method, as well as kits, systems, and devices for carrying out the method. Also provided are therapeutic methods, such as methods for adoptive cell therapy, for the use of cells and compositions prepared by the method, and methods including pharmaceutical compositions for administration to a subject.

[0072] A. Isolation, Isolated Cells, and Other Processing Steps Aspects provided also include methods for isolating multiple cells and cell populations from a sample, and isolated cells, such as enriched cells, produced by such methods. Isolation can include one or more various cell preparation and separation steps, including separation based on one or more properties such as size, density, sensitivity or resistance to a particular reagent, and / or affinity for an antibody or other binding partner, such as immunological affinity. In some aspects, isolation is performed sequentially and / or simultaneously in a single process flow using the same apparatus or equipment. In some aspects, the isolation, culture, and / or manipulation of different populations is performed from the same starting composition or material, such as from the same sample.

[0073] In some aspects, a plurality of cell populations are isolated within the same closed system or apparatus, and / or within the same tank or set of tanks, such as the same (or the same set of) units, chambers, columns, such as magnetic separation columns, tubes, tubing sets, culture or cultivation chambers, culture vessels, processing units, cell separation vessels, centrifuge chambers. For example, in some cases, the isolation of a plurality of cell populations is performed in a system or apparatus using a single column or set of columns, and / or a single or the same isolation or separation vessel or set of vessels, such as a single tube, or tubing set, without the need to transfer cell populations, compositions, or suspensions from one vessel, such as a tubing set, to another.

[0074] In some aspects, such methods are achieved by using simultaneous or sequential selection in which a plurality of different cell populations, such as CD4+ cells or CD8+ cells, are selected, enriched, and / or isolated from a sample, such as a sample containing primary human T cells, in a single process stream, for example, in a closed system. In one aspect, a sample containing cells is subjected to selection by simultaneous enrichment of both the CD4+ population and the CD8+ population. In some aspects, performing separation or isolation in the same vessel or set of vessels, such as a tubing set, is achieved by performing sequential positive and negative selection steps, subsequent steps in which the negative and / or positive fractions from the previous step are subjected to further selection, where the entire process is performed within the same tube or tubing set. In one aspect, a sample containing the cells to be selected is subjected to sequential selection, in which a first selection is made to enrich one of the CD4+ population or the CD8+ population, and the cells not selected from the first selection are used as a source of cells for a second selection to enrich the other of the CD4+ population or the CD8+ population. In some aspects, one or more additional selections can be made to enrich subpopulations of one or both of the CD4+ population or the CD8+ population, such as central memory T (T CM ) cells.

[0075] In one particular aspect, the first selection step is carried out using beads labeled with a CD4 binding molecule, such as an antibody (or a second reagent that recognizes such a molecule), and the positive and negative fractions from the first selection step are retained, and for example, further positive or negative selection of the negative fraction is carried out by using beads labeled with a CD8 binding molecule to enrich for CD8+ cells, and optionally, central memory CD8 from the CD8+ fraction + T cells and / or a subpopulation of cells expressing one or more of the markers CD62L, CD45RA, CD45RO, CCR7, CD27, CD127, or CD44 are selected. In some embodiments, the order of selection can be reversed. In some embodiments, CD4 selection is always carried out first, and in a second selection, from the negative fraction (CD4-), for example CD45RA + and one or more negative selections of CD14, and / or one or more positive selections of CD62L, CCR7, and / or other markers expressed on central memory cells enrich the CD8+ fraction.

[0076] In some embodiments, multiple cell populations, such as CD4 + T cell populations and CD8 + T cell populations are isolated to produce, for example, a culture starting composition containing multiple cell populations. The culture starting composition typically contains cells at a starting culture ratio designed to result in a specific desired output ratio of two or more cell types, such as a specific ratio of CD4 + to CD8 + after one or more incubation, culture, cultivation, and / or manipulation steps. The desired output ratio in some embodiments is a ratio designed to be optimal for administration of the cells to a patient, for example, in adoptive cell therapy.

[0077] In some aspects, isolating multiple populations within a single column or column set, and / or within a single or same isolation or separation tank or set of tanks such as the same tube or tubing set, or using the same separation matrix or medium or reagent such as the same magnetic matrix, affinity label solid support, or antibody or other binding partner, includes features that rationalize the isolation, resulting in, for example, a reduction in cost, time, complexity, need for sample handling, resources, reagents, or use of equipment. In some aspects, such features are advantageous in that they minimize the cost, efficiency, time, and / or complexity associated with the method and / or avoid potential harm to cell products such as harm caused by infection, contamination, and / or temperature changes.

[0078] In some embodiments, the isolated cell populations obtained for use in the methods herein are sterile. Microbial contamination of cell separation products can, in some cases, lead to infection of recipient subjects such as immunocompromised recipient patients who may not be able to fight the infection. In some embodiments, the cells, cell populations, and compositions are produced under Good Manufacturing Practice (GMP) conditions. In some embodiments, GMP conditions include stringent batch testing. In certain embodiments, histocompatibility testing is performed prior to transplantation, for example, to avoid mismatches of human leukocyte antigens (HLA) and prevent problems such as graft-versus-host disease. In some embodiments, the provided methods reduce handling by individual users and automate various steps. This can, in some aspects, promote consistency in treatment and safety by increasing the consistency of isolated cell populations and compositions and reducing errors.

[0079] 1. Cells and Cell Populations In some embodiments, the method includes the steps of selecting, isolating and / or enriching a cell sample, such as a primary human cell sample. The isolated cell population typically includes a plurality of cell populations, usually blood or blood-derived cells such as hematopoietic cells, leukocytes (white blood cells), peripheral blood mononuclear cells (PBMC), and / or cells of the immune system, such as myeloid or lymphoid cells, such as lymphocytes, typically cells of innate or adaptive immunity such as T cells and / or NK cells. In some embodiments, the sample is an apheresis sample and the leukapheresis sample. In some embodiments, the selection, isolation and / or enrichment can include positive or negative selection of cells from the sample.

[0080] In some embodiments, the sample is a sample containing primary human T cells such as CD4+ T cells and CD8+ T cells. In certain embodiments, the sample is CD4 + cell population and CD8 + A sample from which a plurality of T cell populations such as T cell populations are isolated. Thus, in some embodiments, the isolation includes positive selection for cells expressing CD4 or CD8 and / or negative selection for cells expressing non-T cell markers such as myeloid cell markers or B cell markers, e.g., CD14, CD19, CD56, CD20, CD11b, and / or CD16.

[0081] In some embodiments, the sample is a sample containing a plurality of populations including a T cell population such as the total T cells or a CD4+ population and an NK cell population. Among the T cell populations that can be enriched, isolated and / or selected, there are included subpopulations of CD4+ T cells and / or CD8+ T cells, including unfractionated T cells, unfractionated CD4+ cells, unfractionated CD8+ cell populations, and subpopulations of T cells generated by enrichment or depletion of specific subtypes of cells or cells based on a specific surface marker expression profile.

[0082] For example, T cells that can be enriched, isolated and / or selected (e.g., CD4 +T cells or CD8 + Among the subtypes of (T cells), subtypes defined by function, activation state, maturity, differentiation ability, proliferation, recirculation, localization, and / or persistence ability, antigen specificity, type of antigen receptor, presence of a specific organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation are included.

[0083] Among the subtypes and subpopulations of T cells and / or CD4+ T cells and / or CD8+ T cells that can be enriched, isolated, and / or selected, there are naive T (TN) cells, effector T cells (TEFF), stem cell memory T (TSCM) cells, central memory T (TCM) cells, effector memory T (TEM) cells, or memory T cells such as terminally differentiated effector memory T cells and their subtypes, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and helper T cells such as delta / gamma T cells.

[0084] In some embodiments, one or more T cell populations enriched, isolated, and / or selected from a sample by the provided method are cells in which one or more specific markers, such as surface markers, are positive (marker+) or highly expressed (marker high), or one or more markers are negative (marker−) or relatively lowly expressed (marker low). In some cases, such markers are absent or relatively lowly expressed on T cells of a particular population (e.g., non-memory cells), but are present or relatively highly expressed on T cells of a particular other population (e.g., memory cells). In one embodiment, cells (e.g., CD8+ cells or T cells, such as CD3+ cells) are enriched (i.e., positively selected) for cells that are positive for CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, or express a high surface level thereof, and / or depleted (e.g., negatively selected) for cells that are positive for CD45RA or express a high surface level thereof. In some embodiments, cells are enriched or depleted for cells that are positive for CD122, CD95, CD25, CD27, and / or IL7-Rα (CD127), or express a high surface level thereof. In some examples, CD45RO-positive (or CD45RA-negative) and CD62L-positive cells of CD8+ T cells are enriched.

[0085] In some embodiments, a CD4+ T cell population and a CD8+ T cell subset, such as a subset enriched for central memory (TCM) cells.

[0086] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0087] 2. Sample Cells and cell populations are typically isolated from a sample, such as a biological sample, e.g., from a subject, such as a subject having a particular disease or condition, or in need of, or to whom a cell therapy will be administered, or derived from a sample obtained from or originating from a subject. In some aspects, the subject is a human, such as a patient in need of a particular therapeutic intervention, such as adoptive cell therapy for which cells are isolated, processed, and / or manipulated therefor. Thus, in some embodiments, the cells are primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps such as separation, centrifugation, genetic manipulation (e.g., transduction using a viral vector), washing, and / or incubation. A biological sample can be a sample obtained directly from a biological origin or a processed sample. Biological samples include, without limitation, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissues as well as organ samples, including processed samples derived therefrom.

[0088] 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 related to cell therapy, e.g., adoptive cell therapy, include samples from autologous and allogeneic origins.

[0089] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are of heterologous origin, e.g., obtained from mouse, rat, non-human primate, and pig.

[0090] 3. Cell Processing, Preparation, and Separation Not Based on Affinity In some embodiments, the isolation of cells or populations involves one or more preparation steps and / or cell separation steps that are not based on affinity. In some examples, the cells are incubated in the presence of washes, centrifugation, and / or one or more reagents such that, for example, unwanted components are removed, desired components are enriched, and cells sensitive to a particular reagent are lysed or removed. In some examples, the cells are separated based on one or more properties such as density, adhesiveness, size, sensitivity and / or resistance to particular components.

[0091] In some examples, cells from the circulating blood of a subject are obtained by, for example, apheresis or leukapheresis. The sample contains, in some aspects, lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and, in some aspects, cells other than red blood cells and platelets.

[0092] In some embodiments, the blood cells collected from a subject are washed such that, for example, the plasma fraction is removed and the cells are placed 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 semi - automated "flow - through" centrifugation (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, after washing, the cells are resuspended in a variety of biocompatible buffers such as Ca ++ / Mg ++ - free PBS. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in the medium.

[0093] In some embodiments, the method includes density-based cell separation methods such as lysing red blood cells and preparing white blood cells from peripheral blood by centrifugation on a Percoll or Ficoll gradient.

[0094] 4. Separation Based on Affinity and / or Marker Profile In some embodiments, the isolation method includes separating different cell types based on the expression or presence of one or more specific molecules in the cell, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is separation based on affinity or immunoaffinity. For example, in some instances, the isolation involves incubating cells and cell populations based on the expression or expression level of one or more markers of the cell, typically cell surface markers, with an antibody or binding partner that specifically binds to such a marker, followed by a separation that usually includes a washing step and separation of cells that have bound to the antibody or binding partner from cells that have not bound to the antibody or binding partner.

[0095] Such separation steps can be based on positive selection where cells that have bound to the reagent are retained for further use and / or negative selection where cells that have not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some instances, negative selection can be particularly useful when separation is best performed based on markers expressed by cells other than the desired population due to the unavailability of antibodies that specifically identify cell types in a heterogeneous population.

[0096] Separation need not result in 100% enrichment or depletion of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular cell type, such as a cell type expressing a marker, represents an increase in the number or percentage of such cells, but need not result in the complete absence of cells not expressing the marker. Similarly, negative selection, removal, or depletion of a particular cell type, such as a cell type expressing a marker, represents a decrease in the number or percentage of such cells, but need not result in the complete removal of all such cells. For example, in some instances, selection of one of the CD4+ population or the CD8+ population enriches that population, i.e., either the CD4+ population or the CD8+ population, but may also contain some remainder or small percentage of the other unselected cells, and the unselected cells may, in some cases, still be present in the enriched population and include the other of the CD4 population or the CD8 population.

[0097] In some examples, multiple rounds of separation steps are performed where a fraction that has been positively or negatively selected in one step is then subjected to another separation step such as a subsequent positive or negative selection. In some examples, for instance, cells expressing multiple markers can be simultaneously depleted in a single separation step by incubating the cells with multiple antibodies or binding partners each specific for a marker targeted for negative selection. Similarly, positive selection of multiple cell types can be performed simultaneously by incubating the cells with multiple antibodies or binding partners expressed on the various cell types.

[0098] For example, in some instances, one or more surface markers such as CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + , and / or CD45RO +Certain subpopulations of T cells, such as T cells that are positive or cells that express them at high levels, are isolated by positive or negative selection techniques.

[0099] For example, CD3 + , CD28 + T cells can be positively selected using CD3 / CD28 conjugate magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0100] In some embodiments, isolation is performed 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 incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are expressed (marker + ) or expressed at a relatively higher level (marker high ) on the cells to be positively or negatively selected, respectively.

[0101] In some embodiments, T cells are isolated from a PBMC sample by negative selection of markers such as CD14 that are expressed on non-T cells such as B cells, monocytes, or other white blood cells. In some embodiments, the method includes isolation, selection, and / or enrichment of CD4+ and CD8+ cells. In one example, to enrich CD4 + cells by negative selection, a cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CD11b, CD16, and HLA-DR. In one example, enrichment of the CD8 + population by negative selection is performed by depletion of cells expressing CD14 and / or CD45RA. In some aspects, positive selection for CD4 and positive selection for CD8, such as CD4 + or CD8 + selection steps are CD4 + helper T cells and CD8 +It is used to isolate cytotoxic T cells. Such selections in some aspects are performed simultaneously and in other aspects are performed sequentially in either order.

[0102] In some aspects, the method includes a first positive selection for CD4+ cells, where the cells not selected from the first selection (CD4- cells) are used as a source of cells for a second positive selection to enrich for CD8+ cells. In some aspects, the method includes a first positive selection for CD8+ cells, where the cells not selected from the first selection (CD8- cells) are used as a source of cells for a second positive selection to enrich for CD4+ cells. Such CD4 + populations and CD8 + populations can be further sorted into subpopulations by positive or negative selection for markers expressed on or relatively more highly expressed on one or more naive cells, memory cells, and / or effector T cell subpopulations.

[0103] In some embodiments, CD4+ cells are further enriched or depleted of naive, central memory, effector memory, and / or central memory stem cells by positive or negative selection based on surface antigens associated with each population. CD4 + T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations having cell surface antigens. CD4 + lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO - , CD45RA + , CD62L + , CD4 + T cells. In some embodiments, central memory CD4 + cells are CD62L + and CD45RO + . In some embodiments, effector CD4 + cells are CD62L- and CD45RO.

[0104] In some embodiments, CD8 + cells are further enriched or depleted of naive, central memory, effector memory, and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (T CM ) cells is performed to increase efficacy such as improving long-term survival, expansion, and / or engraftment after administration, and the efficacy is particularly strong in some aspects 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, enriching T CM in CD8 + T cells and combining with CD4 + T cells further enhances efficacy.

[0105] In embodiments, memory T cells are present in both the CD62L + subset and the CD62L- subset of peripheral blood lymphocytes. For example, using anti-CD8 and anti-CD62L antibodies, PBMCs can be enriched or depleted of the CD62L-CD8 + fraction and / or the CD62L + CD8 + fraction. + can be enriched or depleted.

[0106] In some embodiments, enrichment of central memory T (T CM ) cells is based on CD45RO, CD62L, CCR7, CD28, CD3, CD27 and / or CD127 positive or high surface expression; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B.

[0107] In some embodiments, the provided method includes isolation, selection, and / or enrichment of CD8+ cells from a sample, such as by positive selection based on surface expression of CD8. In some embodiments, the method can further include enriching central memory T (T CM ) cells. In one aspect, central memory T (T CM ) cells can be further enriched by selecting one or more markers expressed on central memory T (T CM ) cells, such as one or more of CD45RO, CD62L, CCR7, CD28, CD3, CD27, and / or CD127, from the enriched CD8+ cells. The selection can be performed before or after isolation, selection, and / or enrichment of CD4+ cells. Such selection in some aspects is performed simultaneously and in other aspects is performed sequentially in either order.

[0108] In some aspects, the method includes a first positive selection for CD4+ cells, where the cells not selected from the first selection (CD4− cells) are used as a source of cells for a second selection to enrich CD8+ cells, and the enriched or selected CD8+ cells are used in a third selection to further enrich cells expressing one or more markers expressed on central memory T (T CM ) cells, such as by a third selection to enrich CD45RO+, CD62L+, CCR7+, CD28+, CD3+, CD27+, and / or CD127+ cells. In some aspects, the method includes a first positive selection for CD8+ cells, where the cells not selected from the first selection (CD8− cells) are used as a source of cells for a second selection to enrich CD4+ cells, and the CD8+ cells enriched or selected from the first selection are also used in a third selection to further enrich cells expressing one or more markers expressed on central memory T (T CM ) cells, such as by a third selection to enrich CD45RO+, CD62L+, CCR7+, CD28+, CD3+, CD27+, and / or CD127+ cells.

[0109] In one aspect, T CM CD8-enriched + Isolation of the population is performed by depletion of cells expressing CD4, CD14, CD45RA, and by positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment of central memory T (T CM ) cells begins and is performed on the negative fraction of cells selected based on CD4 expression, and the negative fraction is subjected to negative selection based on the expression of CD14 and CD45RA and positive selection based on CD62L. Such selection in some aspects is performed simultaneously and in other aspects sequentially in either order. In some aspects, the same CD4-expression-based selection step used to prepare a CD8 + cell population or subpopulation is also used to generate a CD4 + cell population or subpopulation, whereby both the positive and negative fractions from the CD4-based separation are optionally retained after one or more additional positive or negative selection steps and used in subsequent steps of the method.

[0110] In a specific example, a sample of PBMCs or other white blood cell sample is subjected to CD4 + cell selection, where both the negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA or CD19 and positive selection based on the marker characteristics of central memory T cells such as CD62L or CCR7, where the positive and negative selections are performed in either order.

[0111] In some embodiments, for example, a method of isolating, selecting, and / or enriching cells by positive or negative selection based on the expression of one or more cell surface markers, by any of the methods described above, can include selection based on immunoaffinity. In some embodiments, selection based on immunoaffinity involves contacting a sample containing cells, such as primary human T cells containing CD4+ and CD8+ cells, with an antibody or binding partner that specifically binds to one or more cell surface markers. In some embodiments, the antibody or binding partner is conjugated to a solid support or matrix such as a sphere or bead, e.g., a microbead, nanobead, magnetic bead, or paramagnetic bead containing agarose, that enables the separation of cells for positive and / or negative selection. In some embodiments, the sphere or bead can be packed into a column for performing immunoaffinity chromatography, in which a sample containing cells such as primary human T cells containing CD4+ and CD8+ cells is contacted with the matrix of the column and subsequently eluted or released therefrom.

[0112] a. Immunoaffinity beads For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher (Copyright) Humana Press Inc., Totowa, NJ).

[0113] In some aspects, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetic-responsive material, such as magnetic-responsive particles like paramagnetic beads or microparticles. The magnetic-responsive material, e.g., particles, is typically directly or indirectly bound to a binding partner, such as an antibody, that specifically binds to a molecule, e.g., a surface marker, present on one or more types of cells or cell populations that it is desirable to separate, e.g., for negative or positive selection. Such beads are known and, in some aspects, are commercially available from various sources, including Dynabeads® (Life Technologies, Carlsbad, CA), MACS® beads (Miltenyi Biotec, San Diego, CA), or Streptamer® bead reagents (IBA, Germany).

[0114] In some embodiments, the magnetic particles or beads comprise a magnetic-responsive material bound to a specific binding member, such as an antibody or other binding partner. There are numerous well-known magnetic-responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday U.S. Patent No. 4,452,773 and European Patent Specification EP452342B, which are incorporated herein by reference. Colloidal-sized particles, such as those described in Owen U.S. Patent No. 4,795,698 and Liberti et al. U.S. Patent No. 5,200,084, are other examples.

[0115] The incubation typically results in the molecule, such as an antibody or binding partner bound to the magnetic particles or beads, or a secondary antibody or other reagent that specifically binds to such an antibody or binding partner, specifically binding to cell surface molecules (if present) on the cells in the sample.

[0116] In some aspects, the sample is placed in a magnetic field, and cells conjugated with magnetoresponsive or magnetizable particles are attracted to a magnet and separated from unlabeled cells. For positive selection, the cells attracted to the magnet are retained; for negative selection, the cells 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, further processed, or subjected to additional separation steps.

[0117] In certain embodiments, the magnetoresponsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, the magnetic particles are bound to cells via a coating of a primary antibody specific for one or more markers. In certain embodiments, instead of beads, cells are labeled with a primary antibody or binding partner and then magnetic particles coated with a secondary antibody or other binding partner specific for the cell type (e.g., streptavidin) are added. In certain embodiments, magnetic particles coated with streptavidin are used with biotinylated primary or secondary antibodies.

[0118] In some embodiments, the magnetoresponsive particles are left bound to the cells to be subsequently incubated, cultured, and / or manipulated; in some aspects, the particles are left bound to the cells for administration to a patient. In some embodiments, the magnetizable or magnetoresponsive 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, antibodies conjugated to magnetizable particles or cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0119] In some embodiments, affinity-based selection is magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). The magnetic-activated cell sorting (MACS) system enables high-purity selection of cells to which magnetic particles are bound. In certain embodiments, MACS operates in such a way that non-target and target species are eluted sequentially after application of an external magnetic field. That is, cells bound to the magnetic particles are retained in place and unbound species are eluted. Next, after this first elution step is complete, the species that were captured within the magnetic field and had their elution blocked are released in such a way that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous cell population.

[0120] In some embodiments, affinity-based selection involves, in part, Streptamers (registered trademark), which are magnetic beads, such as 1-2 μM nanobeads or microbeads, conjugated to a binding partner affinity reagent, such as an antibody, via a streptavidin mutant, such as Strep-Tactin® or Strep-Tactin XT® (see, for example, U.S. Patent No. 6,103,493, International Publication PCT Application WO / 2013011011, WO2014 / 076277). In some embodiments, the streptavidin mutant is functionalized, coated, and / or immobilized on the beads.

[0121] In some embodiments, the streptavidin variant exhibits a higher binding affinity for a peptide ligand comprising an amino acid sequence shown in any of SEQ ID NOs: 1-6, such as SEQ ID NO: 5 and / or SEQ ID NO: 6 (e.g., Strep-tag II®), than an unmodified or wild-type streptavidin such as the unmodified or wild-type streptavidin shown in SEQ ID NO: 11 or SEQ ID NO: 14. In some embodiments, the streptavidin variant exhibits a binding affinity as an affinity constant for such a peptide that is 5-fold, 10-fold, 50-fold, 100-fold, or 200-fold or more greater than the binding affinity of wild-type streptavidin for the same peptide.

[0122] The streptavidin mutant protein contains one or more amino acid differences compared to an unmodified streptavidin such as wild-type streptavidin or a fragment thereof. The term "unmodified streptavidin" refers to the starting polypeptide on which one or more modifications are made. In some embodiments, the starting or unmodified polypeptide can be the wild-type polypeptide shown in SEQ ID NO: 11. In some embodiments, the unmodified streptavidin is a fragment of wild-type streptavidin that is truncated at the N-terminus and / or C-terminus. Such minimal streptavidin includes any that starts at the N-terminus from the region of amino acid positions 10-16 of SEQ ID NO: 11 and terminates at the C-terminus in the region of amino acid positions 133-142 of SEQ ID NO: 11. In some embodiments, the unmodified streptavidin has the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the unmodified streptavidin as shown in SEQ ID NO: 14 can further contain an N-terminal methionine at the position corresponding to Ala13 in the numbering shown in SEQ ID NO: 11. References to residue numbers in the streptavidin provided herein refer to the numbering of residues in SEQ ID NO: 11.

[0123] The terms "streptavidin mutant protein", "streptavidin variant" or variations thereof refer to streptavidin proteins that contain one or more amino acid differences compared to unmodified or wild-type streptavidin such as that shown in SEQ ID NO:11 or SEQ ID NO:14. The one or more amino acid differences can be amino acid mutations such as the exchange (substitution), insertion or deletion of one or more amino acids. In some embodiments, the streptavidin mutant protein can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid differences compared to wild-type or unmodified streptavidin. In some embodiments, the amino acid exchange (substitution) is a conservative or non-conservative mutation. The streptavidin mutant protein containing one or more amino acid differences exhibits a binding affinity as a dissociation constant greater than 2.7×10 4 M -1 for the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also called Strep-tag® shown in SEQ ID NO:5). In some embodiments, the streptavidin variant exhibits a binding affinity as a dissociation constant greater than 1.4×10 4 M -1 for the peptide ligand (Trp Ser His Pro Gln Phe Glu Lys; also called Strep-tag® II shown in SEQ ID NO:6). In some embodiments, the binding affinity can be determined by methods known in the art such as any of the following.

[0124] In some embodiments, the streptavidin mutant protein comprises mutations at one or more of residues 44, 45, 46, and / or 47. In some embodiments, the streptavidin variant comprises the residues Val44 - Thr45 - Ala46 - Arg47 as shown in the exemplary streptavidin mutant protein set forth in SEQ ID NO:12 or SEQ ID NO:15. In some embodiments, the streptavidin mutant protein comprises the residues Ile44 - Gly45 - Ala - 46 - Arg47 as shown in the exemplary streptavidin mutant protein set forth in SEQ ID NO:13 or 16. In some embodiments, the streptavidin mutant protein exhibits the amino acid sequence set forth in SEQ ID NO: 12, 13, 15 or 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:12, 13, 15 or 16, and shows a binding affinity greater than 2.7×10 4 M -1 for the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also called Strep-tag® shown in SEQ ID NO:5), and / or greater than 1.4×10 4 M -1 for the peptide ligand (Trp Ser His Pro Gln Phe Glu Lys; also called Strep-tag® II shown in SEQ ID NO:6).

[0125] In some embodiments, the streptavidin mutant protein is a variant described in International Publication PCT Application WO2014 / 076277. In some embodiments, the streptavidin mutant protein contains at least two cysteine residues in the region of amino acid positions 44-53 in relation to the amino acid positions shown in SEQ ID NO:11. In some embodiments, the cysteine residues are present at positions 45 and 52, creating a disulfide bridge that links these amino acids. In such an embodiment, amino acid 44 is typically glycine or alanine, amino acid 46 is typically alanine or glycine, and amino acid 47 is typically arginine. In some embodiments, the streptavidin mutant protein contains at least one mutation or amino acid difference in the region of amino acid residues 115-121 in relation to the amino acid positions shown in SEQ ID NO:11. In some embodiments, the streptavidin mutant protein contains at least one mutation at amino acid positions 117, 120 and 121 and / or deletions of amino acids 118 and 119 and at least a substitution at amino acid position 121.

[0126] In some embodiments, the streptavidin mutant protein is such that the resulting streptavidin mutant protein has a binding affinity for the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also called the Strep-tag® shown in SEQ ID NO:5) greater than 2.7×10 4 M -1 and / or for the peptide ligand (Trp Ser His Pro Gln Phe Glu Lys; also called the Strep-tag® II shown in SEQ ID NO:6) greater than 1.4×10 4 M -1 As long as it exhibits a greater binding affinity, any combination of the above mutations can be included.

[0127] In some embodiments, the binding affinity of the streptavidin mutant for the peptide ligand binding reagent is 5×10 4 M -1 、1×105 M -1 、 5×10 5 M -1 、 1×10 6 M -1 、 5×10 6 M -1 or 1×10 7 M -1 but greater than, generally 1×10 13 M -1 、 1×10 12 M -1 or 1×10 11 M -1 and less than.

[0128] In some embodiments, the streptavidin variant also binds, without limitation, to other streptavidin ligands such as biotin, iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, HABA (hydroxyazobenzene-benzoic acid) or / and dimethyl-HABA. In some embodiments, the streptavidin mutant protein has a binding affinity for another streptavidin ligand such as biotin or desthiobiotin that is greater than the binding affinity of the streptavidin mutant protein for the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also called Strep-tag® shown in SEQ ID NO:5) or the peptide ligand (Trp Ser His Pro Gln Phe Glu Lys; also called Strep-tag® II shown in SEQ ID NO:6).

[0129] In some embodiments, the streptavidin mutant protein is a multimer. The multimer can be produced using any method known in the art, such as any of those described in U.S. Patent Application Publication No. 2004 / 0082012. In some embodiments, the oligomer or polymer of the mutant protein can be prepared by introducing carboxyl residues into a polysaccharide, such as dextran. In some aspects, the streptavidin mutant protein is then coupled in a second step via the primary amino groups of internal lysine residues and / or the free N-terminus to carboxyl groups within the dextran backbone using conventional carbodiimide chemistry reactions. In some embodiments, the coupling reaction is carried out at a molar ratio of about 60 moles of streptavidin mutant per mole of dextran. In some embodiments, the oligomer or polymer can also be obtained by cross-linking via a bifunctional linker such as glutaraldehyde or by other methods known in the art.

[0130] In some aspects, immunoaffinity beads such as Streptamer® or other immunoaffinity beads can contain antibodies produced by or derived from hybridomas such as: OKT3 (αCD3), 13B8.2 (αCD4), OKT8 (αCD8), FRT5 (αCD25), DREG56 (αCD62L), MEM56 (αCD45RA). In some embodiments, any of the above antibodies can contain one or more mutations within the framework of the heavy and light chain variable regions without targeting the highly variable CDR regions. Examples of such antibodies include, in some aspects, the anti-CD4 antibodies described in U.S. Patent No. 7,482,000 and Bes et al. (2003) J. Biol. Chem., 278:14265-14273. In some embodiments, antigen-binding fragments such as Fab fragments can be generated from such antibodies using methods known in the art such as amplification and cloning of the hypervariable sequences of the heavy and light chains, which in some aspects can be combined with sequences encoding appropriate constant domains. In some embodiments, the constant domain is human subclass IgG1 / κ. Such antibodies can be fused at the carboxy terminus to a streptavidin-binding molecule that is a peptide as shown in SEQ ID NO:10. Examples of such antibodies are described in Stemberget et al. (2102) PLoS One, 7:35798 and international PCT application WO2013 / 011011.

[0131] In some embodiments, an antibody that specifically binds to a cell surface marker associated with or coated on beads or other surfaces is a full-length antibody or a (Fab) fragment, F(ab')2 fragment, Fab' fragment, Fv fragment, variable heavy chain (V H) Its antigen-binding fragments include those from the variable region, single-chain variable fragments (scFv), and single-chain antibody fragments including single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. In some embodiments, the antibody is a Fab fragment. In some embodiments, the antibody can be monovalent, bivalent, or multivalent. In some embodiments, an antibody such as Fab is a multimer. In some embodiments, an antibody such as a Fab multimer forms a multivalent complex with a cell surface marker.

[0132] In some embodiments, an antibody such as Fab related to Streptamer represents a specific kinetic measure of binding affinity (e.g., dissociation constant K D , association constant K A , off-rate, or other rate parameter of binding affinity). Such measures can be determined using any binding assay known to those skilled in the art. In certain examples, affinity-based biosensor techniques are utilized for measuring binding affinity. Exemplary biosensor techniques include, for example, Biacore technologies, BioRad ProteOn, Reichert, GWC Technologies, IBIS SPIR Imaging, Nomadics SensiQ, Akubio RAPid, ForteBio Octet, IAsys, Nanofilm, and others (see, for example, Rich et al. (2009) Analytical Biochemistry, 386:194-216). In some embodiments, the binding affinity is determined by fluorescence titration or titration calorimetry.

[0133] In some embodiments, an antibody such as Fab has a dissociation time of about 0.5×10 -4 sec -1 , about 1×10 -4 sec -1 , about 2×10 -4 sec -1 , about 3×10 -4 sec -1 , about 4×10 -4 sec-1 , about 5×10 -4 sec -1 , about 1×10 -3 sec -1 , about 1.5×10 -3 sec -1 , about 2×10 -3 sec -1 , about 3×10 -3 sec -1 , about 4×10 -3 sec -1 , about 5×10 -3 sec -1 , about 1×10 -2 sec -1 , or about 5×10 -1 sec -1 or greater than or equal to k 0ff represents the rate (also called the dissociation rate constant). The individual k 0ff rates can determine the rate at which the antibody reagent can dissociate from the interaction between the reagent and the cell via its binding to the cell surface marker (see, for example, International Publication PCT Application WO / 2013011011). For example, in some scenarios, the range of k 0ff can be selected within a range according to the individual application or use of the selected or enriched cells, including factors such as the desire to remove bound antibodies from the cell surface, the time to culture or incubate the cells, and the sensitivity to cells and other factors. In one aspect, the antibody has a high k -4 sec -1 rate greater than, for example, 4.0×10 0ff , such that as a result, after disruption of the multivalent binding complex, most of the antibody can be removed within 1 hour. This is because in some scenarios considering the half-life T1 / 2 of the complex, assuming the effect of rebinding can be ignored by sufficient dilution, the concentration of the complex decreases to 25% of the initial concentration within 56 minutes). In another aspect, for an antibody with a lower k0ff rate, for example, 1.0×10 -4 sec -1 , dissociation can take longer, for example, about or approximately 212 minutes or about 3 and a half hours, to remove 75% of the antibody from the surface.

[0134] In some embodiments, an antibody such as a Fab has a dissociation constant (Ka) with respect to binding to a cell surface marker on a cell that can range from about 10 -2 M to about 10 -8 M, or from about 10 -2 M to about 10 -9 M, or from about 10 -2 M to about 0.8×10 -9 M, or from about 10 -2 M to about 0.6×10 -9 M, or from about 10 -2 M to about 0.4×10 -9 M, or from about 10 -2 M to about 0.3×10 -9 M, or from about 10 -2 M to about 0.2×10 -9 , or from about 10 -2 M to about 0.15×10 -9 M, or from about 10 -2 to about 10 -10 . In some embodiments, the dissociation constant (Ka) with respect to binding to a cell surface marker on a cell ranges from about 10 -7 M to about 10 -10 M, or from about 10 -7 M to about 0.8×10 -9 M, or from about 10 -7 M to about 0.6×10 -9 M, from about 10 -7 M to about 0.3×10 -9 M, from 1.1×10 -7 M to about 10 -10 M, or from about 1.1×10 -7 M to about 0.15×10 -9 M, or from about 1.1×10 -7 M to about 0.3×10 -9 M, or from about 1.1×10 -7 M to about 0.6×10 -9 M, or from about 1.1×10 -7 M to about 0.8×10 -9 M.

[0135] In some embodiments, an antibody, such as an immunoaffinity reagent like a Fab, is directly or indirectly linked to a peptide ligand, such as a peptide ligand that can bind to a streptavidin variant (see, e.g., U.S. Patent No. 5,506,121). In some embodiments, such a peptide comprises an amino acid sequence shown in any of SEQ ID NOs: 1-6. In some embodiments, an antibody, such as an immunoaffinity reagent like a Fab, is directly or indirectly linked to a peptide ligand comprising the amino acid sequence shown in SEQ ID NO: 6.

[0136] In some embodiments, an antibody, such as an immunoaffinity reagent like a Fab, is directly or indirectly fused to a peptide sequence comprising a contiguous arrangement of at least two streptavidin binding modules, wherein the distance between the two modules is at least 0 and less than or equal to 50 amino acids, one binding module has 3-8 amino acids and comprises at least the sequence His-Pro-Xaa (SEQ ID NO: 1), where Xaa is glutamine, asparagine, or methionine, and the other binding module has the sequence of the same or a different streptavidin peptide ligand as shown in SEQ ID NO: 3 (see, e.g., International Publication PCT Application WO02 / 077018; U.S. Patent No. 7,981,632). In some embodiments, the peptide ligand directly or indirectly fused to the immunoaffinity reagent, such as an antibody like a Fab, comprises a sequence having a formula shown in either SEQ ID NO: 7 or 8. In some embodiments, the peptide ligand has an amino acid sequence shown in any of SEQ ID NOs: 9, 10, or 17-19.

[0137] Alternatively, other streptavidin-binding peptides known in the art can be used, such as those described by Wilson et al. (Proc. Natl. Acad. Sci. USA 98 (2001), 3750-3755). In some embodiments, the peptide is fused to the N-terminus and / or C-terminus of the protein.

[0138] In some embodiments, an antibody such as a Fab fused to a peptide ligand capable of binding to a streptavidin variant is contacted with a streptavidin variant containing beads in order to coat the beads with the antibody. In some embodiments, the coated beads can be used in the enrichment and selection methods described herein by contacting such beads with a sample containing the cells to be enriched or selected.

[0139] In some embodiments, the binding between the peptide ligand binding partner and the streptavidin variant protein binding reagent is reversible. In some embodiments, the binding between the peptide ligand binding partner and the streptavidin variant protein binding reagent is high as described above, but lower than the binding affinity of the streptavidin binding reagent for biotin or a biotin analog. Thus, in some embodiments, biotin (vitamin H) or a biotin analog can be added to compete for the binding and disrupt the binding interaction between the streptavidin variant protein binding reagent on the beads and the peptide ligand binding partner associated with an antibody that specifically binds to a cell marker on the surface. In some embodiments, the interaction can be reversed in the presence of a low concentration of biotin or analog, such as 0.1 mM to 10 mM, 0.5 mM to 5 mM, or 1 mM to 3 mM, such as generally at least or at least about 1 mM or at least 2 mM, such as 2.5 mM or about 2.5 mM. In some embodiments, incubation in the presence of a competitor such as biotin or a biotin analog releases the beads from the selected cells.

[0140] b. Immunoaffinity chromatography In some embodiments, affinity-based selection uses immunoaffinity chromatography. The immunoaffinity chromatography method includes, in some aspects, one or more chromatography matrices described in U.S. Patent Application Publication No. 2015 / 0024411. In some embodiments, the chromatography method is a fluid chromatography, typically liquid chromatography. In some embodiments, the chromatography can be carried out in a flow-through mode in which a fluid sample containing the cells to be isolated is added, for example, by gravity flow or by a pump to one end of a column containing the chromatography matrix, where the fluid sample exits the column at the other end of the column. Additionally, in some aspects, the chromatography can be carried out in an "up-down" mode in which a fluid sample containing the cells to be isolated is added by pipette to one end of a column containing a chromatography matrix filled, for example, in a pipette tip, where the fluid sample enters the chromatography matrix / pipette tip and exits from the other end of the column. In some embodiments, the chromatography can also be carried out in a batch mode in which the chromatography material (stationary phase) is incubated with the sample containing the cells while, for example, shaking, rotating or repeatedly contacting, and the fluid sample is removed, for example, by pipette.

[0141] In some embodiments, the chromatography matrix is a stationary phase. In some embodiments, the chromatography is column chromatography. In some embodiments, any suitable chromatography material can be used. In some embodiments, the chromatography matrix has a solid or semi-solid form. In some embodiments, the chromatography matrix can include a polymer resin, a metal oxide, or a metalloid oxide. In some embodiments, the chromatography matrix is a non-magnetic material or a non-magnetizable material. In some embodiments, the chromatography matrix is a cross-linked gel in the form of derivatized silica or a natural polymer such as a polysaccharide. In some embodiments, the chromatography matrix is an agarose gel. Agarose gels for use in chromatography matrices are known in the art and, in some instances, include Sepharose materials such as Superflow™ agarose or Superflow™ Sepharose®, which are commercially available with different bead diameters and pore sizes. In some embodiments, the chromatography matrix is a specific cross-linked agarose matrix to which dextran is covalently bound, such as Sephadex®, Superdex®, or Sephacryl® in some instances, which are available with different bead diameters and pore sizes and are known in the art.

[0142] In some embodiments, the chromatography matrix is made from a synthetic polymer such as polyacrylamide, a styrene-divinylbenzene gel, an acrylic acid / diol or acrylamide / diol copolymer, a polysaccharide / agarose copolymer such as a polyacrylamide / agarose composite, a polysaccharide, and N,N'-methylenebisacrylamide, or derivatized silica coupled with a synthetic or natural polymer.

[0143] In some embodiments, a chromatography matrix, such as agarose beads or other matrices, has a size of at least or at least about 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm or 150 μm or greater. The exclusion limit of the size exclusion chromatography matrix is selected to be less than the maximum width of the target cells in the sample, such as T cells. In some embodiments, the volume of the matrix is at least 0.5 mL, 1 mL, 1.5 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL or more. In some embodiments, the chromatography matrix is packed within a column.

[0144] In some embodiments, the chromatography matrix, which is an immunoaffinity chromatography matrix, comprises an affinity reagent such as an antibody or an antigen-binding fragment such as a Fab immobilized thereon. The antibody or antigen-binding fragment such as a Fab can be any of the above, including antibodies known in the art, antibodies having a specific k 0ff rate and / or antibodies having a specific dissociation constant (Ka).

[0145] In some embodiments, the affinity reagent such as an antibody or an antigen-binding fragment such as a Fab is immobilized. In some embodiments, the immunoaffinity reagent such as an antibody or an antigen-binding fragment such as a Fab is fused or linked to a binding partner that interacts with a binding reagent immobilized on the matrix. In some embodiments, the binding capacity of the chromatography matrix is sufficient or capable of adsorbing at least 1×107 cells / mL, 5×107 cells / mL, 1×108 cells / mL, 5×108 cells / mL, or 1×109 cells / ml or more, where the cells are cells expressing cell surface markers that are specifically recognized by an affinity reagent such as an antibody or a Fab.

[0146] In some embodiments, the interaction between the binding reagent and the binding partner forms a reversible bond such that the binding of the antibody to the matrix is reversible. In some embodiments, the reversible bond can be mediated by a streptavidin mutant binding partner and a binding reagent immobilized on the matrix that is streptavidin, a streptavidin analog or mutant protein, avidin or an avidin analog or mutant protein.

[0147] In some embodiments, the reversible binding of an affinity reagent such as an antibody or an antigen-binding fragment such as a Fab is through the interaction of a peptide ligand binding reagent and a streptavidin mutant protein as described above with respect to the immunoaffinity beads. In the context of a chromatography matrix, the matrix, such as agarose beads or other matrix, is functionalized or conjugated with a streptavidin mutant protein such as any of those shown in any of the above, e.g., SEQ ID NO:12, 13, 15 or 16. In some embodiments, an antibody or an antigen-binding fragment such as a Fab is directly or indirectly fused or linked to a peptide ligand capable of binding to a streptavidin mutant such as any of the above. In some embodiments, the peptide ligand is any of the above, such as a peptide comprising an amino acid sequence shown in any of SEQ ID NO:1-10 or 17-19. In some embodiments, a chromatography matrix column is contacted with such an affinity reagent such as an antibody or an antigen-binding fragment such as a Fab to immobilize or reversibly bind the affinity reagent to the column.

[0148] In some embodiments, an immunoaffinity chromatography matrix can be used in the enrichment and selection methods described herein by contacting the matrix with a sample containing the cells to be enriched or selected. In some embodiments, the selected cells are eluted or released from the matrix by disrupting the binding partner / binding reagent interaction. In some embodiments, the binding partner / binding reagent interaction is mediated by the interaction between a peptide ligand and a streptavidin mutant, and the selected cells can be released due to the presence of reversible binding. For example, in some embodiments, the binding between the peptide ligand binding partner and the streptavidin mutant protein binding reagent is high as described above, but lower than the binding affinity of the streptavidin binding reagent for biotin or a biotin analog. Thus, in some embodiments, biotin (vitamin H) or a biotin analog is added to compete for binding and disrupt the binding interaction between the streptavidin mutant protein binding reagent on the matrix and the peptide ligand binding partner associated with the antibody specifically binding to the cell marker on the surface. In some embodiments, the interaction can be reversed in the presence of a low concentration of biotin or an analog, generally at least or at least about 1 mM or at least 2 mM, such as 2.5 mM or about 2.5 mM, in the presence of 0.1 mM to 10 mM, 0.5 mM to 5 mM or 1 mM to 3 mM. In some embodiments, elution in the presence of a competitor such as biotin or a biotin analog releases the selected cells from the matrix.

[0149] In some embodiments, the immunoaffinity chromatography in the provided method is performed using at least two chromatographic matrix columns that are operably connected, wherein an antibody, such as a Fab, an affinity agent or binder for one of CD4 or CD8, is coupled to a first chromatographic matrix in a first selection column, and an antibody, such as a Fab, an affinity agent or binder for the other of CD4 or CD8, is coupled to a second chromatographic matrix in a second selection column. In some embodiments, the at least two chromatographic matrix columns are present within a closed system or device, such as a sterile closed system or device.

[0150] In some embodiments, a closed system or device is also provided herein that includes at least two chromatographic matrix columns that are operably connected, wherein an antibody, such as a Fab, an affinity agent or binder for one of CD4 or CD8, is coupled to a first chromatographic matrix in a first selection column, and an antibody, such as a Fab, an affinity agent or binder for the other of CD4 or CD8, is coupled to a second chromatographic matrix in a second selection column. Examples of such systems and methods are shown in FIGS. 1A and 1B, and in the Examples.

[0151] In some embodiments, the closed system is automated. In some embodiments, the components associated with the system can include an integrated microcomputer, a peristaltic pump, and various valves, such as pinch valves, or stopcocks, for controlling the flow of fluid between the various parts of the system. The integrated computer in some aspects controls all of the components of the device and commands the system to perform repetitive procedures in a standardized sequence. In some embodiments, the peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures a controlled flow of buffer through the system.

[0152] Referring to FIGS. 1A and 1B, in some embodiments, a first affinity matrix 3 including a first affinity agent or binder in a first selection column 1 is operably connected to a second affinity matrix 4 including a second affinity agent or binder in a second selection column 2 via tubing and valve 13 such that cells that pass through the first affinity chromatography matrix and are not bound to the first affinity agent or binder thereon can enter into the second affinity matrix 4. (ii) operably connected to an output container such as a culture tank 12 to collect such cells from the first affinity matrix after eluting and releasing the selected cells bound to the first affinity agent or binder on the first affinity matrix. In some embodiments, the second affinity matrix 4 including the second affinity agent or binder in the second selection column 2 is also operably connected to an output container such as a culture tank 12 to collect such cells from the second affinity matrix after eluting and releasing the selected cells bound to the second affinity agent or binder thereon. In some embodiments, the second selection column 2 is operably connected to an output container such as a culture tank 12 via a removal chamber 9 containing a binding reagent such as a streptavidin mutant that can bind with high affinity, such as greater than 10 -10 M -1 The second selection column 2 is operably connected to an output container such as a culture tank 12 via a removal chamber 9 containing a binding reagent such as a streptavidin mutant that can bind with high affinity, such as greater than.

[0153] In some embodiments, the sizes of the first selection column 1 and the second selection column 2, such as length and / or diameter, can be the same or different. In some embodiments, the size of one of the first column 1 or the second column 2, such as length and / or diameter, is at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 5 times, 7 times, 8 times, 9 times, 10 times or more larger than the size of the other column.

[0154] In some embodiments, a first affinity matrix 3 comprising a first affinity agent or binder in a first selection column 1 is operably connected via tubing and valves 13 to a third affinity matrix 17 comprising a third affinity agent or binder in a third selection column 15, such that selected cells bound to the first affinity agent or binder on the first affinity matrix can, for example, be eluted and released from the first affinity matrix and then such cells can enter into the third affinity matrix. In some embodiments, the first selection column 1 is operably connected to the third selection column 15 via a removal chamber 9 containing a binding reagent such as a streptavidin mutant that can bind with a higher affinity than 10 -10 M -1 to biotin. In some embodiments, the third affinity matrix 17 comprising a third affinity agent or binder in the third selection column 15 is also operably connected to an output container such as a culture tank 12 to collect such cells from the third affinity matrix after eluting and releasing the selected cells bound to the third affinity agent or binder (and previously bound to the first affinity agent or binder) from the third affinity matrix (and previously the first affinity matrix), for example. In some embodiments, the third selection column 15 is operably connected to an output container such as a culture tank 12 via a removal chamber 9 containing a binding reagent such as a streptavidin mutant that can bind with a higher affinity than 10 -10 M -1 to biotin.

[0155] In some embodiments, the sizes of the first selection column 1 and the third selection column 15, such as length and / or diameter, can be the same or different. In some embodiments, the size of one of the first column 1 or the third column 15, such as length and / or diameter, is at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 5 times, 7 times, 8 times, 9 times, 10 times, or more larger than the size of the other column.

[0156] In some embodiments, the first selection column 1 is operably connected to a storage reservoir containing the cell sample 5, for example via tubing, valves, and a pump 8, to provide the cell sample into the first selection column.

[0157] In some embodiments, the first selection column 1 is also operably connected to a wash reservoir 6 containing a wash buffer and / or an elution reservoir 7 containing an eluate, for example via tubing and valves, to pass the wash buffer or elution buffer through the first chromatographic matrix in the first selection column. In some embodiments, due to the operable connection between the first and second selection columns, the wash buffer and / or elution buffer can operably enter the second column. In some embodiments, due to the operable connection between the first selection column and the third selection column, the wash buffer and / or elution buffer can operably enter the third selection column.

[0158] In some embodiments, the second selection column 2 is also operably connected to a wash reservoir 6 containing a wash buffer and / or an elution reservoir 7 containing an eluate, for example via tubing and valves, to pass the wash buffer or elution buffer through the first chromatographic matrix in the first selection column.

[0159] The washing buffer can be any physiological buffer compatible with cells, such as phosphate buffered saline. In some embodiments, the washing buffer contains bovine serum albumin, human serum albumin, or recombinant human serum albumin, for example, at a concentration of 0.1% - 5% or 0.2% - 1%, for example, 0.5% or about 0.5%. In some embodiments, the eluate is a biotin analog such as biotin or desthiobiotin at, for example, at least or at least about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, or 5 mM.

[0160] In some embodiments, the first affinity matrix 3 in the first selection column 1 is operably connected via tubing and valves to a first affinity reagent reservoir (e.g., a Fab reservoir) 18 that contains a first affinity agent or binder, such as an antibody for immobilization on the first affinity matrix, e.g., a Fab. In some embodiments, the second affinity matrix 4 in the second column 2 is operably connected via tubing and valves to a second affinity agent or binder reservoir (e.g., a Fab reservoir) 19 that contains a second affinity agent or binder, such as an antibody for immobilization on the second affinity matrix, e.g., a Fab. In some embodiments, the third affinity matrix 17 in the third selection column 15 is operably connected via tubing and valves to a third affinity agent or binder reservoir (e.g., a Fab reservoir) 20 that contains a third affinity agent or binder, such as an antibody for immobilization on the second affinity matrix, e.g., a Fab.

[0161] In some embodiments, the first and / or second affinity reagents specifically bind to CD4 or CD8, where the first and second affinity reagents are not the same. In some embodiments, the third affinity reagent specifically binds to a marker on naive T cells, resting T cells, or central memory T cells, or to a marker that is CD45RO, CD62L, CCR7, CD28, CD3, CD27, and / or CD127.

[0162] 5. Enrichment and ratio of the generated composition In some embodiments, by making the first and second selections using the above method, from a sample, a first population of cells expressing a first cell surface marker and a second population of cells expressing a second cell surface marker are enriched, respectively. In certain examples, the first and / or second population of enriched cells can be a cell population enriched for CD4+ cells, and the other of the enriched cell populations, i.e., the other of the first or second population of cells, can be a population enriched for CD8+. As described above, in some embodiments, a third, fourth or subsequent selection can be made to enrich for further cell subpopulations, such as a subpopulation of CD4+ cells and / or a subpopulation of CD8+ cells, from a population of cells enriched in a previous first, second, or subsequent enrichment.

[0163] In some embodiments, the method produces an enriched cell composition containing first and second populations of enriched cells, such as a cell population enriched for CD4+ cells and a cell population enriched for CD8+ cells. In some embodiments, the enriched cell composition is referred to as a starting culture composition and is used in subsequent processing steps, such as subsequent processing steps involving incubation, stimulation, activation, manipulation and / or formulation of the enriched cells. In some embodiments, after further processing steps, such as processing steps involving incubation, stimulation, activation, manipulation and / or formulation, in some aspects, an output composition can be generated that contains genetically engineered cells containing CD4+ cells and CD8+ cells expressing a genetically engineered antigen receptor.

[0164] In some embodiments, the enriched cell composition is the enriched cells from the starting sample, where the number of cells in the starting sample is at least as large as the desired number of cells in the enriched composition such as the starting composition for culturing. In some embodiments, the number of cells in the starting sample is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000% or greater than the desired number of cells in the enriched composition. In some examples, the desired number of cells in the enriched population, including enriched CD4+ cells, CD8+ cells or subpopulations thereof, is at least 1×10 6 cells, 2×10 6 cells, 4×10 6 cells, 6×10 6 cells, 8×10 6 cells, 1×10 7 cells, 2×10 7 cells, 4×10 7 cells, 6×10 7 cells, 8×10 7 cells, 1×10 8 cells, 2×10 8 cells, 4×10 8 cells, 6×10 8 cells, 8×10 8 cells, 1×10 9 cells, or more. In some embodiments, the number of cells in the starting sample is at least 1×10 8 cells, 5×10 8 cells, 1×10 9 cells, 2×10 9 cells, 3×10 9 cells, 4×10 9 cells, 5×10 9 cells, 6×10 9 cells, 7×10 9 cells, 8×10 9 cells, 9×10 9 cells, 1×10 10 cells, or more.

[0165] In some embodiments, the yield of the first and / or second population or subpopulation in the enriched composition, i.e., the number of cells in the same population or subpopulation after enrichment compared to the number of the cell population or cell subpopulation in the starting sample, is from 10% to 100%, such as from 20% to 80%, from 20% to 60%, from 20% to 40%, from 40% to 80%, from 40% to 60%, or from 60% to 80%. In some embodiments, the yield of the first and / or second cell population or subpopulation thereof is less than 70%, less than 60%, less than 50%, less than 40%, less than 30% or less than 20%.

[0166] In some embodiments, the purity of the first and / or second cell population or its cell subpopulation in the enriched composition, i.e., the percentage of cells that are positive for the selected cell surface marker relative to all cells in the enriched cell population, is at least 90%, 91%, 92%, 93%, 94%, and generally at least 95%, 96%, 97%, 98%, 99% or more.

[0167] In some embodiments, an enriched cell composition such as a starting culture composition includes the ratio of CD4+ cells to CD8+ cells at the starting culture ratio. The starting culture ratio is the ratio or number of cells included in the starting culture composition designed such that two types of cells or isolated cell populations result in the desired output ratio or dose, such as the ratio or dose for administration to a patient, at the end of the incubation and / or manipulation steps or other processing steps, and / or at the time of thawing and / or immediately prior to administration to the subject, or within the allowable error rate or tolerance thereof. In embodiments of the methods provided herein, the selection for the first and / or second selection, or subpopulations thereof, can be performed in a manner that results in the selected starting culture ratio. Examples of such methods are described in the following and in the examples.

[0168] a. Starting culture ratio and number In some embodiments, CD4 + or its subpopulation and CD8 +The starting ratio of culturing with the cells or a subpopulation thereof is between 10:1 or about 10:1 and 1:10 or about 1:10, between 5:1 or about 5:1 and 1:5 or about 1:5, or between 2:1 or about 2:1 and 1:2 or about 1:2. In some embodiments, the starting ratio of culturing CD4+ cells or a subpopulation thereof with CD8+ cells or a subpopulation thereof is 1:1 or about 1:1.

[0169] In some embodiments, the starting ratio of culturing CD4+ and CD8+ cells, or subpopulations thereof, is different from the ratio of CD4+ cells to CD8+ cells or subpopulations thereof in a sample from a subject. In some embodiments, the ratio of CD4+ T cells to CD8+ T cells in a sample such as a blood sample from a subject is CD4+ cells:CD8+ cells between 1:1 and 14:1, and generally is reported to be CD4+ cells:CD8+ cells between about 1.5:1 and about 2.5:1. In some embodiments, the ratio of CD4+ T cells to CD8+ T cells in a sample such as a blood sample is CD4+ cells:CD8+ cells of about 2:1. In some embodiments, the ratio of CD4+ T cells to CD8+ T cells in a sample such as a blood sample is about 1:1. (See, for example, Amadori, A et al., Nature Med. 1: 1279-1283, 1995; Chakravarti, A., Nature Med. 1: 1240-1241, 1995; Clementi, M., et al., Hum. Genet. 105: 337-342, 1999). In some embodiments, the ratio of the subject is CD4+ cells:CD8+ cells less than 1:1 (see, for example, Muhonen, T. J Immunother Emphasis Tumor Immunol. 1994 Jan;15(1):67-73). In some embodiments, the starting ratio of culturing CD4+ cells and CD8+ cells 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 100%, at least 125%, at least 150, at least 200%, at least 300%, at least 400%, or at least 500% greater or less than the ratio of CD4+ cells to CD8+ cells in a sample from a subject.

[0170] In some embodiments, the ratio of CD4+ T cells to CD8+ T cells in a sample from a subject is determined before making the first and / or second selection. Based on a particular ratio of CD4+ T cells to CD8+ T cells in the subject (which may vary between subjects), a particular selection pattern can be individualized for the subject, for example by sizing a chromatography column or selecting the amount or concentration of an immunoaffinity reagent, to achieve a desired or selected starting ratio for culturing. The relative levels or frequencies of various cell populations in a subject can be determined based on assessing the surface expression of one or more markers present in such a population or subpopulation. Several well-known methods for assessing the expression level of surface markers or proteins, such as detection by affinity-based methods, e.g., immunoaffinity-based methods, can be used, for example, by flow cytometry, e.g., in the context of cell surface proteins.

[0171] Depending on the circumstances, the starting ratio for culturing appropriate for a particular cell type may vary, for example, as an example, depending on the particular disease, condition, or previous treatment of the subject from which the cells are derived, and / or the particular antigen specificity of the cells, the relative representation between various subpopulations, e.g., effector cells vs. memory cells vs. naive cells of a particular type of cell (e.g., CD8 + T cells), and / or one or more conditions under which the cells are incubated, such as the medium, stimulant, culture time, buffer, oxygen content, carbon dioxide content, antigen, cytokine, antibody, and other components. Thus, a cell type that is typically or generally known to grow or expand more rapidly than another cell type may not always have such properties in all circumstances. Thus, in some aspects, the starting ratio for culturing is determined in connection with an assessment of the phenotype or condition of the cell or the subject from which the cell is derived and / or empirical basis, based on the known capabilities of the cell type in normal or typical circumstances.

[0172] In some embodiments, the seeding ratio is based on knowledge of one or more of these characteristics for a particular cell type that is known or determined to be at that concentration. In some embodiments, CD4 in the seeding composition + cells and CD8 + cells, the ratio of CD4 + to CD8 + is respectively 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more or less, greater or smaller than the desired output ratio of CD4

[0173] to CD8. In some embodiments, for example, CD4+ cells are known to grow or expand to a lesser extent or at a slower rate than CD8+ cells when incubated under certain stimulation conditions, depending on the situation. See, for example, Foulds et al. (2002) J Immunol. 168(4): 1528-1532; Caggiari et al. (2001) Cytometry. 46(4) 233-237; Hoffman et al. (2002) Transplantation. 74(6): 836-845; and Rabenstein et al. (2014) J Immunol. Published online on March 17, 2014, doi: 10.4049 / jimmunol.1302725, prior to printing. Thus, in some examples, the ratio of CD4 + cells to CD8 + cells in the seeding composition is 10 times or 100 times the desired output ratio. For example, if a 1:1 (or 50% / 50%) CD4 / CD8 output ratio is desired, then the CD4 + population and CD8 +The population, in one example, can be included in the starting culture composition at a ratio of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1, for example, to account for differences in growth rates over a particular period. Depending on the difference between the rate or increase or proliferation of one of CD4+ cells or CD8+ cells, or a subpopulation thereof, and the other, the technician can empirically determine the starting culture ratio to achieve the desired output ratio according to the individual stimulation or activation conditions.

[0174] The starting culture ratio is not necessarily the same as, or approximate to, the desired output ratio. For example, at 1:1, or within a particular error thereof, of CD4 + and CD8 + If it is desired to administer engineered (e.g., CAR-expressing) T cells, the starting culture ratio of CD4 + and CD8 + cells is often not 1:1. In some embodiments, the starting culture ratio that yields the desired output ratio varies depending on, for example, the source of the cells, the cell type to be cultured, the patient to whom the cells are to be administered, one or more subjects from which the cells are isolated or derived, the disease or condition of such subjects, the disease to be treated, the culture conditions, and other parameters such as whether such subjects have any diseases or conditions.

[0175] In some embodiments, the starting culture ratio is based on the composition of each subpopulation of cells. In certain embodiments, the starting culture ratio is based on the length of time the cell populations are cultured before they are genetically engineered. In some embodiments, the starting culture ratio is based on the growth rate of each cell population.

[0176] As another aspect, in some embodiments, the method further includes determining a starting culture ratio or number. In some embodiments, the provided method includes methods and steps for determining the appropriate ratio, dosage, and number of cells, cell types, and cell populations. For example, CD4 + / CD8 +Methods are provided for determining the ratios of cells, populations, and / or subpopulations and for determining the appropriate dosages of such cells and subtypes. In some embodiments, methods are provided for determining the appropriate ratio or number of cell types or cultured cell populations to be included in a composition, such as a starting culture composition, to achieve a desired result. In some aspects, such ratio or number is designed for use in an incubation or manipulation step to achieve a desired output ratio or dosage. In some embodiments, methods are provided for determining the desired ratio of cells, types, or populations for administration to a subject or patient and / or the number of such cells.

[0177] In some embodiments, the selected starting culture ratio is based on the relative ability of different cell types or populations to survive and / or the rate of growth or expansion of each cell population or cell type (such as CD4 + cells to CD8 + cells, etc.) in culture when incubated under such conditions. Thus, in some aspects, the growth rate, survival, and / or output ratio are measured or evaluated at one or more specific time points, such as after test incubation, for example after incubation, and / or after cryopreservation or freezing steps, and / or after thawing following such procedures, immediately prior to administration, such as at the bedside, to determine the ratio optimal for starting culture. Any of several well-known methods for determining in vitro or ex vivo cell growth rate or survival include flow cytometry methods such as labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) or a similar fluorescent dye, followed by incubation, and subsequently evaluating fluorescence intensity by flow cytometry and / or evaluating the binding of cells to annexin V or other compounds that recognize appropriate markers on or in the cells and / or the uptake of a DNA interchelating agent such as propidium iodide or 7AAD, and using flow cytometry to evaluate uptake and / or the stage of the cell cycle as a measure of growth or apoptosis.

[0178] In some embodiments, the starting culture ratio is determined by incubating two isolated cell populations, e.g., cell subpopulations, in different ratios in a test composition under specific test conditions and evaluating one or more results such as the output ratio achieved after a specific period of time. In some aspects, the conditions are stimulatory conditions such as conditions that approximate the conditions under which the starting culture composition is to be incubated for the culture stage and / or the manipulation stage. For example, in some aspects, the test composition is administered in the presence of one or more of, e.g., the same stimulant, medium, buffer, gas content, and / or in the same type of container or tank, and / or for the same or approximately the same amount of time as the parameters to be used for the incubation stage and / or the manipulation stage to prepare or produce the final composition such as the manipulated composition for administration.

[0179] Exemplary test ratios for the test starting culture composition can include 90% / 10%, 80% / 20%, 70% / 30%, 60 / 40%, 50 / 50%, 40 / 60%, 30 / 70%, 20% / 80%, and 10% / 90% or about those ratios, or 1:1.5 or about 1:1.5 to 0.1:1, 0.5:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:1.1, 1:1.2., 1:1.3, 1:1.4, 1:1.5 or more, or 1:0.1, 1:0.4, 1:0.7, 1:0.8, 1:0.8, 1.1:1, 1.2:1, 1.3:1, 1.4:1, or 1.5;1 or more.

[0180] In some situations, the appropriate starting culture ratio of CD4 + cells to CD8 + cells to achieve the desired CD4:CD8 ratio at the end of production is the presence and / or percentage of naive, effector, and various memory compartments represented in a particular isolated composition, such as the CD4 + fraction and / or CD8 +It is determined based on subpopulations of the delineation. Such an evaluation can be by, for example, flow cytometry, by determining the presence or level of various surface markers on the cells.

[0181] In some embodiments, the starting culture ratio is based on the phenotype of each cell population. In certain embodiments, the starting culture ratio is based on culture conditions (e.g., medium composition, growth factors, stimulants, and / or the presence and / or absence of other agents, temperature, aeration conditions, etc.).

[0182] In some embodiments of the methods described herein, the starting culture ratio is CD4 + :CD8 + to produce an output composition comprising the ratio of cells or the number or total number of such subtypes, which is within about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% of the desired ratio or dose (including any range between these values). In some embodiments of the methods described herein, the starting culture ratio is CD4 + cells:CD8 + to produce an output composition having the desired ratio of cells or a dose of such cells for at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the time (including any range between these values). In certain embodiments of the method, the starting culture ratio is such that in the output composition, CD4 + cells to CD8 + cells ratio is produced that is within 20% of the desired output ratio for at least 80% of the time. In some embodiments of the methods described herein, the output composition is within the tolerance of the desired output ratio, the ratio of CD4 + cells to CD8 + cells, and the tolerance is determined, as described above, for example, by administering CD4 + cells and CD8 + cells to one or more subjects at multiple ratios.

[0183] b. Output ratio and dosage In some embodiments, the method yields an output composition after one or more processing steps of the starting composition, such as incubation, stimulation, activation, manipulation, and / or formulation of the cells. In some embodiments, the method is carried out to achieve or yield a desired ratio of CD4 + cells and CD8 + cells or subpopulations thereof, which is between 2:1 or about 2:1 to 1:5 or about 1:5, 2:1 or about 2:1 to 1:2 or about 1:2, 1.5:1 or about 1.5:1 to 1:5 or about 1:5, 1.5:1 or about 1.5:1 to 1:2 or about 1:2, or 1:1 or about 1:1 to 1:2 or about 1:2. In some embodiments, the desired output ratio of CD4 + cells to CD8 + cells in the output composition is 1:1 or about 1:1.

[0184] In some embodiments, the method produces or generates an output composition, such as a composition containing genetically engineered CD4+ T cells and CD8+ T cells or subpopulations thereof, wherein the output ratio of CD4+ cells and CD8+ cells or subpopulations thereof in the composition is between 2:1 or about 2:1 to 1:5 or about 1:5, 2:1 or about 2:1 to 1:2 or about 1:2, 1.5:1 or about 1.5:1 to 1:5 or about 1:5, 1.5:1 or about 1.5:1 to 1:2 or about 1:2, or 1:1 or about 1:1 to 1:2 or about 1:2. In some embodiments, the method produces or generates an output composition containing an output ratio of CD4 + cells to CD8 + cells of 1:1 or about 1:1.

[0185] In some embodiments, the output ratio of CD4+ T cells to CD8+ T cells is a ratio desired as part of the dosage of T cells for immunotherapy, such as in relation to adoptive immunotherapy.

[0186] In some embodiments, a desired dosage, such as a desired number of cells or a desired output ratio of a cell type or population (e.g., a ratio optimal for therapeutic administration to a patient), is determined. In some embodiments, the method determines a margin of error or tolerance, i.e., a range of variation in the ratio in a given composition, e.g., an engineered composition, from the desired output ratio, but still achieving a desired result, such as an acceptable level of safety in a subject or patient, or effectiveness in treating a particular disease or condition or other therapeutic effect.

[0187] In some embodiments, the desired dosage, ratio, and / or margin of error depend on the disease or condition to be treated, the subject, the source of the cells, e.g., whether the cells are from a subject having a particular condition or disease, and whether the cells are for autologous or allogeneic transplantation, e.g., whether the subject from whom the cells are isolated is intended to receive the cells in adoptive cell therapy, and / or whether the subject has received or is receiving another treatment and / or the like. In some embodiments, the ratio, number, and / or tolerance or error depend on one or more other properties of the cells, such as growth rate, viability, expression of specific markers or secretion of factors such as cytokines, or a particular subpopulation isolated prior to the incubation and manipulation steps. In some examples, the desired ratio and / or margin of error or difference may vary depending on the age, gender, health status, and / or weight of the subject, biomarkers as indicators of disease traits, and treatments to be administered concurrently or previously administered to the subject.

[0188] In some embodiments, the desired ratio, dosage, and / or margin of error are determined by administering to a test subject various test compositions containing different ratios or different numbers of the cell type or population of interest, and subsequently evaluating one or more results or parameters, such as safety, therapeutic effectiveness, in vivo concentration or localization of the cells, and / or other desired outcome parameters.

[0189] In some embodiments, the subject of the test is a non-human animal, such as a normal animal or a disease animal model, such as a disease or condition to be treated by administration of cells. In some embodiments, various test ratios for administration to the subject are, for example, one cell type or subtype (e.g., CD4 + T cells or its subtypes) and another cell type (e.g., CD8 + T cells or NK cells or their subtypes), as well as 90% / 10%, 80% / 20%, 70% / 30%, 60 / 40%, 50 / 50%, 40 / 60%, 30 / 70%, 20% / 80%, and 10% / 90% or approximately those values, expressed as provisional values. The ratios can be expressed as relative percentages, or in any other form such as the ratios 0.1:1, 0.5:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:1.1, 1:1.2., 1:1.3, 1:1.4, 1:1.5 or approximately those values, or more than 1:1.5 or approximately more than 1:1.5, or 1:0.1, 1:0.4, 1:0.7, 1:0.8, 1:0.8, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5;1 or more, or approximately those values or approximately those values or more (including any range between these values). In some embodiments, the subject of the test is a human. In some embodiments, the desired ratio is the average, mean, or median ratio between subjects having a particular optimal effect. In some aspects, the desired ratio is the ratio that achieves an optimal balance of safety and efficacy. In some aspects, the desired ratio or dose is the ratio or dose that achieves the highest efficacy among all test ratios or doses but still maintains the safety threshold. In some aspects, the desired ratio or dose is the ratio or dose that achieves the highest level of safety while maintaining the efficacy threshold or being within the efficacy range. In some cases, the optimal ratio or dose is such that the ratio between one cell type and another is between 1:1 and 1:2, or between 10 4 and 10 9 per kg body weight or between 10 5 and 10 6 per kg body weight, expressed as a range.

[0190] In some embodiments, the tolerance is determined based on the deviation from the desired average test subjects that was monitored to evaluate the therapeutic efficacy and / or safety of each percentage combination. In some embodiments, the tolerance is within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio (including any value between these ranges).

[0191] 6. Exemplary methods of selecting or enriching cells a. Single-step process and / or simultaneous selection using immunomagnetic beads In some embodiments, the separation and / or steps are performed using immunomagnetic beads. In some embodiments, a cell sample containing CD4+ and CD8+ cells, such as a primary human T cell sample, is contacted with magnetic beads containing a first immunoaffinity reagent that binds to CD4 or CD8 and magnetic beads containing a second immunoaffinity reagent that binds to the other of CD4 or CD8. The separation and / or steps can occur simultaneously and / or sequentially.

[0192] In some embodiments, contacting the cells with the magnetic beads is done simultaneously, and enrichment of cells containing surface markers CD4 and CD8 is also done simultaneously. In some such scenarios, the method includes contacting cells of a sample containing primary human T cells with a first immunoaffinity reagent that specifically binds to CD4 and a second immunoaffinity reagent that specifically binds to CD8 in an incubation composition under conditions where the immunoaffinity reagents specifically bind to CD4 molecules and CD8 molecules on the cell surfaces in the sample, and generating an enriched composition containing CD4+ and CD8+ cells at the starting culture ratio by recovering the cells bound to the first and / or second immunoaffinity reagents.

[0193] In some embodiments, the first and / or second immunoaffinity reagent is present in the incubation composition at a sub-optimal yield concentration, wherein the enriched composition produces a composition enriched in CD4+ T cells and CD8+ T cells by containing less than 70% of the total CD4+ cells in the incubation composition and / or less than 70% of the CD8+ cells in the incubation composition.

[0194] In some embodiments, a sub-optimal yield concentration of an affinity reagent is a concentration less than the concentration used or required to achieve the optimal or maximum yield of bound cells in a given selection or enrichment that 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 having a marker targeted by the reagent, or to which the reagent is specific, or to which the reagent is specific and capable of binding, compared to the total number of cells in the incubation). A sub-optimal yield concentration is generally, in such a process or step, the concentration or amount of the reagent that achieves a yield of less than 70% of the bound cells upon recovery of the cells bound to the reagent. In some embodiments, a yield of 50%, 45%, 40%, 30%, or 25% or about that value or less is achieved by a sub-optimal concentration. Concentration can be expressed in terms of the number or mass of particles or surfaces per cell and / or the mass or number of molecules of a drug (e.g., an antibody such as an antibody fragment) per cell.

[0195] For example, in some embodiments, a sub-optimal yield concentration is 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9 per cell, or 20×10 9Less than 30 μM or less than about 30 μM of the agent (e.g., antibody) per cell. In some embodiments, the sub-optimal yield concentration is 1×10 cells per cell in the incubation composition 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9 per cell, and less than 30 μM or less than about 30 μM of the agent (e.g., antibody); in some embodiments, the sub-optimal yield concentration is 1×10 cells per cell in the incubation composition 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9 per cell, and less than 20 μM or less than about 20 μM of the agent (e.g., antibody); in some embodiments, the sub-optimal yield concentration is 1×10 cells per cell in the incubation composition 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9 per cell, and less than 10 μM or less than about 10 μM of the agent (e.g., antibody); in some embodiments, the sub-optimal yield concentration is 1×10 cells per cell in the incubation composition 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9less than 15 μM or less than about 15 μM of the agent (e.g., antibody) per cell; in some embodiments, the sub-optimal yield concentration is 1×10 cells in the incubation composition 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9 per cell, less than 10 μM or less than about 10 μM of the agent (e.g., antibody); in some embodiments, the sub-optimal yield concentration is 1×10 cells in the incubation composition 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9 less than 5 μM or less than about 5 μM of the agent (e.g., antibody) per cell; in some embodiments, the sub-optimal yield concentration is 1×10 cells in the incubation composition 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9 less than 1 μM or less than about 1 μM of the agent (e.g., antibody) per cell; in some embodiments, the sub-optimal yield concentration is 1×10 cells in the incubation composition 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9less than 0.5 μM or less than about 0.5 μM of the agent (e.g., antibody) per cell; in some embodiments, the sub-optimal yield concentration is 1×10 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9 per cell, less than 0.2 μM or less than about 0.2 μM of the agent (e.g., antibody).

[0196] In some embodiments, the sub-optimal yield concentration is 1×10 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9 per cell, and the total amount of beads, particles, surface, or reagent is less than 15 mg or less than about 15 mg; in some embodiments, the sub-optimal yield concentration is 1×10 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9 per cell, 15×10 9 per cell, or 20×10 9 per cell, and the total amount of beads, particles, surface, or reagent is less than 10 mg or less than about 10 mg; in some embodiments, the sub-optimal yield concentration is 1×10 9 per cell, 2×10 9 per cell, 3×10 9 per cell, 4×10 9 per cell, 5×10 9 per cell, 10×10 9per piece, 15×10 9 per piece, or 20×10 9 per piece, the entire amount of beads, particles, surface, or reagent is less than 5 mg or about 5 mg; in some embodiments, the sub-optimal yield concentration is 1×10 9 cells per piece, 2×10 9 cells per piece, 3×10 9 cells per piece, 4×10 9 cells per piece, 5×10 9 cells per piece, 10×10 9 cells per piece, 15×10 9 cells per piece, or 20×10 9 per piece, the entire amount of beads, particles, surface, or reagent is less than 4 mg or about 4 mg; in some embodiments, the sub-optimal yield concentration is 1×10 9 cells per piece, 2×10 9 cells per piece, 3×10 9 cells per piece, 4×10 9 cells per piece, 5×10 9 cells per piece, 10×10 9 cells per piece, 15×10 9 cells per piece, or 20×10 9 per piece, the entire amount of beads, particles, surface, or reagent is less than 3 mg or about 3 mg; in some embodiments, the sub-optimal yield concentration is 1×10 9 cells per piece, 2×10 9 cells per piece, 3×10 9 cells per piece, 4×10 9 cells per piece, 5×10 9 cells per piece, 10×10 9 cells per piece, 15×10 9 cells per piece, or 20×10 9 per piece, the entire amount of beads, particles, surface, or reagent is less than 2 mg or about 2 mg; in some embodiments, the sub-optimal yield concentration is 1×10 9 cells per piece, 2×10 9 cells per piece, 3×10 9 cells per piece, 4×10 9 cells per piece, 5×10 9Per unit, 10×10 9 Per unit, 15×10 9 Per unit, or 20×10 9 Per unit, the entire amount of beads, particles, surface, or reagent is less than 1 mg or about less than 1 mg; in some embodiments, the sub - optimal yield concentration is, in the incubation composition, cells 1×10 9 Per cell, 2×10 9 Per cell, 3×10 9 Per cell, 4×10 9 Per cell, 5×10 9 Per cell, 10×10 9 Per cell, 15×10 9 Per cell, or 20×10 9 Per cell, the entire amount of beads, particles, surface, or reagent is less than 0.5 mg or about less than 0.5 mg.

[0197] In some embodiments, for example, when working at sub - optimal yield concentrations for each or one or more of two or more selection reagents having affinity for two or more markers or cells, one or more such reagents are used at a higher concentration than the other in order to bias the ratio of the cell types recognized by that reagent compared to the cell type recognized by the other one or more such reagents. For example, a reagent that specifically binds to a marker for which it is desired to bias the ratio can be included at a concentration (e.g., drug or mass per cell) that is half, 1 - fold, 2 - fold, 3 - fold, 4 - fold, 5 - fold, 10 - fold or more increased compared to one or more others, depending on how much the ratio is desired to be increased.

[0198] In some embodiments, achieving a sub - optimal yield concentration to bias one or both cell populations can achieve the desired, or selected, starting culture ratio. In some embodiments, the selection is at least cells 1×10 9 Per cell, 2×10 9 Per cell, 3×10 9 Per cell, 4×10 9 Per cell, 5×10 9 Per cell, 6×10 9 Per cell, 7×10 9 Per cell, 8×109 cells, 1 x 10 10 cells, 2 x 10 10 cells, 3 x 10 10 cells, 4 x 10 10 cells, and 5 x 10 10 cells or more, and is performed on a sample containing CD4+ cells and CD8+ cells. In some embodiments, the high cell number is sufficient to ensure saturation of the immunoaffinity reagent in the sample for cells expressing a marker such as CD4 or CD8 to which the reagent specifically binds.

[0199] In some embodiments, when working in the sub-optimal range and / or with a sufficient number of cells to achieve saturation of the reagent, the amount of immunoaffinity reagent is proportional to the approximate yield of enriched cells. In one embodiment, for achieving an initial culture ratio of CD4+ cells to CD8+ cells of about or approximately 1:1, the selection of CD4+ cells and CD8+ cells can be performed at the same or nearly the same sub-optimal yield concentration of the immunoaffinity reagent for CD4 and CD8, respectively. In another exemplary embodiment, for achieving an initial culture ratio of CD4+ cells to CD8+ cells of about or approximately 2:1, the selection of CD4+ cells can be performed at a sub-optimal yield concentration of the immunoaffinity reagent for CD4 that is about or approximately two-fold greater compared to the sub-optimal yield concentration of the immunoaffinity reagent for CD8. Considering the above illustrations, it is within the scope of one of ordinary skill in the art to empirically select or choose the appropriate amount or concentration of the immunoaffinity reagent according to the desired or selected initial culture ratio of the generated composition containing enriched or selected cells.

[0200] In some embodiments, the separation and / or step is performed using magnetic beads to which the immunoaffinity reagent is reversibly bound, for example, via the interaction of a peptide ligand with the streptavidin mutant protein described above. An example of such magnetic beads is Streptamers®. In some embodiments, the separation and / or step is performed using magnetic beads such as those commercially available from Miltenyi Biotec.

[0201] In some aspects, the separation and / or other steps are performed for the automated separation of cells at clinical scale levels in a closed sterile system. The components can include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the device and commands the system to execute repetitive procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for a selection column. The peristaltic pump controls the flow rate through the tubing set and, together with the pinch valves, ensures a controlled flow of buffer and a continuous suspension of cells through the system. In some aspects, the separation and / or other steps are performed using a CliniMACS system (Miltenyi Biotec).

[0202] In some embodiments, automated separation using a CliniMACS system or the like uses antibody-coupled magnetizable particles supplied in a sterile, pyrogen-free solution in some aspects. In some embodiments, after the cells are labeled with magnetic particles, the cells are washed and excess particles are removed. Next, the cell preparation bag is connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing including a pre-column and a separation column and is single-use only. After initiation of the separation program, the system automatically adds the cell sample onto the separation column. The labeled cells are retained within the column while the unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population for use in the methods described herein is unlabeled and not retained within the column. In some embodiments, the cell population for use in the methods described herein is labeled and retained within the column. In some embodiments, the cell population for use in the methods described herein is eluted from the column after removal of the magnetic field and collected within the cell collection bag.

[0203] In certain embodiments, the separation and / or other steps are performed using a system comprising a cell processing unit that enables automated washing and fractionation of cells by centrifugation. In some aspects, the separation and / or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). A system having a cell processing unit can also include an onboard camera and image recognition software that determines optimal cell fractionation endpoints by discerning macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into red blood cells, white blood cells, and plasma layers. A cell processing system such as the CliniMACS Prodigy system can also include an integrated cell cultivation chamber that accomplishes cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. The input ports can enable aseptic removal and replenishment of the media, and the cells can be monitored using an integrated microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0204] In some embodiments, the cell populations described herein are collected and enriched (or depleted) via flow cytometry in which cells stained with multiple cell surface markers are carried by a fluid flow. In some embodiments, the cell populations described herein are collected and enriched (or depleted) via preparative scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by use of a microelectromechanical systems (MEMS) chip in combination with a FACS-based detection system (see, e.g., WO2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In either case, the cells can be labeled with multiple markers and it is possible to isolate the T cell subsets detailed herein with high purity.

[0205] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, the separation can be based on binding to a fluorescently labeled antibody. In some instances, separation of cells based on the binding of an antibody or other binding partner specific for one or more cell surface markers is carried out in a fluid flow by fluorescence-activated cell sorting (FACS) including preparative scale (FACS) and / or by a microelectromechanical systems (MEMS) chip in combination with, for example, a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0206] b. Single step flow and / or sequential selection using immunoaffinity chromatography In some embodiments, the first selection or enrichment of the cell population and the second selection and / or enrichment of the cell population are performed using affinity-based reagents each comprising at least first and second affinity chromatography matrices having antibodies immobilized on their surfaces. In some embodiments, one or both of the first and / or second selections can use multiple affinity chromatography matrices and / or antibodies, where the multiple matrices and / or antibodies used for the same selection, i.e., the first selection or the second selection, are connected in series. In some embodiments, the one or more affinity chromatography matrices employed for the first and / or second selections can adsorb, or are capable of selecting or enriching, at least about 50×10 6 cells / mL, 100×10 6 cells / mL, 200×10 6 cells / mL or 400×10 6 cells / mL. In some embodiments, the adsorption capacity can be adjusted based on the diameter and / or length of the column. In some embodiments, the seeding ratio of the selected or enriched composition is achieved by choosing a sufficient amount of matrix and / or in a sufficient relative amount to achieve the expected seeding ratio based, for example, on the adsorption capacity of one or more columns for selecting the cells.

[0207] In an exemplary embodiment, the adsorption capacity of the one or more matrices is the same between the first and second selections, e.g., 1×10 8 cells or about 1×10 8cells / mL, wherein enrichment or selection of cells in the first and second selections results in a composition containing CD4+ and CD8+ cells at a starting culture ratio of about 1:1. In another exemplary embodiment, the adsorption capacity of one or more matrices used in one of the first or second selections is at least 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold or more than the adsorption capacity of one or more matrices used in the other of the first or second selections, such that cells selected with a greater adsorption capacity, such as CD4+ or CD8+ cells, are present in the starting culture ratio in an amount that is at least 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold or more than the other cell population. Selecting or choosing an appropriate volume, diameter or number of affinity matrix chromatography columns for the first and / or second selections according to the desired or selected starting culture ratio of the resulting composition containing the enriched or selected cells is within the scope of a person skilled in the art.

[0208] Exemplary steps for performing the selection by the provided method are shown in Example 2. In some embodiments, such steps achieve a desired starting culture ratio in the enriched or generated composition.

[0209] In some embodiments, the first and / or second selections in the provided method include a step of first enriching one of CD4+ or CD8+ cells, and then enriching a cell subpopulation based on surface expression of a marker, such as CD28, CD62L, CCR7, CD127 or CD27, that is expressed on, for example, resting T cells, naive T cells or central memory T cells. In some embodiments, the first and / or second selections include enriching CD8+ cells, and the selection is central memory T (T CM) further comprising enriching cells, wherein the other of the first and / or second selection comprises enriching CD4+ cells. In some embodiments, the method is performed to enrich or select CD4+ cells and to enrich or select cell subpopulations that are CD8+ / CD28+, CD8+ / CD62L+, CD8+ / CCR7+, CD8+ / CD127+ or CD8+ / CD27+. In some embodiments, the first selection comprises enriching CD8+ cells and the second selection comprises enriching CD4+ cells, wherein the first selection comprising cells enriched for CD8+ cells is central memory T (T CM ) cells, or further comprising enriching cells expressing a marker that is CD28, CD62L, CCR7, CD127 or CD27, thereby generating a composition such as a culture starting composition containing CD4+ cells and central memory T (T CM ) cells or enriched CD8+ cells expressing a marker that is CD28, CD62L, CCR7, CD127 or CD27. In some embodiments, the first selection comprises enriching CD4+ cells and the second selection comprises enriching CD8+ cells, wherein the second selection comprising cells enriched for CD8+ cells is central memory T (T CM ) cells, or further comprising enriching cells expressing a marker that is CD28, CD62L, CCR7, CD127 or CD27, thereby generating a composition such as a culture starting composition containing CD4+ cells, and central memory T (T CM ) cells or enriched CD8+ cells expressing a marker that is CD28, CD62L, CCR7, CD127 or CD27.

[0210] In some such embodiments, with further enrichment of the cell subpopulation to achieve a starting ratio of CD4+ cells or its subpopulations to CD8+ cells or its subpopulations, the adsorption capacity of one or more column matrices is adjusted to account for the difference in the frequency of the subpopulation, i.e., the frequency of CD4+ or CD8+ cells enriched in cells expressing markers that are resting cells, naive cells, central memory cells or CD28, CD62L, CCR7, CD127 or CD27, compared to the cell frequency of each CD4+ or CD8+ parental population in the starting sample from the subject. The relative levels or frequencies of the various cell populations in the subject can be determined based on assessing the surface expression of one or more markers present in such populations or subpopulations. Several well-known methods for assessing the expression level of surface markers or proteins, such as detection by affinity-based methods, e.g., methods based on immunoaflinity, can be used by flow cytometry, for example, in the context of cell surface proteins.

[0211] In an exemplary aspect, the sample is enriched in CD4+ and CD8+ / CD62L+ cells such that a ratio of CD4+ to CD8+ of 1:1 is obtained. In this exemplary aspect, the first selection can involve enriching CD8+ cells using a column having an adsorption capacity adjusted with respect to the relative frequencies of CD4+ cells and CD8+ / CD62L+ cells known to be present in the sample, or using a ratio generally estimated to be in such a sample. For example, the CD62L+ subpopulation of CD8+ cells collected from a human subject can sometimes be about 25% of the total fraction of CD8+ T cells. See, for example, Maldonado, Arthritis Res Ther. 2003; 5(2): R91-R96. In such an aspect, the column can be arranged to collect 4 times more CD8+ cells than CD4+ cells to produce a 1:1 starting culture ratio of CD4+ cells to the CD8+ subpopulation further comprising CD62L+ cells. Thus, assuming similar adsorption capacity and efficiency for each selection column, the CD8+ column can be about or approximately 4 times larger than the CD4 selection column or the CD62L selection column. The size of the column can also be adjusted for the expected yield. For example, if each column is 80% efficient, the size of each column can be adjusted to account for the efficiency of each subsequent selection.

[0212] For example, a desired or selected starting culture composition can be a composition containing 200×10 6 CD4+ cells and 200×10 6 CD8+ / CD62L+ cells. In this example, assuming the ratios presented above, the sample to be enriched can contain at least or about 200×10 6 CD4+ cells and at least or about 800×10 6 CD8+ cells (approximately 25% (200×10 6 cells) of which can also be CD62L+). Each 2 mL of the selection matrix can contain about or approximately 200×10 6Assuming that it is possible to enrich the cells, a CD8 selection column with an 8 mL selection matrix can bind approximately or about 800×10 6 cells of CD8+. On the other hand, the flow-through fraction passes through the CD4 selection column. A CD4 selection column with a 2 mL selection matrix can bind approximately or about 200×10 6 cells of CD4+. CD8+ cells can be further enriched for CD62L by eluting the CD8+ cells in a CD62L selection column. In this exemplary embodiment, the CD62L selection column can accommodate a 2 mL selection matrix and thus enrich approximately or about 200×10 6 cells of CD8+ / CD62L+. By eluting from the CD4 and CD8 / CD62L columns into the culture tank, a starting culture composition or a composition having an approximate or about 1:1 starting culture ratio can be obtained.

[0213] In another exemplary embodiment, the sample is enriched for CD4+ and CD8+ / CCR7+ cells, resulting in a 1:1 ratio of CD4+ to CD8+. In this exemplary embodiment, the first selection can involve using a column having an adsorption capacity adjusted with respect to the relative frequency of CD4+ cells to CD8+ / CCR7+ cells known to be present in the sample, or using a ratio generally estimated to be in such a sample, to enrich for CD8+ cells. For example, the CCR7+ subpopulation of CD8+ cells collected from a human subject can sometimes be about 60% of the total fraction of CD8+ T cells. See, for example, Chen, Blood. 2001 Jul 1;98(1):156-64. The columns can be arranged to collect 3 1 / 3 times more CD8+ cells than CD4+ cells to produce a 1:1 starting culture ratio. Assuming similar adsorption capacity and efficiency for each selection column, the CD8+ column is about or approximately 3 1 / It can be three times larger. The size of the column can also be adjusted for the expected yield. For example, if each column is 80% efficient, the size of each column can be adjusted to account for the efficiency of each subsequent selection.

[0214] For example, a desired starting culture can contain 200×10 6 cells of CD4+ and 200×10 6 cells of CD8+ / CCR7+. In this example, assuming the ratios presented above, the sample to be enriched can contain at least 200×10 6 cells of CD4+ and at least or about 6.6×10 6 cells of CD8+, approximately 60% (200×10 6 cells) of which can also be CCR7+. Assuming that each 2 mL of the selection matrix can enrich 200×10 6 cells, a CD8 selection column with a selection matrix of 3 1 / 3 mL can bind approximately or about 6.6x10 6 cells of CD8+, while the flow-through fraction passes through the CD4 selection column. A CD4 selection column with a 2 mL selection matrix can bind approximately or about 200×10 6 cells of CD4+. By eluting the CD8+ cells in the CCR7 selection column, CD62L can be further enriched from the CD8+ cells. In this exemplary embodiment, the CCR7 selection column can accommodate 1 mL of the selection matrix and thus enrich approximately or about 200×10 6 cells of CD8+ / CCR7+. The CD4 and CCCR7 columns can be eluted into the culture vessel, yielding a starting culture containing approximately or about 200×10 6 cells of CD4+ and approximately or about 200×10 6 cells of CD8+ / CCR7+, or a starting culture with a 1:1 culture start ratio.

[0215] Depending on the desired or selected starting ratio of the generated composition containing enriched or selected cells, the expected frequency of each subpopulation, the various efficiencies of each selection column, and other factors within the level of skill of the artisan, it is within the level of skill of the artisan to empirically select or choose an appropriate volume, diameter, or number of affinity matrix chromatography columns for the first and / or second and / or third selections, taking into account the above illustrations.

[0216] B. Incubation of Isolated Cells In some embodiments, the provided method includes one or more various steps for incubating isolated cells and cell populations, such as populations isolated according to the methods herein, e.g., an isolated CD4+ T cell population, e.g., an unfractionated CD4+ T cell population or a subpopulation thereof, and an isolated CD8+ T cell population, e.g., an isolated unfractionated CD8+ T cell population or a subpopulation thereof. In some embodiments, the cell population is incubated in a starting culture composition.

[0217] Multiple isolated cell populations, e.g., CD4+ cell populations and CD8+ cell populations (e.g., unfractionated or subpopulations thereof), are typically incubated together with cell populations combined in a starting culture composition within the same culture vessel, such as the same unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culturing or growing cells.

[0218] In some aspects, the cell population or cell type is designed to achieve a particular desired output ratio, or a ratio within a particular range of tolerance of such a desired output ratio, in the starting culture composition, e.g., after an incubation and / or manipulation step, or to do so for a particular percentage of the time, e.g., CD4 + cells to CD8 +It exists in a ratio to the cells. The output ratio can be, for example, the optimal ratio to achieve one or more therapeutic effects when administered to a patient, for example, by adoptive cell therapy. In some aspects, the starting culture ratio is determined empirically, for example, using the determination methods described herein, such that the optimal starting culture ratio to achieve the desired output ratio is determined in a particular situation.

[0219] The incubation stage can include culturing, cultivating, stimulating, activating, propagating, etc. by incubation in the presence of conditions designed to mimic antigen exposure, to induce stimulatory conditions, e.g., to induce the growth, expansion, activation, and / or survival of cells in the population, and / or to pre-stimulate the cells for genetic manipulation, e.g., for the introduction of a genetically engineered antigen receptor.

[0220] The conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, drugs, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other drugs designed to activate the cells. In one example, the stimulatory conditions include one or more drugs, e.g., ligands that initiate or start the TCR / CD3 intracellular signaling cascade in T cells. Such drugs can include antibodies specific for TCR components and / or co-stimulatory receptors, e.g., antibodies bound to a solid support, such as beads, e.g., anti-CD3, anti-CD28, anti-4-1BB, and / or one or more cytokines. Optionally, the expansion method can further include the step of adding anti-CD3 and / or anti-CD28 antibodies (e.g., at a concentration of at least about 0.5 ng / ml) to the medium. Optionally, the expansion method can further include the step of adding IL-2 and / or IL-15 and / or IL-7 and / or IL-21 to the medium (e.g., wherein the concentration of IL-2 is at least about 10 units / ml).

[0221] In some aspects, incubation is performed according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0222] In some embodiments, CD4 + and CD8 + Cell populations, such as populations or subpopulations, are added to a culture starting composition feeder cell, such as non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the starting population to be expanded); and the culture is expanded by incubating (e.g., for a time sufficient to increase the number of T cells). In some aspects, the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000 - 3600 rad to block cell division. In some aspects, the feeder cells are added to the medium prior to the addition of the T cell population.

[0223] In some embodiments, the stimulation conditions include a temperature appropriate for the growth of human T lymphocytes, such as at least about 25 degrees Celsius, usually at least about 30 degrees Celsius, usually 37 degrees Celsius or about 37 degrees Celsius. In some embodiments, during the culture, a temperature change such as from 37 degrees Celsius to 35 degrees Celsius is induced. Optionally, incubation can further include adding non-dividing EBV-transformed lymphoblastoid cells (LCLs) as feeder cells. The LCLs can be irradiated with gamma rays in the range of about 6000 - 10,000 rad. The LCL feeder cells are provided in any suitable amount, such as a ratio of at least about 10:1 LCL feeder cells to starting T lymphocytes, in some aspects.

[0224] In embodiments, antigen-specific CD4+ and CD8 + The population can be obtained by stimulating naive T lymphocytes or antigen - specific T lymphocytes with an antigen. For example, an antigen - specific T cell line or clone can be generated against an antigen by isolating T cells from an infected subject and stimulating the cells in vitro with a cytomegalovirus antigen. Naive T cells can also be used.

[0225] Provisional evaluation and adjustment In some embodiments, the method includes the evaluation and / or adjustment of cells or cell - containing compositions at a time after the start of incubation or culture, such as the time during incubation. The evaluation can include performing one or more measurements of the cell - containing composition or vessel, for example, evaluating the cells for growth rate, viability, phenotype, such as the expression of one or more surface markers or intracellular markers such as proteins or polynucleotides, and / or evaluating the composition or vessel for temperature, media components, oxygen content, or carbon dioxide content, and / or the presence or absence or amount or relative amount of one or more factors, agents, components, and / or subtypes, including cell types. In some embodiments, the evaluation includes determining the ratio between a plurality of, for example, two cell types, such as CD4 + T cells and CD8 + T cells. In some aspects, the evaluation is performed in an automated manner, for example, using the devices described herein, and / or is set prior to the time at which it should be performed at a specific point during incubation. In some aspects, the result of the evaluation, such as the determined provisional ratio of two cell types, indicates that an adjustment, such as the addition or removal of one or more cell types, should be made.

[0226] Adjustments can include adjusting temperature, incubation or the length of time (in hours) during which the step is carried out (incubation period), replenishment, addition and / or removal of one or more components in the composition being incubated, such as a medium or buffer or components thereof, agents, such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, or any cell culture factor or parameter such as a cell or cell type or cell population. In some aspects, removal or addition of various components or other adjustments are carried out in an automated manner, for example using the devices or systems described herein. In some embodiments, the system is programmed such that adjustments are automatically initiated based on a particular readout from a provisional assessment. For example, in some cases, the system or device is programmed to perform one or more assessments at a particular time; in such cases, the system or device can be further programmed such that a particular result of such an assessment, such as a particular ratio of one cell type to another cell type, initiates a particular adjustment, such as the addition of one or more cell types.

[0227] In some aspects, the adjustment is carried out by addition or removal in a manner that does not disrupt the closed environment containing the cells and composition, such as by input valves and / or removal valves designed to add or remove components while maintaining sterility, such as in one or more of the devices or systems described herein.

[0228] In a particular embodiment, CD4 + T cells and CD8 + The provisional ratio of T cells is evaluated during the incubation period. In some embodiments, the evaluation is carried out 1, 2, 3, 4, 5, 6, or 7 days later, such as between 3 and 5 days, and / or at a time when all cells are or are suspected to be in the cell cycle. In some aspects, the provisional ratio so determined is for an isolated population of CD4 + T cells or CD8 +An isolated population of T cells (e.g., a subpopulation such as central memory CD8 + T cells), cells such as CD4 + T cells or CD8 + T cells are shown to be added or enriched to a culture vessel or a composition being incubated. Thus, in some aspects, after evaluation, such addition or removal, typically addition, continues. In some aspects, multiple evaluations and possible adjustments are performed over the course of incubation, e.g., in an iterative manner.

[0229] For example, in some embodiments where cells are engineered to introduce a genetically engineered antigen receptor, incubation in the presence of one or more stimulants continues during the engineering period.

[0230] In some embodiments, the cells are incubated for a total of, or prior to any manipulation, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or about that number of days.

[0231] C. Engineering, Engineered Antigen Receptors, and Engineered Cells In some embodiments, the method includes genetic engineering of isolated and / or incubated cells, such as introducing into the cells a recombinant gene for expression of a receptor useful in adoptive therapy settings, such as a molecule such as an antigen receptor.

[0232] Genes for improving the effectiveness of a therapy, such as by promoting the viability and / or function of the transplanted cells, among the genes for introduction; for example, genes for providing gene markers for cell selection and / or evaluation for assessing in vivo survival or localization; for example, genes for improving safety by making cells sensitive to negative selection in vivo, which are described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications PCT / US91 / 08442 and PCT / US94 / 05601 by Lupton et al., which describe the use of bifunctional selection fusion genes derived from fusing a dominant positive selection marker with a negative selection marker. This can be carried out according to known techniques (e.g., see Riddell et al., U.S. Patent No. 6,040,177, columns 14-17), or its variations that would be apparent to those skilled in the art based on this disclosure.

[0233] The manipulation typically involves the introduction of one or more genes for the expression of a genetically engineered antigen receptor. Such antigen receptors include genetically engineered T cell receptors (TCRs) and their components, as well as functional non-TCR antigen receptors such as chimeric antigen receptors (CARs).

[0234] In some embodiments, the antigen receptor specifically binds to a ligand of a cell or disease to be targeted, such as cancer or other disease or condition, including the ligands described herein for targeting with the provided methods and compositions. Exemplary antigens include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B virus surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, cancer fetal antigen, ROR1, TAG72, VEGF-R2, fetal cancer antigen (CEA), prostate specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, Ephrin B2, CD123, CS-1, c-Met, GD-2, and MAGE A3 and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0235] Antigen receptor In one embodiment, the engineered antigen receptor is a CAR. A CAR typically comprises a genetically engineered receptor that includes an extracellular ligand-binding domain linked to one or more intracellular signaling components. Such molecules typically mimic or approximate signals via natural antigen receptors and / or signals via such receptors together with co-stimulatory receptors.

[0236] In some embodiments, the CAR is engineered to have specificity for a particular marker expressed in a particular cell type to be targeted by adoptive therapy, such as a gamma marker, for example, a particular marker. This is achieved, in some aspects, by including in the extracellular portion of the CAR one or more antigen-binding fragments, domains, or moieties, or one or more antigen-binding molecules such as one or more antibody variable domains and / or antibody molecules. In some embodiments, the CAR comprises one or more antigen-binding portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0237] In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds to a cell surface antigen of a cell or disease to be targeted, such as a tumor cell or cancer cell, such as any target antigen described herein or known in the art.

[0238] In some embodiments, the tumor antigen or cell surface molecule is a polypeptide. In some embodiments, the tumor antigen or cell surface molecule is selectively expressed or overexpressed on tumor cells as compared to non-tumor cells of the same tissue.

[0239] In some embodiments, the CAR binds to a pathogen-specific antigen. In some embodiments, the CAR is specific for a viral antigen (e.g., HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen.

[0240] In some aspects, the antigen-specific binding or recognition component is linked to one or more transmembrane domains and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. Optionally, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, such that interaction with other members of the receptor complex is minimized.

[0241] In some embodiments, the transmembrane domain is derived from either natural or synthetic origin. When of natural origin, in some aspects the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions include regions derived from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., including at least their transmembrane regions). Alternatively, in some embodiments the transmembrane domain is synthetic. In some aspects, synthetic transmembrane domains mainly contain hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0242] In some embodiments, a short-chain oligopeptide or polypeptide linker, such as a linker between 2 to 10 amino acids in length, such as a linker containing glycine and serine, such as a glycine-serine doublet, is present to form a linkage between the transmembrane domain of the CAR and the cytoplasmic signaling domain.

[0243] The CAR generally comprises one or more intracellular signaling components. In some embodiments, the CAR mediates TCR CD3 for T cell activation and cytotoxicity +It includes a lock, an intracellular component of the TCR complex such as the CD3 zeta chain. Thus, in some aspects, the antigen-binding molecule is linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the CAR further includes a part of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.

[0244] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions of immune cells, such as T cells engineered to express the cells. For example, depending on the situation, the CAR induces the function of T cells, such as cytotoxic activity, or T helper activity such as the secretion of cytokines or other factors. In some embodiments, a shortened portion of the intracellular signaling domain of an antigen receptor component or co-stimulatory molecule. Such a shortened portion is used in some aspects in place of the intact immune-stimulatory chain, for example if it transmits an effector function signal. In some embodiments, one or more intracellular signaling domains include the cytoplasmic sequence of the T cell receptor (TCR), and in some aspects, cooperate with such a receptor in a natural situation to initiate signaling after association of the antigen receptor, and / or the cytoplasmic sequence of a co-receptor, and / or any derivative or variant of such a molecule, and / or any synthetic sequence having the same functional ability.

[0245] In the context of natural TCRs, full activation generally requires not only signaling through the TCR but also co-stimulatory signals. Thus, in some embodiments, components for generating secondary or co-stimulatory signals are also included in the CAR to promote full activation. T cell activation is, in some aspects, mediated by two classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and sequences that act antigen-independently to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.

[0246] The primary cytoplasmic signaling sequences can, in some aspects, regulate the primary activation of the TCR complex either stimulatory or inhibitory. Stimulatory primary cytoplasmic signaling sequences can include signaling motifs known as immunoreceptor activation tyrosine motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences include sequences derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule in the CAR includes a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

[0247] In some embodiments, the CAR includes the signaling domain and / or transmembrane portion of co-stimulatory receptors such as CD28, 4-1BB, OX40, DAP10, and ICOS.

[0248] In certain embodiments, the intracellular signaling domain includes the CD28 transmembrane and signaling domain linked to the CD3 intracellular domain. In some embodiments, the intracellular signaling domain includes a chimeric co-stimulatory domain of CD28 and CD137 linked to the CD3 intracellular domain. In some embodiments, the CAR can also include a transduction marker (e.g., tEGFR). In some embodiments, CD8 +The intracellular signaling domain of cytotoxic T cells is CD4 + is the same as the intracellular signaling domain of helper T cells. In some embodiments, CD8 + The intracellular signaling domain of cytotoxic T cells is CD4 + is different from the intracellular signaling domain of helper T cells.

[0249] In some embodiments, the CAR includes two or more costimulatory domains together with an activation domain, such as a primary activation domain, in the cytoplasmic portion. One example is a receptor that includes the intracellular components of CD3-zeta, CD28, and 4-1BB.

[0250] The CAR and its production and introduction can include, for example, those described by the published patent disclosures WO200014257, US6451995, US2002131960, US7446190, US8252592, EP2537416, US2013287748, and WO2013126726, and / or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75.

[0251] In some embodiments, the T cells are modified with a recombinant T cell receptor. In some embodiments, the recombinant TCR is specific for an antigen present on a target cell, such as an antigen expressed on a tumor-specific antigen, a specific cell type associated with an autoimmune or inflammatory disease, or an antigen derived from a viral or bacterial pathogen.

[0252] In some embodiments, T cells are engineered to express a T cell receptor (TCR) cloned from a native T cell. In some embodiments, high affinity T cell clones specific for a target antigen (e.g., a cancer antigen) are identified, isolated from a patient, and introduced into the cells. In some embodiments, TCR clones specific for a target antigen are generated from transgenic mice engineered using human immune system genes (e.g., human leukocyte antigen system or HLA). See, for example, tumor antigens (e.g., see Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808). In some embodiments, phage display is used to isolate TCRs specific for a target antigen (e.g., see Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354).

[0253] In some embodiments, after the T cell clone is obtained, the TCR alpha and beta chains are isolated and cloned into a gene expression vector. In some embodiments, the TCR alpha and beta genes are linked via a picornavirus 2A ribosome skipping peptide such that both chains are co-expressed. In some embodiments, genetic transfer of the TCR is effected via a retroviral or lentiviral vector, or via a transposon (see, e.g., Baum et al. (2006) Molecular Therapy: The Journal of the American Society of Gene Therapy. 13:1050-1063; Frecha et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:1748-1757; and Hackett et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:674-683).

[0254] In some embodiments, genetic transfer is effected by first stimulating the growth of T cells, then transducing the activated cells, and culturing and expanding them to a number sufficient for clinical application.

[0255] In some situations, overexpression of stimulatory factors (such as lymphokines or cytokines) can be toxic to a subject. Thus, in some situations, the engineered cells contain genetic segments that render the cells sensitive to negative selection in vivo, for example, upon administration in adoptive immunotherapy. For example, in some aspects, the cells are engineered so that they can be eliminated as a result of changes in the in vivo state of the patient to whom the cells are administered. A phenotype that allows for negative selection can result from the insertion of a gene that confers sensitivity to an administered agent, such as a compound. Genes that allow for negative selection include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene that confers ganciclovir sensitivity (Wigler et al., Cell II :223, I977); the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0256] In some aspects, the cells are further engineered to promote the expression of inflammatory cytokines, such as cytokines such as IL-2, IL-12, IL-7, IL-15, IL-21.

[0257] Introduction of Genetically Engineered Components Various methods for the introduction of genetically engineered components, such as antigen receptors, such as CARs, are well known and can be used in the provided methods and compositions. Exemplary methods include methods for the transfer of nucleic acids encoding receptors, including transduction with viruses, such as retroviruses or lentiviruses, transposons, and methods via electroporation.

[0258] In some embodiments, the recombinant nucleic acid is introduced into cells using recombinant infectious virus particles such as vectors derived from, for example, Simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is introduced into T cells using a recombinant lentiviral vector or a retroviral vector such as a gamma-retroviral vector (see, for example, Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November; 29(11): 550-557).

[0259] In some embodiments, retroviral vectors, such as retroviral vectors derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus-forming virus (SFFV), or adeno-associated virus (AAV), have long terminal repeats (LTRs). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retrovirus includes those derived from any avian or mammalian cell origin. Retroviruses are typically amphotropic, meaning they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Several exemplary retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A.D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0260] Lentiviral transduction methods are known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0261] In some embodiments, the recombinant nucleic acid is introduced into T cells by electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, the recombinant nucleic acid is introduced into T cells via gene translocation (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (described, e.g., in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[0262] In some embodiments, CD4 + T lymphocytes and CD8 + T lymphocytes are each introduced with the same CAR. In some embodiments, CD4 + T lymphocytes and CD8 + T lymphocytes are each introduced with a different CAR. In some embodiments, the CARs in each of these populations have antigen-binding molecules that specifically bind the same antigen. In some embodiments, the CARs in each of these populations bind to different molecules. In some embodiments, the CARs in each of these populations have different cell signaling modules. In some embodiments, CD4 +T lymphocytes or CD8 + Each of the T lymphocytes is sorted into naive cells, central memory cells, effector memory cells, or effector cells prior to transduction.

[0263] In other aspects, cells such as tumor infiltrating lymphocytes and / or T cells that are cultured in vitro or ex vivo during, for example, an incubation step, such as T cells, are not engineered to express a recombinant receptor, but rather contain a native antigen receptor specific for a desired antigen and promote the expansion of cells having a particular antigen specificity. For example, in some aspects, the cells are generated for adoptive cell therapy by isolation of tumor specific T cells, such as autologous tumor infiltrating lymphocytes (TIL). Direct targeting of human tumors with autologous tumor infiltrating lymphocytes can, in some cases, mediate tumor regression (see Rosenberg SA, et al. (1988) N Engl J Med. 319:1676-1680). In some aspects, the lymphocytes are removed from an excised tumor. In some aspects, such lymphocytes are expanded in vitro. In some aspects, such lymphocytes are cultured with lymphokines (such as IL-2). In some aspects, such lymphocytes mediate the specific lysis of autologous tumor cells, but not allogeneic tumors or autologous normal cells.

[0264] In some aspects, the incubation and / or manipulation steps and / or the method generally results in a desired output ratio (or a ratio within a tolerance or tolerance from such a ratio), or does so within a specified percentage of the time during which the method is performed.

[0265] D. Cryopreservation In some embodiments, the provided method includes steps for freezing, e.g., cryopreserving, the cells either before or after isolation, incubation, and / or manipulation. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and to some extent monocytes in the cell population. In some embodiments, the cells are suspended in a cryopreservation solution after a washing step to remove, e.g., plasma and platelets. In any of a variety of known cryopreservation solutions and parameters may be used in some aspects. One example involves using PBS or other suitable cell cryopreservation medium containing 20% DMSO and 8% human serum albumin (HSA). This is then diluted 1:1 with the medium such that the final concentrations of DMSO and HSA are 10% and 4% respectively. The cells are then frozen at -80 °C at a rate of 1 °C per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0266] II. Kits and systems Systems, devices, and kits useful for performing the provided method are also provided. In one example, a single system is provided that performs one or more of the isolation, cell preparation, separation, e.g., separation based on density, affinity, sensitivity to one or more components, or other properties, washing, processing, incubation, culture, and / or formulation steps of the method. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, e.g., to minimize errors, user handling, and / or contamination. In one example, the system is a system such as that described in International Patent Application Publication No. WO2009 / 072003, or US20110003380A1.

[0267] In some embodiments, a system or apparatus performs one or more, e.g., all, of the isolation, processing, manipulation, and formulation steps in an integrated or self-sufficient system and / or in an automated or programmable manner. In some aspects, a system or apparatus includes a computer and / or computer program in communication with the system or apparatus that enables a user to program, control, evaluate the results of, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0268] A kit for practicing the provided method is also provided. In some embodiments, the kit includes an antibody or other binding partner generally coupled to a solid support for the isolation step of the method, e.g., an affinity-based separation.

[0269] In some embodiments, the kit includes antibodies for positive and negative selection coupled to magnetic beads. In one embodiment, the kit starts with a sample such as a PBMC sample and includes instructions for performing selection based on the expression of a first surface marker recognized by one or more antibodies provided with the kit and for performing the selection by retaining both the positive and negative fractions. In some aspects, the instructions further include instructions for performing one or more additional selection steps starting from the positive and / or negative fractions derived therefrom while maintaining, e.g., in a containment environment for the composition and / or in the same separation vessel.

[0270] In one embodiment, the kit includes anti-CD4, anti-CD14, anti-CD45RA, anti-CD14, and anti-CD62L antibodies coupled to magnetic beads. In one embodiment, the kit starts with a sample such as a PBMC sample, selects based on the expression of CD4, retains both the positive and negative fractions, and further subjects the negative fraction to either negative selection using anti-CD14 and anti-CD45RA antibodies and positive selection using anti-CD62L antibody in either order to perform the selection and includes instructions for performing the selection. Alternatively, the components and instructions are adjusted according to any of the separation embodiments described herein.

[0271] In some embodiments, the kit further includes instructions for moving a population of cells isolated by a selection step into a culture, cultivation, or processing vessel, while maintaining the cells in a self-sufficient system. In some embodiments, the kit includes instructions for moving different isolated cells in a specific ratio.

[0272] III. Cells, compositions, and methods of administration Also provided are cells, cell populations, and compositions (including pharmaceutical and therapeutic compositions) containing such cells and populations produced by the provided methods. Also provided are methods, such as therapeutic methods, for administering the cells and compositions to a subject, e.g., a patient.

[0273] Provided are methods of administering the cells, populations, and compositions, and the use of such cells, populations, and compositions for treating or preventing diseases, conditions, and disorders including cancer. In some embodiments, the cells, populations, and compositions are administered to a subject or patient having a specific disease or condition to be treated by adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells and compositions prepared by the provided methods, such as engineered compositions and end-product compositions after incubation and / or other processing steps, are administered to a subject, such as a subject having or at risk of having a disease or condition. In some aspects, the method thereby treats, e.g., alleviates, one or more symptoms of the disease or condition, such as by reducing the amount of systemic tumor tissue in a cancer expressing an antigen recognized by, e.g., engineered T cells.

[0274] Methods for the administration of cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, methods of adoptive T cell therapy are described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

[0275] In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by autotransplantation, where the cells are isolated and / or otherwise prepared from a subject who is intended to receive the cell therapy or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, such as a patient in need of treatment, and the cells are administered to the same subject after isolation and processing.

[0276] In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by allotransplantation, where the cells are isolated and / or otherwise prepared from a subject other than the subject who is or will ultimately be receiving the cell therapy, such as a first subject. In such embodiments, the cells are then administered to a different subject of the same species, such as a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0277] In some embodiments, the subject to which the cells, cell population, or composition is administered, such as a patient, is a mammal, typically a primate such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be of any suitable age, including infant, child, young, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal such as a rodent.

[0278] Pharmaceutical compositions for use in such methods are also provided.

[0279] Among the diseases, conditions, and disorders treatable using the provided compositions, cells, methods, and uses are tumors, including solid tumors, hematological malignancies, and melanoma, as well as infections and infectious diseases such as those caused by viruses or other pathogens, e.g., HIV, HCV, HBV, CMV, and parasitic diseases. In some embodiments, the disease or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disorder. Such diseases include, without limitation, leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, low-grade B-cell lymphoma, B-cell malignancies, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin (including melanoma) cancer, bone cancer, and brain cancer, ovarian cancer, epithelial cancer, renal cell cancer, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and / or mesothelioma.

[0280] In some embodiments, the disease or condition is, without limitation, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, cytomegalovirus (CMV), Epstein - Barr virus (EBV), adenovirus, BK polyomavirus, or other infectious or infective states. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition such as arthritis, e.g., rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and / or a disease or condition associated with transplantation.

[0281] In some embodiments, the antigen associated with the disease or disorder is selected from the group consisting of orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1 - CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B virus surface antigen, anti - folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP - 2, EGP - 4, EphA2, ErbB2, 3, or 4, FBP, fetal acetylcholine e receptor, GD2, GD3, HMW - MAA, IL - 22R - alpha, IL - 13R - alpha2, kdr, kappa light chain, Lewis Y, L1 - cell adhesion molecule, MAGE - A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY - ESO - 1, MART - 1, gp100, cancer fetal antigen, ROR1, TAG72, VEGF - R2, fetal cancer antigen (CEA), prostate - specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS - 1, c - Met, GD - 2, and MAGE A3 and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0282] In some embodiments, the cells and compositions are administered to a subject in the form of a pharmaceutical composition, such as a composition comprising a cell or cell population and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition additionally comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like. In some embodiments, the agent is administered in the form of a salt, for example a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids such as p-toluenesulfonic acid.

[0283] The choice of carrier in the pharmaceutical composition is determined in part by the particular engineered CAR or TCR, vector, or cell expressing the CAR or TCR, and by the particular method used to administer the host cell expressing the vector or CAR. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of from about 0.0001 wt% to about 2 wt% of the total composition.

[0284] In addition, in some aspects, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or its mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, by Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0285] In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as a cyclodextrin inclusion complex, or as a liposome. Liposomes can be useful for targeting host cells (e.g., T cells or NK cells) to specific tissues. To prepare liposomes, numerous methods are available, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0286] In some aspects, the pharmaceutical composition uses a time-release, delayed-release, and / or sustained-release delivery system such that delivery of the composition occurs before sensitization of the site to be treated and for a time sufficient to cause such sensitization. Many types of release delivery systems are available and known to those of skill in the art. In some aspects, such systems can enhance convenience to the subject and the physician by avoiding repeated administration of the composition.

[0287] In some embodiments, the pharmaceutical composition comprises an amount of cells or cell populations effective to treat or prevent a disease or condition, such as a therapeutically effective amount or a prophylactically effective amount. Thus, in some embodiments, the method of administration comprises administration of the cells and populations in an effective amount. In some embodiments, therapeutic or prophylactic efficacy is monitored by periodic evaluation of the treated subject. For repeated administrations over several days, depending on the condition, the treatment is repeated until the desired suppression of the disease symptoms occurs. However, other modes of administration may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, multiple bolus administrations of the composition, or continuous infusion administration of the composition.

[0288] In some embodiments, the cells are administered in a desired dosage that, in some aspects, includes the desired dosage or number of cells or cell types and / or the desired ratio of cell types. Thus, the dosage of cells, in some embodiments, is based on the desired ratio of individual populations or subtypes, such as the total cell number (or number per kg body weight) and the ratio of CD4+ to CD8+. In some embodiments, the dosage of cells is based on the desired total number of cells (or number per kg body weight) of an individual population or individual cell type. In some embodiments, the dosage is based on a combination of such characteristics, such as the desired total cell number, the desired ratio, and the desired total cell number in an individual population.

[0289] In some embodiments, CD8 + T cells and CD4 + Populations or subtypes of cells, such as T cells, are administered in a desired dosage of the total cells, such as the desired T cell dosage, or within its tolerance. In some aspects, the desired dosage is the desired number of cells, or the desired number of cells per unit body weight of the subject to which the cells are administered, such as cells / kg. In some aspects, the desired dosage is at least the minimum number of cells or the minimum number of cells per unit body weight. In some aspects, between the total cells administered in the desired dosage, an individual population or subtype has the desired output ratio (e.g., CD4 + to CD8 +It is present at, or near, such a ratio, for example, within a specific tolerance or error of such a ratio.

[0290] In some embodiments, the cells are administered at, or within a tolerance of, a desired dose of one or more individual cell populations or cell subtypes, such as a desired dose of CD4+ cells and / or a desired dose of CD8+ cells. In some aspects, the desired dose is the desired number of cells of the subtype or population, or the desired number of such cells per unit body weight of the subject to which the cells are administered, e.g., cells / kg. In some aspects, the desired dose is greater than or equal to the minimum number of cells of the population or subtype, or greater than or equal to the minimum number of cells of the population or subtype per unit body weight.

[0291] Thus, in some embodiments, the dosage is based on a desired fixed dose and desired ratio of total cells and / or based on one or more, e.g., respective desired fixed doses, of individual subtypes or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and the desired ratio of CD4 + cells to CD8 + cells and / or based on the desired ratio of CD4 + and / or CD8 + cells and / or based on a desired fixed or minimum dose of CD4 and / or CD8 cells.

[0292] In certain embodiments, an individual population of cells or cell subtypes is, for example, from about 1 million to about 50 billion (such as about 5 million, about 25 million, about 500 million, about 1 billion, about 5 billion, about 20 billion, about 30 billion, about 40 billion, or a range defined by any two of the foregoing values), for example, from about 10 million to about 100 billion (such as about 20 million, about 30 million, about 40 million, about 60 million, about 70 million, about 80 million, about 90 million, about 10 billion, about 25 billion, about 50 billion, about 75 billion, about 90 billion, or a range defined by any two of the foregoing values), and in some cases from about 100 million to about 50 billion (such as about 120 million, about 250 million, about 350 million, about 450 million, about 650 million, about 800 million, about 900 million, about 3 billion, about 30 billion, about 45 billion) or any value between these ranges, in the range of about 1 million to about 100 billion, and is administered to a subject.

[0293] In some embodiments, the dose of total cells and / or the dose of individual cell subpopulations is between 10 5 and 10 6 cells / kg body weight, such as 1×10 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 cells / kg, or 1×10 6 cells / kg body weight or about that value, between 10 4 or about 10 4 and 10 9 or about 10 9 cells per kilogram (kg) of body weight. For example, in some embodiments, the cells are T cells between 10 5 and 10 6 cells / kg body weight, such as T cells 1×10 5 cells / kg, T cells 1.5×10 5 cells / kg, T cells 2×10 5 cells / kg, or T cells 1×10 6 cells / kg body weight, or about that value, T cells between 10 4 or about 10 4 and 10 9 or about 10 9administered between or within a particular error range of that number of cells per kilogram (kg) of body weight.

[0294] In some embodiments, the cells are CD4 + and / or CD8 + cells are between 10 5 and 10 6 cells per kg of body weight, for example, CD4 + cells and / or CD8 + cells are 1×10 5 cells / kg, CD4 + cells and / or CD8 + cells are 1.5×10 5 cells / kg, CD4 + cells and / or CD8 + cells are 2×10 5 cells / kg, or CD4 + cells and / or CD8 + cells are 1×10 6 cells / kg of body weight, or about that value, CD4 + cells and / or CD8 + cells are 10 4 or about 10 4 and 10 9 or about 10 9 cells per kilogram (kg) of body weight, or within a particular error range of that value, are administered.

[0295] In some embodiments, the cells are CD4 + cells are at least about 1×10 6 and at least about 2.5×10 6 and at least about 5×10 6 and at least about 7.5×10 6 and at least about 9×10 6 cells, and / or CD8+ cells are at least about 1×10 6 and at least about 2.5×10 6 and at least about 5×10 6 and at least about 7.5×10 6 and at least about 9×10 6 cells, and / or T cells are at least about 1×10 6 and at least about 2.5×10 6 and at least about 5×106 , about 7.5×10 6 , or about 9×10 6 cells, or within its specific error range. In some embodiments, the cells are T cells with about 10 8 to 10 12 or about 10 10 to 10 11 cells, CD4 + cells are about 10 8 to 10 12 cells or about 10 10 to 10 11 cells, and / or CD8 + cells are about 10 8 to 10 12 cells or about 10 10 cells to 10 11 cells, or within its specific error range.

[0296] In some embodiments, the cells are administered in a desired output ratio of multiple cell populations or subtypes, such as CD4+ cells and CD8+ cells or subtypes, or within its tolerance range. In some aspects, the desired ratio can be a specific ratio or a range of ratios. For example, in some embodiments, the desired ratio (e.g., CD4 + cells to CD8 +(The ratio with cells) is 5:1 or about 5:1 to 5:1 or about 5:1 (or greater than about 1:5 and less than about 5:1), or 1:3 or about 1:3 to 3:1 or about 3:1 (or greater than about 1:3 and less than about 3:1), for example, 2:1 or about 2:1 to 1:5 or about 1:5 (or greater than about 1:5 and less than about 2:1, for example, about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5 or about that ratio. In some aspects, the tolerance is within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio (including any value between these ranges).

[0297] In some aspects, the cell population and composition are administered to a subject using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, oral, sublingual, or suppository administration. In some aspects, the cell population is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some aspects, the cell population is administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0298] In some aspects, the cell population obtained using the methods described herein is co-administered simultaneously or sequentially in any order, either with one or more additional therapeutic agents or in connection with another therapeutic intervention. Depending on the situation, the cells are co-administered with another treatment at a time sufficiently close so that the cell population enhances or is the reverse of the effect of one or more additional therapeutic agents. In some aspects, the cell population is administered prior to one or more additional therapeutic agents. In some aspects, the cell population is administered after one or more additional therapeutic agents.

[0299] After administration of the cells, in some embodiments, the biological activity of the engineered cell population is measured by any of several known methods, for example. Parameters to be evaluated include engineered or native T cell or other immune cell specific binding to an antigen, for example, in vivo by imaging or ex vivo by, for example, ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying the expression and / or secretion of one or more cytokines such as CD107a, IFNγ, IL-2, and TNF. In some aspects, the biological activity is measured by assessing clinical outcomes, such as a decrease in systemic tumor tissue mass or tumor burden.

[0300] In certain embodiments, the engineered cells are further modified in any number of ways such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population can be conjugated to a targeting moiety either directly or indirectly via a linker. Conjugating a compound, such as a CAR or TCR, to a targeting moiety is known in the art. See, for example, Wadwa et al., J. Drug Targeting 3: 111 (1995), and U.S. Patent No. 5,087,616.

[0301] IV. Definitions As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more".

[0302] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the claimed subject matter. Accordingly, a description in terms of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values within that range. For example, when a range of values is provided, it is understood that each intervening value, between the upper and lower limits of that range, and any other stated value or intervening value in the stated range, is included within the claimed subject matter. These smaller ranges of upper and lower limits may independently be included within the smaller ranges, subject to any specifically excluded limit values in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the claimed subject matter. This applies regardless of the breadth of the range.

[0303] The term "about" as used herein represents the ordinary error range for each respective value readily understood by a person of ordinary skill in the art. Reference to "about" a value or parameter herein includes (and describes) aspects that are directed to that value or parameter itself.

[0304] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more".

[0305] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. The description in range format should be understood as being for convenience and brevity only and should not be construed as a rigid limitation on the scope of the claimed subject matter. Accordingly, a range description should be considered to specifically disclose all the possible sub-ranges and individual numerical values within that range. For example, when a range of values is provided, each intervening value between the upper and lower limits of that range and any other stated value or intervening value in the stated range is understood to be included within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges and are also included within the claimed subject matter, subject to any specifically excluded limit values in the stated range. Where the stated range includes one or both of these limit values, ranges excluding one or both of those included limit values are also included within the claimed subject matter. This applies regardless of the breadth of the range.

[0306] As used herein with respect to amino acid sequences (reference polypeptide sequences), "percent amino acid sequence identity" and "percent identity" are defined as the percentage of amino acid residues in a candidate sequence (e.g., a streptavidin mutant protein) that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences to achieve maximum sequence identity, introducing gaps as necessary, and not considering any conservative substitutions as part of sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0307] An amino acid substitution can involve the replacement of one amino acid in a polypeptide with another. Amino acids can generally be grouped according to the following common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0308] A non-conservative amino acid substitution involves exchanging a member of one of these classes for another.

[0309] As used herein, the term "subject" includes any organism, such as humans and other mammals. Mammals include, without limitation, humans, as well as non-human animals including agricultural animals, sport animals, rodents, and pets.

[0310] As used herein, the term "composition" refers to any mixture of two or more products, substances, or compounds, including cells. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0311] As used herein, "depleting" when referring to one or more specific cell types or cell populations refers to decreasing the number or percentage of the cell type or population, for example, by negative selection based on a marker expressed by the population or cells, compared to the total number of cells in a composition or the volume of the composition, or relative to other cell types, or by positive selection based on a marker not present on the cell population or cells to be depleted. The term does not require complete removal of the cells, cell type, or population from the composition.

[0312] As used herein, "enriching" when referring to one or more specific cell types or cell populations refers to increasing the number or percentage of the cell type or population, for example, compared to the total cell number in a composition or the volume of the composition, or relative to other cell types, by positive selection based on markers expressed by the population or cells, or negative selection based on markers not present on the cell population or cells to be depleted. The term does not require complete removal of other cells, cell types, or populations from the composition, nor does it require that the thus-enriched cells be present at 100% or even near 100% in the enriched composition.

[0313] As used herein, the terms "treating", "treat", and "treatment" refer to the complete or partial remission or reduction of a disease or condition or disorder, or symptom, adverse effect or outcome, or associated phenotype. In certain embodiments, the effect is therapeutic such that it partially or completely cures the disease or condition or adverse symptoms resulting therefrom.

[0314] As used herein, a "therapeutically effective amount" of a compound or composition or combination refers to an amount effective to achieve a desired therapeutic result for a period of time, at a dosage, such as the treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effects of the treatment. The therapeutically effective amount can vary depending on factors such as the medical condition of the subject, age, sex, and weight, and the cell population being administered.

[0315] As used herein, the statement that a cell or cell population is "positive" for a particular marker indicates that a particular marker, typically a surface marker, is detectably present on or in the cell. When referring to a surface marker, the term represents the presence of surface expression detected by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detected by flow cytometry at a level substantially exceeding the staining detected under the same conditions using an isotype-matched control or a fluorescence minus one (FMO) gating control, and / or at a level substantially similar to that of cells known to be positive for the marker, and / or at a level substantially higher than the level for cells known to be negative for the marker.

[0316] As used herein, the statement that a cell or cell population is "negative" for a particular marker indicates that a particular marker, typically a surface marker, is not substantially detectably present on or in the cell. When referring to a surface marker, the term represents the absence of surface expression detected by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detected by flow cytometry at a level substantially exceeding the staining detected under the same conditions using an isotype-matched control or a fluorescence minus one (FMO) gating control, and / or at a level substantially lower than the level for cells known to be positive for the marker, and / or at a level substantially similar to the level for cells known to be negative for the marker as compared to the level for cells known to be negative for the marker.

[0317] In some embodiments, a decrease in the expression of one or more markers is a 1 log decrease in mean fluorescence intensity 10A decrease in the percentage of cells, which represents a loss of and / or a decrease in at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% of the cells and any % between 20 and 100% of the cells, as compared to a reference cell population. In some embodiments, a cell population in which one or more markers are positive represents at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% of the cells and any % between 50 and 100% of the cells that express a marker, as compared to a reference cell population.

[0318] V. Exemplary embodiments Among the embodiments provided herein are the following: 1. (a) Performing a first selection in a closed system, the first selection comprising enriching for either (i) CD4+ cells or (ii) CD8+ cells from a sample containing primary human T cells, thereby generating a first selected population and a non-selected population; and (b) Performing a second selection in a closed system, the second selection comprising enriching for the other of (i) CD4+ cells and (ii) CD8+ cells from the non-selected population, thereby generating a second selected population A method for enriching CD4+ T cells or CD8+ T cells, comprising A method for producing an enriched composition in which CD4+ cells and CD8+ cells are enriched and which contains the cells of the first selected population and the cells of the second selected population. 2. (c) Producing an enriched composition by combining the cells of the first selected population and the cells of the second selected population Further comprising, and / or CD4+ cells and CD8+ cells in the enriched composition are present at the starting ratio of CD4+ cells to CD8+ cells The method of embodiment 1. 3. Combining is performed in a closed system, the method of embodiment 2. 4. (a) A step of making a first selection in a closed system, wherein the first selection comprises enriching, from a sample containing primary human T cells, (i) CD4 + cells and (ii) CD8 + cells, thereby generating an enriched first selected population and a non-selected population; and (b) A step of making a second selection in a closed system, wherein the second selection comprises enriching, from the non-selected population, (i) CD4+ cells and (ii) the other of the CD8+ cells, thereby generating an enriched second selected population; (c) Generating stimulated cells by incubating a starting culture composition containing the cells of the first selected population and the cells of the second selected population under stimulating conditions in a culture vessel; and (d) Introducing a genetically engineered antigen receptor into the stimulated cells generated in (c) A method for producing genetically engineered T cells, comprising thereby generating an output composition containing CD4 + T cells and CD8 + T cells expressing the genetically engineered antigen receptor. 5. Before step (c), further comprising the step of combining the cells of the first and second selected cell populations to produce a starting culture composition, and / or CD4+ cells and CD8+ cells in the starting culture composition are present at the starting ratio of CD4+ cells to CD8+ cells, the method of embodiment 4. 6. Combining is performed in a closed system, the method of embodiment 5. 7. A method according to any one of aspects 1-6, wherein one or more of the steps are performed in an automated manner and / or a closed system is automated. 8. CD4 + cells and CD8 + A method according to any one of aspects 2-7, wherein the starting ratio of CD4 cells to CD8 cells is between 10:1 or about 10:1 and 1:10 or about 1:10, between 5:1 or about 5:1 and 1:5 or about 1:5, or between 2:1 or about 2:1 and 1:2 or about 1:2. 9. A method according to any one of aspects 2-8, wherein the starting ratio of CD4+ cells to CD8+ cells is 1:1 or about 1:1. 10. The sample is obtained from a human subject, CD4 + cells and CD8 + The starting ratio of CD4 cells to CD8 cells is different from the ratio of CD4 cells to CD8 cells in the sample from the subject, and / or + CD4 + cells and CD8 CD4 + cells and CD8 + The starting ratio of CD4 cells to CD8 cells is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% greater than or less than the ratio of CD4 cells to CD8 cells in the sample from the subject, + CD4 + cells and CD8 A method according to any one of aspects 2-6. 11. A method according to any one of aspects 1-10, wherein enriching the cells in the first and / or second selection comprises performing positive or negative selection based on the expression of cell surface markers. 12. A method according to aspect 11, wherein enriching the cells in the first and / or second selection comprises negative selection including depleting cells expressing non-T cell surface markers. 13. A method according to aspect 12, wherein the non-T cell marker comprises CD14. 14. Enriching cells in the first or second selection involves performing multiple positive or negative selection steps based on the expression of one or more cell surface markers to enrich CD4+ cells or CD8+ cells, according to any of the methods of Aspects 1-13. 15. Enriching cells in the first and / or second selection involves a selection based on immunoaffinity, according to any of the methods of Aspects 1-14. 16. Positive selection is performed by contacting the cells with an antibody capable of specifically binding to a cell surface marker and recovering the cells bound to the antibody, or negative selection is performed by recovering the cells not bound to the antibody, such that a selection based on immunoaffinity is carried out, CD4+ cells or CD8+ cells are enriched from the recovered cells, and the antibody is immobilized on magnetic particles, according to the method of Aspect 15. 17. The first selection and the second selection are performed in separate separation tanks operably connected, according to any of the methods of Aspects 1-13. 18. The separation tanks are operably connected by tubing, according to the method of Aspect 17. 19. The selection based on immunoaffinity is carried out by contacting the cells with an antibody immobilized or bound to an affinity chromatography matrix, the antibody being capable of specifically binding to a cell surface marker for positive or negative selection of CD4+ cells or CD8+ cells, according to any of the methods of Aspects 15-18. 20. 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 matrix during contact, and cells expressing the cell surface marker specifically bound by the antibody on the matrix can be recovered from the matrix by disruption of the reversible bond between the binding reagent and the binding partner, according to the method of Aspect 19. 21. The binding partner is selected from biotin, biotin analogs, and peptides capable of binding to the binding reagent, wherein the binding reagent is selected from streptavidin, streptavidin analogs or mutant proteins, avidin, and avidin analogs or mutant proteins, The method of embodiment 17. 22. The binding partner comprises the amino acid sequence shown in SEQ ID NO:6 and / or the binding reagent is a streptavidin mutant protein comprising the amino acid sequence shown in SEQ ID NO:12, 13, 15, or 16, The method of embodiment 21. 23. After contacting the cells in the sample with the affinity chromatography matrix in the first selection and / or the second selection, adding a competing reagent to disrupt the binding between the binding partner and the binding reagent, thereby recovering the selected cells from the matrix The method according to any one of embodiments 19 to 21, further comprising. 24. The method of embodiment 23, wherein the competing reagent is biotin or a biotin analog. 25. One or more antibodies in the first and / or second selection have a dissociation rate constant (k -5 sec -1 greater than 3×10 -5 sec -1 or about 3×10 off ) with respect to the cell surface marker. 26. One or more antibodies in the first and / or second selection have a dissociation constant (K d ) of about 10 -3 to 10 -7 or about 10 -7 to about 10 -10 with respect to the cell surface marker. 27. A method according to any one of aspects 20 to 26, wherein the first and / or second selected chromatographic matrix is packed in a separation tank that is a column. 28. The affinity chromatography matrix is capable of adsorbing and / or selecting at least or at least about 50×10 6 cells / mL, 100×10 6 cells / mL, 200×10 6 cells / mL, or 400×10 6 cells / mL, a method according to any one of aspects 19 to 27. 29. A method according to any one of aspects 19 to 28, wherein the first and second selection steps involve the use of an affinity chromatography matrix, and the matrix used in the first and second selection steps is in a relative amount sufficient to achieve a starting culture ratio. 30. Enriching CD4+ cells involves positive selection based on surface expression of CD4, a method according to any one of aspects 1 to 29. 31. Enriching CD8+ cells involves positive selection based on surface expression of CD8, a method according to any one of aspects 1 to 29. 32. One of the first and second selections, which involves enriching CD8+ cells, enriches central memory T (T CM ) cells, and / or enriches cells expressing a marker selected from CD28, CD62L, CCR7, CD127, and CD27 and further comprises a method according to any one of aspects 1 to 29. 33. The first selection involves enriching CD8+ cells, the second selection involves enriching CD4+ cells, and the first selection enriches central memory T (T CMEnriching cells and / or further comprising enriching cells expressing a marker selected from CD28, CD62L, CCR7, CD127, and CD27, The method of any one of aspects 1 to 32. 34. Central memory T (T CM ) Enriching cells and / or enriching cells expressing a marker is CD8 + Selecting cells expressing CD62L from the first and / or second selected cell populations enriched in cells, and / or CD8 + Selecting cells expressing CD27 from the first and / or second selected cell populations enriched in cells, and / or CD8 + Selecting cells expressing CCR7 from the first and / or second selected cell populations enriched in cells, and / or CD8 + Selecting cells expressing CD28 from the first and / or second selected cell populations enriched in cells, and / or CD8 + Selecting cells expressing CD127 from the first and / or second selected cell populations enriched in cells The method of aspect 32 or aspect 33 or aspect 35 comprising 35. The first selection comprises enriching CD4+ cells, the second selection comprises enriching CD8+ cells, and The second selection further comprises enriching central memory T (T CM ) cells, The method of any one of aspects 1 to 32. 36. (i) The first selection comprises generating a first selected population enriched in CD4+ primary human T cells and a non-selected sample by enriching CD4+ cells by positive selection based on surface expression of CD4; (ii) The second selection includes enriching CD8+ cells and further includes enriching central memory T (T CM ) cells from the second selection sample, wherein enriching the central memory T (T CM ) cells is a negative selection to deplete cells expressing surface markers present on naive T cells and a positive selection for cells expressing surface markers present on central memory T (T ) cells and not present on another memory T cell subpopulation, or CM ) a positive selection for cells expressing surface markers present on central memory T cells and not present on naive T cells and a positive selection for cells expressing surface markers present on central memory T (T ) cells and not present on another memory T cell subpopulation CM including, thereby generating CD8+ primary human T cells enriched in T cells, the method of embodiment 35. CM 37. The marker present on naive T cells includes CD45RA and enriching central memory T (T CM ) cells is a negative selection to deplete cells expressing CD45RA and a positive selection for cells expressing surface markers present on central memory T (T CM ) cells and not present on another memory T cell subpopulation, the method of embodiment 36. 38. The surface marker present on central memory T cells and not present on naive T cells includes CD45RO; enriching central memory T (T CM ) cells is a positive selection for cells expressing CD45RO and a positive selection for cells expressing surface markers present on central memory T (T CM ) cells and not present on another memory T cell subpopulation, the method of embodiment 38. ​​​​​The method of aspect 37. 39. A surface marker that is present on central memory T (T CM ) cells and not present on another memory T cell subset is selected from the group consisting of CD62L, CCR7, CD27, CD127, and CD44, the method according to any one of aspects 36 to 38. 40. A surface marker that is present on central memory T (T CM ) cells and not present on another memory T cell subset is CD62L, the method of aspect 39. 41. The CD8 + population in the enriched composition or the starting culture composition contains at least 50% central memory T (T CM ) cells, or contains less than 20% naive T (T N ) cells, or contains at least 80% CD62L+ cells, the method according to any one of aspects 32 to 40. 42. Contacting the cells of a sample containing primary human T cells with a first immunoaffinity reagent that specifically binds to CD4 and a second immunoaffinity reagent that specifically binds to CD8 in an incubation composition under conditions such that the immunoaffinity reagents specifically bind to the CD4 molecules and CD8 molecules on the cell surface of the sample, respectively, and Generating an enriched composition containing CD4+ cells and CD8+ cells in a starting culture ratio by recovering the cells bound to the first and / or the second immunoaffinity reagent A method for enriching CD4+ T cells and CD8+ T cells, comprising The first and / or the second immunoaffinity reagent is present in the incubation composition at a suboptimal yield concentration, wherein the enriched composition contains less than 70% of all CD4+ cells in the incubation composition and / or less than 70% of CD8+ cells in the incubation composition, thereby producing a composition enriched in CD4+ T cells and CD8+ T cells. 43. (a) A step of enriching primary human T cells from a sample containing primary human T cells, contacting the cells of the sample with a first immunoaffinity reagent that specifically binds to CD4 and a second immunoaffinity reagent that specifically binds to CD8 in an incubation composition under conditions such that the immunoaffinity reagents specifically bind to CD4 molecules and CD8 molecules on the surface of the cells in the sample, respectively; generating an enriched composition containing CD4+ cells and CD8+ cells at a culture starting ratio by recovering the cells bound to the first and / or the second immunoaffinity reagent; wherein the first and / or the second immunoaffinity reagent is present in the incubation composition at a suboptimal yield concentration, and wherein the enriched composition contains less than 70% of all CD4+ cells in the incubation composition and / or less than 70% of CD8+ cells in the incubation composition; and (b) A step of generating stimulated cells by incubating the cells of the enriched composition in the culture starting composition in a culture tank under stimulating conditions, wherein the cells are at the culture starting ratio or substantially at the culture starting ratio; and (c) By introducing a genetically engineered antigen receptor into the stimulated cells of (b), CD4 + T cells and CD8 + generating an output composition containing T cells. A method for producing genetically engineered T cells, comprising: 44. The method according to aspect 42 or aspect 43, wherein the enrichment of primary human T cells is performed in a closed system. 45. The method according to any one of aspects 42 to 44, wherein the first and second immunoaffinity reagents are present in the incubation composition at a suboptimal yield concentration, and wherein the enriched composition contains less than 70% of all CD4+ cells in the incubation composition and less than 70% of all CD8+ cells in the incubation composition. 46. The first immunoaffinity reagent is present in the incubation composition at a suboptimal yield concentration, where the enriched composition comprises less than 60%, less than 50%, less than 40%, less than 30% or less than 20% of the total CD4+ cells in the incubation composition, and / or The second immunoaffinity reagent is present in the incubation composition at a suboptimal yield concentration, where the enriched composition comprises less than 60%, less than 50%, less than 40%, less than 30% or less than 20% of the total CD8+ cells in the incubation composition, A method according to any one of aspects 42 to 45. 47. The sample contains at least 1×10 9 A method according to any one of aspects 42 to 46, wherein the sample contains at least 1×10 48. A method according to any one of aspects 42 to 47, wherein the concentration of one of the first and second immunoaffinity reagents in the incubation composition is greater than the concentration of the other, and the greater concentration results in a higher yield compared to the yield of the other of the CD4+ cells or CD8+ cells in the enriched composition of CD4+ cells or CD8+ cells, thereby generating a starting culture ratio in the enriched composition. 49. The concentration of one of the first and second immunoaffinity reagents is at least 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, or 10-fold greater than the concentration of the other of the first and second immunoaffinity reagents, and / or The higher yield at the enriched concentration is 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, or 10-fold greater, A method according to aspect 48. 50. CD4 + cells and CD8 +The method according to any one of aspects 42 to 49, wherein the starting ratio of culturing the cells is between 10:1 or about 10:1 and 1:10 or about 1:10, between 5:1 or about 5:1 and 1:5 or about 1:5, or between 2:1 or about 2:1 and 1:2 or about 1:2. 51. The method according to any one of aspects 42 to 50, wherein the starting ratio of culturing CD4+ cells and CD8+ cells is 1:1 or about 1:1. 52. CD4 + cells and CD8 + The starting ratio of culturing the cells is different from the ratio of CD4 + cells and CD8 + cells in the sample from the subject, and / or CD4 + cells and CD8 + The starting ratio of culturing the cells is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% greater or smaller than the ratio of CD4 + cells and CD8 + cells in the sample from the subject. The method according to any one of aspects 42 to 51. 53. The method according to any one of aspects 42 to 52, wherein more than 95% or more than 98% of the cells in the starting composition are CD4+ cells and CD8+ cells. 54. The method according to any one of aspects 42 to 53, wherein each of the immunoaffinity reagents comprises an antibody. 55. The method of aspect 54, wherein the antibody is immobilized on the outer surface of the sphere. 56. The method of aspect 55, wherein the sphere is a magnetic bead. 57. The antibody comprises one or more binding partners capable of forming a reversible bond with a binding reagent immobilized on the sphere, wherein the antibody is reversibly immobilized on the sphere. The method further includes the step of recovering selected cells from the spheres by contacting cells in a sample with first and second immunoaffinity reagents and then adding a competing reagent to disrupt the binding between the binding partner and the binding reagent. The method of embodiment 55 or embodiment 56. 58. The binding partner is selected from biotin, a biotin analog, or a peptide capable of binding to the binding reagent. The binding reagent is selected from streptavidin, a streptavidin analog or mutant protein, avidin, an avidin analog or mutant protein. The method of embodiment 57. 59. The binding partner includes a peptide containing the amino acid sequence shown in SEQ ID NO: 6 and / or The binding reagent is a streptavidin mutant protein containing the amino acid sequence shown in SEQ ID NO: 12, 13, 15, or 16. The method of embodiment 58. 60. The binding reagent is a multimer containing one or more monomers of streptavidin or a streptavidin mutant protein, and The binding partner is a peptide containing a continuous arrangement of at least two modules each capable of reversibly binding to at least one monomer of the binding reagent. The method of embodiment 58 or embodiment 59. 61. The method of embodiment 60, wherein the binding partner contains the amino acid sequence shown in any one of SEQ ID NOs: 7 to 10. 62. The method according to any one of embodiments 57 to 61, wherein the competing reagent is biotin or a biotin analog. 63. One or more antibodies in the first and / or second selection are greater than 3×10 -5 sec -1 with respect to the binding between the antibody and the cell surface marker, or about 3×10 -5 sec-1 having a dissociation rate constant (k off ) greater than that of any of the methods of aspects 42-63. 64. One or more antibodies in the first and / or second selection having an affinity with a dissociation constant (Kd) in the range of about 10 -3 to 10 -7 or having an affinity with a dissociation constant in the range of about 10 -7 to about 10 -10 of any of the methods of aspects 42-63. 65. Any of the methods of aspects 42-63, wherein one or more steps are performed in an automated manner and / or a closed system is automated. 66. Determining the ratio of CD4+ T cells to CD8+ T cells in the sample prior to making the first selection and / or the second selection, and Adjusting the first and / or second selection based on the ratio of CD4+ T cells to CD8+ T cells in the sample to produce a composition containing CD4+ cells and CD8+ cells at the starting ratio of culture comprising Any of the methods of aspects 2, 3, and 5-41. 67. The first and / or second selection comprises a selection based on immunoaffinity comprising an affinity chromatography matrix, and the step of adjusting the first and / or second selection comprises selecting an amount of the affinity chromatography matrix sufficient to achieve the starting ratio of culture in the first and / or second selection. The method of aspect 66. 68. Determining the ratio of CD4+ T cells to CD8+ T cells in the sample prior to contacting the cells of the sample with the first and second immunoaffinity reagents; and Selecting the concentration of the first and / or second immunoaffinity reagent based on the ratio of CD4+ T cells to CD8+ T cells in the sample to produce an enriched composition containing CD4+ cells and CD8+ cells at the starting ratio of culture A method according to any of aspects 42 to 65, comprising 69. A CD4 + cell to CD8 + cell ratio of from 2:1 or about 2:1 to 1:5 or about 1:5, producing an output composition comprising the ratio, a method according to any of aspects 1 to 68. 70. CD4 in the output composition + cell to CD8 + cell ratio is 1:1 or about 1:1, a method according to aspect 69. 71. A method according to any of aspects 1 to 70, wherein the sample is obtained from a subject. 72. A method according to aspect 71, wherein the subject is a subject administered with genetically engineered T cells or cells for adoptive cell therapy. 73. A method according to aspect 72, wherein the subject is a subject other than a subject administered with genetically engineered T cells or cells for adoptive therapy. 74. A method according to any of aspects 1 to 73, wherein the sample is a blood or blood-derived sample. 75. A method according to any of aspects 1 to 74, wherein the sample is a leukocyte sample. 76. A method according to any of aspects 1 to 75, wherein the sample is an apheresis, peripheral blood mononuclear cell (PBMC), or leukapheresis sample. 77. A method according to any of aspects 4 to 41 or 43 to 76, wherein the composition is incubated under stimulating conditions in a culture vessel before, during, and / or after introduction of the genetically engineered antigen receptor. 78. A method according to any of aspects 4 to 41 or 43 to 76, wherein the composition is incubated under stimulating conditions in a culture vessel before, during, and after introduction of the genetically engineered antigen receptor. 79. A method according to any of aspects 4 to 41 or 43 to 78, wherein the stimulating conditions include conditions under which the T cells of the composition proliferate. 80. A method according to any one of aspects 4 to 41 or 43 to 79, wherein the stimulating condition comprises an agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex. 81. A method according to aspect 80, wherein one or more components of the TCR complex comprise the CD3 zeta chain. 82. A method according to any one of aspects 4 to 41 or 43 to 81, wherein the stimulating condition comprises the presence of an anti-CD3 antibody and an anti-CD28 antibody, an anti-4-1BB antibody, and / or a cytokine. 83. A method according to aspect 82, wherein the anti-CD3 antibody and / or the anti-CD28 antibody is present on the surface of a solid support. 84. A method according to aspect 83, wherein the cytokine comprises IL-2, IL-15, IL-7, and / or IL-21. 85. A method according to any one of aspects 4 to 41 or 43 to 84, wherein the genetically engineered antigen receptor comprises a T cell receptor (TCR) or a functional non-TCR antigen receptor. 86. A method according to aspect 85, wherein the receptor specifically binds to an antigen expressed by cells of the disease or condition to be treated. 87. A method according to any one of aspects 85 or 86, wherein the antigen receptor is a chimeric antigen receptor (CAR). 88. A method according to aspect 87, wherein the CAR comprises an extracellular antigen recognition domain, and an intracellular signaling domain comprising an ITAM-containing sequence, and an intracellular signaling domain of a T cell co-stimulatory molecule. 89. (a) Producing an output composition comprising CD4 + T cells and CD8 + T cells according to any one of aspects 1 to 88; and (b) Administering the cells of the output composition to a subject A treatment method comprising. 90. The method of aspect 89, wherein the sample from which the cells are isolated is derived from the subject to whom the cells are administered. 91. A composition of cells produced by the method of any one of aspects 1 to 88. 92. The composition of aspect 91, comprising a pharmaceutically acceptable carrier. 93. A treatment method comprising administering to a subject the cell composition of aspect 91 or 92. 94. The method of aspect 93, wherein the genetically engineered antigen receptor specifically binds to an antigen associated with a disease or condition. 95. The treatment method of aspect 94, wherein the disease or condition is cancer. 96. The composition of aspect 91 or 92 for use in treating a disease or condition in a subject. 97. Use of the composition of aspect 91 or 92 for the manufacture of a medicament for treating a disease or disorder in a subject. 98. The composition of aspect 96 or the use of aspect 97, wherein the genetically engineered antigen receptor specifically binds to an antigen associated with a disease or condition. 99. The composition or use of any one of aspects 96 to 98, wherein the disease or condition is cancer. 100. a) A first affinity chromatography matrix comprising a first binder immobilized on a surface, wherein the binder specifically binds to a first cell surface marker present on a first cell, wherein the first affinity chromatography matrix is operably connected to a storage reservoir containing a cell sample via a first operable connection, and the first operable connection enables passage of the cells from the storage reservoir to the first affinity chromatography matrix, the first affinity chromatography matrix is operably connected to an output tank via a second operable connection. a first affinity chromatography matrix; and b) a second affinity chromatography matrix comprising a second binder immobilized on its surface, wherein the second binder specifically binds to a second cell surface marker present on a second cell, and wherein the first affinity chromatography matrix is operably connected to the second affinity chromatography matrix via a third operable connection, and the third operable connection enables cells that have passed through the first affinity chromatography matrix and not bound to the first binder to pass through to the second affinity chromatography matrix, the second affinity chromatography matrix is operably connected to an output tank via a fourth operable connection, and the fourth operable connection enables cells that have bound to and eluted from the first and / or the second affinity chromatography matrix to pass through, the second affinity chromatography matrix is operably connected to a waste tank via a fifth operable connection, and the fifth operable connection enables cells that have passed through the first affinity chromatography matrix and not bound to the first binder and have passed through the second affinity chromatography matrix and not bound to the second binder to pass through, a second affinity chromatography matrix; c) an output tank; and d) a waste tank A closed device system for the purification of target cells, comprising within the closed system, (i) cells that have bound to and been recovered from the first affinity chromatography matrix, and (ii) cells that have passed through the first affinity chromatography matrix, not bound to it, bound to the second chromatography matrix, and been recovered from it configured to enable collection of a single composition within the output tank, a closed device system. 101. One or more of the operable connections include tubing connecting a storage reservoir, a first affinity chromatography matrix, a second affinity chromatography matrix, and / or a culture tank, the closed device of aspect 100. 102. The closed device of aspect 101, wherein the tubing is connected to a stopcock, valve or clamp. 103. (a) The first affinity chromatography matrix is one of a CD4+ affinity chromatography matrix or a CD8+ affinity chromatography matrix, (b) The second affinity chromatography matrix is the other of a CD4+ affinity chromatography matrix or a CD8+ affinity chromatography matrix, The closed device system of any of aspects 100 - 102. 104. d) A third affinity chromatography matrix comprising a third binder immobilized on a surface, wherein the binder specifically binds to a third cell surface marker and the third binder is capable of binding to cells expressing the third cell surface marker, wherein a third operable connection further operably connects the third affinity chromatography matrix to the first matrix, a sixth operable connection operably connects the third affinity chromatography matrix to an output container, such that the sixth operable connection allows cells bound to and recovered from the first matrix and bound to and recovered from the third matrix to pass to the output container, A fifth operable connection further operably connects the third affinity chromatography matrix to a waste container, such that the fifth operable connection enables cells that passed through and did not bind to the third column to be sent to the waste container. The third affinity chromatography matrix A closed device system according to any of aspects 100 - 103, further comprising. 105. d) A third affinity chromatography matrix comprising a third binding agent immobilized on a surface, wherein the binding agent specifically binds to a third cell surface marker and the third affinity chromatography matrix is capable of binding to cells expressing the third cell surface marker, where A second operable connection further operably connects the third affinity chromatography matrix to a first matrix and an output container, such that the second operable connection enables cells that bound to and were recovered from the first matrix and cells that bound to and were recovered from the third matrix to be sent to the output container. The third affinity chromatography matrix A closed device system according to any of aspects 100 - 103, further comprising. 106. The first affinity chromatography matrix comprises a binding agent that specifically binds to CD8, The second affinity chromatography matrix comprises a binding agent that specifically binds to CD4, The third affinity chromatography matrix comprises a binding agent that specifically binds to a marker expressed on central memory T (T CM ) cells. A closed device system according to aspect 104 or 105. 107. The closed device system of embodiment 106, wherein the binder of the third affinity chromatography matrix selectively binds to a cell surface marker selected from CD62L, CD45RA, CD45RO, CCR7, CD27, CD127, and CD44. 108. The closed device system of any one of embodiments 100-107, wherein one or more or all of the affinity chromatography matrices are further operably connected to an elution buffer reservoir containing one or more competing reagents. 109. The closed device system of any one of embodiments 100-107, wherein the competing reagent is one or more of the group consisting of biotin, biotin analogs, and peptides capable of binding to the chromatography matrix. 110. The closed device system of any one of embodiments 100-107, wherein one or more or all of the third, fourth, or sixth operable connections further comprise a competitor removal chamber. 111. The closed device system of embodiment 110, wherein the competitor removal chamber further comprises a binding reagent. 112. The closed device system of embodiment 111, wherein the binding reagent comprises one or more of the group consisting of streptavidin, streptavidin analogs or mutant proteins, avidin, and avidin analogs or mutant proteins. 113. The closed device system of any one of embodiments 100-112, wherein one or more or all of the binders are antibodies. 114. The closed device system of any one of embodiments 100-112, wherein one or more or all of the binders are reversibly bound to the affinity chromatography matrix.

Example

[0319] VI. Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0320] Example 1: Generation of compositions of CD4+ T cells and CD8+ T cells using a single - step process flow by immunomagnetic separation for gene manipulation and adoptive cell therapy In an exemplary method, central memory T cells are enriched from an apheresis product sample, and CD4 + T cell populations and CD8 + T cell populations are isolated, then incubated, manipulated, and subsequently administered to a subject. The isolation procedure is performed using immunomagnetic separation with the CliniMACS® Prodigy system, using a single-step process flow. This procedure is streamlined compared to other methods and uses the CliniMACS® Prodigy device and three separate tubing sets to separate CD4 + cells from the first apheresis fraction and CD8 + cells from the second apheresis fraction and further depleted / enriched.

[0321] The streamlined process is performed using one tubing set for the isolation of CD4 + and CD8 + cell populations, without transferring the cell populations from one reservoir (e.g., tubing set) to another.

[0322] The CD4 + cell population is isolated by incubating the apheresis sample with the CliniMACS® CD4 reagent. The cells are then separated by the CliniMACS® Prodigy device set to run an enrichment program, and both cell fractions (i.e., the immunomagnetically selected enriched CD4 + cell fraction and the pass-through cell fraction) are retained. The enriched (positive) fraction is the isolated CD4 + T cell population. The pass-through (negative) fraction is incubated with the CliniMACS® CD14 reagent and the CliniMACS® CD45RA reagent or CD19 reagent to isolate CD8 +Isolate the T cell population. Set the CliniMACS® Prodigy device to run the depletion program. Next, separate the cell / reagent mixture using the same tubing set as used in the first separation step by the CliniMACS® Prodigy device. CD14 + / CD45RA + Or incubate the passing (negative) fraction depleted of CD14+ / CD45RA or CD14+ / CD19+ cells with the CliniMACS® CD62L reagent. Separate the cell / reagent mixture using the CliniMACS® Prodigy device running the enrichment program using the same tubing set. The positive fraction is an isolated CD8 + cell population enriched for central memory cells.

[0323] Combine the isolated CD4+ population and CD8+ population in the same culture vessel in the state of a culture start composition at a certain culture start ratio. Design the culture start ratio to achieve a specific desired output ratio, or a ratio within a specific range of tolerance of such a desired output ratio, after the incubation stage and / or manipulation stage, or design to do so at a specific percentage of time. Incubate the cells under stimulation conditions using anti-CD3 / anti-CD28 beads in the Prodigy system at 37 °C for 72 hours in the presence of IL-2 (100 IU / mL).

[0324] Optionally and periodically evaluate and / or adjust the cell composition one or more times after the start of incubation or culture, for example at a certain time during incubation. Evaluation includes measuring the growth rate, measuring the viability, determining the expression of one or more surface markers or intracellular markers such as proteins or polynucleotides, and / or adjusting the composition or vessel with respect to the presence or absence or amount or relative amount of the cell type including temperature, medium components, oxygen or carbon dioxide content, and / or one or more factors, drugs, components, and / or subtypes.

[0325] Next, the cells thus incubated are genetically engineered by introducing into the cells a recombinant gene for the expression of a recombinant antigen receptor, such as a chimeric antigen receptor (CAR) or a recombinant TCR. CD4 present in the same composition and tank + cells and CD8 + cells are used for introduction in a contained environment within a CliniMACS® Prodigy device. The method results in an output composition having engineered CD4 + T cells and CD8 + T cells.

[0326] Example 2: Generation of compositions of CD4+ T cells and CD8+ T cells by sequential purification in a closed system This example illustrates an exemplary method of enriching or selecting CD4+ T cell populations and CD8+ T cell populations from an apheresis product sample obtained from a subject. To sequentially subject CD4+ T cell populations and CD8+ T cell populations to positive selection from the same starting sample, the steps are performed using a closed system having multiple chromatography columns.

[0327] A. Enrichment of CD4+ T cell populations and CD8+ T cell populations In the exemplary process shown in FIG. 1A, a series of immunochromatographic selection columns and removal columns are arranged in a closed device 14, which are operably connected to a peristaltic pump 8 via valves 13 for controlling the flow of each other and various tubing lines and liquid phases.

[0328] The first selection column 1 contains a selected volume of an affinity chromatography matrix 3, such as an agarose resin, such as the resin described in U.S. Patent Application Publication No. 2015 / 0024411 for the isolation of T cells, such as having an exclusion limit designed to be larger than the size of T cells, such as an exclusion size of 6×10 6It contains agarose obtained from Agarose Beads Technologies, Madrid, Spain, which has an exclusion size smaller than that of Superflow™ agarose having Dalton. The matrix in the first column is bound to a Strep-Tactin® multimer (e.g., a streptavidin variant represented by any of SEQ ID NO: 12, 13, 15 or 16 (IBA GmbH, Germany), or a streptavidin variant described in International Publication PCT Application WO2014 / 076277). The second selection column 2 contains a selected volume of an affinity chromatography matrix 4, such as an agarose resin, such as the resin as described above, and the matrix is also bound to a Strep-Tactin® multimer.

[0329] The anti-CD8 Fab is added to the first selection column 1 by loading it into the reservoir 18 containing the anti-CD8 Fab fragment and flowing it through the pump 8, tubing, and valve 13, whereby the anti-CD8 Fab is immobilized on Strep-Tactin® on the affinity matrix 3 by a Twin Strep-Tag® (e.g., as shown in SEQ ID NO: 10; IBA GmbH) fused to the carboxy terminus of its heavy chain in the Fab fragment. (See, e.g., U.S. Patent Application Publication No. 2015 / 0024411). In some embodiments, a wash buffer, such as phosphate buffered saline (PBS) containing 0.5% bovine serum albumin, human serum albumin, or recombinant human serum albumin from the wash buffer reservoir 6, is loaded and flowed through the first selection column via operably connected tubing. The flow-through fraction is directed to the waste container 10 via a valve 13 that operably connects the first selection column and the waste container. In some embodiments, the wash step is repeated multiple times.

[0330] Load the reservoir containing anti-CD4 Fab fragment 19 and flow it through pump 8, tubing, and valve 13 to add anti-CD4 Fab to the second selection column 2, whereby anti-CD8 Fab will be immobilized on Streptactin® on the affinity matrix 4 by Twin Strep-Tag®. In some embodiments, wash buffer from wash buffer reservoir 6 is flowed through the second selection column via operably connected tubing. The flow-through fraction is directed to waste container 10 via valve 13 operably connecting the second selection column and the waste container. In some embodiments, the wash step is repeated multiple times.

[0331] The volume of the affinity matrix reagent contained in the first and second selection columns may be the same or different and can be selected based on the desired yield of selection and / or the desired ratio of CD4+ cells to CD8+ cells after selection. The volume so selected is based on the assumption that the average yield selectable per 1 mL of the volume of the packed column is 1×10 8 cells. In an exemplary step, the column volumes are the same, for example, using a 2 mL column for the anti-CD8 Fab fragment and a 2 mL column for the anti-CD4 Fab fragment. The column length and / or column diameter can be selected to fit the desired volume, for example, to achieve the desired starting ratio of CD4+ cells to CD8+ cells in culture. In some embodiments, to adapt the volume of the affinity matrix, multiple columns immobilized with the same Fab reagent can be directly serially added and operably connected to each other via tubing lines.

[0332] To achieve cell selection, an apheresis sample is added to a first selection column 1, whereby CD8+ T cells, if present in the sample, remain bound to the resin 3 of the first column, and the unselected cells (containing CD8- cells) pass through the column. The number of cells in the starting sample used in some examples is chosen to be greater than the number of cells to be selected based on the total volume of the matrix, for example, an amount greater than the capacity of the column for each selection (e.g., more than 200 million CD8+ cells and more than 200 million CD4+ cells, e.g., at least about 10% or 20% more). Next, the flow-through fraction (negative fraction) containing the unselected cells is passed from the first column to a second selection column 2 via operably connected tubing. CD4+ T cells remain bound to the resin 4 in the second column. The flow-through fraction containing the fluid and further unselected cells (negative cells) is directed to a waste container 10 via a valve 13 that operably connects the second selection column and the waste container.

[0333] In some embodiments, the first selection column can alternatively be an anti-CD4 affinity chromatography matrix, and the second selection column can be an anti-CD8 affinity chromatography matrix.

[0334] Washing buffer is passed through and flowed through the first and second columns via operably connected tubing from a washing buffer reservoir 6. The flow-through fraction containing any cells eluted from the first and second columns is directed to a waste container 10 via a valve 13 that operably connects the second selection column and the waste container. In some embodiments, the washing step is repeated multiple times.

[0335] Load a buffer containing a low concentration of biotin or an analog thereof, such as eluate of 2.5 mM desthiobiotin, from the dissolution buffer reservoir 7 and flow it through the first column and the second column via operably connected tubing. In some embodiments, the eluate contains cell culture medium. Direct the passing fraction containing enriched CD4+ and CD8+ cells and residual biotin or analog to the removal chamber 9 to remove the biotin or its analog. The removal chamber 9 is a column of Superflow (TM) Sepharose (R) beads immobilized with Strep-Tactin (R) having a volume sufficient to remove biotin or biotin analog from the sample, for example, a column having a total volume of 6 mL with a binding capacity of 300 nanomoles / mL of biotin. Direct the passing fraction containing the enriched cells positively selected for CD4+ and CD8+ through a valve operably connecting the removal chamber 9 and the culture tank to the culture tank 12 such as a bag. In some embodiments, the elution step is repeated.

[0336] In some embodiments, after performing the elution step, replace the wash buffer with an activation buffer containing a T cell activation reagent (such as anti-CD3, anti-CD28, IL-2, IL-15, IL-7, and / or IL-21), direct it through the removal chamber, and direct the passing fraction to the culture tank. The positive fraction collected in the culture tank is an isolated and combined CD4+ cell population and CD8+ cell population.

[0337] B. Enrichment of CD4+ T cell population and CD8+ T cell population in which cells expressing markers on central memory T (T CM ) cells are enriched In the exemplary process shown in FIG. 1B, a series of immunochromatographic selection columns and removal columns are arranged in the closed device 14, which are operably connected to the peristaltic pump 8 via each other and via valves 13 for controlling the flow of various tubing lines and liquid phases.

[0338] The first selection column 1 and the second selection column 2 are the same as those described above with respect to Example 2A. Additionally, the process further includes a third selection column 15 that houses a selected volume of affinity matrix 17, such as an agarose resin, such as the resin described in U.S. Patent Application Publication No. 2015 / 0024411 for the isolation of T cells, such as the resin described in Example 2A above.

[0339] The anti-CD8 Fab and anti-CD4 Fab described in Example 2A are added to the first column and the second column, respectively. A further Fab fragment against a marker expressed on central memory T (T CM ) cells, such as one of CD28, CD62L, CCR7, CD27 or CD127, is loaded into reservoir 20 and passed through pump 8, tubing, and valve 13 to add the further Fab to the third selection column 15, whereby the further Fab is conjugated to Strep-Tactin® on the affinity matrix 17 by a Twin Strep-Tag® (SEQ ID NO:10; IBA GmbH) that is fused to the carboxy terminus of its heavy chain as described in Example 2A in the Fab fragment. In some embodiments, the wash buffer described in Example 2A is loaded and passed through the third selection column via operably connected tubing. The flow-through fraction is directed to waste container 10 via valve 13 that operably connects the third selection column to the waste container. In some embodiments, the wash step is repeated a plurality of times.

[0340] The volume of the affinity matrix reagent housed in the first selection column, the second and / or third selection columns may be the same or different, depending on the desired yield of the selection, and / or, after the selection, the central memory T (T CM)Cells expressing a marker expressed on the cell can be selected based on the desired ratio of enriched CD4+ cells to CD8+ cells. The volume so selected is based on the assumption that the selectable average yield is 1×10 8 cells per 1 mL of the volume of the packed column. Also, for a particular selected starting culture ratio, e.g., the starting culture ratio of CD4+ cells to a CD8+ cell-containing population enriched for a marker expressed on central memory T (T CM ) cells, such as a population of CD8+ cells enriched based on further selection for one of CD28, CD62L, CCR7, CD27 or CD127, the volume of matrix for selecting the enriched population, e.g., the parental population of CD8+ cells, is large compared to the matrix used to select the other of the CD4+ T cell population or CD8+ T cell population, e.g., the CD4+ population. The volume of matrix used to select for the parental population of a further enriched population, e.g., a matrix containing anti-CD8 Fab fragments, that is greater in amount or degree than the volume of the other matrix or other matrices is selected based on the ratio or percentage of the further enriched cell population in the sample, e.g., CD8+ / CD28+, CD8+ / CD62L+, CD8+ / CCR7+, CD8+ / CD27+ or CD8+ / CD127+, compared to the ratio or percentage of the parental population, e.g., CD8+ cells, present in the sample. This can be estimated based on the average among patients or healthy donors, or it can be measured for a given patient for whom the selection is being made prior to determining the size of the column to be used.

[0341] In an exemplary step, the column volumes used in the step are, for example, a 2 mL column with anti-CD4 Fab fragments, a 6 mL column with anti-CD8 Fab fragments, and a 2 mL column with anti-CD62L Fab fragments. For example, CD4+ cells and central memory T (T CMTo achieve a desired starting culture ratio with CD8+ cells expressing a marker expressed on the cell surface, such as CD8+ / CD28+, CD8+ / CD62L+, CD8+ / CCR7+, CD8+ / CD27+ or CD8+ / CD127+ cells, the column length and / or column diameter can be selected to fit the desired volume. In some embodiments, to adapt the volume of the affinity matrix, multiple columns with the same Fab reagent immobilized thereon can be added directly in series and operably connected to each other via tubing lines.

[0342] To achieve cell selection, an apheresis sample is added to a first selection column 1, whereby CD8+ T cells, if present in the sample, remain bound to the resin of the first column and unselected cells (including CD8− cells) pass through the column. The number of cells in the starting sample used in some examples should be above the number of cells to be selected based on the total volume of the matrix, e.g., an amount greater than the capacity of the column for each selection (e.g., more than 200 million CD8+ / CD62L+ cells and more than 200 million CD4+ cells, e.g., at least about 10% or 20% more). The flow-through fraction (negative fraction) containing the unselected cells is directed through valve 13 operably connecting the first column and the second column to the second column 2. CD4+ T cells remain bound to the resin in the second column. The flow-through fraction containing the fluid and further unselected cells (negative cells) is directed through valve 13 operably connecting the second selection column and the negative fraction container to the first waste container 10.

[0343] Load a wash buffer as described in Example 2A from wash buffer reservoir 6 and pass it through the first and second columns via the valves and tubing operably connecting the columns. The flow-through fraction containing any cells eluted from the first and second columns is directed through valve 13 operably connecting the second selection column and the waste container to the waste container 10. In some embodiments, the wash step is repeated multiple times.

[0344] Load a buffer containing an eluent, such as biotin or an analog thereof, e.g., 2.5 mM desthiobiotin, from the elution buffer reservoir 7, and pass it through the second column 2 via a valve 13 and tubing that operably connects the elution buffer reservoir to the second column. In some embodiments, the elution buffer contains cell culture medium. Direct the flow-through fraction containing enriched CD4+ cells and residual biotin towards a removal chamber 9 that is operably connected to the second column via the valve 13 and tubing, and remove biotin as described in Example 2A. Direct the flow-through fraction containing the enriched cells that have been positively selected for CD4+ towards a culture tank 12, such as a bag, via the valve 13 that operably connects the removal chamber 9 to the culture tank. In some embodiments, the elution step is repeated. In some embodiments, after the elution step, replace the wash buffer with an activation buffer containing a T cell activation reagent (e.g., anti-CD3, anti-CD28, IL-2, IL-15, IL-7, and / or IL-21), and direct it from the wash buffer reservoir towards the second column, the removal chamber, and the culture tank via the valve and tubing. The positive fraction collected in the culture tank is an isolated population of CD4+ cells.

[0345] Load a buffer containing an eluent such as biotin from the elution buffer reservoir 7 and pass it through the first column via a valve 13 and tubing that operably connect the elution buffer reservoir and the first column 1. In some embodiments, the elution buffer contains cell culture medium. Direct the flow-through fraction containing enriched CD8+ cells and residual biotin or its analogs through the valve 13 and tubing to a removal chamber 9 that is operably connected to the first column to remove biotin or biotin analogs as described in Example 2A. Direct the flow-through fraction containing the enriched cells that have been positively selected for CD8+ through the valve 13 that operably connects the removal chamber 9 and the third column to pass through the third column 15. The CD62L+ subset of CD8+ cells remains bound to the resin in the third column. Direct the flow-through fraction containing fluid and further unselected cells (negative cells) through the valve 13 that operably connects the third selection column and the second waste container to the second waste container 11.

[0346] Load a wash buffer as described in Example 2A from the wash buffer reservoir 6 and pass it through the third column via a valve 13 and tubing that operably connect the wash buffer reservoir and the third column 15. Direct the flow-through fraction containing any cells eluted from the third column through the valve 13 that operably connects the third selection column and the second waste container to the second waste container 11. In some embodiments, the wash step is repeated multiple times.

[0347] Load a buffer containing an eluent such as biotin or its analog, e.g., 2.5 mM desthiobiotin, from the elution buffer reservoir 7 and pass it through the third column 15 via a valve 13 and tubing that operably connect the elution buffer reservoir and the third column. In some embodiments, the elution buffer contains cell culture medium. Central memory T (T) expressing one of CD28, CD62L, CCR7, CD27, or CD127 CM)Direct the flow-through fraction containing CD8+-enriched cells and residual biotin or its analogs through valve 13 and tubing to a removal chamber 9 operably connected to a third column to remove biotin or biotin analogs as described in Example 2A. For CD8 and for a marker expressed on the cell such as one of CD28, CD62L, CCR7, CD27 or CD127, positive selection is performed on the enriched T cell memory cells. CM )Direct the flow-through fraction containing the enriched T cell memory cells that have been positively selected for CD8 and for a marker expressed on the cell such as one of CD28, CD62L, CCR7, CD27 or CD127 through valve 13, which operably connects the removal chamber 9 and the culture tank, to a culture tank 12 such as a bag. In some embodiments, the culture tank contains a T cell activation reagent, cell medium, or both. In some embodiments, the elution step is repeated. In some embodiments, after performing the elution step, replace the wash buffer with an activation buffer containing a T cell activation reagent (e.g., anti-CD3, anti-CD28, IL-2, IL-15, IL-7, and / or IL-21) and direct it from the wash buffer reservoir through the valves and tubing into the first and / or third column, the removal chamber, and the culture tank. CD8+ cells and central memory T (T CM )Collect the positive fraction containing cells that are positive for a marker on the cell in the culture tank together with the previously collected CD4+ positive fraction. In some embodiments, the steps of the process can be performed in a different order, for example, first enriching for cells that are positive for a marker on the cell such as one of CD8+ cells and CD28, CD62L, CCR7, CD27 or CD127, and then enriching for CD4+ cells. CM )cells.

[0348] Example 3: Generation of compositions of CD4+ T cells and CD8+ T cells by sequential purification in a closed system for use in gene manipulation and adoptive cell therapy This example describes a procedure for selecting and generating a cell composition containing CD4+ T cells and CD8+ T cells, such as CD4+ T cells and CD8+ T cells present at a certain starting culture ratio, for incubation / activation and transduction in a method related to genetic manipulation of cells for use related to adoptive cell therapy.

[0349] A cell composition generated by selection of CD4+ cells and CD8+ cells, performed as described in either Example 2A (CD4+ and CD8+) or Example 2B (CD8+ enriched for CD4+ and CD62L+), is incubated under stimulating conditions, for example using anti-CD3 / anti-CD28 in the presence of IL-2 (100 IU / mL), for example at 37°C for 72 hours. Next, the stimulated cells are genetically engineered by introducing a recombinant gene for expression of a recombinant antigen receptor, such as a chimeric antigen receptor (CAR) or a recombinant TCR, into the cells, for example by viral transduction. In some embodiments, following the introduction, the cells are generally incubated further at 37°C to, for example, allow cell proliferation.

[0350] The method yields an output composition having engineered CD4 + T cells and CD8 + T cells. In some embodiments, based on the selected volume of the selection column, the ratio of CD4+ cells to CD8+ cells (starting culture ratio) in the composition incubated under stimulating conditions prior to manipulation results in a particular desired output ratio of CD4+ cells to CD8+ cells, or of engineered CD4+ cells to engineered CD8+ cells, or such a ratio within a particular range of tolerance of such a desired output ratio, following the incubation, stimulation, and / or manipulation steps. In some embodiments, such a desired output ratio or ratio within the range of tolerance is achieved at a particular percentage of the allowed time.

[0351] Example 4: Selection of cells using sub - optimal yield concentrations on Fab - coated surfaces Human apheresis-derived PBPC samples in different cell number ranges were gently mixed and incubated for about 30 minutes in a single composition with magnetic microbeads conjugated with anti-CD4 Fab and magnetic microbeads conjugated with anti-CD8 Fab. Following this incubation, elution of the unselected cells and recovery using a...

Claims

**Claim 1** (a) A step of making a first selection in a closed system, wherein the first selection comprises enriching, from a sample containing primary human T cells, either (i) CD4+ cells or (ii) CD8+ cells, whereby the enrichment generates a first selected population and a non-selected population; and (b) A step of making a second selection in a closed system, wherein the second selection comprises enriching, from the non-selected population, the other of (i) CD4+ cells and (ii) CD8+ cells, whereby the enrichment generates a second selected population A method for enriching CD4+ T cells or CD8+ T cells, comprising A method of producing an enriched composition in which CD4+ cells and CD8+ cells are enriched and which contains the cells of the first selected population and the cells of the second selected population. **Claim 2** (c) A step of producing an enriched composition by combining the cells of the first selected population and the cells of the second selected population further comprising and / or the CD4+ cells and CD8+ cells in the enriched composition are present at the starting culture ratio of CD4+ cells to CD8+ cells The method according to claim 1. **Claim 3** The method according to claim 2, wherein the combining is performed in a closed system. **Claim 4** (a) A step of making a first selection in a closed system, wherein the first selection comprises enriching, from a sample containing primary human T cells, (i) CD4 + cells and (ii) CD8 + cells, thereby generating an enriched first selection population and a non-selected population; and (b) A step of making a second selection in a closed system, wherein the second selection comprises enriching, from the non-selected population, the other of (i) CD4+ cells and (ii) CD8+ cells, whereby the enrichment generates a second selected population; (c) A step of generating stimulated cells by incubating a starting culture composition containing the cells of the first selected population and the cells of the second selected population under stimulating conditions in a culture vessel; and (d) A step of introducing a genetically engineered antigen receptor into the stimulated cells generated in (c) A method for producing genetically engineered T cells, comprising Thereby, a method for generating an output composition comprising CD4 + T cells expressing a genetically engineered antigen receptor and CD8 + T cells. **Claim 5** Before step (c), further comprising a step of combining the cells of the first and second selected cell populations to produce a starting culture composition and / or the CD4+ cells and CD8+ cells in the starting culture composition are present at the starting culture ratio of CD4+ cells to CD8+ cells, the method according to claim 4. **Claim 6** The method according to claim 5, wherein the combining is performed in a closed system.

7. The method according to any one of claims 1 to 6, wherein one or more of the steps are performed in an automated manner and / or a closed system is automated.

8. CD4 + cells and CD8 + The method according to any one of claims 2 to 7, wherein the starting ratio of culturing CD4 cells and CD8 cells is between 10:1 or about 10:1 and 1:10 or about 1:10, between 5:1 or about 5:1 and 1:5 or about 1:5, or between 2:1 or about 2:1 and 1:2 or about 1:

2.

9. The method according to any one of claims 2 to 8, wherein the starting ratio of CD4+ cells to CD8+ cells is 1:1 or about 1:

1.

10. The sample is obtained from a human subject, CD4 + cells and CD8 + The starting ratio of culturing the cells is CD4 in a sample from the subject + cells and CD8 + is different from the ratio of the cells and / or CD4 + cells and CD8 + The starting ratio of culturing of cells is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% greater than or less than the ratio of CD4 + cells and CD8 + cells in the sample from the subject, The method according to any one of claims 2 to 6.

11. The method according to any one of claims 1 to 10, wherein enriching the cells in the first and / or second selection comprises performing positive or negative selection based on the expression of cell surface markers.

12. The method according to claim 11, wherein enriching the cells in the first and / or second selection comprises negative selection including depleting cells expressing non-T cell surface markers.

13. The method according to claim 12, wherein the non-T cell marker comprises CD14.

14. The method according to any one of claims 1 to 13, wherein enriching the cells in the first or second selection comprises performing a plurality of positive or negative selection steps based on the expression of one or more cell surface markers to enrich CD4+ cells or CD8+ cells.

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

16. Selection based on immunoaffinity is performed by contacting the cells with an antibody capable of specifically binding to a cell surface marker, recovering the cells bound to the antibody, thereby performing positive selection, or recovering the cells not bound to the antibody, thereby performing negative selection, and CD4+ cells or CD8+ cells are enriched from the recovered cells, and the antibody is immobilized on magnetic particles. The method according to claim 15.

17. The method according to any one of claims 1 to 13, wherein the first selection and the second selection are performed in separate separation tanks operably connected.

18. The method according to claim 17, wherein the separation tanks are operably connected by tubing.

19. The selection based on immune affinity is performed by contacting cells with an antibody immobilized or bound to an affinity chromatography matrix, and the antibody is capable of specifically binding to a cell surface marker to perform positive or negative selection of CD4+ cells or CD8+ cells. The method according to any one of claims 15 to 18.

20. 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 matrix during contact, and cells expressing a cell surface marker specifically bound by the antibody on the matrix can be recovered from the matrix by disrupting the reversible bond between the binding reagent and the binding partner. The method according to claim 19.

21. The binding partner is selected from biotin, biotin analogs, and peptides capable of binding to the binding reagent, the binding reagent is selected from streptavidin, streptavidin analogs or mutant proteins, avidin, and avidin analogs or mutant proteins. The method according to claim 17.

22. The binding partner comprises the amino acid sequence shown in SEQ ID NO: 6, and / or the binding reagent is a streptavidin mutant protein comprising the amino acid sequence shown in SEQ ID NO: 12, 13, 15, or 16. The method according to claim 21.

23. After contacting the cells in the sample with the affinity chromatography matrix in the first selection and / or the second selection, a competing reagent is added to disrupt the bond between the binding partner and the binding reagent, thereby recovering the selected cells from the matrix. The method according to any one of claims 19 to 21, further comprising the step of

24. The method according to claim 23, wherein the competing reagent is biotin or a biotin analog.

25. One or more antibodies in the first and / or second selection have a dissociation rate constant (k -5 sec -1 greater than 3×10 -5 sec -1 or greater than about 3×10 off ), the method according to any one of claims 20 to 24.

26. One or more antibodies in the first and / or second selection have a dissociation constant (K d ) of about 10 -3 to 10 -7 or a range of about 10 -7 to about 10 -10 for the cell surface marker, the method according to any one of claims 20 to 25.

27. The method according to any one of claims 20 to 26, wherein the chromatography matrix for the first and / or the second selection is filled in a separation tank which is a column.

28. The affinity chromatography matrix is also at least or at least about 50×10 6 cells / mL, 100×10 6 cells / mL, 200×10 6 cells / mL, or 400×10 6 The method according to any one of claims 19 to 27, which is capable of adsorbing and / or selecting cells / mL.

29. The method according to any one of claims 19 to 28, wherein the first and second selection steps involve the use of an affinity chromatography matrix, and the matrix used in the first and second selection steps is in a relative amount sufficient to achieve the starting culture ratio.

30. The method according to any one of claims 1 to 29, wherein enriching CD4+ cells involves positive selection based on surface expression of CD4.

31. The method according to any one of claims 1 to 29, wherein enriching CD8+ cells involves positive selection based on surface expression of CD8.

32. One of the first and second selections, which involves enriching CD8+ cells, Enriching central memory T (T CM ) cells, and / or further involves enriching cells expressing a marker selected from CD28, CD62L, CCR7, CD127, and CD27 The method according to any one of claims 1 to 29.

33. The first selection involves enriching CD8+ cells, the second selection involves enriching CD4+ cells, and The first selection further comprises enriching central memory T (T CM ) cells and / or enriching cells expressing a marker selected from CD28, CD62L, CCR7, CD127, and CD27 The method according to any one of claims 1 to 32.

34. Enriching central memory T (T CM ) cells and / or enriching cells expressing a marker is CD8 + Selecting cells expressing CD62L from a first and / or second selected cell population enriched in cells, and / or CD8 + Selecting cells expressing CD27 from a first and / or second selected cell population enriched in cells, and / or CD8 + Selecting cells expressing CCR7 from a first and / or second selected cell population enriched in cells, and / or CD8 + Selecting cells expressing CD28 from a first and / or second selected cell population enriched in cells, and / or CD8 + Selecting cells expressing CD127 from a first and / or second selected cell population enriched in cells The method according to claim 32 or claim 33 or claim 35, including

35. The first selection involves enriching CD4+ cells, the second selection involves enriching CD8+ cells, and The second selection further comprises enriching central memory T (T CM ) cells The method according to any one of claims 1 to 32.

36. (i) The first selection involves enriching CD4+ cells by positive selection based on surface expression of CD4 to generate a first selected population enriched in CD4+ primary human T cells and a non-selected sample; (ii)The second selection includes enriching CD8+ cells and further includes enriching central memory T (T CM ) cells from the second selection sample, where enriching the central memory T (T CM ) cells is Negative selection to deplete cells expressing surface markers present on naive T cells, and positive selection for cells expressing surface markers present on central memory T ( CM CM T) cells and not present on another memory T cell subset, or Positive selection of cells expressing surface markers that are present on central memory T cells and not on naive T cells, and positive selection of cells expressing surface markers that are present on central memory T ( CM CM T) cells and not on another memory T cell subset comprising, thereby generating CM CD8+ primary human T cells enriched with T The method according to claim 35.

37. The marker present on naive T cells includes CD45RA, and Enriching central memory T (T CM ) cells involves negative selection to deplete cells expressing CD45RA, and positive selection for cells expressing surface markers that are present on central memory T (T CM ) cells and not present on another memory T cell subset, The method according to claim 36.

38. The surface marker present on central memory T cells and not present on naive T cells includes CD45RO; Enriching central memory T (T CM ) cells involves positive selection for cells expressing CD45RO and positive selection for cells expressing surface markers that are present on central memory T (T CM ) cells and absent on another memory T cell subset. The method according to claim 37.

39. Central memory T (T CM ) cells and not on another memory T cell subpopulation, the surface marker is selected from the group consisting of CD62L, CCR7, CD27, CD127, and CD44, the method according to any one of claims 36 to 38.

40. Central memory T (T CM ) The method according to claim 39, wherein the surface marker that is present on the cells and not present on another memory T cell subset is CD62L.

41. CD8 in the enriched composition or the culture starting composition + population contains at least 50% central memory T (T CM ) cells, or contains less than 20% naive T (T N ) cells, or contains at least 80% CD62L+ cells, the method according to any one of claims 32 to 40.

42. contacting the cells of a sample containing primary human T cells with a first immunoaffinity reagent that specifically binds to CD4 and a second immunoaffinity reagent that specifically binds to CD8 in an incubation composition under conditions such that the immunoaffinity reagents specifically bind to the CD4 molecules and CD8 molecules on the cell surface in the sample, and Generating an enriched composition comprising CD4+ cells and CD8+ cells at a culture starting ratio by recovering cells bound to the first and / or the second immunoaffinity reagent A method for enriching CD4+ T cells and CD8+ T cells, comprising: The first and / or the second immunoaffinity reagent is present in the incubation composition at a sub-optimal yield concentration, wherein the enriched composition contains less than 70% of all CD4+ cells in the incubation composition and / or less than 70% of CD8+ cells in the incubation composition, thereby producing a composition enriched in CD4+ T cells and CD8+ T cells.

43. (a) Enriching primary human T cells from a sample containing primary human T cells, comprising: Contacting the cells of the sample with a first immunoaffinity reagent that specifically binds to CD4 and a second immunoaffinity reagent that specifically binds to CD8 in an incubation composition under conditions such that the immunoaffinity reagents specifically bind to CD4 molecules and CD8 molecules on the surface of the cells in the sample, respectively; Generating an enriched composition comprising CD4+ cells and CD8+ cells at a culture starting ratio by recovering cells bound to the first and / or the second immunoaffinity reagent; The first and / or the second immunoaffinity reagent is present in the incubation composition at a sub-optimal yield concentration, wherein the enriched composition contains less than 70% of all CD4+ cells in the incubation composition and / or less than 70% of CD8+ cells in the incubation composition; and (b) Generating stimulated cells by incubating the cells of the enriched composition in the culture starting composition in a culture vessel under stimulating conditions, wherein the cells are at the culture starting ratio or substantially at the culture starting ratio; and (c) By introducing a genetically engineered antigen receptor into the stimulated cells of (b), CD4 + T cells and CD8 + Generating an output composition comprising T cells A method for producing genetically engineered T cells.

44. The method according to claim 42 or claim 43, wherein enriching the primary human T cells is performed in a closed system.

45. The method according to any one of claims 42 to 44, wherein the first and second immunoaffinity reagents are present in a sub-optimal yield concentration in the incubation composition, and wherein the enriched composition comprises less than 70% of all CD4+ cells in the incubation composition and less than 70% of all CD8+ cells in the incubation composition.

46. The first immunoaffinity reagent is present in a sub-optimal yield concentration in the incubation composition, and wherein the enriched composition comprises less than 60%, less than 50%, less than 40%, less than 30% or less than 20% of all CD4+ cells in the incubation composition, and / or The second immunoaffinity reagent is present in a sub-optimal yield concentration in the incubation composition, and wherein the enriched composition comprises less than 60%, less than 50%, less than 40%, less than 30% or less than 20% of all CD8+ cells in the incubation composition, The method according to any one of claims 42 to 45.

47. The method according to any one of claims 42 to 46, wherein the sample contains at least 1×10 9 CD3+ T cells.

48. The method according to any one of claims 42 to 47, wherein the concentration of one of the first and second immunoaffinity reagents in the incubation composition is greater than the concentration of the other, and wherein the greater concentration results in a higher yield in the enriched composition of CD4+ cells or CD8+ cells as compared to the yield of the other of CD4+ cells or CD8+ cells, thereby generating a culture start ratio in the enriched composition.

49. The concentration of one of the first and second immunoaffinity reagents is at least 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, or 10-fold greater than the concentration of the other of the first and second immunoaffinity reagents, and / or The higher yield at the enriched concentration is 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, or 10-fold greater, The method according to claim 48.

50. CD4 + cells and CD8 + The method according to any one of claims 42 to 49, wherein the starting ratio of culturing CD4 cells and CD8 cells is between 10:1 or about 10:1 and 1:10 or about 1:10, between 5:1 or about 5:1 and 1:5 or about 1:5, or between 2:1 or about 2:1 and 1:2 or about 1:

2.

51. The method according to any one of claims 42 to 50, wherein the culture start ratio of CD4+ cells to CD8+ cells is 1:1 or about 1:

1.

52. CD4 + cells and CD8 + The starting ratio of culturing the cells is different from the ratio of CD4 + cells and CD8 + cells, and / or CD4 + cells and CD8 + The starting ratio of culturing cells is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% greater or smaller than the ratio of CD4 + cells and CD8 + cells in the sample from the subject, The method according to any one of claims 42 to 51.

53. The method according to any one of claims 42 to 52, wherein more than 95% or more than 98% of the cells in the starting composition for culturing are CD4+ cells and CD8+ cells.

54. The method according to any one of claims 42 to 53, wherein each of the immunoaffinity reagents comprises an antibody.

55. The method according to claim 54, wherein the antibody is immobilized on the outer surface of the sphere.

56. The method according to claim 55, wherein the sphere is a magnetic bead.

57. The antibody comprises one or more binding partners capable of reversibly forming a bond with a binding reagent immobilized on the sphere, wherein the antibody is reversibly immobilized on the sphere, the method further comprising the step of recovering selected cells from the sphere by adding a competing reagent after contacting the cells in the sample with the first and second immunoaffinity reagents to disrupt the bond between the binding partner and the binding reagent. The method according to claim 55 or claim 56.

58. The binding partner is selected from biotin, a biotin analog, or a peptide capable of binding to the binding reagent, the binding reagent is selected from streptavidin, a streptavidin analog or mutant protein, avidin, an avidin analog or mutant protein, The method according to claim 57.

59. The binding partner comprises a peptide comprising the amino acid sequence shown in SEQ ID NO:6, and / or the binding reagent is a streptavidin mutant protein comprising the amino acid sequence shown in SEQ ID NO:12, 13, 15, or 16. The method according to claim 58.

60. The binding reagent is a multimer comprising one or more monomers of streptavidin or a streptavidin mutant protein, and the binding partner is a peptide comprising a continuous arrangement of at least two modules each capable of reversibly binding to at least one monomer of the binding reagent. The method according to claim 58 or claim 59.

61. The method according to claim 60, wherein the binding partner comprises the amino acid sequence shown in any of SEQ ID NOs:7 to 10.

62. The method according to any one of claims 57 to 61, wherein the competing reagent is biotin or a biotin analog.

63. One or more antibodies in the first and / or second selection have a dissociation rate constant (k -5 sec -1 greater than 3×10 -5 sec -1 or greater than about 3×10 off sec ), The method according to any one of claims 42 to 63.

64. One or more antibodies in the first and / or second selection have a dissociation constant (Kd) in the range of about 10 -3 to 10 -7 and an affinity having a dissociation constant in the range of about 10 -7 to about 10 -10 The method according to any one of claims 42 to 63.

65. The method according to any one of claims 42 to 63, wherein one or more steps are performed in an automated manner and / or a closed system is automated. **Claim 66** Determining the ratio of CD4+ T cells to CD8+ T cells in a sample prior to making the first selection and / or the second selection, and Adjusting the first and / or the second selection based on the ratio of CD4+ T cells to CD8+ T cells in the sample so as to produce a composition containing CD4+ cells and CD8+ cells at a starting culture ratio comprising the method according to any one of claims 2, 3 and 5 to 41. **Claim 67** wherein the first and / or the second selection comprises a selection based on immunoaffinity comprising an affinity chromatography matrix, and the step of adjusting the first and / or the second selection comprises selecting an amount of the affinity chromatography matrix sufficient to achieve a starting culture ratio in the first and / or the second selection the method according to claim 66. **Claim 68** Determining the ratio of CD4+ T cells to CD8+ T cells in a sample prior to contacting the cells of the sample with the first and second immunoaffinity reagents; and Selecting the concentration of the first and / or the second immunoaffinity reagent based on the ratio of CD4+ T cells to CD8+ T cells in the sample so as to produce an enriched composition containing CD4+ cells and CD8+ cells at a starting culture ratio comprising the method according to any one of claims 42 to 65. **Claim 69** An output composition comprising a ratio of CD4 + cells to CD8 + cells that is between 2:1 or about 2:1 to 1:5 or about 1:5, the method according to any one of claims 1 to 68. **Claim 70** CD4 in the output composition + cells and CD8 + The method according to claim 69, wherein the ratio of cells is 1:1 or about 1:

1. **Claim 71** The method according to any one of claims 1 to 70, wherein the sample is obtained from a subject. **Claim 72** The method according to claim 71, wherein the subject is a subject to whom genetically engineered T cells or cells for adoptive cell therapy are administered. **Claim 73** The method according to claim 72, wherein the subject is a subject other than a subject to whom genetically engineered T cells or cells for adoptive therapy are administered. **Claim 74** The method according to any one of claims 1 to 73, wherein the sample is a blood or blood-derived sample. **Claim 75** The method according to any one of claims 1 to 74, wherein the sample is a leukocyte sample. **Claim 76** The method according to any one of claims 1 to 75, wherein the sample is an apheresis, peripheral blood mononuclear cell (PBMC), or leukapheresis sample. **Claim 77** The method according to any one of claims 4 to 41 or 43 to 76, wherein the composition is incubated under stimulating conditions in a culture vessel before, during, and / or after introduction of the genetically engineered antigen receptor.

78. The method according to any one of claims 4 to 41 or 43 to 76, wherein the composition is incubated under stimulating conditions in a culture vessel before, during, and after introduction of the genetically engineered antigen receptor.

79. The method according to any one of claims 4 to 41 or 43 to 78, wherein the stimulating conditions include conditions under which T cells of the composition proliferate.

80. The method according to any one of claims 4 to 41 or 43 to 79, wherein the stimulating conditions include an agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex.

81. The method according to claim 80, wherein one or more components of the TCR complex include the CD3 zeta chain.

82. The method according to any one of claims 4 to 41 or 43 to 81, wherein the stimulating conditions include the presence of an anti-CD3 antibody, and an anti-CD28 antibody, an anti-4-1BB antibody, and / or a cytokine.

83. The method according to claim 82, wherein the anti-CD3 antibody and / or the anti-CD28 antibody are present on the surface of a solid support.

84. The method according to claim 83, wherein the cytokine includes IL-2, IL-15, IL-7, and / or IL-21.

85. The method according to any one of claims 4 to 41 or 43 to 84, wherein the genetically engineered antigen receptor includes a T cell receptor (TCR) or a functional non-TCR antigen receptor.

86. The method according to claim 85, wherein the receptor specifically binds to an antigen expressed by cells of the disease or condition to be treated.

87. The method according to any one of claims 85 or 86, wherein the antigen receptor is a chimeric antigen receptor (CAR).

88. The method according to claim 87, wherein the CAR includes an extracellular antigen recognition domain, and an intracellular signaling domain containing an ITAM sequence, and an intracellular signaling domain of a T cell costimulatory molecule.

89. (a)An output composition comprising a CD4 + T cell and a CD8 + producing step; and (b) administering to the subject cells of the output composition A treatment method comprising:

90. The method according to claim 89, wherein the sample from which the cells are isolated is derived from the subject to whom the cells are administered.

91. A composition of cells produced by the method according to any one of claims 1 to 88.

92. The composition according to claim 91, comprising a pharmaceutically acceptable carrier.

93. A treatment method comprising the step of administering the cell composition according to claim 91 or 92 to a subject.

94. The method according to claim 93, wherein the genetically engineered antigen receptor specifically binds to an antigen associated with a disease or condition.

95. The treatment method according to claim 94, wherein the disease or condition is cancer.

96. The composition according to claim 91 or claim 92 for use in treating a disease or condition in a subject.

97. Use of the composition according to claim 91 or claim 92 for the manufacture of a medicament for treating a disease or disorder in a subject.

98. The composition according to claim 96 or the use according to claim 97, wherein the genetically engineered antigen receptor specifically binds to an antigen associated with a disease or condition.

99. The composition or use according to any one of claims 96 to 98, wherein the disease or condition is cancer.

100. a) A first affinity chromatography matrix comprising a first binder immobilized on a surface, wherein the binder specifically binds to a first cell surface marker present on a first cell type, and the first affinity matrix does not comprise a second binder that specifically binds to a second cell surface marker, wherein the first affinity chromatography matrix is operably connected to a storage reservoir containing a cell sample via a first operable connection, and the first operable connection enables cells to pass from the storage reservoir to the first affinity chromatography matrix, the first affinity chromatography matrix is operably connected to an output tank via a second operable connection; The first affinity chromatography matrix; b) A second affinity chromatography matrix comprising a second binder immobilized on a surface, wherein the second binder specifically binds to a second cell surface marker present on a second cell type, wherein The first affinity chromatography matrix is operably connected to a second affinity chromatography matrix via a third operable connection, and the third operable connection enables cells that have passed through the first affinity chromatography matrix and have not bound to the first binder to pass through to the second affinity chromatography matrix. The second affinity chromatography matrix is operably connected to an output tank via a fourth operable connection, and the fourth operable connection enables cells that have bound to and eluted from the first and / or the second affinity chromatography matrix to pass through. The second affinity chromatography matrix is operably connected to a waste tank via a fifth operable connection, and the fifth operable connection enables cells that have passed through the first affinity chromatography matrix and have not bound to the first binder and have passed through the second affinity chromatography matrix and have not bound to the second binder to pass through. A second affinity chromatography matrix; c) An output tank; and d) A waste tank A closed device system for the purification of target cells, comprising within a closed system, (i) cells that have bound to the first affinity chromatography matrix and have been recovered therefrom, and (ii) cells that have passed through the first affinity chromatography matrix, have not bound to it, have bound to the second chromatography matrix, and have been recovered therefrom configured to enable collection in a single composition within the output tank of A closed device system.

101. The closed device according to claim 100, wherein one or more of the operable connections comprise tubing connecting a storage reservoir, a first affinity chromatography matrix, a second affinity chromatography matrix, and / or a culture tank.

102. The closed device according to claim 101, wherein the tubing is connected to a stopcock, a valve, or a clamp.

103. (a)The first affinity chromatography matrix is one of a CD4+ affinity chromatography matrix or a CD8+ affinity chromatography matrix, (b)The second affinity chromatography matrix is the other of a CD4+ affinity chromatography matrix or a CD8+ affinity chromatography matrix, The closed device system according to any one of claims 100 to 102.

104. d) A third affinity chromatography matrix comprising a third binder immobilized on a surface, wherein the binder specifically binds to a third cell surface marker and the third binder is capable of binding to cells expressing the third cell surface marker, where A third operable connection further operably connects the third affinity chromatography matrix to the first matrix, The third affinity chromatography matrix is operably coupled to an output container via a sixth operable connection, such that the sixth operable connection allows cells bound to and recovered from the first matrix and bound to and recovered from the third matrix to pass to the output container, A fifth operable connection further operably connects the third affinity chromatography matrix to a waste container, such that the fifth operable connection allows cells that have passed through and not bound to the third column to be sent to the waste container, The third affinity chromatography matrix The closed device system according to any one of claims 100 to 103, further comprising.

105. d) A third affinity chromatography matrix comprising a third binder immobilized on a surface, wherein the binder specifically binds to a third cell surface marker and the third affinity chromatography matrix is capable of binding to cells expressing the third cell surface marker, where A second operable connection further operably connects the third affinity chromatography matrix to the first matrix and the output container, such that the second operable connection enables cells that have bound to and been recovered from the first matrix and that have bound to and been recovered from the third matrix to be sent to the output container. The third affinity chromatography matrix The closed device system according to any one of claims 100 to 103, further comprising

106. The first affinity chromatography matrix comprises a binder that specifically binds to CD8. The second affinity chromatography matrix comprises a binder that specifically binds to CD4. The third affinity chromatography matrix comprises a binder that specifically binds to a marker expressed on central memory T (T CM ) cells. The closed device system according to claim 104 or 105.

107. The closed device system according to claim 106, wherein the binder of the third affinity chromatography matrix selectively binds to a cell surface marker selected from CD62L, CD45RA, CD45RO, CCR7, CD27, CD127, and CD44.

108. The closed device system according to any one of claims 100 to 107, wherein one or more or all of the affinity chromatography matrices are further operably connected to an elution buffer reservoir containing one or more competing reagents.

109. The closed device system according to any one of claims 100 to 107, wherein the competing reagent is one or more of the group consisting of biotin, biotin analogs, and peptides capable of binding to the chromatography matrix.

110. The closed device system according to any one of claims 100 to 107, wherein one or more or all of the third, fourth, or sixth operable connections further comprise a competitor removal chamber.

111. The closed device system according to claim 110, wherein the competitor removal chamber further comprises a binding reagent.

112. The closed device system according to claim 111, wherein the binding reagent comprises one or more of the group consisting of streptavidin, streptavidin analogs or mutant proteins, avidin, and avidin analogs or mutant proteins.

113. The closed device system according to any one of claims 100 to 112, wherein one or more or all of the binders are antibodies.

114. The closed device system according to claim 113, wherein the antibody is a Fab.

115. The closed device system according to any one of claims 100 to 113, wherein one or more or all of the binders are reversibly bound to an affinity chromatography matrix.

116. The method according to any one of claims 17 to 41 or 54 to 88, wherein the antibody is a Fab.

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