Methods for isolating, culturing, and genetically manipulating 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 priority to U.S. Provisional Patent Application No. 61 / 983,415, filed April 23, 2014, the contents of which are incorporated by reference in their entirety.

[0002] INCORPORATION BY REFERENCE OF SEQUENCE LISTING This application is filed with an electronic sequence listing, which is provided as a file named 735042000240seqlist.txt, created on April 22, 2015, and is 13 kilobytes in size. The information in the electronic sequence listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates in some aspects to methods, cells, and compositions for preparing cells, and compositions for genetic engineering and cell therapy. In some embodiments, streamlined cell preparation methods are provided, for example, for isolating, processing, incubating, and genetic engineering cells and cell populations. Also provided are cells and compositions produced by the methods, and methods for using them. The cells can include immune cells such as T cells, and generally include multiple isolated T cell populations or T cell subtypes. In some aspects, the methods can prepare multiple different cell populations for adoptive therapy using fewer steps and / or resources and / or reduced handling compared to other methods. [Background technology]

[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 and expressing 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 subjects. Improved methods for the preparation (e.g., isolation, processing, culture, and manipulation) of cells for cell therapy use are needed. In particular, methods for the preparation and manipulation of cells, such as multiple isolated cell types or subtypes, with improved efficiency, safety, variability, and conservation of resources are needed. Methods, cells, compositions, kits, and systems that address such needs are provided. Summary of the Invention

[0005] overview Methods for the preparation and manipulation of cells and cell populations, as well as cells and compositions produced by the methods are provided. In some embodiments, the cells can be used for immunotherapy, such as in relation to adoptive immunotherapy methods. In some aspects, the methods provided include the isolation, selection, or enrichment of CD4+ and CD8+ cells or their subpopulations from the same starting sample, such as a single apheresis sample, a leukapheresis sample, or a sample containing peripheral blood mononuclear cells (PBMCs). In some embodiments, the methods include the selection or enrichment of at least two cell populations, such as a CD4+ and a CD8+ population, in a single process flow, in which the negative fraction sample is not discarded from a first selection or enrichment of one of CD4+ or CD8+ in a process, and then a second selection is performed for the other of CD4+ or CD8+ cells. In some embodiments, 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+ and CD8+ cells, is performed simultaneously, e.g., in the same vessel or using the same device, or sequentially as part of a system or device to which the vessels, e.g., columns, chambers used to perform 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 flow, without handling any 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 the different populations is performed from the same starting composition or material, e.g., from the same sample.

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

[0008] In some embodiments, the second selection is carried out 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 used as a base material for further selection to enrich another cell type. Generally, if the T cell subset enriched in the first selection is a CD4+ subset (i.e., the first selection enriches CD4+ cells), the first selection will naturally be designed not to enrich the other subtype of cells to be enriched in the second selection. For example, in some embodiments, the first selection enriches CD4+ cells and does not enrich CD8+ cells, and the second selection enriches CD8+ cells from the negative fraction recovered from the first selection. Similarly, generally, if the T cell subset enriched in the first selection is a CD8+ subset (i.e., the first selection enriches for CD8+ cells), then it will be expected that the first selection will be designed not to enrich for cells of the other subtype that should be enriched in the second selection. For example, in some embodiments, the first selection enriches for CD8+ cells and not for CD4+ cells, and the second selection enriches for CD4+ cells from the negative fraction recovered from the first selection.

[0009] In some embodiments, the methods further involve a third, fourth, etc., further selection, which may enrich for 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 or the unselected population from a given step (e.g., the second selection step) are further enriched. For example, in some embodiments, the selected CD8+ cells are further enriched for CD8+ cell subtypes, such as resting cells or central memory cells.

[0010] In some aspects, (a) providing a culture initiation composition, the composition being produced by performing a first selection in a closed system, the first selection being for isolating CD4 T cells from a sample containing primary human T cells; +the first selected population and the unselected population by enriching for one of CD4+ cells and CD8+ cells, and the second selection in the closed system, the second selection comprising enriching for the other of CD4+ cells and CD8+ cells from the unselected population to generate a second selected population; (b) generating stimulated cells by incubating a culture starting composition comprising the first selected population of cells and the second selected population of cells in a culture vessel under stimulated conditions; and (d) introducing the engineered antigen receptor into the stimulated cells produced in (b). A method for producing engineered T cells is provided, whereby the method comprises producing a CD4 T cell expressing an engineered antigen receptor. + T cells and CD8 + An output composition comprising T cells is generated.

[0011] In some embodiments, methods are also provided that include simultaneous enrichment or selection of a first and a second cell population, such as a CD4+ cell population and a CD8+ cell population. In some embodiments, the methods include contacting cells of a sample containing primary human T cells with a first immunoaffinity reagent that specifically binds CD4 and a second immunoaffinity reagent that specifically binds CD8 in an incubation composition under conditions in which the immunoaffinity reagents specifically bind to CD4 and CD8 molecules, respectively, on the surface of cells in the sample, and recovering cells that are bound to the first and / or second immunoaffinity reagent, thereby generating an enriched composition that includes CD4+ and CD8+ cells. In some embodiments, the method is performed by including a concentration of the first and / or second immunoaffinity reagent in the incubation composition that is a suboptimal yield concentration 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 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 of the CD8+ cells in the incubation composition.

[0012] In some embodiments, a method for enriching CD4+ and CD8+ T cells is provided that includes providing an enriched composition of CD4+ and CD9+ T cells, wherein the enriched composition is produced by 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 where the immunoaffinity reagents specifically bind to CD4 and CD8 molecules, respectively, on the surface of cells in the sample; and recovering cells that are bound to the first and / or second immunoaffinity reagent, thereby generating an enriched composition comprising CD4+ and CD8+ cells at a culture starting ratio, wherein the first and / or second immunoaffinity reagents are present in the incubation composition at a suboptimal yield concentration, wherein the enriched composition contains less than 70% of the total CD4+ cells in the incubation composition and / or less than 70% of the CD8+ in the incubation composition, thereby producing a composition enriched for CD4+ and CD8+ T cells.

[0013] In some of any of the embodiments 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 marker expressed on naive T cells, resting T cells, or central memory T cells. In some embodiments, the solid support is a bead, such as a sphere, e.g., a microbead or nanobead. In some embodiments, the bead can be a magnetic bead. In some embodiments, 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, e.g., spheres or chromatography matrices, capable of forming a reversible bond with a binding reagent immobilized on a solid surface, where 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 a streptavidin, analog or variant set forth in any of SEQ ID NOs: 11-16, or an amino acid sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence set forth in any of SEQ ID NOs: 11-16 and retains 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 or contains a peptide capable of binding to the binding reagent, such as a streptavidin-binding peptide, such as a peptide comprising a sequence set forth in any of SEQ ID NOs:1-10. In some embodiments, the method includes, as part of the first and / or second selection, contacting cells in the sample with a solid support comprising an antibody immobilized thereon, followed by recovering the selected cells from the solid surface by adding a competing reagent to disrupt the binding between the binding partner and the binding reagent. 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, or subpopulations thereof, selected or enriched for CD8+ cells, present at a culture starting ratio. In some embodiments, the culture starting ratio of CD4+ cells to CD8+ cells is between or about 10:1 and 1:10 or about 1:10, between or about 5:1 and 1:5 or about 1:5, or between or about 2:1 and 1:2 or about 1:2, such as 1:1 or about 1:1. It is within the level of a skilled artisan to choose or select sufficient amounts or sufficient relative amounts of immunoaffinity-based reagents, such as antibody-coated beads (e.g., magnetic beads) or one or more affinity chromatography matrices, to achieve or produce a culture starting ratio in a produced composition, such as a culture starting composition, containing cells enriched or selected for CD4, CD8, or subpopulations thereof. 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 a plurality of cell populations or cell types, which in some aspects are included in the composition at a specific ratio or number of cells or cell types. Also provided are methods for optimizing the method, for example, by selecting or determining appropriate ratios and numbers, such as the starting culture ratios and desired output ratios and doses of cell types and populations for use in connection with the method.

[0017] Cells, cell populations, and compositions thereof for use in and produced by the methods are also provided. Systems, devices, apparatus, reagents, compounds, and kits for carrying out the methods are also provided. Methods of therapy and use for the cells and compositions produced by the methods, such as adoptive cell therapy, are also provided.

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

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

[0020] In some embodiments, the method is carried out by: (a) incubating a culture start composition in a culture vessel that contains a plurality of cell populations at a particular culture start ratio or at a particular cell number; and (b) genetically engineering the cells in the culture vessel, such as by introducing an engineered antigen receptor into the cells.

[0021] In some embodiments, the engineered antigen receptor is located within a cell in a culture vessel, such as a distinct cell type or subpopulation in a culture vessel, e.g., within a CD4 + Cells and CD8 + is introduced into cells.

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

[0023] In some embodiments, the isolation comprises isolating CD4 + Primary human T cell populations and / or CD8 + The method comprises or is performed by isolating a primary human T cell population. In some aspects, it comprises isolating a primary human T cell population.+ Depleting or enriching subpopulations of cells and / or CD8 + The method includes the steps of depleting or enriching a subpopulation of cells. Thus, in some aspects, the culture initiation composition comprises isolated CD4 + Primary human T cells and CD8 + Contains primary human T cells.

[0024] In some aspects, the enrichment or depletion step is carried out by immunoaffinity-based selection, such as binding with an antibody or other binding molecule that recognizes a surface marker on cells.In some aspects, the antibody or other molecule is coupled to a magnetic responsive particle or a 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 primary human T cell populations, CD8 + In some embodiments, the method includes depleting or enriching a subpopulation of cells. + Isolation of primary human T cell populations is performed using CD4 + In some aspects, isolating a T cell population includes depleting or enriching a subpopulation of T cells. CM In some aspects, this includes enriching for CD8 + Primary human T cell populations and / or CD4 + Isolation of primary human T cell populations, known as central memory T (T CM In some aspects, this involves enriching for central memory T (T CM Enrichment for central memory T (T) cells can be achieved by negative selection of cells expressing surface markers present on naive T cells, such as CD45RA, or positive selection for cells expressing surface markers present on central memory T cells and not present on naive T cells, such as CD45RO; and / or by selection of surface markers expressing central memory T (T CM) cells and not present on another memory T cell subpopulation.

[0026] In some embodiments, the isolation comprises (i) subjecting the sample to positive selection based on surface expression of CD4 to produce isolated CD4 + (ii) generating a positive fraction and a first negative fraction, the positive fraction being a population of T cells; and (iii) 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 CM ) subjecting the second negative fraction to positive selection based on surface expression of a marker that is present on the surface of the cells and is not present on the surface of another memory T cell subpopulation.

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

[0028] In some aspects, the isolation or selection is performed in the same separation vessel. In some aspects, the isolation includes (i) subjecting the sample to a first selection to isolate CD4 + Primary human T cell populations and CD8 + (ii) generating one of the primary human T cell populations and an unselected sample; and (ii) subjecting the unselected sample to a second selection to obtain CD4 + Primary human T cell populations and CD8 + In some aspects, the method further comprises generating another primary human T cell population. + A primary human T cell population was generated by first selection and identified as CD8 + The primary human T cell population is generated in a second selection. In some aspects, the first and / or second selection comprises multiple positive 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, comprises positive selection based on surface expression of CD62L, CCR7, CD44, or CD27. + T cell populations, or primary human CD8 + Isolation of one or more populations, such as a T cell population, includes negative selection based on surface expression of CD45RA or positive selection based on surface expression of CD45RO.

[0030] In some embodiments, multiple cell populations, e.g., CD4 + Populations and CD8 + Various isolations, such as population isolations, are performed in the same isolation vessel. In some aspects, the isolation vessel is or includes a tube, tubing set, chamber, unit, well, culture vessel, bag, and / or column. In some aspects, the isolation vessel maintains the cells in a contained or sterile environment during isolation.

[0031] In some embodiments, the incubating step is performed under stimulatory conditions. + Primary human T cell populations and CD8 + The primary human T cell population is included at a starting ratio. In some aspects, the starting ratio is a ratio of CD4 + Cells and CD8 + The desired output ratio of T cells (or desired total number of T cells or subpopulations) is designed to obtain a desired output ratio of T cells.

[0032] In some embodiments, the desired output ratio is between or about 5:1 and 1:5 or about 1:5 (or greater than about 1:5 and less than about 5:1), e.g., between or about 1:3 and 3:1 or about 3:1 (or greater than about 1:3 and less than about 3:1), e.g., between or about 2:1 and 1:5 or about 1:5 (or greater than about 1:5 and less than about 2:1), or 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 therein.

[0033] In some aspects, the method comprises detecting CD4 + Cells and CD8 + This results in a ratio of two different cell types or populations, such as a ratio of 5:1 or about 5:1 to 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). full complement), or the ratio is or is about 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.

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

[0035] In some aspects, the method produces a desired output ratio or cell number in the output composition, produces a ratio or number in the output composition that is within a particular tolerance or error range of such desired output ratio or number, and / or produces such ratio or number a particular 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 at least 80% of the time the method is performed.

[0037] In some embodiments, the tolerance and / or desired output ratio or number can be determined by administering different cell types at multiple test ratios or numbers to one or more subjects, e.g., CD4 + Cells and CD8 + By administering cells and evaluating one or more parameters, it is determined or has been determined.In some aspects, determining the desired output ratio or number or tolerance comprises evaluating one or more results after administering to subject.In some aspects, the results include the results selected from the results that show the amelioration of disease symptoms and the results that show safety and / or low or no toxicity.

[0038] In some embodiments, the initiation ratio is CD4 + Primary human T cell populations and / or CD8 +The culture start ratio is selected based on the proliferation rate or viability of various isolated cell types, such as primary human T cell population.In some embodiments, the culture start 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 culture start ratio is selected based on the disease or condition that affects the subject, and / or the treatment that the subject is undergoing, has undergone, or will undergo, such as the concurrent treatment for administration with adoptive cell therapy.In some aspects, the culture start ratio is selected based on the phenotype of one or more cells or cell subtypes that are isolated or cultured, such as the phenotype of CD4+ cell population and / or 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 selecting a culture starting ratio prior to the incubation step. + Primary human T cells and / or isolated CD8 + Selection is performed by measuring the proliferation rate or viability of one or more isolated or incubated cell populations, such as primary human T cells. In some aspects, selection is performed by measuring the proliferation rate or viability of one or more isolated or incubated cell populations, such as primary human T cells. + Primary human T cells and / or isolated CD8 + The phenotype of primary human T cells is evaluated.In some aspects, the phenotype is selected from the expression of surface markers and secretion of cytokines or other factors.In some aspects, the selection is performed by evaluating the origin of the sample, such as when the culture start ratio is selected based on the disease or condition that affects the subject from which the sample is derived.

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

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

[0042] In some aspects, the CD8 + The population had 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 the subject that genetically engineered cells, such as T cells, or cells for adoptive cell therapy, are administered to, or the subject that needs such administration.In other aspects, the subject is the subject other than the subject that genetically engineered cells, such as T cells, or cells for adoptive cell therapy, are administered to, or the subject that does not need such treatment.The sample includes blood and blood-derived samples, such as white blood cell samples, apheresis samples, leukapheresis samples, peripheral blood mononuclear cell (PBMC) samples, and whole blood.

[0044] In some aspects, the stimulating conditions for incubation or operation include conditions that allow the T cells of the culture starter composition to proliferate or expand.For example, in some aspects, incubation is performed in the presence of an agent that can activate one or more intracellular signaling domains of one or more components of the TCR complex, such as the CD3 zeta chain, or can activate activation signaling via such 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 engineered antigen receptor is or comprises 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). In some aspects, the receptor specifically binds to the antigen expressed by the cell of the disease or condition to be treated. In some aspects, the CAR comprises an extracellular antigen recognition domain. In some aspects, it further comprises an intracellular signaling domain, including an ITAM-containing sequence and an intracellular signaling domain of a T cell costimulatory molecule.

[0046] Also provided are cells and compositions, including pharmaceutical compositions, produced by any method or embodiment, 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 cells and compositions in such methods. For example, provided are treatment methods carried out by producing cells according to the cell production method, and administering the cells of the output composition or compositions derived therefrom to a subject. Provided are treatment methods including administering to a subject the cells or compositions provided. In some aspects, the sample from which the cells are isolated is from the subject to which the cells are administered. In some aspects, the sample is from a different subject. Thus, the method includes autologous and allogeneic methods. In some embodiments, the method ameliorates, 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 condition. In some embodiments, cancer includes leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin's 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 carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and / or mesothelioma. [Brief description of the drawings]

[0047] [Figure 1A]1 provides a schematic of one embodiment of a closed system for use in embodiments of the provided methods. 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, which is operably linked via a series of tubing lines to a second chromatography column 2 containing a second matrix 4. The second chromatography column 2 is operably linked to a removal chamber 9. The removal chamber 9 is operably linked to a valve 13 that directs cells and fluids via a series of tubing lines to a waste container 10 or culture vessel 12. The system is enclosed in an enclosure 14. [Figure 1B]1 provides a schematic of one embodiment of a closed system for use in embodiments of the provided methods. 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 housing a first matrix 3. A valve 13 operably connected to the series of tubing directs fluids via the series of tubing. A valve 13 operably connected to the first chromatography column 1 directs cells and fluids to a second chromatography column 2 housing a second matrix 4, which is operably connected to a removal chamber 9. A valve 13 operably connected to the first chromatography column 1 also directs cells and fluids to a removal chamber 9, which is operably connected to a third chromatography column 15 housing 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 primary waste container 10 or culture vessel 12 via a series of tubing lines and valves 13. The system is enclosed in an enclosure 14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Detailed Description Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or terms used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the subject matter of the claims belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or quick 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, referenced in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that the definitions set forth herein are opposite or otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

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

[0051] I. Methods and Systems for Isolating, Cultivating, and Manipulating Cells for Adoptive Therapy Methods for preparing cells, e.g., T cells, for use in therapeutic procedures such as genetic engineering and adoptive cell therapy are provided. Specifically, in some embodiments, the methods involve preparing multiple different cell populations or cell types, e.g., isolated CD4 + and CD8 + Use or generate the composition comprising T cell population and subpopulation.In some embodiments, the method comprises the step of isolating one or more cell populations, generally a plurality of cell populations.Cells are generally isolated from a sample derived from a subject.

[0052] In some aspects, the method is carried out by using simultaneous or sequential selection or enrichment, where multiple 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 carried out using a single process flow, where 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, without discarding any cells from the first population, and then the selection of the second population of cells is carried out.In some aspects, the first and second selections can be carried out simultaneously or sequentially. In some embodiments, the selection method is performed as a single step flow by performing a first selection to enrich a first population of cells, e.g., one of CD4+ and CD8+ cells, from a sample, e.g., a sample containing primary human T cells, and using unselected cells from the first selection as a source of cells for a second selection, such as the other of the CD4+ or CD8+ cells from the sample.

[0053] In some embodiments, one or more further selections of the first or second selected cells can be performed. In some embodiments, the further selection is performed by selecting central memory T (T CM ) enrich for subpopulations of CD4+ or CD8+ cells expressing markers on the cells, and / or enrich for subpopulations of cells expressing CD62L, CD45RA, CD45RO, CCR7, CD27, CD127, or CD44.

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

[0055] For example, in some aspects, one or more steps are performed with multiple cell populations combined in the same composition, or multiple cell populations present in the same vessel, e.g., in the same closed system or device, or in the same vessel, unit, or chamber, such as the same column, e.g., magnetic separation column, tube, tubing set, culture or cultivation chamber, culture vessel, processing unit, cell separation vessel, centrifugation chamber, or using the same separation matrix, medium, and / or reagent, e.g., the same magnetic or magnetic response matrix, particle, or bead, the same solid support, e.g., affinity-labeled solid support, and / or the same antibody and / or other binding partner, e.g., fluorescently labeled antibody and binding partner, for the 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 device, such that the isolation, selection, and / or enrichment methods occur in a single process flow where the unselected cell population from the first selection can be used as a source of cells for the second selection.

[0056] In some embodiments, the isolation or enrichment of one or more specific populations or subpopulations of cells, such as CD4+ T cells and CD8+ T cells to be cultured or engineered for adoptive therapy, provides one or more advantages. For example, isolated CD4 + T cells and CD8 + Engineering cells enriched for multiple different cell populations or cell types, such as populations and subpopulations of T cells, can improve efficacy or reduce or avoid undesired effects. In some aspects, the isolation or enrichment increases the ability of the cells ultimately administered to the subject to persist, expand, become activated, 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) of a T cell population, such as a CD8+ T cell population, is improved or increased. CMEnriching for CD8+ T cells and CD4+ T cells can provide such advantages. In some aspects, one or more such advantages can be achieved by enriching for isolated populations or subpopulations of CD8+ T cells and CD4+ T cells, e.g., T CM Enriched CD8 + Population and CD4 + For example, such advantages may be achieved in some aspects by administering a CD4+ population and a CD8+ population compared to a CD8+ population alone.

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

[0058] In certain embodiments, the isolation or separation is carried out 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, a system is used to perform each of these steps in a closed or sterile environment, for example, to minimize 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 apparatus 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 device performs isolation, such as a selection step or an enrichment step. In some embodiments, an additional system or device, such as a closed system or device, can be used to perform one or more other steps, such as the cell preparation, processing, incubation, culture and / or formulation steps of the method. In some embodiments, the system or device performs one or more, such as all, of the isolation, processing, manipulation and formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or a computer program in communication with the system or device, which allows a user to program, control, evaluate the results and / or adjust various aspects of the processing, isolation, manipulation and formulation steps.

[0060] In some embodiments, the enriched composition, such as the culture initiation composition, is generated by the provided method to contain different cell populations, such as CD4+ and CD8+, so that 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 population of selected, enriched, or isolated cells containing different populations of cells, such as CD4+ and CD8+ cells, in a single step flow. In some aspects, this differs from prior art methods, including prior art methods of processing cells for manipulation for adoptive cell therapy, which typically include separate step flows, such as at least two step flows, for each different cell population. For example, in some aspects of the 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, and 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 reduced complexity, time, cost, and / or resource usage 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 operation of different populations is carried out using the same device or equipment and / or simultaneously.In some aspects, the isolation, culture, and / or operation of different populations is carried out based on the same starting composition.In some aspects, such features of the method reduce the amount of time, method steps, cost, complexity, and / or number of resources compared to the method in which cell populations are isolated, incubated, and / or operated separately in separate vessels, using separate equipment, at separate times, and / or starting with different starting compositions.

[0063] In some aspects, the method of isolating, incubating and manipulating cells produces output compositions in which different cell populations or cell types, such as CD4+ cells and CD8+ cells, are present in desired ratios or within some tolerance of desired ratios.Such ratios include the ratios considered optimal for therapeutic use, such as the output ratios considered appropriate or optimal for administration to patients in relation to adoptive cell therapy.Methods are also provided for administering the cells and compositions prepared by the method to patients, for example in relation to adoptive cell therapy.

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

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

[0066] In some embodiments, cells are administered at or within the tolerance of one or more individual cell populations or subtypes of desired dose, such as desired dose of CD4+ cells and / or 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, for example, per kg.In some aspects, the desired dose is equal to or greater than 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 a desired fixed dose and a desired ratio of total cells, and / or based on one or more, e.g., a desired fixed dose of each, of individual subtypes or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed dose or minimum dose of T cells and CD4 + and CD8 + Based on the desired ratio of cells and / or CD4+ Cells and / or CD8 + Based on a desired fixed or minimum dose of cells.

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

[0069] Also provided is a method for determining a culture start ratio designed to achieve a desired output ratio or dose within a certain tolerance and / or a certain percentage of time to do so. Also provided is a method for evaluating the interim ratio or number of cell populations over the course of various method steps, such as one or more periods during incubation. Also provided is a method for adjusting various conditions, such as culture conditions, based on such evaluation. In some aspects, adjustments are made to ensure that a specific output ratio or dose is achieved or is achieved within a tolerance.

[0070] In certain embodiments, CD4 + T cell populations and CD8 + T cell populations (e.g., CD8 + population) at or near the desired output ratio, and / or T cell and / or CD4 + T cells and CD8 +Streamlined methods are provided for preparing compositions having T cells at or near a desired dose (e.g., number or number per unit of body weight) where cell populations are isolated, incubated, and / or manipulated in combination, which methods are associated with increased efficiency and / or reduced complexity, time, cost, and / or resource use compared to methods where the populations are isolated, incubated, and / or manipulated separately.

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

[0072] A. Isolation, Isolated Cells, and Other Processing Steps Also included in the provided embodiments are methods for isolating a plurality of 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 of size, density, sensitivity or resistance to a particular reagent, and / or affinity for an antibody or other binding partner, e.g., immunoaffinity. In some aspects, isolation is performed sequentially and / or simultaneously in a single process flow using the same device or equipment. In some aspects, 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, multiple cell populations are isolated in the same closed system or device and / or in the same vessel or set of vessels, e.g., the same (or the same set of) units, chambers, columns, e.g., magnetic separation columns, tubes, tubing sets, culture or cultivation chambers, culture vessels, processing units, cell separation vessels, centrifugation chambers. For example, in some cases, the isolation of multiple cell populations is performed in a system or device using a single or the same isolation or separation vessel or set of vessels, such as a single column or set of columns, and / or the same tube or tubing set, without the need to transfer the cell population, composition, or suspension from one vessel, e.g., tubing set, to another vessel.

[0074] In some aspects, such methods are achieved by using simultaneous or sequential selection, in which multiple different cell populations, such as CD4+ or CD8+ cells, are selected, enriched, and / or isolated from a sample, such as a sample containing primary human T cells, in a single step flow, e.g., in a closed system. In one embodiment, a sample containing cells is subjected to selection by simultaneous enrichment of both CD4+ and CD8+ populations. In some aspects, performing separation or isolation in the same vessel or set of vessels, e.g., tubing set, is achieved by performing sequential positive and negative selection steps, the subsequent step of subjecting the negative and / or positive fractions from the previous step to further selection, where all steps are performed in the same tube or tubing set. In one embodiment, a sample containing cells to be selected is subjected to sequential selection, in which a first selection is performed to enrich one of the CD4+ or CD8+ populations, and unselected cells from the first selection are used as a source of cells for a second selection to enrich the other of the CD4+ or CD8+ populations. In some embodiments, the further selection or selections include selection of subpopulations of one or both of the CD4+ or CD8+ populations, such as central memory T (T CM ) can be performed to enrich for cells.

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

[0076] In some embodiments, multiple cell populations, e.g., CD4 + T cell populations and CD8 + The T cell population is isolated, e.g., to produce a culture starting composition containing a plurality of cell populations. The culture starting composition typically comprises, after one or more incubation, culturing, cultivation, and / or manipulation steps, a CD4 + and CD8 + The cells contain a starting culture ratio designed to result in a particular desired output ratio of two or more cell types, such as a particular ratio of 0 to 1. In some embodiments, the desired output ratio is a ratio designed to be optimal for administration of cells to a patient, e.g., in adoptive cell therapy.

[0077] In some aspects, isolating multiple populations in a single or the same isolation or separation vessel or set of vessels, such as a single column or set of columns, and / or the same tube or tubing set, or using the same separation matrix or medium or reagent, such as the same magnetic matrix, affinity labeled solid support, or antibody or other binding partner, comprises features that streamline isolation, resulting in, for example, reduced cost, time, complexity, sample handling requirements, resource, reagent, or equipment use. 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 the cell product, such as harm caused by infection, contamination, and / or temperature changes.

[0078] In some embodiments, the isolated cell population obtained for use in the methods herein is sterile. Microbial contamination of cell separation products can potentially lead to infection of recipient subjects, such as immunocompromised recipient patients who cannot fight infection. In some embodiments, the cells, cell populations, and compositions are produced under GMP (Good Manufacturing Practice) conditions. In some embodiments, GMP conditions include stringent batch testing. In certain embodiments, tissue matching is performed prior to transplantation, for example, to avoid human leukocyte antigen (HLA) mismatches and prevent problems such as graft-versus-host disease. In some embodiments, the methods provided reduce individual user handling and automate various steps, which in some aspects can increase the consistency of isolated cell populations and compositions and reduce errors, thereby promoting consistency of treatment and safety.

[0079] 1. Cells and cell populations In some embodiments, the method includes a step of selecting, isolating and / or enriching a cell sample, such as a primary human cell sample. The isolated cell population typically includes a population of cells, usually blood or blood-derived cells, such as hematopoietic cells, white blood cells (leukocytes), peripheral blood mononuclear cells (PBMCs), and / or cells of the immune system, such as myeloid or lymphoid cells, e.g. 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, such as a 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 a sample containing primary human T cells, such as CD4+ T cells and CD8+ T cells. + Cell populations and CD8 + The sample from which a plurality of T cell populations, such as a population of T cells, are isolated.Thus, in some embodiments, the isolation comprises 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, for example, negative selection for cells expressing CD14, CD19, CD56, CD20, CD11b, and / or CD16.

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

[0082] For example, T cells (e.g., CD4 +T cells or CD8 + Among the subtypes of T cells are those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, type of antigen receptor, presence of specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation.

[0083] Among the subtypes and subpopulations of T cells and / or CD4+ T cells and / or CD8+ T cells that may be enriched, isolated and / or selected are memory T cells and their subtypes, such as 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 terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, innate and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and 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 methods are cells that are positive for (marker+) or express high levels (marker high) of one or more particular markers, such as surface markers, or that are negative for (marker-) or express relatively low levels (marker low) of one or more markers. In some cases, such markers are markers that are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels on certain other populations of T cells (e.g., memory cells). In one embodiment, cells (e.g., CD8+ cells or T cells, e.g., CD3+ cells) are enriched (i.e., positively selected) for cells positive for, or expressing high surface levels of, CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, and / or are depleted (e.g., negatively selected) for cells positive for, or expressing high surface levels of, CD45RA. In some embodiments, cells are enriched or depleted for cells positive for, or expressing high surface levels of, CD122, CD95, CD25, CD27, and / or IL7-Rα (CD127). In some examples, CD8+ T cells are enriched for CD45RO positive (or CD45RA negative) and CD62L positive cells.

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

[0086] In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a monocyte or granulocyte, such as a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil.

[0087] 2. Samples Cells and cell populations are typically isolated from samples such as biological samples, e.g., samples obtained or derived from a subject, such as a subject having a particular disease or condition, or in need of cell therapy, or to whom cell therapy will be administered. In some aspects, the subject is a human, such as a patient who is in need of a particular therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, processed, and / or manipulated. 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 with viral vectors), washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, bodily fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue, and 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 (PBMC), 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 relevant to cell therapy, e.g., adoptive cell therapy, include samples from autologous and allogeneic sources.

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

[0090] 3. Cell processing, preparation, and non-affinity-based separation In some embodiments, the isolation of a cell or population includes one or more preparation steps and / or cell separation steps that are not based on affinity. In some cases, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents to, for example, remove undesired components, enrich for desired components, or lyse or remove cells that are sensitive to a particular reagent. In some cases, cells are separated based on one or more properties, such as density, adhesiveness, size, sensitivity and / or resistance to a particular component.

[0091] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some aspects, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects, contains cells other than red blood cells and platelets.

[0092] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the wash step is accomplished by 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, the cells are washed after washing, e.g., with Ca ++ / Mg ++ The cells are resuspended in a variety of biocompatible buffers, such as free PBS. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

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

[0094] 4. Isolation based on affinity and / or marker profiles In some embodiments, the isolation method comprises the separation of different cell types based on the expression or presence of one or more specific molecules in cells, such as surface markers, for example surface proteins, intracellular markers, or nucleic acids.In some embodiments, any known method for such marker-based separation can be used.In some embodiments, the separation is based on affinity or immunoaffinity.For example, the isolation in some aspects comprises the separation of cells and cell populations based on the expression or expression level of one or more markers of cells, typically cell surface markers, for example by incubating with an antibody or binding partner that specifically binds to such marker, followed by a washing step and usually the separation of the cells that bind with the antibody or binding partner from the cells that do not bind with the antibody or binding partner.

[0095] Such separation step can be based on positive selection, in which the cells that bind to the reagent are retained for further use, and / or negative selection, in which the cells that do not bind to the antibody or binding partner are retained.In some cases, both fractions are retained for further use.In some aspects, 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 does not necessarily result in the enrichment or removal of 100% of a particular cell population or cell 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 does not necessarily result in the complete absence of cells that do not express 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 does not necessarily result in the complete removal of all such cells. For example, in some aspects, the selection of one of the CD4+ or CD8+ populations enriches the population, i.e., either the CD4+ or CD8+ population, but may contain some residual or a small percentage of other unselected cells, and the unselected cells may include the other of the CD4 or CD8 population, which may still be present in the enriched population.

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

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

[0099] For example, CD3 + , CD28 + T cells can be positively selected using CD3 / CD28 conjugated 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 performed by using a marker expressed on the cells being positively or negatively selected, respectively. + ) or expressed at relatively higher levels (markers high ), is accomplished by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers.

[0101] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers, such as CD14, expressed on non-T cells, such as B cells, monocytes, or other white blood cells. In some embodiments, the method includes isolating, selecting, and / or enriching CD4+ cells and CD8+ cells. In one example, negative selection is used to isolate CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, and HLA-DR. In one example, negative selection is used to enrich for CD8 + Enriching the population is performed by depletion of cells expressing CD14 and / or CD45RA. In some aspects, positive selection for CD4 and positive selection for CD8 are used. + or CD8 + The selection stage is CD4 + Helper T cells and CD8 +The selection is used to isolate cytotoxic T cells. In some aspects such selection is performed simultaneously, in other aspects sequentially in any order.

[0102] In some aspects, the method includes a first positive selection for CD4+ cells, where the non-selected cells (CD4- cells) from the first selection 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 non-selected cells (CD8- cells) from the first selection are used as a source of cells for a second positive selection to enrich for CD4+ cells. Such CD4 + Populations and CD8 + The population can be further sorted into subpopulations by positive or negative selection for markers expressed, or expressed to a relatively greater extent, on one or more naive, memory, and / or effector T cell subpopulations.

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

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

[0105] In an embodiment, the memory T cells are CD8 + CD62L on peripheral blood lymphocytes + For example, using anti-CD8 and anti-CD62L antibodies, PBMCs are enriched in CD62L-CD8 + Fraction and / or CD62L + CD8 + Fractions can be enriched or depleted.

[0106] In some embodiments, the central memory T(T CM ) Enrichment of cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, CD27 and / or CD127; 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 methods provided include isolating, selecting and / or enriching CD8+ cells from a sample, such as by positive selection based on surface expression of CD8. In some embodiments, the methods include isolating, selecting and / or enriching CD8+ cells from a sample, such as by positive selection based on surface expression of CD8. CM In one aspect, the enriched CD8+ cells can further include enriching for central memory T (T) cells, such as one or more of CD45RO, CD62L, CCR7, CD28, CD3, CD27, and / or CD127. CM ) cells by selecting one or more markers expressed on the central memory T (T CM ) cells can be further enriched. Selection can be performed before or after isolation, selection and / or enrichment of CD4+ cells. Such selections in some aspects are performed simultaneously, and in other aspects sequentially in any order.

[0108] In some aspects, the method includes a first positive selection for CD4+ cells, where unselected cells (CD4− cells) from the first selection are used as a source of cells for a second selection to enrich for CD8+ cells, and the enriched or selected CD8+ cells are used in a third selection to enrich for central memory T (T CM In some aspects, the method further enriches for cells expressing one or more markers expressed on central memory T (T ) cells. In some aspects, the method includes a first positive selection for CD8+ cells, where the unselected cells (CD8- cells) from the first selection are used as a source of cells for a second selection to enrich for CD4+ cells, and the enriched or selected CD8+ cells from the first selection are also used in a third selection to enrich for central memory T (T ), such as by the third selection to enrich for CD45RO+, CD62L+, CCR7+, CD28+, CD3+, CD27+ and / or CD127+ cells. CM ) cells expressing one or more markers expressed on the cells are further enriched.

[0109] In some cases, T CM Enriched CD8 cells + 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, central memory T (T CM Enrichment of cells is performed beginning with a negative fraction of cells selected on the basis of expression of CD4, and the negative fraction is subjected to negative selection on the basis of expression of CD14 and CD45RA, and positive selection on the basis of CD62L. Such selections in some aspects are performed simultaneously, and in other aspects sequentially in either order. In some aspects, enrichment of cells is performed on CD8 + The selection step based on the expression of the same CD4 used to prepare the cell population or subpopulation may be performed using the CD4 + By also being used to generate cell populations or subpopulations, both the positive and negative fractions from CD4-based separation are retained, optionally after one or more further positive or negative selection steps, and used in subsequent steps of the method.

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

[0111] In some embodiments, the method of isolating, selecting and / or enriching cells, for example by positive or negative selection based on the expression of one or more cell surface markers, for example by any of the methods described above, can include immunoaffinity-based selection. In some embodiments, immunoaffinity-based selection includes 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 bound to a solid support or matrix, such as spheres or beads, for example microbeads, nanobeads, magnetic beads or paramagnetic beads, including agarose, to allow separation of cells for positive and / or negative selection. In some embodiments, the spheres or beads can be packed into a column to perform 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 then 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: SA 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 small magnetizable or magnetically responsive materials, such as magnetically responsive particles or microparticles, such as paramagnetic beads. The magnetically responsive materials, e.g., particles, are usually directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on one or more cells or populations of cells that it is desired to separate, e.g., to negatively or positively select. Such beads are known and, in some aspects, are commercially available from a variety of 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 magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner.There are many known magnetically responsive materials that are used in magnetic separation methods.Suitable magnetic particles include those described in U.S. Patent No. 4,452,773 to Molday and European Patent Specification EP452342B, which are incorporated herein by reference.Colloidal-sized particles, such as those described in U.S. Patent No. 4,795,698 to Owen and U.S. Patent No. 5,200,084 to Liberti et al., are other examples.

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

[0116] In some aspects, the sample is placed in a magnetic field, and the cells that are bound with magnetically responsive or magnetizable particles are attracted to the magnet and separated from unlabeled cells.For positive selection, the cells that are attracted to the magnet are retained; for negative selection, the cells that are not attracted (unlabeled cells) are retained.In some aspects, a combination of positive selection and negative selection is performed during the same selection step, where positive and negative fractions are retained and further processed or subjected to further separation steps.

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

[0118] In some embodiments, the magnetically responsive particles are left attached to the cells to be subsequently incubated, cultured and / or manipulated; in some aspects, the particles are left attached to the cells for administration to the patient. In some embodiments, the magnetizable particles or the magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known, and include, for example, the use of competitive unlabeled antibodies, magnetizable particles, or antibodies conjugated with cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0119] In some embodiments, the affinity-based selection is magnetic activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). The magnetic activated cell sorting (MACS) system allows for high purity selection of cells bound to magnetized particles. In certain embodiments, MACS operates in a manner in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells bound to magnetized particles are kept in place, and unbound species are eluted. Then, after this first elution step is completed, species trapped in the magnetic field and prevented from eluting are released in a manner that allows them to be eluted and collected. In certain embodiments, non-target cells are labeled and depleted from the heterogeneous cell population.

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

[0121] In some embodiments, the streptavidin variants exhibit higher binding affinity for a peptide ligand comprising an amino acid sequence as set forth in any of SEQ ID NOs:1-6, such as, for example, SEQ ID NO:5 and / or SEQ ID NO:6 (e.g., Strep-tag II®), than unmodified or wild-type streptavidin, such as unmodified or wild-type streptavidin as set forth in SEQ ID NO:11 or SEQ ID NO:14. In some embodiments, the streptavidin variants exhibit binding affinity, as an affinity constant, for such peptides 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] A streptavidin mutein contains one or more amino acid differences compared to unmodified streptavidin, such as wild-type streptavidin or a fragment thereof. The term "unmodified streptavidin" refers to a starting polypeptide to which one or more modifications are made. In some embodiments, the starting or unmodified polypeptide can be the wild-type polypeptide set forth in SEQ ID NO:11. In some embodiments, unmodified streptavidin is a fragment of wild-type streptavidin that is truncated at the N-terminus and / or C-terminus. Such minimal streptavidins include any that begin at the N-terminus in the region of amino acid positions 10-16 of SEQ ID NO:11 and end at the C-terminus in the region of amino acid positions 133-142 of SEQ ID NO:11. In some embodiments, unmodified streptavidin has the amino acid sequence set forth in SEQ ID NO:14. In some embodiments, unmodified streptavidin as shown in SEQ ID NO:14 can further include an N-terminal methionine at a position corresponding to Ala13 in the numbering shown in SEQ ID NO:11. References to residue numbers in streptavidin provided herein refer to the residue numbering in SEQ ID NO:11.

[0123] The term "streptavidin mutein", "streptavidin mutant" or variations thereof refers to a streptavidin protein that contains one or more amino acid differences compared to unmodified or wild-type streptavidin, such as the streptavidin 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 one or more amino acid exchanges (substitutions), insertions or deletions. In some embodiments, the streptavidin mutein 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 exchanges (substitutions) are conservative or non-conservative mutations. Streptavidin muteins containing one or more amino acid differences showed a 2.7×10 binding affinity to the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also called Strep-tag® as shown in SEQ ID NO:5). 4 M -1 In some embodiments, the streptavidin variant exhibits binding affinity as an affinity constant greater than 1.4×10 for the peptide ligand (Trp Ser His Pro Gln Phe Glu Lys; also known as Strep-tag® II as shown in SEQ ID NO:6). 4 M -1 The binding affinity is expressed as an affinity constant greater than 0.05. In some aspects, the binding affinity can be determined by methods known in the art, such as any of the following:

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

[0125] In some embodiments, the streptavidin mutein is a variant described in International Publication PCT Application WO2014 / 076277. In some embodiments, the streptavidin mutein comprises at least two cysteine ​​residues in the region of amino acid positions 44-53 relative to the amino acid positions shown in SEQ ID NO:11. In some embodiments, cysteine ​​residues are present at positions 45 and 52 creating a disulfide bridge linking these amino acids. In one such 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 mutein comprises at least one mutation or amino acid difference in the region of amino acid residues 115-121 relative to the amino acid positions shown in SEQ ID NO:11. In some embodiments, the streptavidin mutein comprises at least one mutation at amino acid positions 117, 120, and 121 and / or a deletion of amino acids 118 and 119 and a substitution at least at amino acid position 121.

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

[0127] In some embodiments, the binding affinity of the streptavidin mutant for a peptide ligand binding reagent is greater than or equal to 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 generally around 1×10 13 M -1 , 1×10 12 M -1 or 1×10 11 M -1 is less than.

[0128] In some embodiments, the streptavidin mutants also exhibit binding to other streptavidin ligands, such as, but not limited to, biotin, iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, HABA (hydroxyazobenzene-benzoic acid) or / and dimethyl-HABA. In some embodiments, the streptavidin mutein exhibits a binding affinity for another streptavidin ligand, such as biotin or desthiobiotin, that is greater than the binding affinity of the streptavidin mutein for the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also referred to as Strep-tag® as shown in SEQ ID NO:5) or the peptide ligand (Trp Ser His Pro Gln Phe Glu Lys; also referred to as Strep-tag® II as shown in SEQ ID NO:6).

[0129] In some embodiments, the streptavidin mutant protein is a multimer. The multimer can be generated using any method known in the art, such as any of those described in US Patent Application Publication No. 2004 / 0082012. In some embodiments, the mutant protein oligomer or polymer can be prepared by introducing carboxyl residues into polysaccharides, such as dextran. In some aspects, the streptavidin mutant protein is then coupled in a second step to the carboxyl groups in the dextran backbone via the primary amino groups of internal lysine residues and / or the free N-terminus, using conventional carbodiimide chemistry. 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 glutaric dialdehyde, or by other methods known in the art.

[0130] In some aspects, immunoaffinity beads, such as Streptamers or other immunoaffinity beads, can include 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 include one or more mutations within the framework of the heavy and light chain variable regions without targeting the hypervariable CDR regions. Examples of such antibodies include, in some aspects, the anti-CD4 antibodies described in U.S. Pat. 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 with a streptavidin binding molecule, which 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, antibodies that specifically bind to cell surface markers associated with or coated on beads or other surfaces are full length antibodies, or can be (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, variable heavy chains (V) capable of specifically binding to an antigen. H) regions, single chain antibody fragments including single chain variable fragments (scFv), and 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, the antibody, such as a Fab, is a multimer. In some embodiments, the antibody, such as a Fab multimer, forms a multivalent complex with a cell surface marker.

[0132] In some embodiments, antibodies, such as Fabs related to Streptamers, are characterized by a particular kinetic measure of binding affinity (e.g., the dissociation constant K D , coupling constant K A , dissociation rate (off-rate) or other kinetic parameters of binding affinity. Such a measure can be determined using any binding assay known to the artisan. In certain examples, affinity-based biosensor techniques are utilized as a measure of 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, binding affinity is determined by fluorescence titration or titration calorimetry.

[0133] In some embodiments, an antibody, such as a Fab, has a specific binding affinity of about 0.5×10 for binding to a cell surface marker on a cell. -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 Greater than or equal to k 0ff The dissociation rate (also called the dissociation rate constant) of each k 0ff The rate can determine the rate at which an antibody reagent can dissociate from its interaction with a cell via binding to a cell surface marker (see, e.g., International Publication PCT Application WO / 2013011011). For example, in some aspects, k 0ff The range can be chosen depending on the particular application or use of the selected or enriched cells, including factors such as the desire to remove bound antibody from the cell surface, the time the cells are cultured or incubated, and the sensitivity of the cells and other factors. In one embodiment, the antibody is administered at a concentration of, for example, 4.0×10 -4 sec -1 Higher than 0ff kinetics, so that after disruption of the multivalent binding complex, most of the antibody can be removed within 1 hour, since in some aspects considering the half-life T of the complex, the concentration of the complex decreases to 25% of the initial concentration within 56 minutes, assuming that the effect of rebinding can be neglected by sufficient dilution). In another embodiment, a lower k0ff rate, e.g., 1.0×10 -4 sec -1 For antibodies having the formula: ##STR00013## dissociation may take longer, for example about or approximately 212 minutes or about three 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 binding affinity of about 10 to a cell surface marker on a cell. -2 M~about 10 -8 M, or about 10 -2 M~about 10 -9 M, or about 10 -2 M ~ approx. 0.8×10 -9 M, or about 10 -2 M ~ approx. 0.6×10 -9 M, or about 10 -2 M ~ approx. 0.4×10 -9 M, or about 10 -2 M ~ approx. 0.3×10 -9 M, or about 10 -2 M ~ approx. 0.2×10 -9 , or about 10 -2 M ~ approx. 0.15×10 -9 M, or about 10 -2 ~about 10 -10 In some embodiments, the dissociation constant (Ka) for binding to a cell surface marker on a cell is in the range of about 10 -7 M~about 10 -10 M, or about 10 -7 M ~ approx. 0.8×10 -9 M, or about 10 -7 M ~ approx. 0.6×10 -9 M, about 10 -7 M ~ approx. 0.3×10 -9 M, 1.1×10 -7 M ~ about 10 -10 M, or approximately 1.1 x 10 -7 M ~ approx. 0.15×10 -9 M, or approximately 1.1 x 10 -7 M ~ approx. 0.3×10 -9 M, or approximately 1.1 x 10 -7 M ~ approx. 0.6×10 -9 M, or approximately 1.1 x 10 -7 M ~ approx. 0.8×10 -9 The range may be M.

[0135] In some embodiments, an immunoaffinity reagent, such as an antibody, e.g., a Fab, is directly or indirectly linked to a peptide ligand, such as a peptide ligand capable of binding to a streptavidin mutant (see, e.g., U.S. Pat. No. 5,506,121). In some embodiments, such a peptide comprises an amino acid sequence set forth in any of SEQ ID NOs:1-6. In some embodiments, an immunoaffinity reagent, such as an antibody, e.g., a Fab, is directly or indirectly linked to a peptide ligand comprising an amino acid sequence set forth in SEQ ID NO:6.

[0136] In some embodiments, an immunoaffinity reagent, such as an antibody, e.g., a Fab, is fused directly or indirectly to a peptide sequence comprising a consecutive arrangement of at least two streptavidin binding modules, where the distance between the two modules is at least 0 and no more than 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 same or a different streptavidin peptide ligand sequence 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, such as an antibody, e.g., a Fab, fused directly or indirectly to the immunoaffinity reagent comprises a sequence having a formula as shown in either SEQ ID NO:7 or 8. In some embodiments, the peptide ligand has an amino acid sequence set forth in any of SEQ ID NOs:9, 10 or 17-19.

[0137] Alternatively, other streptavidin-binding peptides known in the art can be used, for example as described in 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 the streptavidin variant is contacted with a streptavidin variant containing bead to coat the bead 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 mutant protein binding reagent is reversible. In some embodiments, the binding between the peptide ligand binding partner and the streptavidin mutant protein binding reagent is high as described above, but is 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 binding and disrupt the binding interaction between the streptavidin mutant protein binding reagent on the bead and the peptide ligand binding partner associated with the antibody that is specifically bound to the cell marker on the surface. In some embodiments, the interaction can be reversed in the presence of low concentrations of biotin or analog, for example 0.1 mM to 10 mM, 0.5 mM to 5 mM, or 1 mM to 3 mM, for example generally at least or at least about 1 mM or at least 2 mM, for example 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, in some aspects, comprises one or more chromatography matrices described in US Patent Application Publication No. 2015 / 0024411. In some embodiments, the chromatography method is fluid chromatography, typically liquid chromatography. In some embodiments, the chromatography can be performed in a flow-through mode, where a fluid sample containing cells to be isolated is added, for example, by gravity flow or by a pump to one end of a column containing a chromatography matrix, where the fluid sample exits the column at the other end of the column. In addition, in some aspects, the chromatography can be performed in an "up-down" mode, where a fluid sample containing cells to be isolated is added, for example, by a pipette to one end of a column containing a chromatography matrix packed in a pipette tip, where the fluid sample enters the chromatography matrix / pipette tip and exits the other end of the column. In some embodiments, chromatography can also be performed in batch mode, where the chromatographic material (stationary phase) is incubated with the cell-containing sample, e.g., with shaking, rotation or repeated contact, and the fluid sample is removed, e.g., by pipette.

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

[0142] In some embodiments, the chromatography matrix is ​​made from synthetic polymers such as polyacrylamide, styrene-divinylbenzene gels, acrylic acid / diol or acrylamide / diol copolymers, polysaccharide / agarose copolymers such as polyacrylamide / agarose composites, polysaccharides and N,N'-methylenebisacrylamide, or derivatized silica coupled with synthetic or natural polymers.

[0143] In some embodiments, the 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 more. The exclusion limit of the size-exclusion chromatography matrix is ​​selected to be less than the maximum width of the target cells, e.g., T cells, in the sample. 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 into 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, may in some aspects be any of the antibodies known in the art, specific k 0ff The antibody may be any of the above, including an antibody with a particular rate and / or an antibody with a particular 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 the binding reagent immobilized on the matrix. In some embodiments, the binding capacity of the chromatography matrix is ​​sufficient to adsorb or can adsorb 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 that express a cell surface marker that is specifically recognized by the affinity reagent, such as an antibody or a Fab.

[0146] In some aspects, 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 aspects, 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 antigen-binding fragment such as a Fab, is via an interaction between a peptide ligand binding reagent and a streptavidin mutant protein, as described above with respect to immunoaffinity beads. In the context of a chromatography matrix, a matrix, such as an agarose bead or other matrix, is functionalized or conjugated with a streptavidin mutant protein, such as any of those described above, e.g., any of those shown in SEQ ID NOs: 12, 13, 15, or 16. In some embodiments, an antibody or 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 those described above. In some embodiments, the peptide ligand is any of those described above, such as a peptide comprising an amino acid sequence shown in any of SEQ ID NOs: 1-10 or 17-19. In some embodiments, a chromatography matrix column is contacted with such an affinity reagent, such as an antibody or 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 is mediated by the interaction of a peptide ligand with a streptavidin mutant, and the selected cells can be released due to the presence of a 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 is lower than the binding affinity of the streptavidin binding reagent to biotin or a biotin analog. Thus, in some embodiments, biotin (vitamin H) or a biotin analog can be 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 that specifically binds to the cell marker on the surface. In some embodiments, the interaction can be reversed in the presence of low concentrations of biotin or analogs, such as in the presence of 0.1 mM to 10 mM, 0.5 mM to 5 mM, or 1 mM to 3 mM, typically at least or at least about 1 mM or at least 2 mM, e.g., 2.5 mM or about 2.5 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 methods is performed using at least two operably connected chromatography matrix columns, where an affinity or binding agent for one of CD4 or CD8, such as an antibody, e.g., a Fab, is coupled to a first chromatography matrix in a first selection column, and an affinity or binding agent for the other of CD4 or CD8, such as an antibody, e.g., a Fab, is coupled to a second chromatography matrix in a second selection column. In some embodiments, the at least two chromatography matrix columns are present in a closed system or device, e.g., a sterile closed system or device.

[0150] In some embodiments, a closed system or device is also provided herein that includes at least two operably connected chromatography matrix columns, where an affinity or binding agent for one of CD4 or CD8, such as an antibody, e.g., a Fab, is coupled to a first chromatography matrix in a first selection column, and an affinity or binding agent for the other of CD4 or CD8, such as an antibody, e.g., a Fab, is coupled to a second chromatography matrix in a second selection column. Examples of such systems and methods are shown in Figures 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 may include an integrated microcomputer, a peristaltic pump, and various valves, such as pinch valves, or stopcocks, to control the flow of fluids between various parts of the system. The integrated computer in some aspects controls all 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 of the entire tubing set and, together with the pinch valves, ensures a controlled flow of buffer through the system.

[0152] 1A and 1B, in some embodiments, a first affinity matrix 3 comprising a first affinity agent or binding agent in a first selection column 1 is i) operably connected to a second affinity matrix 4 comprising a second affinity agent or binding agent in a second selection column 2 via tubing and valve 13 so that cells that have passed through the first affinity chromatography matrix and are not bound to the first affinity agent or binding agent thereon can enter into the second affinity matrix 4, and ii) operably connected to an output container, such as a culture vessel 12, for collecting selected cells that are bound to the first affinity agent or binding agent on the first affinity matrix from the first affinity matrix, e.g., after elution and release of such cells from the first affinity matrix. In some embodiments, a second affinity matrix 4 containing a second affinity or binding agent in the second selection column 2 is also operably connected to an output vessel, such as a culture vessel 12, for collecting selected cells bound to the second affinity or binding agent thereon from the second affinity matrix, e.g., after elution and release of such cells from the second affinity matrix. -10 M -1 A second selection column 2 is operably connected to an output vessel, such as a culture vessel 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 100 mM.

[0153] In some embodiments, the size, e.g., length and / or diameter, of the first selection column 1 and the second selection column 2 can be the same or different. In some embodiments, the size, e.g., length and / or diameter, of one of the first column 1 or the second column 2 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, the first affinity matrix 3 containing the first affinity agent or binding agent in the first selection column 1 is also operably connected via tubing and valve 13 to a third affinity matrix 17 containing a third affinity agent or binding agent in a third selection column 15, such that selected cells bound to the first affinity agent or binding agent on the first affinity matrix can enter the third affinity matrix, e.g., after such cells are eluted and released from the first affinity matrix. In some embodiments, an elution reagent such as biotin is added to the third affinity matrix 17, and the third affinity matrix 17 is operably connected to the first affinity agent or binding agent in the first selection column 15. -10 M -1 The first selection column 1 is operably connected to a third selection column 15 via a removal chamber 9 containing a binding reagent, such as a streptavidin variant, capable of binding with high affinity, such as greater than 100 mM. In some embodiments, a third affinity matrix 17 containing a third affinity agent or binding agent in the third selection column 15 is also operably connected to an output vessel, such as a culture vessel 12, for collecting selected cells bound to the third affinity agent or binding agent on the third affinity matrix (and previously bound to the first affinity agent or binding agent) from the third affinity matrix, e.g., after elution and release of such cells from the third affinity matrix (and previously from the first affinity matrix). In some embodiments, a third affinity matrix 17 containing a third affinity agent or binding agent on the third affinity matrix (and previously bound to the first affinity matrix) is also operably connected to an output vessel, such as a culture vessel 12, for collecting selected cells from the third affinity matrix, e.g., after elution and release of such cells from the third affinity matrix (and previously from the first affinity matrix). -10 M -1 A third selection column 15 is operably connected to an output vessel, such as culture vessel 12, via a removal chamber 9 containing a binding reagent, such as a streptavidin mutant, capable of binding with high affinity, such as greater than 100 mM.

[0155] In some embodiments, the size, e.g., length and / or diameter, of the first selection column 1 and the third selection column 15 can be the same or different. In some embodiments, the size, e.g., length and / or diameter, of one of the first column 1 or the third column 15 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 a cell sample 5, e.g., via tubing, a valve, 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, e.g., via tubing and valves, to a wash reservoir 6 containing a wash buffer and / or an elution reservoir 7 containing an elution solution, respectively, for passing a wash buffer or an elution buffer to the first chromatography matrix in the first selection column. In some embodiments, due to an operable connection between the first and second selection columns, the wash buffer and / or the elution buffer can operably enter into the second column. In some embodiments, due to an operable connection between the first selection column and the third selection column, the wash buffer and / or the elution buffer can operably enter into the third selection column.

[0158] In some embodiments, the second selection column 2 is also operably connected, e.g., via tubing and valves, to a wash reservoir 6 containing a wash buffer and / or an elution reservoir 7 containing an elution solution, for passing the wash buffer or elution buffer, respectively, to the first chromatography matrix in the first selection column.

[0159] The wash buffer can be any physiological buffer compatible with cells, such as phosphate buffered saline. In some embodiments, the wash buffer contains bovine serum albumin, human serum albumin, or recombinant human serum albumin, for example at a concentration of 0.1% to 5% or 0.2% to 1%, for example at 0.5% or about 0.5%. In some embodiments, the eluent is, 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 biotin or a biotin analogue, such as desbiotin.

[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., Fab reservoir) 18 that contains a first affinity agent or binding agent, such as an antibody, e.g., a Fab, for immobilization onto the first affinity matrix. 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 binding agent reservoir (e.g., Fab reservoir) 19 that contains a second affinity agent or binding agent, such as an antibody, e.g., a Fab, for immobilization onto the second affinity matrix. 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 binding agent reservoir (e.g., a Fab reservoir) 20 that contains a third affinity agent or binding agent, such as an antibody, e.g., a Fab, for immobilization onto the second affinity matrix.

[0161] In some embodiments, the first and / or second affinity reagent specifically binds 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 specifically binds to a marker that is CD45RO, CD62L, CCR7, CD28, CD3, CD27, and / or CD127.

[0162] 5. Enrichment and Ratio of the Resulting Compositions In some embodiments, the first and second selections using the above method enrich the sample with a first population of cells expressing a first cell surface marker and a second population of cells expressing a second cell surface marker, respectively. In a particular example, the first and / or second population of enriched cells can be a population of cells 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 performed to enrich further cell subpopulations, such as a subpopulation of CD4+ cells and / or a subpopulation of CD8+ cells, from the population of cells previously enriched in the first, second or subsequent enrichment.

[0163] In some embodiments, the method produces an enriched cell composition containing a first and a second population 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 culture starter composition and is used for 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, an output composition is generated that can contain engineered cells, including CD4+ cells and CD8+ cells expressing an engineered antigen receptor.

[0164] In some embodiments, the enriched cell composition is enriched cells from a starting sample as described above, where the number of cells in the starting sample is at least as high as the desired number of cells in the enriched composition, such as the culture starter composition. 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 higher 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 pieces, 2×10 6 pieces, 4×10 6 pieces, 6×10 6 pieces, 8×10 6 pieces, 1×10 7 pieces, 2×10 7 pieces, 4×10 7 pieces, 6×10 7 pieces, 8×10 7 pieces, 1×10 8 pieces, 2×10 8 pieces, 4×10 8 pieces, 6×10 8 pieces, 8×10 8 pieces, 1×10 9 In some embodiments, the number of cells in the starting sample is at least 1 x 10 8 pieces, 5×10 8 pieces, 1×10 9 pieces, 2×10 9 pieces, 3×10 9 pieces, 4×10 9 pieces, 5×10 9 pieces, 6×10 9 cells, 7 x 10 9 pieces, 8×10 9 pieces, 9×10 9 pieces, 1×10 10 One or more.

[0165] In some embodiments, the yield of the first and / or second population or subpopulation thereof 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 subpopulation in the starting sample, is between 10% and 100%, e.g., between 20% and 80%, between 20% and 60%, between 20% and 40%, between 40% and 80%, between 40% and 60%, or between 60% and 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 cell subpopulations thereof in the enriched composition, i.e., the percentage of cells positive for a select cell surface marker relative to the total 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, the enriched cell composition, such as the culture start composition, comprises a ratio of CD4+ cells to CD8+ cells at a culture start ratio. The culture start ratio is the ratio or number of cells in the culture start composition designed to produce a desired output ratio or dose, such as a ratio or dose for administration to a patient, or within a margin of error or tolerance, at the completion of incubation and / or manipulation or other processing steps, and / or at the time of thawing, and / or immediately before administration to a subject. In embodiments of the methods provided herein, the first and / or second selection, or the selection for the subpopulation, can be performed in a manner that produces a selected culture start ratio. Examples of such methods are described below and in the Examples.

[0168] a. Culture starting ratio and number In some embodiments, CD4 + or a subpopulation and CD8 +The starting ratio of CD4+ cells or subpopulations thereof to CD8+ cells or subpopulations thereof is between at or about 10:1 and 1:10 or about 1:10, between at or about 5:1 and 1:5 or about 1:5, or between at or about 2:1 and 1:2 or about 1:2. In some embodiments, the starting ratio of CD4+ cells or subpopulations thereof to CD8+ cells or subpopulations thereof is 1:1 or about 1:1.

[0169] In some embodiments, the starting ratio of CD4+ to CD8+ cells, or subpopulations thereof, is different from the ratio of CD4+ 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 between 1:1 and 14:1 CD4+ cells:CD8+ cells, and is generally reported to be between about 1.5:1 and about 2.5:1 CD4+ cells:CD8+ cells. In some embodiments, the ratio of CD4+ T cells to CD8+ T cells in a sample, such as a blood sample, is about 2:1 CD4+ cells:CD8+ cells. 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, e.g., 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 subject ratio is less than 1:1 CD4+ cells:CD8+ cells (see, e.g., Muhonen, T. J Immunother Emphasis Tumor Immunol. 1994 Jan;15(1):67-73). In some embodiments, the starting culture ratio of CD4+ cells to 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 the subject.

[0170] In some embodiments, the ratio of CD4+ T cells to CD8+ T cells in a sample from a subject is determined before performing the first and / or second selection. Based on the specific ratio of CD4+ T cells to CD8+ T cells in a subject (which may vary between subjects), a specific selection mode can be individualized for a subject, for example, by sizing a chromatography column or selecting the amount or concentration of immunoaffinity reagents, to achieve a desired or chosen culture starting ratio. The relative levels or frequencies of various cell populations in a subject can be determined based on evaluating the surface expression of one or more markers present in such populations or subpopulations. Several well-known methods for evaluating 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, in the context of cell surface proteins, for example, by flow cytometry.

[0171] In some circumstances, the appropriate culture starting ratio for a particular cell type may depend, for example, on the particular disease, condition, or prior treatment of the subject from which the cells are derived, and / or the particular antigen specificity of the cells, the specific type of cells (e.g., CD8 + The relative expression between T cells and T cells) and / or the situation of one or more conditions that cells are incubated under, such as medium, stimulant, incubation time, buffer, oxygen content, carbon dioxide content, antigen, cytokine, antibody, and other components.Therefore, a cell type that is typically or generally known to grow or increase more rapidly than another cell type may not always have such a property in all situations.Therefore, in some aspects, the culture starting ratio is determined based on the known ability of the cell type in normal or typical situations, combined with the evaluation and / or empirical basis of the phenotype or condition of the cell or the subject from which the cell is derived.

[0172] In some embodiments, the culture initiation 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. + Cells and CD8 + The ratio of CD4 + and CD8 + and a desired output ratio of 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 times greater or less than the desired output ratio.

[0173] In some embodiments, for example, CD4+ cells are known to proliferate or expand to a lesser extent or at a slower rate than CD8+ cells when incubated under certain stimulatory conditions, depending on the circumstances. See, e.g., 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 ahead of print March 17, 2014, doi: 10.4049 / jimmunol.1302725. Thus, in some instances, CD4+ cells in the culture initiation composition may be used as a marker for the proliferation or proliferation of CD8+ cells. + Cells and CD8 + The ratio of CD4 / CD8 cells is 10-fold or 100-fold the desired output ratio. For example, if a 1:1 (or 50% / 50%) CD4 / CD8 output ratio is desired, then CD4 + Populations and CD8 +The populations, in one example, can be included in a culture starting composition in 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 to account for differences in expansion rates over a particular period of time. Depending on the differences in the rate or expansion or proliferation of one of the CD4+ or CD8+ cells, or subpopulations thereof, compared to the other, the artisan can empirically determine the culture starting ratio to achieve the desired output ratio according to the particular stimulation or activation conditions.

[0174] The starting ratio of the culture is not necessarily the same as or even close to the desired output ratio. For example, the CD4 + and CD8 + If it is desired to administer engineered (e.g., CAR-expressing) T cells, CD4 + and CD8 + The starting culture ratio of cells is often not 1: 1. In some embodiments, the starting culture ratio that results in a desired output ratio will vary 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, the subject or subjects from whom the cells were isolated or derived, the disease to be treated, such as what disease or condition such subject has, the culture conditions, and other parameters.

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

[0176] In another aspect, in some embodiments, the method further comprises a step of determining the culture initiation ratio or number. In some embodiments, the provided methods include methods and steps for determining the appropriate ratios, doses, and numbers of cells, cell types, and cell populations. For example, CD4 + / CD8 +Methods are provided for determining the ratio of cells, populations, and / or subpopulations, and for determining the appropriate dose 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 culture starter composition, to achieve a desired result.In some aspects, such ratios or numbers are designed for use in incubation or manipulation steps to achieve a desired output ratio or dose.In some embodiments, methods are provided for determining the desired ratio of cells, types, or populations, and / or the cell numbers thereof, for administration to a subject or patient.

[0177] In some embodiments, the selected culture initiation ratios are determined so that the different cell types or populations exhibit a high chromatin density in culture when incubated under such conditions, with each cell population or cell type (e.g., CD4 + Cells vs. CD8 + The relative ability of the cells (e.g., cells) to survive and / or grow or expand is therefore determined in some aspects. Thus, the growth rate, survival, and / or output ratio is measured or evaluated at one or more specific time points, e.g., at the bedside, after a test incubation, e.g., after incubation, and / or after a cryopreservation or freezing step, and / or after thawing after such a procedure, immediately before administration, to determine the optimal ratio for starting the 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 similar fluorescent dyes, followed by incubation, followed by evaluating fluorescence intensity by flow cytometry, and / or evaluating binding of cells to Annexin V or other compounds that recognize markers on or in the appropriate cells and / or uptake of DNA-reciprocal chelators such as propidium iodide or 7AAD, and evaluating uptake and / or cell cycle stage as a measure of proliferation or apoptosis by flow cytometry.

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

[0179] Exemplary test ratios for the test culture initiation composition can include 90% / 10%, 80% / 20%, 70% / 30%, 60 / 40%, 50 / 50%, 40 / 60%, 30 / 70%, 20% / 80%, and 10% / 90% or about ratios thereof, or 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 than 1:1.5 or about 1:1.5.

[0180] In some situations, CD4 to CD8 ratios are used to achieve the desired CD4:CD8 ratio at the end of production. + Cells vs. CD8 + The appropriate culture initiation ratio of cells depends on the CD4+ / CD5+ / CD6+ expression in a particular isolated cell product, such as the presence and / or percentage of naive, effector, and various memory compartments represented in a particular isolated composition. + Fraction and / or CD8 +Such assessment can be by determining the presence or levels of various surface markers on the cells, for example, by flow cytometry.

[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 the culture conditions (e.g., medium composition, the presence and / or absence of growth factors, stimulants, and / or other agents, temperature, aeration conditions, etc.).

[0182] In some embodiments of the methods described herein, the initiation ratio is CD4 + :CD8 + An output composition is produced that includes a ratio of cells or a number of such subtypes or a total number of cells that 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 ranges between these values). + Cell: CD8 + The output composition has a desired ratio or dose of 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 ranges between these values). In certain embodiments of the method, the culture starting ratio produces CD4 + Cells vs. CD8 + In some embodiments of the methods described herein, the output composition produces a ratio of CD4 cells that is within a tolerance of the desired output ratio. + Cells and CD8 + The ratio of CD4 cells to CD4+ cells is included, and the tolerance is as above. + Cells and CD8 + This was determined by administering cells to one or more subjects in multiple ratios.

[0183] b. Output ratio and dose In some embodiments, the method produces an output composition after one or more processing steps of the culture initiation composition, such as incubation, stimulation, activation, manipulation and / or formulation of the cells. In some embodiments, the method produces an output composition after one or more processing steps of the culture initiation composition, such as incubation, stimulation, activation, manipulation and / or formulation of the cells. In some embodiments, the method produces an output composition after one or more processing steps of the culture initiation composition, such as incubation, stimulation, activation, manipulation and / or formulation of the cells. In some embodiments, the method produces an output composition after one or more processing steps of the culture initiation composition, such as incubation, stimulation, activation, manipulation and / or formulation of the cells. In some embodiments, the method produces an output composition after one or more processing steps of the culture initiation composition, such as incubation, stimulation, activation, manipulation and / or formulation of the cells. + Cells and CD8 + In some embodiments, the ratio of CD4 + Cells and CD8 + The desired output ratio to cells is 1:1 or about 1:1.

[0184] In some embodiments, the method produces or generates an output composition, such as a composition containing 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. + Cells and CD8 + An output composition is produced or generated that contains the output ratio with the cells.

[0185] In some embodiments, the output ratio of CD4+ T cells to CD8+ T cells is the ratio desired as part of an immunotherapeutic T cell dosage, e.g., in the context of adoptive immunotherapy.

[0186] In some embodiments, a desired cell number of a cell type or population and / or a desired dosage, such as a desired output ratio (e.g., an optimal ratio for therapeutic administration to a patient), is determined. In some embodiments, the method includes a step for determining an allowable error or tolerance from the desired output ratio or administration ratio or dose, i.e., a margin of error within which the ratio in a given composition, e.g., an engineered composition, may vary from the desired output ratio, but still achieve a desired result, such as an acceptable degree of safety in a subject or patient, or efficacy in treating a particular disease or condition, or other therapeutic effect.

[0187] In some embodiments, the desired dose, ratio and / or tolerance depends on the disease or condition to be treated, the subject, the source of the cells, e.g., whether the cells are from a subject with 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 also going to receive the cells in adoptive cell therapy, and / or whether the subject has undergone or has undergone another treatment and / or the same of such treatment. In some embodiments, the ratio, number and / or tolerance or difference depends on one or more other properties of the cells, such as proliferation rate, viability, expression of a particular marker or secretion of a factor such as a cytokine, or the particular subpopulation isolated before the incubation and manipulation steps. In some examples, the desired ratio and / or tolerance or difference may vary depending on the age, sex, health and / or weight of the subject, biomarkers as indicators of disease traits, treatments to be administered to the subject simultaneously or previously administered.

[0188] In some embodiments, the desired ratios, doses, and / or tolerances are determined by administering to a test subject various test compositions, each containing different ratios or different numbers of the cell types or populations of interest, and subsequently evaluating one or more results or parameters, such as parameters indicative of safety, therapeutic efficacy, in vivo concentration or localization of cells, and / or other desired results.

[0189] In some embodiments, the test subject is a non-human animal, such as a normal animal or an animal model of a disease, such as a disease or condition to be treated by administration of cells. In some embodiments, the various test ratios for administration to the test subject are, for example, a single cell type or subtype (e.g., CD4 + T cells or subtypes) and another cell type (e.g., CD8 + T cells or NK cells or subtypes thereof), and expressed as provisional values, 90% / 10%, 80% / 20%, 70% / 30%, 60 / 40%, 50 / 50%, 40 / 60%, 30 / 70%, 20% / 80%, and 10% / 90%, or approximately therein. The ratio can be expressed as a relative percentage or in any other format, such as a ratio of 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 about, or more than 1:1.5 or more than about 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 about, or more than (including any range between these values). In some embodiments, the test subject is a human. In some embodiments, the desired ratio is the average, mean, or median ratio between test subjects that has a particular optimal effect. In some aspects, the desired ratio is the ratio that achieves an optimal balance between safety and efficacy. In some aspects, the desired ratio or dose is one that achieves the highest efficacy of all tested ratios or doses, yet still maintains a threshold of safety. In some aspects, the desired ratio or dose is one that achieves the highest degree of safety, while maintaining a threshold of efficacy or within the range of efficacy. In some cases, the optimal ratio or dose is between 1:1 and 1:2 of one cell type to another, or 10 per kg of body weight. 4 From 10 9 Between pieces or 10 5 From 10 6 It is expressed as a range such as between individuals.

[0190] In some embodiments, the margin of error is determined based on the deviation from the desired average test subject monitored to assess the therapeutic efficacy and / or safety of each percentage combination, hi some embodiments, the margin of error 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 values ​​between these ranges).

[0191] 6. Exemplary Methods for Selecting or Enriching Cells a. Single step flow 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+ cells and CD8+ cells, such as a primary human T cell sample, is contacted with magnetic beads that contain a first immunoaffinity reagent that binds to CD4 or CD8 and 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, the contacting of the cells with the magnetic beads is performed simultaneously, with simultaneous enrichment of cells comprising the surface markers CD4 and CD8. In some such aspects, the method includes contacting cells of a sample containing primary human T cells with a first immunoaffinity reagent that specifically binds CD4 and a second immunoaffinity reagent that specifically binds CD8 in an incubation composition under conditions in which the immunoaffinity reagents specifically bind to CD4 and CD8 molecules, respectively, on the surface of cells in the sample, and recovering cells that are bound to the first and / or second immunoaffinity reagent, thereby generating an enriched composition comprising a starting ratio of CD4+ and CD8+ cells.

[0193] In some embodiments, the first and / or second immunoaffinity reagents are present in the incubation composition at a suboptimal yield concentration, where the enriched composition contains 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, thereby producing a composition enriched in CD4+ T cells and CD8+ T cells.

[0194] In some embodiments, a suboptimal yield concentration of an affinity reagent is a concentration less than the concentration used or required to achieve an optimal or maximum yield of bound cells in a given selection or enrichment involving incubating cells with the reagent and recovering or separating the cells bound to the reagent (where "yield" refers, for example, to the number of such recovered or selected cells that are targeted by the reagent or that bear a marker for which the reagent is specific or to which the reagent is specific and can bind, compared to the total number of cells in the incubation). A suboptimal yield concentration is generally a concentration or amount of reagent that achieves a yield of 70% or less of bound cells upon recovery of the cells bound to the reagent in such a step or stage. In some embodiments, a yield of 50%, 45%, 40%, 30%, or 25% or less or about that value is achieved by a suboptimal concentration. The concentration can be expressed in terms of the number or mass of particles or surfaces per cell and / or the number of mass or molecules of agent (e.g., antibody, such as antibody fragment) per cell.

[0195] For example, in some embodiments, a suboptimal yield concentration is 1×10 cells in an incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9In some embodiments, the suboptimal yield concentration is less than or about 30 μM of agent (e.g., antibody) per cell. In some embodiments, the suboptimal yield concentration is less than or about 1×10 cells in the incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 in some embodiments, a suboptimal yield concentration is less than or about 30 μM of agent (e.g., antibody) per cell in the incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 20 μM of agent (e.g., antibody) per cell; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in the incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 10 μM of agent (e.g., antibody) per cell; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in an incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9in some embodiments, a suboptimal yield concentration is less than or about 15 μM of agent (e.g., antibody) per cell in the incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 10 μM of agent (e.g., antibody) per cell; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in the incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 5 μM of agent (e.g., antibody) per cell; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in an incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 1 μM of agent (e.g., antibody) per cell; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in an incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 pcs or 20 x 10 9or less than about 0.5 μM of agent (e.g., antibody) per cell; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in an incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 Each has less than or about 0.2 μM of drug (e.g., antibody).

[0196] In some embodiments, the suboptimal yield concentration is 1×10 cells in the incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 15 mg of total beads, particles, surfaces, or reagents per unit; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in the incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 10 mg of total beads, particles, surfaces, or reagents per unit; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in an incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 5 mg of total beads, particles, surfaces, or reagents per unit; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in an incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 4 mg of total beads, particles, surfaces, or reagents per unit; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in an incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 3 mg of total beads, particles, surfaces, or reagents per unit; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in an incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 or less than about 2 mg of total beads, particles, surfaces, or reagents per unit; in some embodiments, a suboptimal yield concentration is less than about 1×10 cells in an incubation composition. 9 Each piece: 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 less than or about 1 mg of total beads, particles, surfaces, or reagents per unit; in some embodiments, a suboptimal yield concentration is less than or about 1 mg of total beads, particles, surfaces, or reagents per unit; in some embodiments, a suboptimal yield concentration is less than or about 1 mg of total beads, particles, surfaces, or reagents per unit; 9 Per, 2 x 10 9 Each piece: 3 x 10 9 Each piece: 4 x 10 9 Each piece: 5 x 10 9 Per piece, 10 x 10 9 Per piece, 15 x 10 9 per piece or 20 x 10 9 Each bead, particle, surface, or total reagent weighs less than or about 0.5 mg.

[0197] In some embodiments, for example, when working with suboptimal 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 to bias the ratio of cell types recognized by that reagent compared to cell types recognized by the other such reagent or 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., agent or mass per cell) that is increased by half, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more compared to the other one or more, depending on how much it is desired to increase the ratio.

[0198] In some embodiments, achieving a suboptimal yield concentration to bias one or both cell populations can achieve a desired or selected culture starting ratio. In some embodiments, the selection involves culturing at least 1×10 cells. 9 pieces, 2×10 9 pieces, 3×10 9 pieces, 4×10 9 pieces, 5×10 9 pieces, 6×10 9 pieces, 7×10 9 pieces, 8×109 pieces, 1×10 10 pieces, 2×10 10 pieces, 3×10 10 pieces, 4×10 10 Pieces are 5 x 10 10 The immunoaffinity reagent is used to specifically bind to cells expressing a marker, such as CD4 or CD8, from a sample that is a sample containing CD4+ and CD8+ cells, such that the sample contains a high number of cells, such as 10 or more. 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 suboptimal range and / or with enough cells to achieve saturation of the reagent, the amount of immunoaffinity reagent is proportional to the approximate yield of enriched cells. In one embodiment, to achieve a starting culture ratio of about or about 1:1 between CD4+ and CD8+ cells, the selection of CD4+ and CD8+ cells can be performed at the same or nearly the same suboptimal yield concentration of immunoaffinity reagent for CD4 and CD8, respectively. In another exemplary embodiment, to achieve a starting culture ratio of about or about 2:1 between CD4+ and CD8+ cells, the selection of CD4+ cells can be performed at a suboptimal yield concentration of immunoaffinity reagent for CD4 that is about or about 2-fold greater compared to the suboptimal yield concentration of immunoaffinity reagent for CD8. In view of the above illustration, it is within the level of the artisan to empirically select or choose the appropriate amount or concentration of immunoaffinity reagent depending on the desired or chosen starting culture ratio of the resulting composition containing enriched or selected cells.

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

[0201] In some aspects, the separation and / or other steps are performed for automated separation of cells at a clinical scale level 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 instrument and commands the system to perform repetitive procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate through the tubing set and, together with the pinch valve, ensures a controlled flow of buffer through the system and continuous suspension of the cells. In some aspects, the separation and / or other steps are performed using the CliniMACS system (Miltenyi Biotic).

[0202] In some embodiments, automated separation, such as using the CliniMACS system, uses antibody-coupled magnetizable particles, which in some aspects are provided in a sterile, non-pyrogenic solution. In some embodiments, after labeling the cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which in turn is connected to a bag containing a buffer and a cell collection bag. The tubing set is made of pre-assembled sterile tubing, including a pre-column and a separation column, and is single-use. After starting the separation program, the system automatically loads the cell sample onto the separation column. The labeled cells are retained in 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 is not retained in the column. In some embodiments, the cell population for use in the methods described herein is labeled and is retained in 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 in a cell collection bag.

[0203] In certain embodiments, the separation and / or other steps are performed using a system with a cell processing unit that allows for automated washing and fractionation of cells by centrifugation. In some aspects, the separation and / or other steps are performed using the CliniMACS Prodigy system (Miltenyi Biotec). A system with a cell processing unit can also include an on-board camera and image recognition software that determines optimal cell fractionation endpoints by distinguishing between macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into red blood cell, white blood cell and plasma layers. Cell processing systems 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. An input port can allow for aseptic removal and replenishment of culture medium, and cells can be monitored using an integrated microscope. See, e.g., 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 in a fluid stream. 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 using a microelectromechanical system (MEMS) chip in combination with a FACS-based detection system (see, for example, WO2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets 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, separation can be based on binding to fluorescently labeled antibody.In some examples, cell separation based on binding of antibody or other binding partner specific to one or more cell surface markers is carried out in fluid flow by fluorescence activated cell sorting (FACS), including preparative scale (FACS) and / or microelectromechanical system (MEMS) chips combined with, for example, flow cytometry detection system.Such methods allow positive and negative selection based on multiple markers simultaneously.

[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 immunoaffinity-based reagents that include at least a first and a second affinity chromatography matrix, respectively, with antibodies immobilized on the surface. 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 in the first and / or second selections can be used to immobilize at least about 50×10 cells. 6 pieces / mL, 100×10 6 pieces / mL, 200×10 6 / mL or 400 x 10 6 It is possible to adsorb or select or enrich 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 starting culture ratio of the selected or enriched composition is achieved by choosing a sufficient amount of matrix and / or a sufficient relative amount to achieve the expected starting culture ratio based on, for example, the adsorption capacity of one or more columns for selecting cells.

[0207] In an exemplary embodiment, the adsorptive capacity of one or more matrices is the same between the first and second selections, e.g., 1×10 cells for both. 8 or about 1 x 10 8cells / mL, where the enrichment or selection of cells in the first and second selection results in a composition containing CD4+ cells and CD8+ cells at a starting culture ratio of about 1:1. In another exemplary embodiment, the adsorptive capacity of the matrix or matrices used in either the first or second selection is at least 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or more times the adsorptive capacity of the matrix or matrices used in the other of the first or second selection, thereby resulting in a starting culture ratio in which the selected cells with the greater adsorptive capacity, e.g., CD4+ cells or CD8+ cells, are present in an amount that is at least 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or more times that of the other cell population in the starting culture ratio. Depending on the desired or selected culture starting ratio of the resulting composition containing the enriched or selected cells, it is within the level of the skilled artisan to select or choose an appropriate volume, diameter or number of affinity matrix chromatography columns for the first and / or second selection.

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

[0209] In some embodiments, the first and / or second selection in the provided methods comprises first enriching for either CD4+ or CD8+ cells, and then enriching for a cell subpopulation based on surface expression of a marker, e.g., a marker expressed on resting T cells, naive T cells, or central memory T cells, e.g., CD28, CD62L, CCR7, CD127, or CD27. In some embodiments, the first and / or second selection comprises enriching for CD8+ cells, which selection comprises enriching for central memory T (T CM) cells, wherein the other of the first and / or second selection comprises enriching for CD4+ cells. In some embodiments, the method is performed to enrich or select for CD4+ cells and to enrich or select for a cell subpopulation that is CD8+ / CD28+, CD8+ / CD62L+, CD8+ / CCR7+, CD8+ / CD127+ or CD8+ / CD27+. In some embodiments, the first selection comprises enriching for CD8+ cells and the second selection comprises enriching for CD4+ cells, wherein the first selection comprising cells enriched for CD8+ cells comprises enriching for central memory T (T CM ) cells, or cells expressing a marker that is CD28, CD62L, CCR7, CD127, or CD27, thereby enriching for CD4+ cells and central memory T (T CM In some embodiments, the first selection comprises enriching for CD4+ cells and the second selection comprises enriching for CD8+ cells, where the second selection comprising cells enriched for CD8+ cells is enriched for central memory T (T CM ) cells or cells expressing a marker that is CD28, CD62L, CCR7, CD127 or CD27, thereby enriching for CD4+ cells and central memory T (T CM ) cells or a composition, such as a culture starting composition, is generated that contains CD8+ cells or cells that are enriched for cells expressing a marker that is CD28, CD62L, CCR7, CD127 or CD27.

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

[0211] In one exemplary embodiment, the sample is enriched for CD4+ and CD8+ / CD62L+ cells to obtain a 1:1 ratio of CD4+ to CD8+. In this exemplary embodiment, the first selection can include enriching for CD8+ cells using a column with an adsorption capacity adjusted for the relative frequency of CD4+ cells and CD8+ / CD62L+ cells known to be present in the sample, or using the ratios that are approximately estimated to be present 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 population of CD8+ T cells. See, for example, Maldonado, Arthritis Res Ther. 2003; 5(2): R91-R96. In one such embodiment, the column can be arranged to collect 4 times more CD8+ cells than CD4+ cells to generate a 1:1 culture starting ratio of CD4+ cells to a CD8+ subpopulation that further includes 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 or CD62L selection columns. The size of the columns can also be adjusted for expected yield. For example, if each column is only 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 culture starting composition may be added at 200×10 6 CD4+ cells and 200 × 10 6 In this example, assuming the ratios presented above, the sample to be enriched may contain at least or about 200×10 CD8+ / CD62L+ cells. 6 CD4+ cells and at least or about 800 x 10 6 CD8+ cells (approximately 25% of them (200 × 10 6 Each 2 mL of selection matrix can contain approximately 200 x 10 cells (which may also be CD62L+). 6Assuming that a CD8 selection column with 8 mL of selection matrix can enrich for approximately 800 x 10 6 10 CD8+ cells while the flow-through is passed over a CD4 selection column. A CD4 selection column with 2 mL of selection matrix can bind approximately or approximately 200×10 6 The 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 2 mL of selection matrix, thus binding approximately or approximately 200×10 6 The starting culture composition or a composition having about or approximately a 1:1 culture starting ratio can be obtained by elution from a CD4 and CD8 / CD62L column into a culture vessel.

[0213] In another exemplary embodiment, the sample is enriched for CD4+ and CD8+ / CCR7+ cells to produce a 1:1 CD4+ to CD8+ ratio. In this exemplary embodiment, the first selection can include enriching for CD8+ cells using a column with an adsorption capacity adjusted for the relative frequency of CD4+ cells to CD8+ / CCR7+ cells known to be present in the sample, or using a ratio that is approximately estimated to be present in such a sample. 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, e.g., Chen, Blood. 2001 Jul 1;98(1):156-64. The column is enriched for 30% more CD8+ cells than CD4+ cells to produce a 1:1 starting culture ratio. 1 Assuming similar adsorption capacity and efficiency for each selection column, the CD8+ column will collect approximately 3 / 4 times as many CD8+ cells as the CD4 or CCR7 selection columns. 1The size of the columns can also be adjusted for expected yields. For example, if each column is only 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 may consist of 200 x 10 6 CD4+ cells and 200 × 10 6 In this example, assuming the ratios presented above, the sample to be enriched contains at least 200×10 CD8+ / CCR7+ cells. 6 CD4+ cells and at least or about 6.6 x 10 6 The CD8+ cells can be contained in the 100-well plate, and approximately 60% of them (200 × 10 6 ) can also be CCR7+. Each 2 mL of selection matrix contains 200 × 10 6 Assuming that one can enrich for 3 cells, the selection matrix 1 / 3mL of CD8 selection column will yield approximately 6.6x10 6 10 CD8+ cells while the flow-through is passed over a CD4 selection column. A CD4 selection column with 2 mL of selection matrix can bind approximately or approximately 200×10 6 The CD8+ cells can be further enriched for CD62L by eluting the CD8+ cells into a CCR7 selection column. In this exemplary embodiment, the CCR7 selection column can accommodate 1 mL of selection matrix, thus binding approximately or approximately 200×10 6 The CD4 and CCR7 columns are enriched for CD8+ / CCR7+ cells, which can be eluted into a culture vessel and yield approximately or about 200×10 6 CD4+ cells and approximately or about 200 × 10 6 A starting culture containing 100 CD8+ / CCR7+ cells, or a 1:1 culture starting ratio, is obtained.

[0215] It is within the level of skill of the artisan to empirically select or choose the appropriate volume, diameter or number of affinity matrix chromatography columns for the first and / or second and / or third selections in light of the above examples, depending on the desired or chosen culture starting ratio of the resulting composition containing the enriched or selected cells, the expected frequency of each subpopulation, the varying efficiency of each selection column, and other factors within the level of skill of the artisan.

[0216] B. Incubation of isolated cells In some embodiments, the provided methods include one or more of various steps for incubating isolated cells and cell populations, such as populations isolated according to the methods herein, e.g., steps for incubating 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 populations are incubated in a culture starting composition.

[0217] Multiple isolated cell populations, e.g., a CD4+ cell population and a CD8+ cell population (e.g., unfractionated or subpopulations thereof), are typically incubated with the combined cell populations in a culture starting composition in 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, cell populations or cell types are provided in a culture starting composition with a culture starting ratio, e.g., CD4+ / CD5+ / CD6+ / CD7+ / CD8+ / CD9+ / CD10+, designed to achieve a particular desired output ratio after incubation and / or manipulation steps, or a ratio that is within a particular range of tolerance of such desired output ratio, or designed to do so for a particular percentage of time. + Cells and CD8 +The output ratio is present in a ratio with cells. The output ratio can be, for example, an optimal ratio for achieving one or more therapeutic effects when administered to a patient, for example, by adoptive cell therapy. In some aspects, the culture start ratio is empirically determined, for example, using the determination method described herein, to determine the optimal culture start ratio for achieving a desired output ratio in a particular situation.

[0219] The incubation step can include culturing, growing, stimulating, activating, propagating, including by incubation in the presence of stimulatory conditions, e.g., conditions designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic manipulation, such as for the introduction of an engineered antigen receptor.

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

[0221] In some aspects, the incubation is carried out according to techniques such as those described in U.S. Pat. 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 + A cell population, such as a population or subpopulation, is expanded by adding to a culture starting composition feeder cells, 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 incubating the culture (e.g., for a time sufficient to expand 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 radiation 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 stimulatory conditions include a temperature suitable for the growth of human T lymphocytes, for example at least about 25 degrees Celsius, usually at least about 30 degrees Celsius, usually at or about 37 degrees Celsius. In some embodiments, a change in temperature is caused during the culture, such as from 37 degrees Celsius to 35 degrees Celsius. Optionally, the incubation can further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL can be gamma irradiated in the range of about 6000-10,000 rad. The LCL feeder cells are provided in any suitable amount, such as in a ratio of LCL feeder cells to starting T lymphocytes of at least about 10:1 in some aspects.

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

[0225] Interim assessment and adjustments In some embodiments, the method includes evaluating and / or adjusting the cells or the composition containing the cells at a time after the start of incubation or culture, such as during the incubation.Evaluating can include taking one or more measurements of the composition or vessel containing the cells, such as evaluating the cells for growth rate, viability, phenotype, expression of one or more surface or intracellular markers, such as proteins or polynucleotides, and / or evaluating the composition or vessel for temperature, medium 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 cell types, including subtypes.In some embodiments, evaluating can include measuring the CD4 + T cells and CD8 + The present invention relates to a method for determining the intermediate ratio of a plurality of, for example, two cell types, such as T cells. In some aspects, the evaluation is performed in an automated manner, for example, using a device described herein, and / or is set ahead of time to be performed at a specific time during incubation. In some aspects, the result of the evaluation, such as the determined interim ratio of two cell types, indicates that adjustment should be made, such as adding or removing one or more cell types.

[0226] The adjustment can include adjusting any cell culture factor or parameter, such as temperature, the length of time that the incubation or step is performed (incubation period), the supplementation, addition and / or removal of one or more components in the composition being incubated, such as medium or buffer or components thereof, drugs, such as nutrients, amino acids, antibiotics, ions, and / or stimulants, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, or cells or cell types or cell populations. In some aspects, the removal or addition of various components or other adjustments are performed in an automated manner, such as using a device or system described herein. In some embodiments, the system is programmed to automatically initiate an adjustment based on a particular readout from an interim evaluation. For example, in some cases, the system or device is programmed to perform one or more evaluations at a particular time; in such cases, the system or device can be further programmed such that a particular result of such an evaluation, such as a particular ratio of one cell type to another, initiates a particular adjustment, such as the addition of one or more cell types.

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

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

[0229] In some embodiments, where cells are engineered, for example to introduce an engineered antigen receptor, incubation in the presence of one or more stimulatory agents continues during the engineering phase.

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

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

[0232] Among the genes for introduction are those for improving the efficacy of the therapy, such as by promoting the viability and / or function of the transferred cells; for providing genetic markers for selection and / or evaluation of cells, e.g., to evaluate in vivo survival or localization; for improving safety, e.g., by making cells susceptible to negative selection in vivo, as described by Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also publications PCT / US91 / 08442 and PCT / US94 / 05601 by Lupton et al., which describe the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. This can be done according to known techniques (see, e.g., Riddell et al., U.S. Pat. No. 6,040,177, columns 14-17), or variations thereon that will be apparent to those of skill in the art based on the present disclosure.

[0233] The engineering typically involves the introduction of one or more genes for the expression of engineered antigen receptors, including 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 the 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, antifolate 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-1 3R-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, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic 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 CAR.CAR usually comprises an engineered receptor that comprises an extracellular ligand-binding domain linked to one or more intracellular signaling components.Such molecules typically mimic or approximate the signal through natural antigen receptors and / or the signal through such receptors together with co-stimulatory receptors.

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

[0237] In some embodiments, a 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 aspects, the tumor antigen or cell surface molecule is a polypeptide. In some aspects, the tumor antigen or cell surface molecule is selectively expressed or overexpressed on tumor cells 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 with one or more transmembrane domains and intracellular signaling domains.In some embodiments, CAR comprises a transmembrane domain fused with the extracellular domain of CAR.In one embodiment, the transmembrane domain that naturally associates with one of the domains in CAR is used.In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.

[0241] In some embodiments, the transmembrane domain is derived from either natural or synthetic origin. If the origin is natural, in some aspects the domain is derived from any membrane-bound or transmembrane protein. The transmembrane region comprises a region derived from (i.e., at least includes) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain comprises mainly 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 oligopeptide or polypeptide linker, e.g., a linker between 2 and 10 amino acids in length, such as a linker comprising glycine and serine, e.g., a glycine-serine dyad, is present to form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0243] CARs generally comprise one or more intracellular signaling components. In some embodiments, CARs comprise a TCR CD3 T cell that mediates T cell activation and cytotoxicity. +The CAR comprises an intracellular component of the TCR complex, such as a CD3-zeta chain, for example. Thus, in some aspects, the antigen binding molecule is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the CAR further comprises a portion of one or more additional molecules, such as Fc receptor gamma, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR comprises a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor gamma 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, in some situations, the CAR induces a function of the T cell, such as cytotoxic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or a costimulatory molecule. Such a truncated portion is used in some aspects instead of an intact immunostimulatory chain, for example, if it transmits an effector function signal. In some embodiments, the one or more intracellular signaling domains include the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also include the cytoplasmic sequence of a co-receptor that acts in concert with such receptor in the natural context to initiate signal transduction after antigen receptor engagement, and / or any derivative or variant of such molecule, and / or any synthetic sequence with the same functional capability.

[0245] In the context of natural TCR, full activation generally requires not only signal transduction through TCR but also costimulatory signal.Thus, in some embodiments, CAR also includes components for generating secondary or costimulatory signal to promote full activation.In some aspects, T cell activation has been described as being mediated by two classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation through TCR (primary cytoplasmic signaling sequences), and sequences that act antigen-independently to provide secondary or costimulatory signal (secondary cytoplasmic signaling sequences).In some aspects, CAR includes one or both of these signaling components.

[0246] In some aspects, the primary cytoplasmic signaling sequence can regulate the primary activation of TCR complex either stimulatory or inhibitory.The primary cytoplasmic signaling sequence that acts stimulatory can contain a signaling motif known as immunoreceptor tyrosine-based activation motif or ITAM.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 comprises a cytoplasmic signaling domain, a part thereof, or a sequence derived from CD3 zeta.

[0247] In some embodiments, the CAR comprises the signaling domain and / or transmembrane portion of a costimulatory receptor such as CD28, 4-1BB, OX40, DAP10, and ICOS.

[0248] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 intracellular domain. In some embodiments, the intracellular signaling domain comprises a CD28 and CD137 chimeric costimulatory domain linked to a CD3 intracellular domain. In some embodiments, the CAR can also comprise a transduction marker (e.g., tEGFR). In some embodiments, the CD8 +The intracellular signaling domain of cytotoxic T cells is CD4 + The intracellular signaling domain of the CD8 helper T cell is the same as that of the CD8 + The intracellular signaling domain of cytotoxic T cells is CD4 + It is distinct from the intracellular signaling domain of helper T cells.

[0249] In some embodiments, the CAR comprises two or more costimulatory domains together with an activation domain, e.g., 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] CARs and their production and introduction can include, for example, those described by 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 engineered with a recombinant T cell receptor. In some embodiments, the recombinant TCR is specific for an antigen, typically an antigen present on a target cell, such as a tumor-specific antigen, an antigen expressed on a particular 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 natural T cell. In some embodiments, high affinity T cell clones against a target antigen (e.g., a cancer antigen) are identified, isolated from a patient, and introduced into the patient. In some embodiments, TCR clones against a target antigen are generated from transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system or HLA). See, e.g., tumor antigens (see, e.g., 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 against target antigens (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354).

[0253] In some embodiments, after a 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 ribosomal skipping peptide, so that both chains are co-expressed. In some embodiments, genetic transfer of the TCR is achieved 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; an Hackett et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:674-683).

[0254] In some embodiments, gene transfer is accomplished by first stimulating the growth of T cells, then transducing the activated cells and expanding them in culture to sufficient numbers for clinical application.

[0255] In some circumstances, overexpression of stimulatory factors (e.g., lymphokines or cytokines) can be toxic to subjects.Thus, in some circumstances, engineered cells contain gene segments that make cells susceptible to negative selection in vivo, for example, when administered in adoptive immunotherapy.For example, in some aspects, cells are engineered to be able to eliminate the cells as a result of changes in the in vivo conditions of the patient to whom the cells are administered.Negatively selectable phenotypes can result from the insertion of genes that confer sensitivity to administered agents, for example, compounds. Negatively selectable genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers sensitivity to ganciclovir (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 expression of inflammatory cytokines, such as IL-2, IL-12, IL-7, IL-15, IL-21.

[0257] Introducing genetically engineered ingredients Various methods for introducing 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 the method for transferring the nucleic acid encoding the receptor, including the method via viral, such as retroviral or lentiviral transduction, transposon, and electroporation.

[0258] In some embodiments, the recombinant nucleic acid is transferred into the cell using a recombinant infectious viral particle, such as a vector derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is transferred into the T cell using a recombinant lentiviral or retroviral vector, such as a gamma retroviral vector (see, e.g., 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 have long terminal repeats (LTRs), for example, retroviral vectors derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell origin. Retroviruses are typically amphotropic, meaning that they can infect host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the gag, pol and / or env sequences of the retrovirus. Several exemplary retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (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 transferred into the T cell 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 transferred into the T cell via gene transposition (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 for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-enhanced biolistic 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 + The same CAR is introduced into each of the T lymphocytes. In some embodiments, the CD4 + T lymphocytes and CD8 + A different CAR is introduced into each of the T lymphocytes. In some embodiments, the CARs in each of these populations have an antigen binding molecule that specifically binds to 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, the 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 embodiments, cells such as tumor-infiltrating lymphocytes and / or T cells, e.g., T cells, cultured in vitro or ex vivo, e.g., during the incubation step, are not engineered to express recombinant receptors, but rather contain native antigen receptors specific for the desired antigen, promoting the expansion of cells with a particular antigen specificity. For example, in some embodiments, cells are produced for adoptive cell therapy by isolation of tumor-specific T cells, e.g., autologous tumor-infiltrating lymphocytes (TILs). Direct targeting of human tumors using autologous tumor-infiltrating lymphocytes can mediate tumor regression in some cases (see Rosenberg SA, et al. (1988) N Engl J Med. 319:1676-1680). In some embodiments, lymphocytes are removed from resected tumors. In some embodiments, such lymphocytes are expanded in vitro. In some embodiments, such lymphocytes are cultured with lymphokines (e.g., IL-2). In some aspects, such lymphocytes mediate specific lysis of autologous tumor cells, but not allogeneic tumor or autologous normal cells.

[0264] In some aspects, the incubating and / or manipulating steps and / or the method generally produce a desired output ratio (or a ratio that is within an acceptable error or tolerance of such ratio), or do so within a particular percentage of the time that the method is performed.

[0265] D. Cryopreservation In some embodiments, the provided methods include a step 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 freezing solution, e.g., after a washing step to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters can be used. One example involves using PBS or other suitable cell freezing media containing 20% ​​DMSO and 8% human serum albumin (HSA). This is then diluted 1:1 with media such that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to -80°C at 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

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

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

[0268] Kits for carrying out the provided methods are also provided. In some embodiments, the kits include an antibody or other binding partner, typically coupled to a solid support, for the isolation, e.g., immunoaffinity-based, separation step of the method.

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

[0270] In one embodiment, the kit comprises anti-CD4, anti-CD14, anti-CD45RA, anti-CD14, and anti-CD62L antibodies coupled to magnetic beads. In one embodiment, the kit comprises instructions to perform selection starting from a sample such as a PBMC sample, selecting based on expression of CD4, retaining both positive and negative fractions, and subjecting the negative fraction to further negative selection using anti-CD14 antibody, anti-CD45RA antibody, and positive selection using anti-CD62L antibody, in either order. Alternatively, the components and instructions are arranged according to any of the separation embodiments described herein.

[0271] In some embodiments, the kit further comprises instructions for transferring cells of the population isolated by the selection step into a culture, cultivation, or processing vessel while maintaining the cells in a self-contained system, hi some embodiments, the kit comprises instructions for transferring the 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 the cells and populations produced by the provided methods. Methods, e.g., methods of treatment, for administering the cells and compositions to a subject, e.g., a patient, are also provided.

[0273] Methods of administering cells, populations, and compositions, and the use of such cells, populations, and compositions for treating or preventing diseases, conditions, and disorders, including cancer, are provided. In some embodiments, the cells, populations, and compositions are administered to subjects or patients with a particular 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 the engineered compositions and the finished compositions after incubation and / or other processing steps, are administered to subjects, such as subjects with or at risk of a disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate, one or more symptoms of a disease or condition, for example by reducing tumor burden in cancers expressing an antigen recognized by engineered T cells.

[0274] Methods for administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions.For example, methods for 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 carried out by autologous transfer, in which cells are isolated and / or otherwise prepared from the subject who will receive cell therapy or from a sample derived from such a subject.Thus, in some aspects, cells are derived from a subject, such as a patient who needs 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 carried out by allogeneic transfer, in which cells are isolated and / or otherwise prepared from a subject other than the subject that will receive or ultimately receive cell therapy, such as the first subject.In such embodiments, the cells are then administered to a different subject of the same species, such as the 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 aspects, the subject, e.g., the patient, to whom the cell, cell population, or composition is administered is a mammal, typically a primate such as a human. In some aspects, the primate is a monkey or an ape. The subject can be male or female and can be of any suitable age, including infants, juveniles, young adults, adults, and geriatric subjects. In some aspects, 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 for treatment with the provided compositions, cells, methods, and uses are tumors, including solid tumors, hematological malignancies, and melanomas, as well as infectious diseases such as infections with 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 disease. Such diseases include, but are not limited to, leukemia, lymphoma, such as chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin's 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 carcinoma, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and / or mesothelioma.

[0280] In some embodiments, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiencies, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus, etc. 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 transplant-related disease or condition.

[0281] In some embodiments, the antigen associated with the disease or disorder is orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B virus surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-A. lufa, 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, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic 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 pharma- ceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition additionally comprises other pharma- ceutical active agents or drugs, such as chemotherapeutic agents, e.g., 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, e.g., a pharma- ceutically acceptable salt. Suitable pharma- ceutically 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 from 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, e.g., 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 mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight 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 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, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0285] In certain embodiments, pharmaceutical compositions are formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or as liposomes.Liposomes can be useful for targeting host cells (e.g., T cells or NK cells) to specific tissues.Many methods are available for preparing liposomes, such as those described in 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 embodiments, pharmaceutical compositions use time-release, delayed release, and / or sustained release delivery systems, so that the delivery of the composition occurs before the sensitization of the site to be treated and at a sufficient time to cause the sensitization.Many types of release delivery systems are available and known to those skilled in the art.In some aspects, such systems can increase the convenience of subjects and physicians by avoiding repeated administration of the composition.

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

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

[0289] In some embodiments, CD8 + T cells and CD4 + A population or subtype of cells, such as T cells, is administered at a desired dose of total cells, such as a desired T cell dose, or within a tolerance thereof. In some aspects, the desired dose is a desired number of cells, or a 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 equal to or greater than a minimum number of cells or a minimum number of cells per unit of body weight. In some aspects, among the total cells administered at a desired dose, an individual population or subtype is administered at a desired output ratio (e.g., CD4 + and CD8 +(ratio of) or near, e.g., within a certain tolerance or error of such ratio.

[0290] In some embodiments, cells are administered at 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, or within a tolerance thereof.In some aspects, the desired dose is the desired cell number 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, for example, per kg.In some aspects, the desired dose is equal to or greater than the minimum cell number of a population or subtype, or equal to or greater than the minimum cell number of a population or subtype per unit of 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 of the individual subtypes or subpopulations, e.g., a desired fixed dose of each. Thus, in some embodiments, the dosage is based on a desired fixed dose or minimum dose of T cells and a desired ratio of CD4 + Cells and CD8 + Based on the desired ratio of cells and / or CD4 + and / or CD8 + Based on a desired fixed or minimum dose of cells.

[0292] In certain embodiments, a distinct population of cells or subtypes of cells may be, for example, 1 million to about 50 billion (e.g., 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, about 10 million to about 100 billion (e.g., about 20 million, about 30 million, about 40 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, or a range defined by any two of the foregoing values). The subject is administered about 1 million to about 100 billion, such as about 0 billion, about 25 billion, about 50 billion, about 75 billion, about 90 billion, or a range defined by any two of the foregoing values), in some cases about 100 million to about 50 billion (e.g., 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.

[0293] In some embodiments, the dose of total cells and / or the dose of individual cell subpopulations is 10 5 From 10 6 Between 1 x 10 5 pieces / kg, 1.5×10 5 pieces / kg, 2×10 5 Pieces / kg or 1×10 6 pcs / kg body weight or about its value, such as 10 4 or about 10 4 From 10 9 or about 10 9 For example, in some embodiments, the cells are in the range of between 10 T cells / kilogram (kg) of body weight. 5 From 10 6 Between 1 x 10 T cells / kg body weight, for example 5 cells / kg, T cells 1.5×10 5 cells / kg, T cells 2×10 5 / kg or 1 x 10 T cells 6 10 T cells / kg body weight, or approximately that value 4 or about 10 4 From 10 9 or about 10 91 piece / kilogram (kg) of body weight or within a specified margin of error thereof.

[0294] In some embodiments, the cells are CD4 + and / or CD8 + Cells are 10 5 From 10 6 Between cells / kg body weight, e.g., CD4 + Cells and / or CD8 + 1 × 10 cells 5 pieces / kg, CD4 + Cells and / or CD8 + 1.5 × 10 cells 5 pieces / kg, CD4 + Cells and / or CD8 + 2 × 10 cells 5 cells / kg or CD4 + Cells and / or CD8 + 1 × 10 cells 6 CD4 count / kg body weight, or approximately that value + Cells and / or CD8 + Cells are 10 4 or about 10 4 From 10 9 or about 10 9 It is administered between 100 mg / kg body weight or within a specified margin of error.

[0295] In some embodiments, the cells are CD4 + Approximately 1 x 10 cells 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9 × 10 6 and / or at least about 1×10 CD8+ cells. 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9 × 10 6 and / or at least about 1×10 T cells 6 , about 2.5×10 6 , about 5×106 , about 7.5×10 6 , or about 9 × 10 6 In some embodiments, the cells are administered at about 10 T cells. 8 From 10 12 Between or about 10 10 From 10 11 Between individuals, CD4 + Approximately 10 cells 8 From 10 12 Between or about 10 10 From 10 11 Between individuals and / or CD8 + Approximately 10 cells 8 From 10 12 Between or about 10 10 pcs to 10 11 The dose is administered between the individual or within a certain margin of error.

[0296] In some embodiments, cells are administered at a desired output ratio, or within a tolerance range, of multiple cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios. For example, in some embodiments, the desired ratio (e.g., CD4 + Cells and CD8 +The ratio of the cells to the total number of cells is preferably between 5:1 or about 5:1 and 5:1 or about 5:1 (or greater than about 1:5 and less than about 5:1), or 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: Less than 1, e.g., 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 ratios thereof. 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 values ​​between these ranges).

[0297] The cell populations and compositions, in some embodiments, are administered to a subject using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0298] In some embodiments, the cell population obtained by using the method described herein is co-administered with one or more additional therapeutic agents or in association with another therapeutic intervention, either simultaneously or sequentially in any order.In some circumstances, the cell population is co-administered with another treatment close enough in time that the cell population enhances the effect of one or more additional therapeutic agents or vice versa.In some embodiments, the cell population is administered before one or more additional therapeutic agents.In some embodiments, 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, for example, by any of several known methods. Parameters to be evaluated include specific binding of engineered or natural T cells or other immune cells to antigens, for example, in vivo by imaging, or ex vivo, for example, by 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, for example, the cytotoxicity assays described in 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 evaluating clinical outcomes, such as reduction in tumor burden or tumor mass.

[0300] In certain embodiments, engineered cells are further modified in any number of ways to increase their therapeutic or prophylactic efficacy.For example, the engineered CAR or TCR expressed by the population can be conjugated to targeting moiety either directly or indirectly via linker.The practice of conjugating a compound, such as CAR or TCR, to targeting moiety is known in the art.See, for example, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995) and U.S. Patent No. 5,087,616.

[0301] IV. Definition 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 the form of a range. Descriptions in the form of a range should be understood to be merely for convenience and brevity, and should not be considered as inflexible limitations on the scope of the claimed subject matter. Thus, the description of a range should be considered to have specifically disclosed all 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 limit of that range, and any other stated or intervening value in the stated range, is included within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also included within the claimed subject matter, subject to any specifically excluded limit value in the stated range. If a stated range includes one or both of these limits, then ranges excluding one or both of those included limits are also included within the claimed subject matter. This is true regardless of the breadth of the range.

[0303] The term "about" as used herein refers to the normal error range for each value that is readily apparent to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) aspects directed to the 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 the form of a range. Descriptions in the form of a range should be understood to be merely for convenience and brevity, and should not be considered as inflexible limitations on the scope of the claimed subject matter. Thus, the description of a range should be considered to have specifically disclosed all 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 limit of that range, and any other stated or intervening value in the stated range, is included within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also included within the claimed subject matter, subject to any specifically excluded limit value in the stated range. If a stated range includes one or both of these limits, then ranges excluding one or both of those included limits are also included within the claimed subject matter. This is true regardless of the breadth of the range.

[0306] As used herein, "percent (%) amino acid sequence identity" and "percent identity" when used in reference to amino acid sequence (reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (e.g., 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 percentage, introducing gaps as necessary, and not considering any conservative substitutions as part of sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0307] Amino acid substitutions may 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] Non-conservative amino acid substitutions involve exchanging a member of one of these classes for another class.

[0309] As used herein, a subject includes any organism, such as humans and other mammals. Mammals include, but are not limited to, humans, and non-human animals, including farm animals, sports animals, rodents, and pets.

[0310] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may 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 reducing the number or percentage of a cell type or population, for example, by negative selection based on a marker expressed by the population or cells, or positive selection based on a marker not present on the cell population or cells to be depleted, relative to the total number of cells in the composition or the volume of the composition, or relative to other cell types. The term does not require complete removal of the cells, cell types, or populations from the composition.

[0312] "Enriching" as used herein when referring to one or more particular cell types or cell populations refers to increasing the number or percentage of a cell type or population, for example, relative to the total number of cells in a composition or the volume of a composition, or relative to other cell types, such as by positive selection based on a marker expressed by the population or cells, or negative selection based on a marker not present on the cell population or cells to be depleted. The term does not require the complete removal of other cells, cell types, or populations from the composition, nor does it require that the cells so enriched are present at or even close to 100% in the enriched composition.

[0313] As used herein, the terms "treatment," "treat," and "treating" refer to the complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or a phenotype associated therewith. In certain embodiments, the effect is therapeutic, such as partially or completely curing a 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, such as treating a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effects of the treatment, at the dosage required for the period of time. The therapeutically effective amount may vary depending on factors such as the condition, age, sex, and weight of the subject, and the cell population to be administered.

[0315] As used herein, the statement that a cell or cell population is "positive" for a particular marker refers to the presence of a particular marker, typically a surface marker, detectable on or in cells.When referring to a surface marker, the term refers to 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, where staining is detectable by flow cytometry at a level that is substantially greater than the staining detected by isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions, and / or at a level that is substantially similar to the level of cells known to be positive for the marker, and / or at a level that is substantially higher than the level of 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 refers to the substantial absence of detectable specific marker, typically surface marker, on or in cells.When referring to surface marker, this term refers to 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, where staining is not detected by flow cytometry at a level substantially greater than the staining detected by isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions, 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.

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

[0318] V. Illustrative Embodiments Among the aspects provided herein are the following: 1. (a) performing a first selection in a closed system, the first selection comprising enriching one of (i) CD4+ cells and (ii) CD8+ cells from a sample containing primary human T cells, whereby the enrichment generates a first selected population and an unselected population; and (b) performing a second selection in the closed system, the second selection comprising enriching the other of (i) CD4+ cells and (ii) CD8+ cells from the unselected population, whereby said enrichment generates a second selected population. 1. A method for enriching CD4+ T cells or CD8+ T cells, comprising: A method in which CD4+ cells and CD8+ cells are enriched and an enriched composition is produced comprising said first selected population of cells and said second selected population of cells. 2. (c) Producing an enriched composition by combining cells of the first selected population and cells of the second selected population. and / or the CD4+ cells and CD8+ cells in the enriched composition are present at a culture starting ratio of CD4+ cells to CD8+ cells; The method of embodiment 1. 3. The method of embodiment 2, wherein the combining is performed in a closed system. 4. (a) performing a first selection in a closed system, the first selection comprising: (i) selecting from a sample containing primary human T cells a CD4 + cells and (ii) CD8 + enriching one of the cells, whereby enrichment produces a first selected population and an unselected population; and (b) performing a second selection in the closed system, the second selection comprising enriching the other of (i) CD4+ cells and (ii) CD8+ cells from the unselected population, whereby the enrichment generates a second selected population; (c) generating stimulated cells by incubating a culture starting composition comprising cells of the first selected population and cells of the second selected population in a culture vessel under stimulated conditions; and (d) introducing a genetically engineered antigen receptor into the stimulated cells produced in (c). 1. A method for producing engineered T cells, comprising: Thereby, CD4 expressing a genetically engineered antigen receptor + T cells and CD8 + generating an output composition comprising T cells. 5. The method of embodiment 4, further comprising, prior to step (c), combining cells of the first and second selected cell populations to produce a culture starting composition, and / or wherein the CD4+ cells and CD8+ cells in the culture starting composition are present in a culture starting ratio of CD4+ cells to CD8+ cells. 6. The method of embodiment 5, wherein the combining is performed in a closed system. 7. The method of any of embodiments 1-6, wherein one or more of the steps are performed in an automated manner and / or the closed system is automated. 8. CD4 + Cells and CD8 + The method of any of aspects 2 to 7, wherein the initiation ratio of cells to be cultured 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 of any of aspects 2 to 8, wherein the initiation culture ratio of CD4+ cells to CD8+ cells is 1:1 or about 1:1. 10. A sample is obtained from a human subject; CD4 + Cells and CD8 + The starting ratio of cells was determined by the CD4 + Cells and CD8 + Different ratios of cells and / or CD4 + Cells and CD8 + The starting ratio of cells was determined by the CD4 + Cells and CD8 + at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% greater or less than the ratio of The method according to any one of aspects 2 to 6. 11. The method of any of embodiments 1-10, wherein enriching the cells in the first and / or second selection comprises performing positive or negative selection based on expression of a cell surface marker. 12. The method of embodiment 11, wherein enriching for cells in the first and / or second selection comprises a negative selection comprising depleting cells expressing non-T cell surface markers. 13. The method of embodiment 12, wherein the non-T cell marker comprises CD14. 14. The method of any of embodiments 1-13, wherein enriching for cells in the first or second selection comprises performing multiple positive or negative selection steps based on expression of one or more cell surface markers to enrich for CD4+ cells or CD8+ cells. 15. The method of any of embodiments 1-14, wherein enriching the cells in the first and / or second selection comprises immunoaffinity-based selection. 16. The method of embodiment 15, wherein immunoaffinity based selection is performed by contacting the cells with an antibody capable of specifically binding to a cell surface marker and positively selecting by recovering cells that bind to the antibody, or negatively selecting by recovering cells that do not bind to the antibody, and enriching for CD4+ or CD8+ cells from the recovered cells, and the antibody is immobilized on a magnetic particle. 17. The method of any of embodiments 1-13, wherein the first selection and the second selection are carried out in separate separation vessels that are operably connected. 18. The method of embodiment 17, wherein the separation vessels are operably connected by tubing. 19. The method of any of embodiments 15 to 18, wherein the immunoaffinity-based selection is performed 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+ or CD8+ cells. 20. The antibody further comprises one or more binding partners capable of forming a reversible bond with a binding reagent immobilized on a 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 disruption of the reversible bond between the binding reagent and the binding partner. The method of embodiment 19. twenty one. the binding partner is selected from biotin, a biotin analogue, and a peptide capable of binding to the binding agent; the binding reagent is selected from streptavidin, streptavidin analogues or mutant proteins, avidin, and avidin analogues or mutant proteins; The method of embodiment 17. twenty two. the binding partner comprises the amino acid sequence shown in SEQ ID NO:6, and / or the binding agent is a streptavidin mutein comprising the amino acid sequence shown in SEQ ID NO: 12, 13, 15, or 16; The method of embodiment 21. twenty three. After contacting the cells in the sample with the affinity chromatography matrix in the first selection and / or the second selection, recovering the selected cells from the matrix by adding a competing reagent to disrupt the binding between the binding partner and the binding reagent. 22. The method of any one of embodiments 19 to 21, further comprising: twenty four. The method of embodiment 23, wherein the competing reagent is biotin or a biotin analogue. twenty five. The antibody or antibodies in the first and / or second selections have a binding and cell surface marker affinity of 3×10 -5 sec -1 Greater than or about 3 x 10 -5 sec -1 The dissociation rate constant (k off 25. The method of any one of embodiments 20 to 24, 26. The antibody or antibodies in the first and / or second selection have a low dissociation constant (K d ) is about 10 -3 ~10 -7 in the range of about 10 -7 ~about 10 -10 26. The method of any of embodiments 20 to 25, wherein the affinity is in the range of 27. The method of any of embodiments 20 to 26, wherein the first and / or second selected chromatography matrices are packed in a separation vessel that is a column. 28. The affinity chromatography matrix has at least about 50×10 6 cells / mL, 100×10 6 cells / mL, 200×10 6 cells / mL, or 400 x 10 6 The method of any of embodiments 19 to 27, wherein the cells / mL can be adsorbed and / or selected. 29. 29. The method of any of embodiments 19 to 28, wherein the first and second selection steps comprise 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 culture starting ratio. 30. The method of any of embodiments 1-29, wherein enriching for CD4+ cells comprises positive selection based on surface expression of CD4. 31. The method of any of embodiments 1-29, wherein enriching for CD8+ cells comprises positive selection based on surface expression of CD8. 32. One of the first and second selections comprising enriching for CD8+ cells, Central Memory T(T CM ) enriching cells; and / or Enriching for cells expressing a marker selected from CD28, CD62L, CCR7, CD127, and CD27. 30. The method of any of embodiments 1-29, further comprising: 33. the first selection comprises enriching for CD8+ cells, the second selection comprises enriching for CD4+ cells, and The first choice is Central Memory T(T CM) cells, and / or enriching for cells expressing a marker selected from CD28, CD62L, CCR7, CD127, and CD27; The method of any one of embodiments 1 to 32. 34. Central Memory T(T CM ) enriching for cells and / or enriching for cells expressing a marker, CD8 + selecting cells expressing CD62L from the enriched first and / or second selected cell populations; and / or CD8 + selecting cells expressing CD27 from the enriched first and / or second selected cell populations; and / or CD8 + selecting cells expressing CCR7 from the enriched first and / or second selected cell populations; and / or CD8 + selecting cells expressing CD28 from the enriched first and / or second selected cell populations; and / or CD8 + Selecting cells expressing CD127 from the enriched first and / or second selected cell populations. The method of embodiment 32, or embodiment 33, or embodiment 35, comprising: 35. the first selection comprises enriching for CD4+ cells, the second selection comprises enriching for CD8+ cells, and The second choice is Central Memory T(T CM ) enriching the cells, The method of any one of embodiments 1 to 32. 36. (i) the first selection comprises enriching for CD4+ cells by positive selection based on surface expression of CD4, thereby generating a first selection population enriched in CD4+ primary human T cells and an unselected sample; (ii) a second selection step includes enriching for CD8+ cells and isolating central memory T (T CM ) cells, wherein the central memory T (T CM ) enriching cells, Negative selection to deplete cells expressing surface markers present on naive T cells and central memory T (T CM ) positive selection for cells expressing a surface marker that is present on the cells and not present on another memory T cell subpopulation, or Positive selection for cells expressing surface markers present on central memory T cells and absent on naive T cells, and central memory T (T CM ) positive selection for cells expressing a surface marker that is present on the cells and not present on another memory T cell subpopulation , whereby T CM generating cell-enriched CD8+ primary human T cells; The method of embodiment 35. 37. Markers present on naive T cells include CD45RA; and Central Memory T(T CM ) cells by negative selection to deplete cells expressing CD45RA and central memory T (T CM ) positive selection for cells expressing a surface marker that is present on the cells and not present on another memory T cell subpopulation, The method of embodiment 36. 38. Surface markers present on central memory T cells and absent on naive T cells include CD45RO; Central Memory T(T CM Enriching for CD45RO-expressing cells results in positive selection for cells expressing central memory T (T CM ) positive selection for cells expressing a surface marker that is present on the cells and not present on another memory T cell subpopulation, The method of embodiment 37. 39. Central Memory T(T CM 39. The method of any of embodiments 36-38, wherein the surface marker that is present on the memory T cell and not present on another subpopulation of memory T cells is selected from the group consisting of CD62L, CCR7, CD27, CD127, and CD44. 40. Central Memory T(T CM 40. The method of embodiment 39, wherein the surface marker that is present on the memory T cell subpopulation and not present on another memory T cell subpopulation is CD62L. 41. CD8 in the enriched composition or culture-initiating composition + The group is the central memory T(T CM ) cells or naïve T (T N 41. The method of any of embodiments 32-40, wherein the IL-16+ / IL-16+ cells comprise less than 20% CD62L+ cells, or comprise at least 80% CD62L+ cells. 42. contacting cells of a sample containing primary human T cells with a first immunoaffinity reagent that specifically binds CD4 and a second immunoaffinity reagent that specifically binds CD8 in an incubation composition under conditions where the immunoaffinity reagents specifically bind to CD4 and CD8 molecules, respectively, on the surface of cells in the sample; generating an enriched composition comprising CD4+ and CD8+ cells at the starting culture ratio by recovering cells that bind to said first and / or said second immunoaffinity reagent. 1. A method for enriching CD4+ T cells and CD8+ T cells, comprising: The method of producing a composition enriched in CD4+ T cells and CD8+ T cells, wherein the first and / or the second immunoaffinity reagent are present in an incubation composition at a suboptimal yield concentration, wherein the enriched composition contains 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. 43. (a) enriching primary human T cells from a sample containing primary human T cells, contacting cells of the sample with a first immunoaffinity reagent that specifically binds CD4 and a second immunoaffinity reagent that specifically binds CD8 in an incubation composition under conditions where the immunoaffinity reagents specifically bind to CD4 and CD8 molecules, respectively, on the surface of cells in the sample; recovering cells that bind to said first and / or said second immunoaffinity reagent to generate an enriched composition comprising CD4+ cells and CD8+ cells at a starting culture ratio; wherein the first and / or the second immunoaffinity reagent are present in the incubation composition at a suboptimal yield concentration, and wherein the enriched composition contains 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; and (b) generating stimulated cells by incubating cells of the enriched composition in a culture starting composition under stimulatory conditions in a culture vessel, the cells being at or substantially at the culture starting ratio; and (c) A CD4+ cell expressing a genetically engineered antigen receptor is produced by introducing the genetically engineered antigen receptor into the stimulated cells of (b). + T cells and CD8 + Producing an output composition comprising T cells 2. A method for producing genetically engineered T cells, comprising: 44. The method of embodiment 42 or embodiment 43, wherein enriching for primary human T cells is performed in a closed system. 45. The method of any of embodiments 42-44, wherein the first and second immunoaffinity reagents are present in the incubation composition at suboptimal yield concentrations, and wherein the enriched composition comprises less than 70% of the total CD4+ cells in the incubation composition and less than 70% of the total CD8+ cells in the incubation composition. 46. the first immunoaffinity reagent is present in the incubation composition at a suboptimal yield concentration, whereby 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, wherein 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; The method of any one of aspects 42 to 45. 47. The sample contains at least 1 × 10 CD3+ T cells. 9 47. The method of any one of aspects 42 to 46, 48. The method of any of embodiments 42-47, wherein a concentration of one of the first and second immunoaffinity reagents in the incubation composition is greater than the other, where the greater concentration results in a higher yield of CD4+ cells or CD8+ cells in the enriched composition as compared to the yield of the other of the CD4+ cells or CD8+ cells, respectively, 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 of embodiment 48. 50. CD4 + Cells and CD8 +50. The method of any of embodiments 42-49, wherein the starting ratio of cells to be cultured 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 of any of aspects 42 to 50, wherein the initiation culture ratio of CD4+ cells to CD8+ cells is 1:1 or about 1:1. 52. CD4 + Cells and CD8 + The starting ratio of cells was determined by the CD4 + Cells and CD8 + different ratio to cells and / or CD4 + Cells and CD8 + The starting ratio of cells was determined by the CD4 + Cells and CD8 + at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% greater or smaller than the ratio of cells; The method of any one of aspects 42 to 51. 53. The method of any of embodiments 42-52, wherein greater than 95% or greater than 98% of the cells in the culture starting composition are CD4+ cells and CD8+ cells. 54. The method of any of embodiments 42 to 53, wherein each of the immunoaffinity reagents comprises an antibody. 55. The method of embodiment 54, wherein the antibody is immobilized on the outer surface of the sphere. 56. The method of embodiment 55, wherein the spheres are magnetic beads. 57. the antibody comprises one or more binding partners capable of forming a reversible bond with a binding reagent immobilized on a sphere, wherein the antibody is reversibly immobilized on the sphere; the method further comprising the step of recovering selected cells from the spheres after contacting the cells in the sample with the first and second immunoaffinity reagents by 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 analogue, or a peptide capable of binding to a binding agent; the binding reagent is selected from streptavidin, a streptavidin analogue or mutant protein, avidin, an avidin analogue or mutant protein, The method of embodiment 57. 59. the binding partner comprises a peptide comprising the amino acid sequence shown in SEQ ID NO:6; and / or the binding agent is a streptavidin mutein comprising 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 comprising one or more monomers of streptavidin or a streptavidin mutein; and the binding partner is a peptide comprising a consecutive 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 comprises an amino acid sequence set forth in any of SEQ ID NOs:7-10. 62. The method of any of embodiments 57-61, wherein the competitive reagent is biotin or a biotin analogue. 63. One or more of the antibodies in the first and / or second selection have a binding affinity of 3×10 -5 sec -1 or approximately 3 × 10 -5 sec-1 The dissociation rate constant (k off The method of any one of embodiments 42 to 63, 64. The one or more antibodies in the first and / or second selection are -3 ~10 -7 or approximately 10 -7 ~about 10 -10 The method of any of embodiments 42 to 63, wherein the antibody has an affinity with a dissociation constant in the range of 65. The method of any of embodiments 42-63, wherein one or more steps are performed in an automated manner and / or the closed system is automated. 66. determining the ratio of CD4+ T cells to CD8+ T cells in the sample prior to performing 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 comprising CD4+ cells and CD8+ cells at a starting culture ratio. Including, The method according to any one of embodiments 2, 3 and 5 to 41. 67. the first and / or second selection comprises an immunoaffinity-based selection comprising an affinity chromatography matrix; adjusting the first and / or second selection comprises selecting a sufficient amount of the affinity chromatography matrix to achieve a culture starting ratio in the first and / or second selection; The method of embodiment 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 a concentration of said first and / or said second immunoaffinity reagent based on the ratio of CD4+ T cells to CD8+ T cells in said sample to produce an enriched composition comprising CD4+ cells and CD8+ cells at an initial culture ratio. The method of any one of embodiments 42 to 65, comprising: 69. CD4 between 2:1 or about 2:1 and 1:5 or about 1:5 + Cells and CD8 + The method of any of embodiments 1-68, producing an output composition comprising a ratio of cells. 70. CD4 in the output composition + Cells and CD8 + The method of embodiment 69, wherein the ratio of cells is 1:1 or about 1:1. 71. The method of any of embodiments 1-70, wherein the sample is obtained from a subject. 72. The method of embodiment 71, wherein the subject is a subject receiving engineered T cells or cells for adoptive cell therapy. 73. The method of embodiment 72, wherein the subject is a subject other than the subject receiving the engineered T cells or cells for adoptive therapy. 74. The method of any of embodiments 1-73, wherein the sample is blood or a blood-derived sample. 75. The method of any of embodiments 1-74, wherein the sample is a white blood cell sample. 76. The method of any of embodiments 1-75, wherein the sample is an apheresis, peripheral blood mononuclear cell (PBMC), or leukapheresis sample. 77. The method of any of embodiments 4-41 or 43-76, wherein incubating the composition in the culture vessel under stimulatory conditions is performed before, during, and / or after introducing the engineered antigen receptor. 78. The method of any of embodiments 4-41 or 43-76, wherein incubating the composition in the culture vessel under stimulatory conditions occurs before, during, and after introducing the engineered antigen receptor. 79. The method of any of embodiments 4-41 or 43-78, wherein the stimulatory conditions comprise conditions under which T cells of the composition proliferate. 80. The method of any of embodiments 4-41 or 43-79, wherein the stimulatory conditions comprise an agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex. 81. The method of embodiment 80, wherein the one or more components of the TCR complex comprise a CD3 zeta chain. 82. The method of any of embodiments 4-41 or 43-81, wherein the stimulatory conditions comprise the presence of an anti-CD3 antibody, and an anti-CD28 antibody, an anti-4-1BB antibody, and / or a cytokine. 83. The method of embodiment 82, wherein the anti-CD3 antibody and / or the anti-CD28 antibody are present on the surface of a solid support. 84. The method of embodiment 83, wherein the cytokines comprise IL-2, IL-15, IL-7, and / or IL-21. 85. The method of any of embodiments 4-41 or 43-84, wherein the engineered antigen receptor comprises a T cell receptor (TCR) or a functional non-TCR antigen receptor. 86. The method of embodiment 85, wherein the receptor specifically binds to an antigen expressed by cells of the disease or condition to be treated. 87. The method of any of embodiments 85 or 86, wherein the antigen receptor is a chimeric antigen receptor (CAR). 88. The method of embodiment 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 costimulatory molecule. 89. (a) a CD4 antibody according to any one of claims 1 to 88 + T cells and CD8 + producing an output composition comprising T cells; and (b) administering cells of the output composition to a subject. A method of treatment comprising: 90. The method of embodiment 89, wherein the sample from which the cells are isolated is derived from the subject to which the cells will be administered. 91. A composition of cells produced by the method of any of embodiments 1 to 88. 92. The composition of embodiment 91, comprising a pharma- ceutically acceptable carrier. 93. A method of treatment comprising administering to a subject a composition of cells of embodiment 91 or 92. 94. The method of embodiment 93, wherein the engineered antigen receptor specifically binds to an antigen associated with the disease or condition. 95. The method of treatment of embodiment 94, wherein the disease or condition is cancer. 96. The composition of embodiment 91 or embodiment 92 for use in treating a disease or condition in a subject. 97. Use of the composition of embodiment 91 or embodiment 92 for the manufacture of a medicament for treating a disease or disorder in a subject. 98. The composition of embodiment 96 or the use of embodiment 97, wherein the engineered antigen receptor specifically binds to an antigen associated with the disease or condition. 99. The composition or use of any of embodiments 96-98, wherein the disease or condition is cancer. 100. a) a first affinity chromatography matrix comprising a first binding agent immobilized on its surface, the binding agent specifically binding 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, the first operable connection permitting passage of cells from the storage reservoir to the first affinity chromatography matrix; the first affinity chromatography matrix is ​​operably connected to an output reservoir via a second operable connection; a first affinity chromatography matrix; and b) a second affinity chromatography matrix comprising a second binding agent immobilized on its surface, the second binding agent specifically binding to a second cell surface marker present on a second cell, wherein: the first affinity chromatography matrix is ​​operably connected to a second affinity chromatography matrix via a third operable connection, the third operable connection allowing cells that have passed through the first affinity chromatography matrix and that have not bound to the first binding agent to be passed to the second affinity chromatography matrix; the second affinity chromatography matrix is ​​operably connected to an output vessel via a fourth operable connection, the fourth operable connection allowing passage of cells bound to and eluted from the first and / or second affinity chromatography matrix; the second affinity chromatography matrix is ​​operably linked to a waste reservoir via a fifth operable connection, the fifth operable connection permitting the passage of cells that passed through the first affinity chromatography matrix and did not bind to the first binding agent and that passed through the second affinity chromatography matrix and did not bind to the second binding agent; A second affinity chromatography matrix; c) an output tank; and d) Waste tank A closed apparatus system for purification of target cells, comprising: In a closed system, (i) cells bound to and recovered from said first affinity chromatography matrix; and (ii) cells that pass through and do not bind to the first affinity chromatography matrix and that bind to and are recovered from the second chromatography matrix. configured to allow collection in the output vessel in a single composition; Closed equipment system. 101. The closed device of embodiment 100, wherein one or more of the operable connections comprises tubing connecting the storage reservoir, the first affinity chromatography matrix, the second affinity chromatography matrix, and / or the culture vessel. 102. The closed apparatus of embodiment 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; A closed device system according to any one of embodiments 100 to 102. 104. d) a third affinity chromatography matrix comprising a third binding agent immobilized on its surface, the binding agent specifically binding to a third cell surface marker, the third binding agent being capable of binding to cells expressing the third cell surface marker, wherein: a third operably connecting 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 allows cells that have passed through and not bound to the third column to be directed to the waste container; Third Affinity Chromatography Matrix The closed apparatus system of any of embodiments 100 to 103, further comprising: 105. d) a third affinity chromatography matrix comprising a third binding agent immobilized on its surface, the binding agent specifically binding to a third cell surface marker, the third affinity chromatography matrix being capable of binding to cells expressing the third cell surface marker, wherein a second operable connection further operably connects the third affinity chromatography matrix to the first matrix and to an output container, such that the second operable connection allows cells bound to and recovered from the first matrix and cells bound to and recovered from the third matrix to be delivered to the output container; Third Affinity Chromatography Matrix The closed apparatus system of any of embodiments 100 to 103, further comprising: 106. the first affinity chromatography matrix comprises a binding agent that specifically binds to CD8; a second affinity chromatography matrix comprising a binding agent that specifically binds CD4; A third affinity chromatography matrix is ​​provided for the central memory T (T CM ) a binding agent that specifically binds to a marker expressed on a cell, The closed device system of embodiment 104 or 105. 107. The closed device system of embodiment 106, wherein the binding agent 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 apparatus system of any 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 competitive reagents. 109. The closed apparatus system of any of embodiments 100-107, wherein the competitive reagent is one or more of the group consisting of biotin, a biotin analogue, and a peptide capable of binding to a chromatography matrix. 110. The closed apparatus system of any 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 apparatus system of embodiment 110, wherein the competitor removal chamber further comprises a binding reagent. 112. 112. The closed apparatus system of embodiment 111, wherein the binding reagent comprises one or more of the group consisting of streptavidin, a streptavidin analogue or mutant protein, avidin, and an avidin analogue or mutant protein. 113. The closed device system of any of embodiments 100-112, wherein one or more or all of the binding agents are antibodies. 114. The closed apparatus system of any of embodiments 100-112, wherein one or more or all of the binding agents are reversibly bound to an affinity chromatography matrix. EXAMPLES

[0319] VI. Working 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 CD4+ and CD8+ T cell compositions using a single step flow by immunomagnetic separation for genetic manipulation and adoptive cell therapy In one exemplary method, apheresis product sample is isolated from which central memory T cells are enriched for CD4 + T cell populations and CD8 + The T cell population is isolated, then incubated, manipulated, and subsequently administered to the subject. The isolation procedure is performed using a single step flow by immunomagnetic separation using the CliniMACS® Prodigy system. The procedure is streamlined compared to other methods, using the CliniMACS® Prodigy device and three separate tubing sets to isolate CD4 + Cells were isolated from the first apheresis fraction and identified as CD8 + Cells are separated from the second apheresis fraction and further depleted / enriched.

[0321] The streamlined process is CD4 + and CD8 + Isolation of a cell population is accomplished using one tubing set without transferring the cell population from one vessel (eg, tubing set) to another.

[0322] CD4 was detected by incubating the apheresis sample with CliniMACS® CD4 Reagent. + The cell populations are then isolated. The cells are then separated by a CliniMACS® Prodigy device set to run the enrichment program, and both cell fractions (i.e., immunomagnetically selected enriched CD4 + The enriched (positive) fraction is the fraction containing the isolated CD4 + The flow-through (negative) fraction was incubated with CliniMACS® CD14 and CliniMACS® CD45RA or CD19 reagents to identify CD8 +Isolate the T cell population. Set the CliniMACS® Prodigy device to run the depletion program. The cell / reagent mixture is then separated by the CliniMACS® Prodigy device using the same tubing set used in the first separation step. CD14 + / CD45RA + The flow-through (negative) fraction, depleted of CD14+ / CD19+ cells, is incubated with CliniMACS® CD62L reagent. Using the same tubing set, the cell / reagent mixture is separated using a CliniMACS® Prodigy device running the enrichment program. The positive fraction is isolated CD8 enriched for central memory cells. + It is a cell mass.

[0323] The isolated CD4+ and CD8+ populations are combined in a culture starting composition at a culture starting ratio in the same culture vessel. After incubation and / or manipulation steps, the culture starting ratio is designed to achieve a particular desired output ratio, or a ratio that is within a particular margin of error of such desired output ratio, or is designed to do so for a particular percentage of time. The cells are incubated under stimulatory conditions such as using anti-CD3 / anti-CD28 beads in the Prodigy system in the presence of IL-2 (100 IU / mL) for 72 hours at 37°C.

[0324] The cell composition is evaluated and / or adjusted one or more times after the start of incubation or culturing, for example at certain times during incubation, and optionally periodically. Evaluation includes measuring the growth rate, measuring viability, determining the phenotype, for example the expression of one or more surface or intracellular markers such as proteins or polynucleotides, and / or adjusting the composition or bath for the presence or absence or amount or relative amounts of one or more factors, agents, components, and / or cell types, including subtypes.

[0325] The cells so incubated are then 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. + Cells and CD8 + The transfer is carried out in a contained environment within the CliniMACS® Prodigy device using engineered CD4 + T cells and CD8 + Producing an output composition comprising T cells.

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

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

[0328] The first selection column 1 contains a selected volume of affinity chromatography matrix 3, such as an agarose resin, such as a resin as described in US Patent Application Publication No. 2015 / 0024411 for the isolation of T cells, with an exclusion limit designed to be larger than the size of a T cell, for example an exclusion size of 6×10 6The first column contains agarose, such as that obtained from Agarose Beads Technologies, Madrid, Spain, having a smaller exclusion size than Superflow™ agarose, having 100 daltons. The matrix in the first column is coupled to Strep-Tactin® multimers (e.g., comprising a streptavidin variant shown in any of SEQ ID NOs: 12, 13, 15 or 16 (IBA GmbH, Germany), or a streptavidin variant described in PCT application WO2014 / 076277). The second selection column 2 contains a selected volume of affinity chromatography matrix 4, such as an agarose resin, such as that described above, which matrix is ​​also coupled to Strep-Tactin® multimers.

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

[0330] The anti-CD4 Fab is added to the second selection column 2 by loading a reservoir containing anti-CD4 Fab fragments 19 and flowing through pump 8, tubing, and valve 13, whereby the anti-CD8 Fab becomes immobilized to the Strep-Tactin® on the affinity matrix 4 by the 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 is directed to waste container 10 via valve 13 operably connecting the second selection column to the waste container. In some embodiments, the wash step is repeated multiple times.

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

[0332] To achieve cell selection, the apheresis sample is added to the first selection column 1, whereby CD8+ T cells, if present in the sample, remain bound to the resin 3 of the first column, and unselected cells (containing CD8- cells) pass through the column. The number of cells in the starting sample used in some examples is chosen to exceed the number of cells to be selected based on the total volume of the matrix, for example, a volume 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, for example, at least about 10% or 20% more). The flow-through fraction containing unselected cells (negative fraction) is then passed from the first column to the second selection column 2 via operably connected tubing. The 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 the waste container 10 via a valve 13 operably connecting the second selection column to 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] From wash buffer reservoir 6, wash buffer is flushed through the first and second columns via operably connected tubing. The flow-through, containing any cells washed out of the first and second columns, is directed to waste container 10 via valve 13 operably connecting the second selection column to the waste container. In some embodiments, the wash step is repeated multiple times.

[0335] From the elution buffer reservoir 7, a buffer containing an eluent at a low concentration of biotin or an analogue thereof, such as 2.5 mM desthiobiotin, is loaded and passed through the first and second columns via operably connected tubing. In some embodiments, the eluent contains cell culture medium. The flow-through containing the enriched CD4+ and CD8+ cells and residual biotin or analogue is directed to a removal chamber 9 to remove the biotin or analogue. The removal chamber 9 is a column of Strep-Tactin®-immobilized Superflow™ Sepharose® beads, such as a column with a total volume of 6 mL with a binding capacity of 300 nmoles of biotin / mL, with a volume sufficient to remove the biotin or biotin analogue from the sample. The flow-through containing the enriched cells positively selected for CD4+ and CD8+ is directed to a culture vessel 12, such as a bag, via a valve operably connecting the removal chamber 9 and the culture vessel. In some embodiments, the elution step is repeated.

[0336] In some embodiments, after the elution step, 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) is directed through the removal chamber to replace the wash buffer, and the flow-through is directed to the culture vessel. The positive fraction collected in the culture vessel is the isolated and combined CD4+ and CD8+ cell populations.

[0337] B. Central Memory T(T CM ) Enrichment of CD4+ and CD8+ T cell populations, enriched for cells expressing markers on the cells. In the exemplary process shown in FIG. 1B, a series of immunochromatographic selection and removal columns are arranged in a closed apparatus 14, which are operably connected to each other and to a peristaltic pump 8 via various tubing lines and valves 13 for controlling the flow of 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. In addition, the process further includes a third selection column 15 containing 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] Anti-CD8 Fab and anti-CD4 Fab described in Example 2A are added to the first and second columns, respectively. Central memory T (T CM ) A reservoir 20 containing additional Fab fragments against a marker expressed on the cells is loaded and flowed through pump 8, tubing, and valve 13 to add additional Fab to the third selection column 15, whereby the additional Fab becomes conjugated to Strep-Tactin® on affinity matrix 17 by Twin Strep-Tag® (SEQ ID NO:10; IBA GmbH) fused to the carboxy terminus of its heavy chain as described in Example 2A on the Fab fragment. In some embodiments, a wash buffer as described in Example 2A is loaded and flowed through the third selection column via operably connected tubing. The flow through is directed to waste container 10 via valve 13 operably connecting the third selection column to the waste container. In some embodiments, the wash step is repeated multiple times.

[0340] The volumes of affinity matrix reagent contained in the first selection column, the second and / or the third selection column may be the same or different and may be determined according to the desired yield of the selection and / or the amount of affinity matrix reagent in the central memory T (T CM) Cells expressing a marker expressed on the cells can be selected based on the desired ratio of enriched CD4+ cells to CD8+ cells. The volume so selected is such that the average selectable yield is 1×10 cells per mL of packed column volume. 8 It is based on the assumption that the number of cells in a given starting culture is 100. Also, the specific starting ratio of CD4+ cells and central memory T (T CM For a 1:1 starting culture ratio with a CD8+ cell-containing population enriched for a marker expressed on the .) cells (e.g., a population of CD8+ cells enriched based on further selection for one of CD28, CD62L, CCR7, CD27, or CD127), the volume of the matrix for selecting the enriched population, e.g., the parent population of CD8+ cells, is greater than the matrix used to select the CD4+ T cell population or the other of the CD8+ T cell populations, e.g., the CD4+ population. The amount or extent to which the volume of the matrix, e.g., the matrix containing anti-CD8 Fab fragments, used to select for the parent population of the further enriched population is greater than the volume of the other matrix or matrices is chosen based on the proportion 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 proportion or percentage of the parent population, e.g., CD8+ cells, present in the sample. This can be estimated based on averages among patients or healthy donors, or it can be measured for a given patient for which a selection is being made before determining the size of column to use.

[0341] In an exemplary process, the column volumes used in the process 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. CM) To achieve a desired starting ratio of enriched CD8+ cells, e.g., CD8+ / CD28+, CD8+ / CD62L+, CD8+ / CCR7+, CD8+ / CD27+ or CD8+ / CD127+ cells, expressing a marker expressed on the cells, the column length and / or column diameter can be chosen to accommodate the desired volume. In some embodiments, to accommodate the volume of the affinity matrix, multiple columns with the same Fab reagent immobilized can be added in direct succession and operably connected to each other via tubing lines.

[0342] To achieve cell selection, the apheresis sample is added to the 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 is chosen to exceed the number of cells to be selected based on the total volume of the matrix, e.g., greater than the capacity of the column for each selection (e.g., greater than 200 million CD8+ / CD62L+ cells and greater than 200 million CD4+ cells, e.g., at least about 10% or 20% more). The flow-through fraction (negative fraction) containing unselected cells is directed to pass through to the second column 2 via valve 13 operably connecting the first and second columns. CD4+ T cells remain bound to the resin in the second column. The flow-through, containing the fluid and additional unselected cells (negative cells), is directed to a first waste container 10 via a valve 13 operably connecting the second selection column and the negative container.

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

[0344] A buffer containing an eluent, such as biotin or an analogue thereof, e.g., 2.5 mM desthiobiotin, is loaded from an elution buffer reservoir 7 and passed through the second column 2 via a valve 13 and tubing operably connecting the elution buffer reservoir to the second column. In some embodiments, the elution buffer contains cell culture medium. The flow-through containing enriched CD4+ cells and residual biotin is directed to a removal chamber 9 operably connected to the second column via a valve 13 and tubing to remove biotin as described in Example 2A. The flow-through containing enriched cells positively selected for CD4+ is directed to a culture vessel 12, such as a bag, via a valve 13 operably connecting the removal chamber 9 to the culture vessel. In some embodiments, the elution step is repeated. In some embodiments, after the elution step, 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) is replaced with the wash buffer and directed from the wash buffer reservoir through the valve and tubing to the second column, the removal chamber, and the culture vessel. The positive fraction collected in the culture vessel is the isolated CD4+ cell population.

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

[0346] A wash buffer, as described in Example 2A, is loaded from wash buffer reservoir 6 and flushed through the third column via valve 13 and tubing operably connecting the wash buffer reservoir to third column 15. The flow-through, containing any cells washed from the third column, is directed to second waste container 11 via valve 13 operably connecting the third selection column to a second waste container. In some embodiments, the wash step is repeated multiple times.

[0347] A buffer containing an elution agent, such as biotin or an analog thereof, e.g., 2.5 mM desthiobiotin, is loaded from elution buffer reservoir 7 and allowed to flow through third column 15 via valve 13 and tubing operably connecting 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 is eluted. CMThe flow-through, containing enriched CD8+ cells and residual biotin or analogues thereof, is directed to removal chamber 9, which is operably connected to a third column via valve 13 and tubing, to remove biotin or biotin analogues as described in Example 2A. For CD8, and for central memory T (T) such as one of CD28, CD62L, CCR7, CD27 or CD127. CM The flow-through containing enriched T cell memory cells that have been positively selected for a marker expressed on the depletion chamber 9 is directed to a culture vessel 12, such as a bag, via a valve 13 operably connecting the depletion chamber 9 and the culture vessel. In some embodiments, the culture vessel contains a T cell activation reagent, cell culture medium, or both. In some embodiments, the elution step is repeated. In some embodiments, after the elution step, 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) replaces the wash buffer and is directed from the wash buffer reservoir through the valve and tubing to the first and / or third columns, the depletion chamber, and into the culture vessel. CD8+ cells and central memory T (T) cells, such as one of CD28, CD62L, CCR7, CD27, or CD127, are depleted. CM The positive fraction containing cells positive for a marker on CD4+ cells is collected in a culture vessel along with the previously collected CD4+ positive fraction. In some embodiments, the process steps include, for example, collecting CD8+ cells and cells positive for a marker on central memory T (T CM ) cells that are positive for the marker on the cells, followed by enrichment for CD4+ cells.

[0348] Example 3: Generation of compositions of CD4+ and CD8+ T cells by sequential purification in a closed system for use in genetic manipulation and adoptive cell therapy This example describes a procedure for selecting and generating a cell composition containing CD4+ and CD8+ T cells, e.g., CD4+ and CD8+ T cells present at a culture starting ratio, for incubation / activation and transduction in methods involving genetic engineering of cells for use in the context of adoptive cell therapy.

[0349] The composition of cells generated by the selection of CD4+ 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 stimulatory conditions, for example, using anti-CD3 / anti-CD28 in the presence of IL-2 (100 IU / mL), for 72 hours, for example, at 37°C. The stimulated cells are then genetically engineered by introducing into the cells, for example, by viral transduction, a recombinant gene for the expression of a recombinant antigen receptor, such as a chimeric antigen receptor (CAR) or a recombinant TCR. In some embodiments, following introduction, the cells are further incubated, typically at 37°C, for example, to allow the cells to grow.

[0350] The method comprises administering to a subject an engineered CD4 + T cells and CD8 + Produces an output composition having T cells. In some embodiments, based on the chosen volume of the selection column, the ratio of CD4+ cells to CD8+ cells in the composition incubated under stimulatory conditions prior to manipulation (start-of-culture ratio) produces, following incubation, stimulation and / or manipulation steps, a particular desired output ratio of CD4+ cells to CD8+ cells or engineered CD4+ cells to engineered CD8+ cells, or such ratio within a particular tolerance of such desired output ratio. In some embodiments, such desired output ratio or ratio within a tolerance is achieved a particular percentage of time with allowance.

[0351] Example 4: Selection of cells using suboptimal yield concentrations of Fab-coated surfaces Human apheresis-derived PBPC samples with a range of different cell numbers were incubated with magnetic microbeads conjugated with anti-CD4 Fab and magnetic microbeads conjugated with anti-CD8 Fab in a single composition for approximately 30 minutes with gentle mixing. This incubation was followed by elution of unselected cells and recovery using a magnetic field, performed as described above. Incubations with 0.05-1.5 mL of each microbead reagent per million cells produced low yields of CD4+ or CD8+ cells, respectively (cells recovered for each selection compared to positive cells in incubation, which generally ranged from 15-70% in this study). On average, greater yields were observed with higher reagent concentrations per cell when such suboptimal yield concentrations were used.

[0352] Thus, ...

Claims

1. 1. A composition comprising a dose of genetically engineered CD4+ and CD8+ T cells for use in a subject with an autoimmune or inflammatory disease, comprising: the CD4+ and CD8+ T cells are in a ratio of between 5:1 and 1:5; the specific dose of CD4+ and CD8+ T cells is between 1×10 6 cells and 1×10 9 cells; and The CD4+ and CD8+ T cells are genetically engineered to express an antigen receptor that recognizes CD19. composition.

2. The particular dose of CD4+ and CD8+ T cells is 5×10 6 ~500×10 6 The composition of claim 1 , wherein the cell is a human cell.

3. The composition of claim 1, wherein the specific dose of CD4+ and CD8+ T cells is between 1 x 106 cells and 25 x 106 cells.

4. The composition according to any one of claims 1 to 3, wherein the specific dose of CD4+ and CD8+ T cells is 5 x 10 6 cells.

5. The composition according to any one of claims 1 to 3, wherein the specific dose of CD4+ and CD8+ T cells is 10 x 10 6 cells.

6. The particular dose of CD4+ and CD8+ T cells is 20×10 6 ~60×10 6 The composition of claim 1 or 2, which is a cell.

7. 7. The composition of any one of claims 1 to 6, wherein the autoimmune or inflammatory disease is selected from the group consisting of rheumatoid arthritis, type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and transplant-related diseases or conditions.

8. The composition according to any one of claims 1 to 7, wherein the autoimmune or inflammatory disease is multiple sclerosis.

9. The composition according to any one of claims 1 to 7, wherein the autoimmune or inflammatory disease is scleroderma.

10. The composition according to any one of claims 1 to 7, wherein the autoimmune disease or inflammatory disease is rheumatoid arthritis.

11. The composition of any one of claims 1 to 10, wherein the antigen receptor comprises a chimeric antigen receptor (CAR).

12. The composition of claim 11, wherein the CAR comprises an Fv that binds to CD19, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory signaling domain and a CD3 zeta signaling domain.

13. The composition of claim 12, wherein the costimulatory signaling domain is a 4-1BB signaling domain.

14. The composition of claim 12, wherein the costimulatory signaling domain is a CD28 signaling domain.

15. The composition of any one of claims 1 to 14, wherein the CD4+ and CD8+ are autologous to the subject.

16. 1. Use of a composition comprising a dose of engineered T cells in the manufacture of a medicament for treating a subject having an autoimmune or inflammatory disease, comprising CD4+ and CD8+ T cells in a ratio between 5:1 and 1:5; the specific dose of CD4+ and CD8+ T cells is between 1×10 6 cells and 1×10 9 cells; and The CD4+ and CD8+ T cells are genetically engineered to express an antigen receptor that recognizes CD19. use.

17. The particular dose of CD4+ and CD8+ T cells is 5×10 6 ~500×10 6 The use according to claim 16, wherein the cell is a single cell.

18. 17. The use according to claim 16, wherein the particular dose of CD4+ and CD8+ T cells is between 1x106 cells and 25x106 cells.

19. The use according to any one of claims 16 to 18, wherein the specific dose of CD4+ and CD8+ T cells is 5x106 cells.

20. The use according to any one of claims 16 to 18, wherein the specific dose of CD4+ and CD8+ T cells is 10x106 cells.

21. The particular dose of CD4+ and CD8+ T cells is 20×10 6 ~60×10 6 18. The use according to claim 16 or 17, wherein the cell is a single cell.

22. 22. The use according to any one of claims 16 to 21, wherein the autoimmune or inflammatory disease is selected from the group consisting of rheumatoid arthritis, type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and transplant-related diseases or conditions.

23. The use according to any one of claims 16 to 22, wherein the autoimmune or inflammatory disease is multiple sclerosis.

24. The use according to any one of claims 16 to 22, wherein the autoimmune or inflammatory disease is scleroderma.

25. The use according to any one of claims 16 to 22, wherein the autoimmune or inflammatory disease is rheumatoid arthritis.

26. The use according to any one of claims 16 to 25, wherein the antigen receptor comprises a chimeric antigen receptor (CAR).

27. The use of claim 26, wherein the CAR comprises an Fv that binds to CD19, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory signaling domain and a CD3 zeta signaling domain.

28. 28. The use of claim 27, wherein the costimulatory signaling domain is a 4-1BB signaling domain.

29. 28. The use of claim 27, wherein the costimulatory signaling domain is a CD28 signaling domain.

30. The use according to any one of claims 16 to 29, wherein the CD4+ and CD8+ cells are autologous to the subject.

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