Methods for t cell transduction

Abstract

To provide methods for transduction of T cells.SOLUTION: In some embodiments, the provided methods include transduction of T cells by incubation with a retroviral vector particle, e.g., lentiviral vector, in which the cells have been selected for CCR7+ expression. The provided methods improve the process for genetically engineering T cells by increasing transduction frequency and / or by reducing the variability in transduction frequency among biological samples. Also provided are resulting cells transduced with a recombinant or heterologous gene, and compositions thereof. In some embodiments, the provided cells and compositions can be used in methods of adoptive immunotherapy.SELECTED DRAWING: Figure 2B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 967,005, filed January 28, 2020, entitled "METHODS FOR T CELL TRANSDUCTION," the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The contents of the following ASCII text file submission are incorporated herein by reference in their entirety: A Sequence Listing in Computer Readable Format (CRF) is provided as a file named 73504_2023140_SEQLIST.txt. This file was created on January 27, 2021, and is 53,345 bytes in size.

[0003] Field The present disclosure provides a method for transducing T cells. In some embodiments, the provided method includes transducing T cells by incubating cells selected for CCR7+ expression with retroviral vector particles, such as lentiviral vectors. In some embodiments, such a method improves the process for genetically modifying T cells by increasing the transduction frequency and / or reducing the variability of the transduction frequency between biological samples. The resulting cells transduced with recombinant genes or heterologous genes, such as genes encoding chimeric receptors, such as chimeric antigen receptors, or other recombinant antigen receptors, such as transgenic T cell receptors, and compositions thereof, are also provided. In some embodiments, the provided cells and compositions can be used in adoptive immunotherapy methods. [Background technology]

[0004] background A variety of strategies are available for transducing T cell populations in vitro, including strategies for transducing T cells in vitro for use in adoptive cellular immunotherapy or cancer therapy. Improved strategies are needed for transducing cell populations in vitro for research, diagnostic, and therapeutic purposes, thereby increasing transduction frequencies and increasing consistency between biological samples. Methods are provided that meet this need. Summary of the Invention

[0005] overview Provided herein is a method for increasing the transduction frequency of primary T cells, the method comprising: (a) selecting primary T cells that are positive for surface expression of CCR7 from a biological sample comprising a population of primary T cells, thereby generating an input population that is enriched for CCR7+ primary T cells; (b) optionally, incubating the input population under stimulatory conditions, thereby generating a stimulated composition, the stimulatory conditions comprising the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and (c) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with T cells of the input cell population, or optionally with T cells of the stimulated composition, thereby generating a population of transduced cells.

[0006] Also provided herein is a method for increasing the transduction frequency of primary T cells, the method comprising: (a) selecting primary T cells that are positive for surface expression of CCR7 from a biological sample comprising a population of primary T cells, thereby generating an input population that is enriched for CCR7+ primary T cells; (b) incubating the input population under stimulatory conditions, thereby generating a stimulated composition, the stimulatory conditions comprising the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and (c) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with the T cells of the stimulated composition, thereby generating a population of transduced cells.

[0007] Also provided herein is a method for increasing the transduction frequency of primary T cells, comprising: (a) selecting primary T cells that are positive for surface expression of CCR7 from a biological sample comprising a population of primary T cells, thereby generating an input population that is enriched for CCR7+ primary T cells; and (b) incubating viral vector particles that comprise a heterologous polynucleotide encoding a recombinant protein with T cells of the input cell population, thereby generating a population of transduced cells.

[0008] Also provided herein is a method for increasing the transduction frequency of primary T cells, comprising: (a) incubating an input primary T cell population enriched for CCR7+ T cells under stimulatory conditions, thereby generating a stimulated composition, the stimulatory conditions comprising the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and (b) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with the T cells of the stimulated composition, thereby generating a population of transduced cells.

[0009] Also provided herein is a method for increasing the transduction frequency of primary T cells, comprising incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with T cells of an input primary T cell population that has been enriched for CCR7+ T cells, thereby generating a population of transduced cells.

[0010] In some of any such embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the input population are CCR7+ primary T cells. In some of any such embodiments, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the input population are CCR7+ primary T cells. In some of any such embodiments, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the input population are CCR7+ primary T cells.

[0011] In some of any such embodiments, the selecting step does not include selecting cells that are (a) CCR7+ and CD45RO+, or (b) CCR7+ and CD27+, or (c) CCR7+ and CD45RA-, or (d) CCR7+ and CD62L+, or (e) CCR7+ and CD45RA+, or (f) CCR7+ and CD62L-. In some of any such embodiments, the input population is not enriched for T cells that are (a) CCR7+ and CD45RO+, or (b) CCR7+ and CD27+, or (c) CCR7+ and CD45RA-, or (d) CCR7+ and CD62L+, or (e) CCR7+ and CD45RA+, or (f) CCR7+ and CD62L-. In some of any such embodiments, the input population is not enriched for CCR7+ and CD45RO+ T cells, optionally where less than 85% of the total cells in the input population are CCR7+ and CD45RO+ T cells. In some of any such embodiments, the input population is not enriched for CCR7+ and CD27+ T cells, optionally where less than 85% of the total cells in the input population are CCR7+ and CD27+ T cells. In some of any such embodiments, the input population is not enriched for CCR7+ and CD45RA- T cells, optionally where less than 85% of the total cells in the input population are CCR7+ and CD45RA- T cells. In some of any such embodiments, the input population is not enriched for CCR7+ and CD62L+ T cells, optionally where less than 85% of the total cells in the input population are CCR7+ and CD62L+ T cells. In some of any such embodiments, the input population is not enriched for CCR7+ and CD45RA+ T cells, and optionally, less than 85% of the total cells in the input population are CCR7+ and CD45RA+ T cells. In some of any such embodiments, the input population is not enriched for CCR7+ and CD62L- T cells, and optionally, less than 85% of the total cells in the input population are CCR7+ and CD62L- T cells.

[0012] In some of any such embodiments, the biological sample is a blood sample. In some of any such embodiments, the biological sample is a leukapheresis sample.

[0013] In some of any such embodiments, the T cells are unfractionated T cells, or enriched or isolated CD3+ T cells, or enriched or isolated CD4+ T cells, or enriched or isolated CD8+ T cells.

[0014] In some of any such embodiments, the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells or CD8+ T cells. In some of any such embodiments, the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells. In some of any such embodiments, the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD8+ T cells. In some of any such embodiments, the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells and CD8+ T cells. In some of any such embodiments, the ratio of CD4+ T cells to CD8+ T cells is at or about 1:1, 1:2, 2:1, 1:3, or 3:1. In some of any such embodiments, the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD3+ T cells.

[0015] In some of any such embodiments, the input population is 100×10 6 ~500×10 6 In some of any such embodiments, the input population comprises 200 x 10 total T cells. 6 ~400×10 6Total T cells, optionally 300 x 10 6 or approximately 300 x 10 6 In some of any such embodiments, the total T cells are viable T cells.

[0016] In some of any such embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of the cells of the stimulated composition (i) express a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB; (ii) comprise intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-gamma, and TNF-alpha; (iii) are in the G1 phase or later of the cell cycle; and / or (iv) have proliferative capacity.

[0017] In some of any such embodiments, the stimulatory reagent comprises a primary agent that specifically binds to a member of the TCR complex, optionally specifically binds to CD3. In some of any such embodiments, the stimulatory reagent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, optionally the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS. In some of any such embodiments, the primary and / or secondary agent comprise antibodies, and optionally the stimulatory reagent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody or antigen-binding fragment thereof.

[0018] In some of any such embodiments, the primary agent and / or secondary agent are present on the surface of a solid support. In some of any such embodiments, the solid support is or includes beads. In some of any such embodiments, the primary agent and secondary agent are reversibly bound to the surface of an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules. In some of any such embodiments, each of the plurality of streptavidin or streptavidin mutein molecules contains a Val at a sequence position corresponding to positions 44-47 in streptavidin in the sequence of amino acids set forth in SEQ ID NO:34. 44 -Thr 45 -Ala 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47In some of any such embodiments, each of the plurality of streptavidin molecules or streptavidin mutein molecules is or comprises: a) a sequence of amino acids set forth in SEQ ID NO:35 or 56, or b) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the sequence of amino acids set forth in SEQ ID NO:35 or 56, or c) a functional fragment of a) or b) that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide. In some of any such embodiments, each of the plurality of streptavidin or streptavidin mutein molecules is a streptavidin mutein molecule, and each of the plurality of streptavidin mutein molecules has: a) a sequence of amino acids set forth in any of SEQ ID NOs: 36, 41, 48-50, or 53-55; b) a sequence of amino acids set forth in any of SEQ ID NOs: 36, 41, 48-50, or 53-55; and (c) a functional fragment of a) or b) that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide; and optionally each of the plurality of streptavidin mutein molecules is or comprises a sequence of amino acids corresponding to SEQ ID NO:36 or SEQ ID NO:36, 41, 48-50, or 53-55, and It is or comprises the amino acid sequence shown in SEQ ID NO:41.

[0019] In some of any such embodiments, the population of transduced cells comprises at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cells that express the recombinant protein. In some of any such embodiments, the population of transduced cells comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cells that express the recombinant protein.

[0020] In some of any such embodiments, the percentage of cells in the population of transduced cells that express the recombinant protein is at least 0.5-fold, at least 1-fold, at least 1.5-fold, or at least 2-fold greater compared to a cell composition that has not been enriched for CCR7+ primary T cells by the selection step.

[0021] In some of any such embodiments, incubating the viral vector particles comprises subjecting the viral vector particles to spinoculation with an input population. In some of any such embodiments, incubating the viral vector particles comprises subjecting the viral vector particles to spinoculation with a stimulated composition.

[0022] In some of any such embodiments, the spinoculation step comprises rotating the viral vector particles and the input population in an internal cavity of a centrifugation chamber, the rotation being between 500 g and 2500 g, 500 g and 2000 g, 500 g and 1600 g, 500 g and 1000 g, 600 g and 1600 g, 600 g and 1000 g, 1000 g and 2000 g, or 1000 g and 1600 g, or about 500 g and The centrifugal force is at a relative centrifugal force at the inner surface of the cavity side wall of 2500g, 500g to 2000g, 500g to 1600g, 500g to 1000g, 600g to 1600g, 600g to 1000g, 1000g to 2000g, or 1000g to 1600g, or at least 600g, 800g, 1000g, 1200g, 1600g, or 2000g, or at least about 600g, 800g, 1000g, 1200g, 1600g, or 2000g. In some of any such embodiments, the spinoculation step comprises rotating the viral vector particles and the stimulated composition in an internal cavity of a centrifuge chamber, the rotation being between 500 g and 2500 g, 500 g and 2000 g, 500 g and 1600 g, 500 g and 1000 g, 600 g and 1600 g, 600 g and 1000 g, 1000 g and 2000 g, or 1000 g and 1600 g, or about 500 g and The centrifugal force is at a relative centrifugal force at the inner surface of the cavity side wall of 2500g, 500g to 2000g, 500g to 1600g, 500g to 1000g, 600g to 1600g, 600g to 1000g, 1000g to 2000g, or 1000g to 1600g, or at least 600g, 800g, 1000g, 1200g, 1600g, or 2000g, or at least about 600g, 800g, 1000g, 1200g, 1600g, or 2000g.

[0023] In some of any such embodiments, the spinoculation step is for a time period that is greater than or about 5 minutes, greater than or about 10 minutes, greater than or about 15 minutes, greater than or about 20 minutes, greater than or about 30 minutes, greater than or about 45 minutes, greater than or about 60 minutes, greater than or about 90 minutes, or greater than or about 120 minutes, or between 5 and 60 minutes, 10 and 60 minutes, 15 and 60 minutes, 15 and 45 minutes, 30 and 60 minutes, or 45 and 60 minutes, inclusive.

[0024] In some of any such embodiments, the method further comprises contacting the input population and / or viral vector particles with a transduction adjuvant during at least a portion of the incubating step. In some of any such embodiments, the method further comprises contacting the primed composition and / or viral vector particles with a transduction adjuvant during at least a portion of the incubating step. In some of any such embodiments, the method further comprises contacting the primed composition and / or viral vector particles with a transduction adjuvant during at least a portion of the incubating step.

[0025] In some of any such embodiments, the method further comprises contacting the input population and / or viral vector particles with a transduction adjuvant during at least a portion of the incubating step.

[0026] In some of any such embodiments, the contacting step is performed before, simultaneously with, or after subjecting the viral vector particles to spinoculation with the input population. In some of any such embodiments, the contacting step is performed before, simultaneously with, or after subjecting the viral vector particles to spinoculation with the stimulated composition.

[0027] In some of any such embodiments, at least a portion of the incubation of viral vector particles is carried out at or about 37°C ± 2°C. In some of any such embodiments, said at least a portion of the incubation of viral vector particles is carried out after spinoculation. In some of any such embodiments, said at least a portion of the incubation of viral vector particles is carried out for less than or equal to about 2, 4, 12, 18, 24, 30, 36, 48, 60, or 72 hours. In some of any such embodiments, said at least a portion of the incubation of viral vector particles is carried out for at or about 24 hours. In some of any such embodiments, the total duration of the incubation of viral vector particles is less than or equal to 12, 24, 36, 48, or 72 hours.

[0028] In some of any such embodiments, the viral vector particle is a lentiviral vector particle. In some of any such embodiments, the lentiviral vector particle is replication-deficient. In some of any such embodiments, the viral vector particle is pseudotyped with a viral envelope glycoprotein. In some of any such embodiments, the viral envelope glycoprotein is VSV-G.

[0029] In some of any such embodiments, the viral vector particles are incubated at a multiplicity of infection of less than or about 20.0 or less than or about 10.0. In some of any such embodiments, the viral vector particles are incubated at a multiplicity of infection of, or about, 1.0 IU / cell to 10 IU / cell or 2.0 U / cell to 5.0 IU / cell, or the viral vector particles are incubated at a multiplicity of infection of, or about, 1.0 IU / cell to 10 IU / cell or 2.0 U / cell to 5.0 IU / cell, or the viral vector particles are at a multiplicity of infection of, or about, 1.6 IU / cell, 1.8 IU / cell, 2.0 IU / cell, 2.4 IU / cell, 2.8 IU / cell, 3.2 IU / cell, 3.6 IU / cell, 4.0 IU / cell, 5.0 IU / cell, 6.0 IU / cell, 7.0 IU / cell, 8.0 IU / cell, 9.0 IU / cell, or 10.0 IU / cell. The cells are incubated at a multiplicity of infection of 1.1 IU / cell, 3.2 IU / cell, 3.6 IU / cell, 4.0 IU / cell, 5.0 IU / cell, 6.0 IU / cell, 7.0 IU / cell, 8.0 IU / cell, 9.0 IU / cell, or 10.0 IU / cell.

[0030] In some of any such embodiments, the stimulated composition comprises at least 50×10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately at least 50 × 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately 50 x 10 6 cells, 100×10 6 cells or 200 x 10 6 In some of any such embodiments, the stimulated composition comprises 50×10 cells, inclusive. 6 or about 50 x 10 6 cells ~300×10 6 or approximately 300 x 10 6In some of any such embodiments, the stimulated composition comprises 100×10 cells, inclusive. 6 or about 100 x 10 6 cells ~200×10 6 or approximately 200 x 10 6 Contains cells.

[0031] In some of any such embodiments, the input population is at least 50 x 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately at least 50 × 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately 50 x 10 6 cells, 100×10 6 cells or 200 x 10 6 In some of any such embodiments, the input population comprises 50 x 10 cells, inclusive. 6 or about 50 x 10 6 cells ~300×10 6 or approximately 300 x 10 6 In some of any such embodiments, the input population comprises 100 x 10, inclusive. 6 or about 100 x 10 6 cells ~200×10 6 or approximately 200 x 10 6 Contains cells.

[0032] In some of any such embodiments, the T cells incubated with the viral particles are at least 50 x 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately at least 50 × 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately 50 x 10 6 cells, 100×10 6 cells or 200 x 10 6In some of any such embodiments, the T cells incubated with the viral particles comprise 50 x 10, inclusive. 6 or about 50 x 10 6 cells ~300×10 6 or approximately 300 x 10 6 In some of any such embodiments, the T cells incubated with the viral particles comprise 100 x 10, inclusive. 6 or about 100 x 10 6 cells ~200×10 6 or approximately 200 x 10 6 Contains cells.

[0033] In some of any such embodiments, the recombinant protein is an antigen receptor. In some of any such embodiments, the antigen receptor is a transgenic T cell receptor (TCR). In some of any such embodiments, the antigen receptor is a chimeric antigen receptor (CAR). In some of any such embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain comprising an ITAM. In some of any such embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to a target antigen, an intracellular signaling domain comprising an ITAM, and a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some of any such embodiments, the intracellular signaling domain comprises the intracellular domain of the CD3 zeta (CD3ζ) chain. In some of any such embodiments, the CAR further comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some of any such embodiments, the transmembrane domain comprises the transmembrane portion of CD28. In some of any such embodiments, the intracellular signaling domain further comprises an intracellular signaling domain of a T cell costimulatory molecule, hi some of any such embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.

[0034] In some of any such embodiments, the antigen receptor specifically binds to an antigen associated with a disease or condition or specifically binds to a universal tag, hi some of any such embodiments, the disease or condition is cancer, an autoimmune disease or disorder, or an infectious disease.

[0035] In some of any such embodiments, the population of transduced cells comprises T cells that have been transduced with a heterologous polynucleotide.

[0036] In some of any such embodiments, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of T cells in a population of transduced cells are transduced with a heterologous polynucleotide. In some of any such embodiments, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of T cells in a population of transduced cells are transduced with a heterologous polynucleotide. In some of any such embodiments, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of T cells transduced with a heterologous polynucleotide are CCR7+.

[0037] In some of any such embodiments, the method further comprises recovering or isolating the transduced T cells produced by the method from the population of transduced cells.

[0038] In some of any such embodiments, the variation in the percentage of T cells in a population of transduced cells that are transduced with the heterologous polynucleotide among multiple populations of transduced cells is 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less.

[0039] In some of any such embodiments, the method is carried out in vitro or ex vivo.

[0040] Also provided herein is a composition comprising a population of transduced cells produced by the method of any one of the embodiments provided herein. In some of any such embodiments, the composition further comprises a cyropreservant. [Brief explanation of the drawings]

[0041] [Figure 1] FIG. 1 is a graph depicting the results of a vector titration experiment demonstrating the variability in maximal transducible frequencies of T cells among six donors. [Figure 2A] Figures 2A and 2B show the results of a study evaluating transduction frequencies among T cell subsets from samples obtained from three healthy donors. In each of Figures 2A and 2B, the upper left panel represents the T cell composition before activation and transduction (input composition or population or selected composition or population), the upper right panel represents the resulting transduction frequencies in those T cell subsets, and the lower panel represents the proportion of transduced T cells represented by each population. [Figure 2B] See legend to Figure 2A. [Figure 3A] Figures 3A and 3B show the results of a larger-scale study evaluating transduction frequencies among T cell subsets from samples obtained from six healthy donors. In each of Figures 3A and 3B, the upper left panel represents the T cell composition before activation and transduction (input composition or population or selected composition or population), the upper right panel represents the resulting transduction frequencies in those T cell subsets, and the lower panel represents the proportion of transduced T cells represented by each population. [Figure 3B] See legend to Figure 3A. [Figure 4]Figure 4 shows the results of a study evaluating the frequency of selected CD4 and CD8 T cell subpopulations that are CCR7+ and CCR7- by flow cytometry in samples obtained from patients with relapsed / refractory large B-cell lymphoma (n=145). The boxes in Figure 4 indicate the interquartile range, and the whiskers indicate the full range. DETAILED DESCRIPTION OF THE INVENTION

[0042] Detailed Description Methods are provided for increasing the transduction frequency of primary T cells by selecting or otherwise obtaining a population of primary T cells enriched for surface expression of CCR7 prior to or in conjunction with performing the transduction.

[0043] Generally, retroviral-based vectors, such as lentiviral vectors, can be used to stably integrate a gene of interest into cells. Among primary cells, T cells are particularly difficult to transduce with retroviral-based vectors. In some cases, transduction efficiency can be improved by first activating the cells with stimulatory agents (e.g., anti-CD3 / anti-CD28). Activation has been shown to increase LDL receptor expression in some cases, thereby enhancing lentiviral vector uptake. Typically, T cells are activated for at least one day (sometimes up to three days or more) prior to transduction for use in adoptive T cell therapy. For example, lentiviral transduction protocols for T cells typically require at least 24 hours of activation prior to transduction (Amirache et al. (2014) Blood, 123:1422-1424). In some instances, available procedures for preparing genetically modified T cells for adoptive immunotherapy may require sequential ex vivo steps of selection, activation, transduction, and expansion.

[0044] Current transduction methods may not be entirely satisfactory, particularly in the context of adoptive cell therapy. For example, as shown herein, transduction frequencies can vary widely between cell populations from different subjects, and the effect is donor-dependent. Variations in transduction frequencies can further lead to variability in the administration of therapeutic cell compositions, for example, because the total number of cells that would need to be administered to achieve a threshold number of cells positive for a heterologous gene (e.g., a recombinant receptor such as a chimeric antigen receptor) can vary significantly between different subjects, or because the frequency of cells positive for a heterologous gene varies if the administration strategy is based on a threshold number of total cells. In addition, variability in transduction frequencies can also have a strong impact on pharmaceutical preparation and manufacturing processes, such as the time to cell harvest, if the transduction frequency is a metric used to monitor the success of the modification process at one or more steps of the method.

[0045] The findings described herein demonstrate that certain subpopulations of T cells exist that are more likely to be transduced than others. This may explain the variability in transduction frequencies resulting from the variability of such subpopulations between different subjects, resulting in discrepancies in transduction frequencies among multiple cell populations from different subjects as well as lower transduction frequencies in some cell populations. Specifically, the provided methods are based on the finding that CCR7 expression varies significantly among cell populations from different subjects, with CCR7+ T cells exhibiting higher transduction frequencies than CCR7- T cells. Thus, the provided methods are advantageous in that they include a step of selecting primary T cells positive for surface expression of CCR7 for use in transduction, or otherwise obtaining primary T cells enriched for CCR7+ T cells, thereby increasing the transduction frequency of a cell population and simultaneously reducing the variability in transduction frequencies among cell populations from different patients.

[0046] The provided methods involve transducing an input population (hereinafter also referred to as an input composition) of cells enriched and / or selected for CCR7-positive cells. In some embodiments, the provided methods involve selecting primary T cells that are positive for surface expression of CCR7 from a population of primary T cells, e.g., as described in Section IA, thereby generating an input population enriched for CCR7+ primary T cells. In some embodiments, the input population enriched for CCR7+ primary T cells is incubated with viral vector particles containing a heterologous gene (encoding a heterologous or recombinant protein) under conditions to transduce cells within the population. Optionally, an input population enriched for CCR7+ primary T cells is first stimulated under stimulatory conditions, such as by incubation in the presence of a stimulatory reagent (e.g., anti-CD3 / anti-CD28) capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules, and then incubated with viral vector particles under conditions to transduce cells in the population.

[0047] In some embodiments, the methods provided involve selecting primary T cells that are positive for surface expression of CCR7 from a composition of cells that have been previously incubated under stimulatory conditions, thereby generating a stimulated population enriched for CCR7+ primary T cells, and subsequently transducing the population, as described herein, e.g., in Section IB.

[0048] Thus, also provided herein is a method for increasing the transduction frequency of primary T cells, comprising: (a) selecting primary T cells that are positive for surface expression of CCR7 from a biological sample comprising a population of primary T cells, thereby generating an input population that is enriched for CCR7+ primary T cells; (b) incubating the input population under stimulatory conditions, thereby generating a stimulated composition, wherein the stimulatory conditions comprise the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and (c) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with the T cells of the stimulated composition, thereby generating a population of transduced cells.

[0049] Also provided herein is a method for increasing the transduction frequency of primary T cells, comprising: (a) incubating an input primary T cell population enriched for CCR7+ T cells under stimulatory conditions, thereby generating a stimulated composition, wherein the stimulatory conditions comprise the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and (b) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with the T cells of the stimulated composition, thereby generating a population of transduced cells.

[0050] All publications mentioned in this application, including patent documents, scientific articles, and databases, are incorporated herein in their entirety for all purposes to the same extent as if each individual publication was individually incorporated herein by reference. To the extent that a definition set forth herein conflicts or is otherwise inconsistent with a definition set forth in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0051] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0052] I. Methods for T Cell Transduction Provided herein is a method for increasing the transduction frequency of cells, for example, primary T cells, comprising the steps of selecting cells, for example, primary T cells, that are positive for CCR7 surface expression from a cell composition, and incubating or contacting the selected cells with retroviral vector particles, for example, lentiviral vector particles.In some embodiments, the method further comprises incubating the primary T cells under stimulating conditions, as described herein, for example, in Section IB.In some embodiments, the primary T cells are incubated under stimulating conditions after selecting cells that are positive for CCR7 surface expression.In some embodiments, the primary T cells are incubated under stimulating conditions before selecting cells that are positive for CCR7 surface expression.In some aspects, the cell composition is a composition of primary cells, for example, a biological sample, obtained from a subject, and in some cases, a subpopulation or subset of cells is selected and / or enriched.The characteristics of the composition are defined.

[0053] Also provided herein is a method for increasing the transduction frequency of primary T cells, comprising: (a) selecting primary T cells that are positive for surface expression of CCR7 from a biological sample containing a population of primary T cells, thereby generating an input population enriched for CCR7+ primary T cells; (b) incubating the input population under stimulatory conditions, thereby generating a stimulated composition; and (c) incubating viral vector particles containing a heterologous polynucleotide encoding a recombinant protein with the T cells of the stimulated composition, thereby generating a population of transduced cells. In some embodiments, the stimulatory conditions include the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules.

[0054] Also provided herein are methods for increasing the transduction frequency of primary T cells, comprising incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with T cells of an input cell population enriched for CCR7+ primary T cells, thereby generating a population of transduced cells. In some embodiments, prior to incubation, the cells of the input cell population are incubated under stimulatory conditions.

[0055] Also provided herein is a method for increasing the transduction frequency of primary T cells, comprising: (a) incubating an input primary T cell population enriched for CCR7+ T cells under stimulatory conditions, thereby generating a stimulated composition; and (b) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with the T cells of the stimulated composition, thereby generating a population of transduced cells. In some embodiments, the stimulatory conditions comprise the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules.

[0056] In any portion of the cells provided, incubation under stimulatory conditions can result in activation or stimulation of cells in the cell population, or can lead to activation or stimulation of cells in the cell population, and / or can activate or stimulate signals, e.g., signals arising from TCRs and / or co-receptors, in cells of the cell population, e.g., CD4+ T cells.

[0057] In some embodiments, the cells contain one or more nucleic acids (e.g., polynucleotides) that have been genetically modified by the methods provided, e.g., as described in Section ID, and thereby express recombinant or genetically modified products of such nucleic acids (e.g., polynucleotides). In some embodiments, the nucleic acids (e.g., polynucleotides) are heterologous, i.e., not normally present in a cell or a sample obtained from the cell, e.g., a nucleic acid obtained from another organism or cell that is not normally found in the cell being modified and / or the organism from which such cell is derived. In some embodiments, the nucleic acids (e.g., polynucleotides) are not naturally occurring, and are not found in nature, e.g., include a chimeric combination of nucleic acids encoding various domains from multiple different cell types.

[0058] The processing step of the method may include any one or more of a number of cell processing steps, alone or in combination. In certain embodiments, the processing step includes transducing cells with a retroviral vector, e.g., a viral vector particle containing a recombinant product encoding the cell for expression. The method may additionally and / or alternatively include other processing steps, such as steps for isolating, separating, selecting, washing, suspending, diluting, concentrating, and / or formulating the cells. Optionally, the method may also include an ex vivo step for culturing (e.g., stimulating the cells to induce cell proliferation and / or activation). In another example, the step for stimulating or activating the cells is carried out in vivo after administration of the cells to a subject, following antigen recognition and / or administration of one or more agents to boost or enhance the expansion, activation, and / or proliferation of the cells in the subject. In some embodiments, the method includes isolating cells from a subject, preparing, processing, culturing, and / or modifying them, and reintroducing them into the same subject before or after cryopreservation.

[0059] In some embodiments, the method includes sequential processing steps in which cells, e.g., primary cells, are first isolated (e.g., selected or separated) from a biological sample, the selected cells are incubated with viral vector particles for transduction, and the transduced cells are formulated into a composition. Optionally, the transduced cells are activated, expanded, or propagated ex vivo, such as by stimulation in the presence of a stimulatory reagent, e.g., anti-CD3 / anti-CD28, and / or one or more recombinant T cell stimulatory cytokines, e.g., IL-2, IL-7, and / or IL-25. In some embodiments, the activation or stimulation step is performed before or after the primary cells undergo one or more selection steps, e.g., selection of cells positive for surface expression of CCR7. In some embodiments, the method can include one or more processing steps of washing, suspending, diluting, and / or concentrating the cells, which can be performed before, during, simultaneously with, or after one or more of the isolation, e.g., separation or selection, transduction, stimulation, and / or formulation steps.

[0060] In some embodiments, one or more or all of the processing steps, such as isolation, selection and / or enrichment, processing, incubation associated with transduction and modification, and formulation steps, are performed using a system, device, or apparatus in an integrated or stand-alone system, and / or in an automated or programmable manner. In some aspects, the system or apparatus includes a computer and / or computer program in communication with the system or apparatus, which a user can use to program, control, evaluate the outcome of, and / or adjust various aspects of the processing, isolation, modification, and formulation steps. In one example, the system is the system described in International Patent Application Publication No. WO 2009 / 072003 or US 20110003380 A1. In one example, the system is the system described in International Publication No. WO 2016 / 073602.

[0061] In some embodiments, with respect to cell preparation, processing, and / or incubation associated with the provided transduction methods, one or more of the cell processing steps can be performed in the inner cavity of a centrifuge chamber, e.g., a substantially rigid chamber that is generally cylindrical and can rotate about an axis of rotation, which can provide certain advantages compared to other available methods. In some embodiments, all processing steps are performed in the same centrifuge chamber. In some embodiments, one or more processing steps are performed in different centrifuge chambers, e.g., multiple centrifuge chambers of the same type. Such methods include any of the methods described in International Publication No. WO 2016 / 073602.

[0062] Exemplary centrifuge chambers include those manufactured and sold by Biosafe SA, such as those used with the Sepax® and Sepax® 2 systems, such as the A-200 / F and A-200 centrifuge chambers, and various kits for use with such systems. Exemplary chambers, systems, and processing equipment and cabinets are described, for example, in U.S. Pat. No. 6,123,655, U.S. Pat. No. 6,733,433, and U.S. Patent Application Publication No. US 2008 / 0171951, and International Patent Application Publication No. WO 00 / 38762, the contents of each of which are incorporated herein by reference in their entirety. Depending on the specific process (e.g., dilution, washing, transduction, formulation), it is within the level of skill of one of ordinary skill in the art to select a specific kit appropriate for that process. Exemplary kits for use with such systems include, but are not limited to, disposable kits sold by BioSafe SA under the product names CS-430.1, CS-490.1, CS-600.1, or CS-900.2. An exemplary method for transduction using a centrifuge chamber is described in International Patent Publication No. WO 2016 / 073602.

[0063] In some embodiments, the system is integrated with and / or associated with other equipment, such as equipment for operating, automating, controlling, and / or monitoring aspects of the various processing steps performed in the system. The equipment, in some embodiments, is housed within a cabinet. In some embodiments, the equipment includes a cabinet that includes a housing that houses control circuitry, a centrifuge, a cover, a motor, a pump, sensors, a display, and a user interface. Exemplary devices are described in U.S. Patent No. 6,123,655, U.S. Patent No. 6,733,433, and US 2008 / 0171951.

[0064] In some embodiments, the system includes a series of containers such as bags, tubing, stopcocks, clamps, connectors, and a centrifuge chamber. In some embodiments, the containers such as bags include one or more containers, such as bags, containing cells to be transduced and viral vector particles, either in the same container or separate containers, e.g., in the same bag or separate bags. In some embodiments, the system further includes one or more containers, such as bags, containing media, e.g., diluents and / or wash solutions, that are drawn into the chambers and / or other components to dilute, resuspend, and / or wash components and / or compositions during the method. The containers can be connected to one or more locations in the system, e.g., locations corresponding to input lines, diluent lines, wash lines, waste lines, and / or output lines.

[0065] In some embodiments, the system, e.g., a closed system, is sterile. In some embodiments, all connections of the system components, e.g., connections between tubing lines and containers via connectors, are made under sterile conditions. In some embodiments, the connections are made under laminar flow. In some embodiments, the connections are made using a sterile connection device that creates a sterile connection, such as a sterile weld, between the tubing and container. In some embodiments, the sterile connection device achieves the connection under thermal conditions high enough to maintain sterility, e.g., at a temperature of at least 200°C, e.g., at least 260°C or 300°C.

[0066] In some embodiments, the system is disposable, such as a disposable kit. In some embodiments, the disposable kit can be used in multiple cycles of one or more processes, for example, in a process that is performed continuously or semi-continuously, for example, at least 2, 3, 4, or 5 times or more. In some embodiments, the system, such as a disposable kit, is used to process cells from a single patient.

[0067] The centrifuge chamber generally can rotate around a rotation axis, and the cavity is typically coaxial with the chamber. In some embodiments, the centrifuge chamber further includes a movable member, such as a piston, that can move (e.g., axially) within the chamber to change the volume of the cavity. Thus, in certain embodiments, the inner cavity is surrounded by the side and end walls of the chamber and the movable member, and has a variable volume that can be adjusted by moving the movable member. The movable member can be made of a rigid material, a substantially or generally rigid material, a flexible material, or a combination thereof.

[0068] The chamber also generally includes one or more openings, such as one or more inlets, one or more outlets, and / or one or more inlets / outlets, that can allow for the introduction and expression of liquid and / or gas into and out of the cavity. In some cases, an opening can be a port through which both the introduction and expression of liquid and / or gas occurs. In some cases, the one or more inlets can be separate or distinct from the one or more outlets. The one or more openings can be in one of the end walls. In some embodiments, liquid and / or gas can be introduced into and / or expressed from the cavity by movement of a movable member to increase and / or decrease the volume of the cavity. In other embodiments, liquid and / or gas can be introduced into and / or expressed from the cavity by tubing lines or other flow paths connected to or disposed in connection with the openings, for example, by arranging the lines or flow paths to be connected to and under the control of a pump, syringe, or other mechanism that can be automatically controlled.

[0069] In some embodiments, the chamber is part of a closed system, such as a sterile system, with tubing lines and various other additional components, such as fittings and a lid, within which processing steps are performed. Thus, in some embodiments, the provided methods and / or steps thereof are performed in a completely closed or semi-closed environment, e.g., a closed or semi-closed sterile system, which facilitates production of cells for therapeutic administration to a subject without the need for a separate sterile environment, e.g., a biosafety cabinet or biosafety room. The method, in some embodiments, is performed automatically or partially automatically.

[0070] In some embodiments, the chamber is associated with a centrifuge that can achieve rotation of the chamber, for example, rotation around its rotation axis. Rotation can be performed before, during, and / or after incubation in one or more of the processing steps. Thus, in some embodiments, one or more of the various processing steps are performed under rotation, for example, with a specific force. The chamber is typically capable of vertical rotation or generally vertical rotation, such that the chamber is in a vertical state during centrifugation, with the side walls and axis being vertical or generally vertical, and the end walls being horizontal or generally horizontal.

[0071] In aspects of the method, the processes need not be performed in the same closed system, e.g., in the same centrifuge chamber, but can be performed under different closed systems, e.g., different centrifuge chambers, and in some embodiments, such different centrifuge chambers are at respective points in the method associated with the same system, e.g., associated with the same centrifuge. In some embodiments, all processing steps are performed in one closed system, with all or part of each one or more processing steps being performed in the same or different centrifuge chambers.

[0072] A. Sample and Cell Preparation The cell is generally a eukaryotic cell, for example, a mammalian cell, typically a human cell. In some embodiments, the cell is derived from blood, bone marrow, lymph, or lymphoid organs, and is a cell of the immune system, for example, a cell of innate or adaptive immunity, for example, a cell of the myeloid or lymphoid system, for example, a lymphocyte, typically a T cell and / or a NK cell. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In some embodiments, the cell is derived from a biological sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a leukapheresis sample. In some embodiments, the cell is a T cell.

[0073] The cells are typically primary cells, e.g., isolated directly from a subject and / or isolated and frozen from a subject. In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the total T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, differentiation potential, expansion potential, recirculation potential, localization potential, and / or persistence potential, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation. With respect to the subject being treated, the cells can be allogeneic and / or autologous. The methods include off-the-shelf methods. In some aspects, e.g., in the case of off-the-shelf techniques, the cells are pluripotent and / or multipotent, e.g., stem cells, e.g., induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from a subject, preparing, processing, culturing, and / or modifying them, and reintroducing them into the same subject, before or after cryopreservation.

[0074] Subtypes and subpopulations of T cells, and / or CD4+ T cells, and / or CD8+ T cells include naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (T SCM ), Central Memory T(T CM ), Effector Memory T(T EM ) or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, endogenous and inducible 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.

[0075] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0076] 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 a xenogeneic source, e.g., mouse, rat, non-human primate, and pig.

[0077] In some embodiments, cells can be isolated from a sample, such as a biological sample, for example, obtained from a subject or derived from a subject.In some embodiments, the subject from which cells are isolated has a disease or condition, or is in need of cell therapy, or is a subject to which cell therapy is administered.In some embodiments, the subject is a human being who needs a particular therapeutic intervention, such as adoptive cell therapy, for which cells are isolated, processed and / or modified.

[0078] Therefore, in some embodiments, the cell is a primary cell, for example, a primary human cell. Samples include tissues, body fluids, and other samples directly collected from subjects, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic modification (e.g., transduction by viral vector), washing, and / or incubation. Biological samples can be samples directly obtained from biological sources or processed samples. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples, and also include processed samples derived therefrom.

[0079] In some aspects, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is an apheresis product or leukapheresis product, or is derived from an apheresis product or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. In the context of cell therapy, such as adoptive cell therapy, samples include samples from autologous and allogeneic sources.

[0080] In some examples, cells are obtained from the subject's circulating blood, for example, by apheresis or leukapheresis. The sample, in some aspects, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and / or platelets, and in some aspects, cells other than red blood cells and platelets.

[0081] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in a buffer or medium appropriate 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 lacks many or all divalent cations. In some aspects, the washing step is performed by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are washed after washing, e.g., by filtration with Ca. ++ / Mg ++The cells are resuspended in a variety of biocompatible buffers, such as PBS, free of erythrocytes, etc. In certain embodiments, a component of the blood cell sample is removed and the cells are suspended directly in culture medium.

[0082] In some embodiments, prior to cell enrichment and / or selection, the sample is contacted with and / or contains serum or plasma, such as human serum or plasma. In some embodiments, the serum or plasma is autologous to the subject from whom the cells were obtained. In some embodiments, the serum or plasma is present in the sample at a concentration of at least 10% (v / v), or at least about 10% (v / v), at least 15% (v / v), or at least about 15% (v / v), at least 20% (v / v), at least 25% (v / v), at least 30% (v / v), at least 35% (v / v), or at least about 35% (v / v), or at least 40% (v / v). In some embodiments, the sample containing the primary cells is contacted with or contains an anticoagulant prior to cell selection and / or transduction, hi some embodiments, the anticoagulant is or contains free citrate ions, e.g., anticoagulant citrate dextrose solution A (ACD-A).

[0083] In some embodiments, the input composition is serum-free and / or substantially serum-free. In particular embodiments, the input composition is incubated and / or contacted in serum-free medium. In some embodiments, the serum-free medium is a defined and / or well-defined cell culture medium. In certain embodiments, the serum-free medium is a controlled culture medium that has been processed, e.g., filtered to remove inhibitors and / or growth factors. In some embodiments, the serum-free medium contains protein. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and / or attachment factors. In some embodiments, the serum-free medium contains protein, e.g., albumin, e.g., bovine serum albumin, human serum albumin, and / or recombinant albumin. In some embodiments, the serum-free medium contains a basal medium, e.g., DMEM or RPMI 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and an energy source. In some embodiments, serum-free media is supplemented with, for example, but not limited to, albumin, chemically defined lipids, growth factors, insulin, cytokines, and / or antioxidants, etc. In some embodiments, serum-free media is formulated to support the growth, proliferation, health, and homeostasis of certain cell types, such as immune cells, T cells, and / or CD4+ and CD8+ T cells.

[0084] In some embodiments, prior to cell selection and / or enrichment, the sample or cells in the sample can be rested or held before further processing steps. In some embodiments, the sample is maintained or held at a temperature of 2°C to 8°C or about 2°C to 8°C for up to 48 hours, e.g., up to 12 hours, 24 hours, or 36 hours.

[0085] In some embodiments, the preparation method includes a step for freezing, e.g., cryopreserving, the cells before or after isolation, selection, and / or enrichment, and / or stimulation, and / or activation, and / or incubation for transduction and modification. In some embodiments, the freezing and subsequent thawing step removes granulocytes, and to some extent, monocytes, from the cell population. In some embodiments, the cells are suspended in a freezing solution, for example, after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters can be used in some aspects. In one example, PBS containing 20% ​​DMSO and 8% human serum albumin (HSA) or other suitable cell freezing medium is used. This is then diluted 1:1 with medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then typically frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0086] In some embodiments, cell isolation involves one or more preparation steps and / or non-affinity-based cell separation steps. In some instances, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, or lyse or remove cells that are sensitive to a particular reagent. In some instances, cells are separated based on one or more properties, such as density, adherence, size, sensitivity and / or resistance to a particular component.

[0087] In some embodiments, the methods involve the preparation of white blood cells from peripheral blood by density-based cell separation methods, such as lysis of red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0088] In some embodiments, the isolation method involves separating different cell types based on the expression or presence of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, in the cells. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is affinity- or immunoaffinity-based. For example, in some aspects, the separation involves separating cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, in the cells, e.g., by incubation with an antibody or binding partner that specifically binds to the marker, typically followed by a washing step and separation of cells bound to the antibody or binding partner from cells that do not bind to the antibody or binding partner.

[0089] Such separation steps can be based on positive selection, in which cells that bind to the reagent are retained for further use, and / or negative selection, in which cells that do not bind to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection is particularly useful when antibodies that specifically identify a cell type in a heterogeneous population are not available, and separation is best performed based on markers expressed by cells other than the desired population.

[0090] Separation does not necessarily result in 100% enrichment or removal of a specific cell population or cells that express a specific marker.For example, positive selection or enrichment of a specific type of cell, such as cells that express a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express that marker.Similarly, negative selection, removal or depletion of a specific type of cell, such as cells that express a marker, refers to reducing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0091] In some instances, multiple rounds of separation steps are performed, in which positively or negatively selected fractions from one step are subjected to another step, such as subsequent positive or negative selection. In some instances, cells expressing multiple markers can be simultaneously depleted in a single separation step, such as by incubating cells with multiple antibodies or binding partners specific to the markers targeted for negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on different cell types.

[0092] For example, in some aspects, specific subpopulations of T cells, such as cells that are positive for or express high levels of one or more surface markers, e.g., CD4 + , CD8 + , CD3+, CD28+, and / or CCR7+ T cells are isolated by positive or negative selection techniques.

[0093] For example, CD3 + , CD28 + T cells can be positively selected using anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0094] In some embodiments, the population of T cells, e.g., primary T cells, are unfractionated T cells, or are enriched or isolated CD3+ T cells, or are enriched or isolated CD4+ T cells, or are enriched or isolated CD8+ T cells.

[0095] In some embodiments, isolation is performed by enriching for a particular cell population by positive selection, or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is performed by using a marker expressed on the cells to be positively or negatively selected, respectively. + ) or expressed at relatively high levels (markers high) This is achieved by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers.

[0096] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, e.g., CD14. + or CD8 + The selection process involves CD4 + Helper T cells and CD8 + It is used to isolate cytotoxic T cells. + and CD8 + The population can be further sorted into subpopulations by positive or negative selection for markers expressed on, or relatively highly expressed in, one or more naive, memory, and / or effector T cell subpopulations.

[0097] 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 can result in increased efficacy, e.g., improved long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, T CM Enriched CD8 + T cells and CD4 + Combining with T cells further enhances efficacy.

[0098] In some embodiments, naive T (T N) cells or central memory T(T CM Enrichment of cells is based on positive or high surface expression of one or more of CCR7, CD4, CD8, and CD3. In some aspects, such selection is performed simultaneously, and in other aspects, sequentially, in either order. In some aspects, CD8 + Cell population or CD8 + The same CD4 expression-based selection process used to prepare the cell subpopulations was used to select CD4 + Cell population or CD4 + It is also used to generate cell subpopulations, and thus both the positive and negative fractions from CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more further positive or negative selections.

[0099] In one specific example, the sample of PBMCs or other white blood cell sample is CD4 + The cells are subjected to selection for CD8+ cells, or CD3+ cells, or CD4+ and CD8+ cells, where both the negative and positive fractions are retained.

[0100] In one example, negative selection results in CD4 +To enrich cells, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are attached to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for cell separation for positive and / or negative selection. 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 (eds. S.A. Brooks and U. Schumacher, © Humana Press Inc., Totowa, NJ)).

[0101] In some embodiments, a composition, e.g., an input population, comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells or CD8+ T cells. In some embodiments, a composition, e.g., an input population, comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells. In some embodiments, a composition, e.g., an input population, comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD8+ T cells. In some embodiments, a composition, e.g., an input population, comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells and CD8+ T cells. In some embodiments, the ratio of CD4+ T cells to CD8+ T cells is, or is about, 1:1, 1:2, 2:1, 1:3, or 3:1. In some embodiments, the composition, e.g., the input population, comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD3+ T cells.

[0102] In some embodiments, the methods involve the use of a cell population of primary T cells that is enriched for CCR7+ T cells, e.g., an input population that is enriched for CCR7+ primary T cells. In some embodiments, a population enriched for cells positive for surface expression of CCR7 is selected or obtained from a biological sample. In some embodiments, the selection step comprises selecting primary T cells positive for surface expression of CCR7 from a biological sample containing a population of primary T cells, thereby generating a cell population enriched for CCR7+ primary T cells. In some aspects, a cell population enriched for CCR7+ primary T cells is referred to as an "input population enriched for CCR7+ primary T cells." In some embodiments, the selection is a positive selection by selecting or isolating CCR7-positive cells from the biological sample. In some embodiments, the selection is a negative selection by removing or depleting CCR7-negative cells from the biological sample. Selection can be performed before or after incubating the cells under stimulatory conditions, e.g., as described herein in Section IB.

[0103] In certain embodiments, negative expression, e.g., negative expression of a particular protein, e.g., negative expression of CCR7, i.e., CCR7-, refers to expression below background expression levels, e.g., using standard techniques, e.g., techniques involving antibody staining with a control antibody not specific for that protein, e.g., an isotype antibody. In certain embodiments, negative expression is below background expression levels when detected by a suitable technique for assessing protein or gene expression, such as, but not limited to, immunohistochemistry, immunofluorescence, or flow cytometry-based techniques. In some embodiments, positive expression, e.g., positive expression of a particular protein, e.g., positive expression of CCR7, i.e., CCR7+, refers to or includes surface expression of a protein at an amount, level, or concentration above background, e.g., using standard techniques, e.g., techniques involving antibody staining with a control antibody not specific for that protein, e.g., an isotype antibody. In certain embodiments, positive expression is above background expression levels when detected by a suitable technique for assessing protein or gene expression, such as, but not limited to, immunohistochemistry, immunofluorescence, or flow cytometry-based techniques. In certain embodiments, the amount, frequency, or percentage of cells negative or positive for protein expression, eg, surface expression, in a sample, composition, or population is determined by flow cytometry.

[0104] In certain embodiments, before selecting, isolating, or enriching CCR7+ T cells from a biological sample, T cells, such as CD3+, or a subset of T cells, such as CD4+ or CD8+ T cells, are selected, isolated, or enriched from the biological sample. In some embodiments, T cells, such as CD3+, or a subset of T cells, such as CD4+ or CD8+ T cells, are selected, isolated, or enriched from the enriched CCR7+ T cell population. In certain embodiments, the step of selecting, isolating, or enriching T cells, such as CD3+, or a subset of T cells, such as CD4+ or CD8+ T cells, involves positively selecting CD3, CD4, or CD8 positive cells from the sample.

[0105] In certain embodiments, (1) CD4+ T cells are enriched, selected, or isolated from a biological sample, thereby producing an enriched population of CD4+ T cells and an unselected population enriched for CD4− cells; (2) CD8+ T cells are enriched, selected, or isolated from the enriched unselected population of CD4− cells, thereby producing an enriched population of CD8+ T cells; and (3) CCR7+ T cells are selected or isolated from the enriched CD4+ and CD8+ T cell population, thereby producing an enriched population of CCR7+CD4+ and CCR7+CD8+ T cells. In certain embodiments, (1) CD8+ T cells are enriched, selected, or isolated from a biological sample, thereby producing an enriched population of CD8+ T cells and an unselected population enriched for CD8− cells; (2) CD4+ T cells are enriched, selected, or isolated from the enriched unselected population of CD4− cells, thereby producing an enriched population of CD4+ T cells; and (3) CCR7+ T cells are selected or isolated from the enriched CD4+ and CD8+ T cell population, thereby producing an enriched population of CCR7+CD4+ and CCR7+CD8+ T cells.

[0106] In certain embodiments, CD4+ T cells are enriched, selected, or isolated from the biological sample, thereby generating an enriched population of CD4+ T cells, and then CCR7+ cells are selected or isolated from the enriched population of CD4+ T cells, thereby generating an enriched population of CCR7+ CD4+ T cells. In certain embodiments, CD8+ T cells are enriched, selected, or isolated from the biological sample, thereby generating an enriched population of CD8+ T cells, and then CCR7+ cells are identified or selected from the enriched population of CD8+ T cells, thereby generating enriched CD57- CD8+ T cells.

[0107] In certain embodiments, CD3+ T cells are enriched, selected, or isolated from a biological sample, thereby generating an enriched population of CD3+ T cells, and then CCR7+ cells are identified or selected from the enriched population of CD3+ T cells, thereby generating enriched CCR7+CD8+ T cells.

[0108] In some embodiments, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the input population enriched for CCR7+ primary T cells are CCR7+ primary T cells. In some embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of an input population enriched for CCR7+ primary T cells are CCR7+ primary T cells. In some embodiments, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of an input population enriched for CCR7+ primary T cells are CCR7+ primary T cells. In some embodiments, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of an input population enriched for CCR7+ primary T cells are CCR7+ primary T cells. In some embodiments, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of an input population enriched for CCR7+ primary T cells are CCR7+ primary T cells.

[0109] In some embodiments, the selection step does not include selecting cells that are (a) CCR7+ and CD45RO+, or (b) CCR7+ and CD27+, or (c) CCR7+ and CD45RA-, or (d) CCR7+ and CD62L+, or (e) CCR7+ and CD45RA+, or (f) CCR7+ and CD62L-.

[0110] In some embodiments, the input population is not enriched for T cells that are (a) CCR7+ and CD45RO+, or (b) CCR7+ and CD27+, or (c) CCR7+ and CD45RA-, or (d) CCR7+ and CD62L+, or (e) CCR7+ and CD45RA+, or (f) CCR7+ and CD62L-. In some embodiments, less than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the input population are (a) CCR7+ and CD45RO+, or (b) CCR7+ and CD27+, or (c) CCR7+ and CD45RA-, or (d) CCR7+ and CD62L+, or (e) CCR7+ and CD45RA+, or (f) CCR7+ and CD62L- T cells. In some embodiments, the input population is not enriched for CCR7+ and CD45RO+ T cells, and optionally, less than 85% of the total cells in the input population are CCR7+ and CD45RO+ T cells. In some embodiments, the input population is not enriched for CCR7+ and CD27+ T cells, optionally, less than 85% of the total cells in the input population are CCR7+ and CD27+ T cells. In some embodiments, the input population is not enriched for CCR7+ and CD45RA- T cells, optionally, less than 85% of the total cells in the input population are CCR7+ and CD45RA- T cells. In some embodiments, the input population is not enriched for CCR7+ and CD62L+ T cells, optionally, less than 85% of the total cells in the input population are CCR7+ and CD62L+ T cells. In some embodiments, the input population is not enriched for CCR7+ and CD45RA+ T cells, optionally, less than 85% of the total cells in the input population are CCR7+ and CD45RA+ T cells. In some embodiments, the input population is not enriched for CCR7+ and CD62L- T cells, and optionally, less than 85% of the total cells in the input population are CCR7+ and CD62L- T cells.

[0111] In some aspects, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as a magnetically responsive particle or microparticle, e.g., a paramagnetic bead (e.g., Dynalbeads or MACS beads). The magnetically responsive material, e.g., a magnetically responsive particle, is generally attached, directly or indirectly, to a binding partner, such as an antibody, that specifically binds to a molecule, such as a surface marker, present on one or more cells or cell populations to be separated, e.g., negatively or positively selected.

[0112] 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. Numerous magnetically responsive materials are known for use in magnetic separation methods. Suitable magnetic particles include those described in U.S. Patent No. 4,452,773 to Molday and European Patent Specification EP 452342 B, which are incorporated herein by reference. Other examples include 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.

[0113] Incubation is generally carried out under conditions in which the antibody or binding partner attached to the magnetic particle or magnetic bead, or a molecule that specifically binds to such an antibody or binding partner, e.g., a secondary antibody or other reagent, specifically binds to the cell surface molecule, if present, on cells in the sample.

[0114] In some aspects, when the sample is placed in a magnetic field, cells with magnetically responsive or magnetizable particles attached are attracted to the magnet and separated from unlabeled cells. In the case of positive selection, cells attracted to the magnet are retained, and in the case of negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, in which case the positive and negative fractions are retained and either further processed or subjected to additional separation steps.

[0115] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are attached to cells via coating with a primary antibody specific for one or more markers. In certain embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic beads coated with a cell type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used together with biotinylated primary or secondary antibodies.

[0116] In some embodiments, the magnetically responsive particles remain attached to the cells for subsequent incubation, culture, and / or modification, and in some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, the magnetizable particles or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known, including, for example, the use of competitive unlabeled antibodies and the use of magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0117] In some embodiments, affinity-based selection is by magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic-activated cell sorting (MACS) systems allow for high-purity selection of cells with attached magnetized particles. In certain embodiments, MACS operates in a mode where non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells attached to magnetized particles are held in place while unattached species are eluted. Then, after this first elution step is complete, species that were trapped by the magnetic field and prevented from elution are released in some way so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous cell population.

[0118] In certain embodiments, the isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method. In some aspects, a system is used to perform each of these steps in a closed or sterile environment, e.g., to minimize errors, user handling, and / or contamination. In one example, the system is a system described in International Patent Application Publication No. WO 2009 / 072003 or US 20110003380 A1. In one example, the system is a system described in International Patent Application Publication No. WO 2016 / 073602.

[0119] In some embodiments, the methods involve selection of cells, wherein all or part of the selection is carried out within the inner cavity of a centrifuge chamber, e.g., under centrifugal rotation. In some embodiments, incubation of the cells with a selection reagent, e.g., an immunoaffinity-based selection reagent, is carried out within the centrifuge chamber.

[0120] For example, immunoaffinity-based selection can rely on favorable energetic interactions between the cells to be separated and molecules, e.g., antibodies or other binding partners on a solid surface such as a particle, that specifically bind to a marker on the cells. In certain available methods for affinity-based separation using particles such as beads, the particles and cells are incubated in a container such as a tube or bag with shaking or mixing at a certain cell density-to-particle (e.g., bead) ratio to help promote energetically favorable interactions. Such approaches may not be ideal for use in large-scale production, for example, because they may require the use of large volumes to maintain an optimal or desired cell-to-particle ratio while maintaining a desired cell number. Therefore, such approaches may require batch or batch-based processing, which can require increased time, steps, and handling, increasing costs and the risk of user error.

[0121] In some embodiments, performing such a selection step, or portions thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads), within the cavity of a centrifuge chamber allows the user to control certain parameters, such as the volume of various solutions and the addition and timing of solutions during processing, which can be advantageous compared to other available methods. For example, if the volume of liquid within the cavity can be reduced during incubation, the concentration of the particles (e.g., bead reagents) used for selection, and therefore the chemical potential of the solution, can be increased without affecting the total number of cells within the cavity. This can result in enhanced pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, performing the incubation step within the chamber, e.g., when associated with the systems, circuits, and controls described herein, allows the user to achieve agitation of the solution at desired points during incubation, which can also improve interactions.

[0122] In some embodiments, at least a portion of the selection step is performed in a centrifuge chamber, including incubation of the cells with a selection reagent. In some aspects of such processes, a volume of cells is mixed with a desired affinity-based selection reagent in an amount that is much less than the amount typically used when performing a similar selection in a tube or container according to the manufacturer's instructions for the same number and / or volume of cells. In some embodiments, one or more selection reagents are used in an amount that is 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 50% or less, 60% or less, 70% or less, or 80% or less of the amount of the same selection reagent used to select cells in a tube-based or container-based incubation according to the manufacturer's instructions for the same number and / or volume of cells.

[0123] Incubation with one or more selection reagents, for example, as part of a selection method that can be performed within the chamber cavity, can include using one or more selection reagents to select for one or more different cell types based on the expression or presence of one or more specific molecules, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, within or on cells. In some embodiments, any known method using one or more selection reagents for such marker-based selection can be used. In some embodiments, the one or more selection reagents result in a separation that is affinity- or immunoaffinity-based separation. For example, selection in some aspects involves incubation with one or more reagents for separating cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, on the cells, e.g., by incubation with an antibody or binding partner that specifically binds the marker, typically followed by a washing step and separation of cells bound to the antibody or binding partner from cells that do not bind to the antibody or binding partner.

[0124] In some embodiments, for selection, e.g., for immunoaffinity-based cell selection, the cells are incubated in the cavity of the chamber in a composition that also contains a selection buffer with a selection reagent, e.g., a molecule that specifically binds to a surface marker on the cells to be enriched and / or depleted but not to surface markers on other cells in the composition, e.g., an antibody, optionally coupled to a polymer or surface, e.g., a scaffold such as beads, e.g., magnetic beads, e.g., magnetic beads coupled to monoclonal antibodies specific for CD4 and CD8. In some embodiments, as described, the selection reagent is added to the cells in the cavity of the chamber in a substantially reduced amount (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or less) compared to the amount of selection reagent that would typically be used or required to achieve approximately the same or similar efficiency of selection of the same number of cells or the same volume of cells when selection is performed by shaking or rotation in a tube. In some embodiments, incubation is, including incubation of reagents, for example, 10 mL to 200 mL, e.g., at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL, or about at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL. In some embodiments, the selection buffer is added to the cells and selection reagent to achieve a target volume of about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL, or about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL. In some embodiments, the selection buffer and selection reagent are premixed before addition to the cells. In some embodiments, the selection buffer and selection reagent are added to the cells separately.In some embodiments, selection incubations are performed under intermittent, mild mixing conditions, which help promote energetically favorable interactions, thereby achieving high selection efficiency while simultaneously reducing the total amount of selection reagent used.

[0125] In some embodiments, the total duration of incubation with the selection reagent is between 5 minutes and 6 hours, or about 5 minutes and 6 hours, such as between 30 minutes and 3 hours, for example at least 30 minutes, 60 minutes, 120 minutes, or 180 minutes, or about at least 30 minutes, 60 minutes, 120 minutes, or 180 minutes.

[0126] In some embodiments, incubation is generally carried out under mixing conditions, e.g., generally with relatively low force or low speed, e.g., a speed less than that used to pellet cells, e.g., a speed of 600 rpm to 1700 rpm, or about 600 rpm to 1700 rpm (e.g., 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or about 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm), e.g., a speed at which the RCF at the sample or wall of the chamber or other container is about 80 g to 100 g (e.g., 80 g, 85 g, 90 g, 95 g, or 100 g, or about 80 g, 85 g, 90 g, 95 g, or 100 g, or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, rotation is performed using a repeat interval of such slow rotation followed by a rest period, e.g., rotation and / or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, e.g., rotation for approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.

[0127] In some embodiments, such a process is carried out within a completely closed system integrated with the chamber. In some embodiments, this process (and in some aspects one or more additional steps, e.g., a pre-wash step to wash a cell-containing sample, e.g., an apheresis sample) is carried out automatically, with cells, reagents, and other components being drawn into the chamber, pushed out of the chamber, and centrifugation being achieved at the appropriate times, such that the washing and binding steps are completed in a single closed system using an automated program.

[0128] In some embodiments, after incubation and / or mixing of the cells with one and / or more selection reagents, the incubated cells are subjected to separation to select cells based on the presence or absence of one or more of the specific reagents. In some embodiments, further selection is performed outside the centrifugation chamber. In some embodiments, separation is performed in the same closed system in which the centrifugation chamber is present and in which the incubation of the cells with the selection reagents is performed. In some embodiments, after incubation with the selection reagents, the incubated cells, including cells to which the selection reagents are bound, are squeezed out of the centrifugation chamber and transferred, for example, from the centrifugation chamber to a system for immunoaffinity-based cell separation. In some embodiments, the system for immunoaffinity-based separation is or contains a magnetic separation column. In some embodiments, one or more other processing steps, such as washing, can be performed in the chamber prior to separation.

[0129] In some aspects, separation and / or other steps are performed using a CliniMACS system (Miltenyi Biotic), for example, for automated separation of cells at clinical scale in a closed and sterile system. Components can include an embedded microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. The embedded computer, in some aspects, controls all components of the instrument and directs 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 selected column. The peristaltic pump controls the flow rate through the tubing set and, together with the pinch valves, ensures a controlled flow of buffer through the system and continuous suspension of the cells.

[0130] In some aspects, the CliniMACS system uses antibody-bound magnetizable particles supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling of cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which is further connected to a bag containing buffer and a cell collection bag. The tubing set consists of a pre-assembled sterile tubing system including a pre-column and a separation column and is disposable. After starting the separation program, the system automatically applies the cell sample to the separation column. Labeled cells are retained in the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations used in the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations used in the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations used in the methods described herein are eluted from the column after removal of the magnetic field and collected in a cell collection bag.

[0131] In certain embodiments, separation and / or other steps are performed using the CliniMACS Prodigy system (Miltenyi Biotec). In some aspects, the CliniMACS Prodigy system is equipped with a cell processing unit that allows for automated cell washing and fractionation by centrifugation. The CliniMACS Prodigy system can also include an integrated camera and image recognition software that determines the optimal cell fractionation endpoint by identifying the macroscopic layers of the source cell product. For example, peripheral blood can be automatically separated into red blood cell, white blood cell, and plasma layers. The CliniMACS Prodigy system can also include an integrated cell culture chamber for performing cell culture protocols, such as cell differentiation and expansion, antigen loading, and long-term cell culture. An inlet port can allow for aseptic removal and replenishment of media, 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.

[0132] In some embodiments, the cell populations described herein are collected and enriched (or depleted) by flow cytometry, in which cells stained for multiple cell surface markers are entrained in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) by preparative-scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by using a microelectromechanical systems (MEMS) chip in conjunction with a FACS-based detection system (see, e.g., WO 2010 / 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 highly pure, distinct T cell subsets.

[0133] In some embodiments, antibodies or binding partners are 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 a fluorescently labeled antibody. In some examples, cell separation based on binding of antibodies or other binding partners specific to one or more cell surface markers is performed in a fluid stream, for example, by fluorescence-activated cell sorting (FACS), for example, on a preparative scale combined with a flow cytometry detection system (FACS) and / or a microelectromechanical system (MEMS) chip. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0134] In some embodiments, the provided retroviral particles are capable of transducing stimulated and / or activated T cells. In certain embodiments, the provided retroviral particles are capable of transducing resting T cells. In some embodiments, the input composition comprises a plurality of cells, e.g., immune cells, e.g., T cells, that are non-cycling and / or quiescent and / or resting cells, or where a majority of the cells in the population to be transduced, e.g., greater than 50%, 60%, 70%, 80%, 80% or more, are non-cycling and / or quiescent and / or resting cells. In some embodiments, the input composition comprises at least 40%, 50%, 60%, 70%, 80%, 90% or more of the T cells in the population are resting T cells, e.g., T cells that lack T cell activation markers, such as surface markers or intracellular cytokines or other markers, and / or are in the G0 or G0G cell cycle. 1a In some embodiments, the cells are in the G0, G0 / G1 phase of the cell cycle. 1a or in the G1 phase.

[0135] In some embodiments, the cells to be transduced were incubated under stimulatory conditions prior to transduction. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of the cells to be transduced (i) express surface markers selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB, (ii) comprise intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-gamma, and TNF-alpha, (iii) are in the G1 phase or later of the cell cycle, and / or (iv) have proliferative capacity.

[0136] B. Activation and stimulation In some embodiments, the provided methods are used in conjunction with incubating cells under stimulatory conditions. In some embodiments, the stimulatory conditions include conditions that activate or stimulate and / or are capable of activating or stimulating a signal in a cell, e.g., a CD4+ T cell, e.g., a signal generated from a TCR and / or a coreceptor. In some embodiments, the stimulatory conditions include one or more steps of culturing, cultivating, incubating, activating, or expanding the cell with and / or in the presence of a stimulatory reagent, e.g., a reagent that activates or stimulates and / or is capable of activating or stimulating a signal in the cell. In some embodiments, the stimulatory reagent stimulates and / or activates the TCR and / or a coreceptor. In certain embodiments, the stimulatory reagent is a reagent provided herein, e.g., one described in Section IB-1.

[0137] In certain embodiments, one or more compositions of enriched T cells are incubated under stimulatory conditions prior to genetic modification of cells, for example, before transfection and / or transduction of cells, for example, by the methods or techniques provided herein, for example, by the methods or techniques described in Sections IC and ID. In certain embodiments, the composition of enriched T cells incubated under stimulatory conditions is an input composition. In certain embodiments, the cells of the input composition have been previously isolated, selected, enriched, or obtained from a biological sample. In certain embodiments, the cells from the input composition have been previously cryogenically frozen and stored, and are thawed prior to incubation.

[0138] In some embodiments, the provided methods are used in conjunction with one or more processing steps, including stimulating cells, e.g., cells from an input composition. In certain embodiments, incubation can occur prior to or in conjunction with genetic modification, e.g., the genetic modification resulting from a transduction embodiment described herein, e.g., a method described in Section ID. In some embodiments, stimulation results in activation and / or proliferation of cells, e.g., prior to modification, e.g., prior to transduction.

[0139] In some embodiments, the processing step includes incubation of cells, such as input cells and / or cells of an input composition, and the incubation step can include culture, cultivation, stimulation, activation, and / or proliferation of the cells. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, and / or to mimic antigen exposure, and / or to prime cells for genetic modification, e.g., introduction of a recombinant antigen receptor.

[0140] In certain embodiments, cells, e.g., cells of the input composition, are cultured at a concentration of 5×10 under stimulatory conditions, e.g., in the presence of a stimulatory agent. 7 cells / mL, 4×10 7 cells / mL, 3×10 7 cells / mL, 2×10 7 cells / mL, 1×10 7 cells / mL, 9×10 6 cells / mL, 8×10 6 cells / mL, 7×10 6 cells / mL, 6×10 6 cells / mL, 5×10 6 cells / mL, 4×10 6 cells / mL, or 3 x 10 6 cells / mL or approximately 5 x 10 7 cells / mL, 4×10 7 cells / mL, 3×10 7 cells / mL, 2×10 7 cells / mL, 1×10 7 cells / mL, 9×10 6 cells / mL, 8×10 6 cells / mL, 7×10 6 cells / mL, 6×10 6 cells / mL, 5×10 6 cells / mL, 4×10 6 cells / mL, or 3 x 10 6 In certain embodiments, the cells are incubated at a density of less than 5 x 10 cells / mL. 6 In some embodiments, the cells are incubated at a density of less than 1 x 10 cells / mL. 3 cells / mL~1×10 9 cells / mL, 1×10 4 cells / mL~1×10 8 cells / mL, 1×10 5 cells / mL~1×10 7 cells / mL, 5×10 5 cells / mL~1×10 7 cells / mL, 1×10 6 cells / mL~5×10 6 cells / mL, or 3 x 10 6 cells / mL~5×10 6In certain embodiments, the cells are incubated at a density of 1 x 10 cells / mL. 6 cells / mL, 1.5×10 6 cells / mL, 2×10 6 cells / mL, 2.5×10 6 cells / mL, 3×10 6 cells / mL, 3.5×10 6 cells / mL, 4×10 6 cells / mL, 4.5×10 6 cells / mL, or 5 x 10 6 cells / mL, or approximately 1 x 10 6 cells / mL, 1.5×10 6 cells / mL, 2×10 6 cells / mL, 2.5×10 6 cells / mL, 3×10 6 cells / mL, 3.5×10 6 cells / mL, 4×10 6 cells / mL, 4.5×10 6 cells / mL, or 5 x 10 6 In certain embodiments, the cells are incubated at a density of 3 x 10 cells / mL. 6 cells / mL or approximately 3 x 10 6 Incubated at a density of 10 ...

[0141] In certain embodiments, indicators of viability include, but are not limited to, indicators of cell replication, mitochondrial function, energy balance, membrane integrity, and cell death. In certain embodiments, indicators of viability further include indicators of oxidative stress, metabolic activation, metabolic stability, enzyme induction, enzyme inhibition, and interaction with cell membrane transporters. In some embodiments, viable cells include cells undergoing normal functional cellular processes and / or cells that have not undergone or are not in the process of undergoing necrosis or programmed cell death. In some embodiments, viability can be assessed by cellular redox potential, cell membrane integrity, or mitochondrial activity or function. In some embodiments, viability is the absence of specific molecules associated with cell death or the absence of signs of cell death in assays. In certain embodiments, cell viability can be detected, measured, and / or assayed by many conventional means. Non-limiting examples of such viability assays include, but are not limited to, dye uptake assays (e.g., calcein AM assay), XTT cell viability assays, and dye exclusion assays (e.g., trypan blue, eosin, or propidium dye exclusion assays). Viability assays are useful for determining cell dose, cell composition, and / or the number or percentage (e.g., frequency) of viable cells in a cell sample.

[0142] In certain embodiments, apoptosis markers can include any known marker associated with apoptosis, including the expression of a gene, protein, or activated protein, or the appearance of features associated with apoptosis, such as blebbing and / or nuclear disruption. In certain embodiments, the apoptosis marker is a marker associated with apoptosis, which can include, but is not limited to, pro-apoptotic factors known to initiate apoptosis, members of the death receptor pathway, activated members of the mitochondrial (intrinsic) pathway, Bcl-2 family members such as Bax, Bad, and Bid, Fas, and FADD, the presence of nuclear shrinkage (e.g., monitored by microscopy), the presence of chromosomal DNA fragmentation (e.g., the presence of chromosomal DNA ladders), or markers associated with apoptosis assays such as TUNEL staining and Annexin V staining. In some embodiments, the marker of apoptosis is caspase expression, such as the expression of activated caspase-1, caspase-2, caspase-3, caspase-7, caspase-8, caspase-9, caspase-10, and / or caspase-13. In some embodiments, the marker of apoptosis is Annexin V. In certain embodiments, the apoptosis marker is active caspase-3.

[0143] In some embodiments, 1 x 10 5 Or about 1 x 10 5 ~500,000×10 6 or approximately 500,000 x 10 6 cells, 1 x 10 6 Or about 1 x 10 6 ~50,000×10 6 or approximately 50,000 x 10 6 cells, 10 x 10 6 Or about 10 x 10 6 ~5,000×10 6 or approximately 5,000 x 10 6 cells, 1 x 10 6 Or about 1 x 10 6 ~1,000×10 6 Or about 1,000 x 106 cells, 50 x 10 6 Or about 50 x 10 6 ~5,000×10 6 or approximately 5,000 x 10 6 cells, 10 x 10 6 Or about 10 x 10 6 ~1,000×10 6 Or about 1,000 x 10 6 cells, 100 x 10 6 Or about 100 x 10 6 ~2,500×10 6 Or about 2,500 x 10 6 cells, 100 x 10 6 Or about 100 x 10 6 ~500×10 6 Or about 500 x 10 6 cells, 200 x 10 6 Or about 200 x 10 6 ~400×10 6 Or about 400 x 10 6 In certain embodiments, at least 50 x 10 cells, e.g., cells of an input composition, are incubated, e.g., under stimulatory conditions, e.g., in the presence of a stimulatory agent. 6 cells, 100 x 10 6 cells, 150 x 10 6 cells, 200 x 10 6 cells, 250 x 10 6 cells, 300 x 10 6 cells, 350 x 10 6 cells, 400 x 10 6 cells, 450 x 10 6 cells or 500 x 10 6 cells, or 50 x 10 6 cells, 100 x 10 6 cells, 150 x 10 6 cells, 200 x 10 6 cells, 250 x 10 6 cells, 300 x 10 6 cells, 350 x 10 6 cells, 400 x 10 6 cells, 450 x 10 6cells or 500 x 10 6 cells, or approximately 50 x 10 6 cells, 100 x 10 6 cells, 150 x 10 6 cells, 200 x 10 6 cells, 250 x 10 6 cells, 300 x 10 6 cells, 350 x 10 6 cells, 400 x 10 6 cells, 450 x 10 6 cells or 500 x 10 6 The cells are incubated, for example, under stimulatory conditions. In some embodiments, the cells are viable cells. In certain embodiments, the cells are negative for apoptotic markers, such as Annexin V or active caspase 3. In certain embodiments, the cells are or comprise CD4+ T cells and CD8+ T cells.

[0144] In some embodiments, 1 x 10 5 Or about 1 x 10 5 ~25,000×10 6 or approximately 25,000 x 10 6 , 1×10 6 Or about 1 x 10 6 ~25,000×10 6 or approximately 25,000 x 10 6 , 10×10 6 Or about 10 x 10 6 ~2,500×10 6 Or about 2,500 x 10 6 , 1×10 6 Or about 1 x 10 6 ~500×10 6 Or about 500 x 10 6 , 50×10 6 Or about 50 x 10 6 ~2,500×10 6 Or about 2,500 x 10 6 , 10×10 6 Or about 10 x 10 6 ~500×10 6 Or about 500 x 10 6, 100×10 6 Or about 100 x 10 6 ~500×10 6 Or about 500 x 10 6 , 200×10 6 Or about 200 x 10 6 ~400×10 6 Or about 400 x 10 6 , 50×10 6 Or about 50 x 10 6 ~300×10 6 Or about 300 x 10 6 CD4+ T cells, e.g., CD4+ T cells of the Input Composition, are incubated, e.g., under stimulatory conditions, e.g., in the presence of a stimulatory reagent. In certain embodiments, at least 25 x 10 6 , 50×10 6 , 75×10 6 , 100×10 6 , 125×10 6 , 150×10 6 , 175×10 6 , 200×10 6 , 225×10 6 Or 250 x 10 6 pieces, or 25 x 10 6 , 50×10 6 , 75×10 6 , 100×10 6 , 125×10 6 , 150×10 6 , 175×10 6 , 200×10 6 , 225×10 6 Or 250 x 10 6 pieces, or approximately 25 x 10 6 , 50×10 6 , 75×10 6 , 100×10 6 , 125×10 6 , 150×10 6 , 175×10 6 , 200×10 6 , 225×10 6 Or 250 x 10 6The CD4+ T cells are incubated, e.g., under stimulatory conditions. In some embodiments, the CD4+ T cells are viable CD4+ T cells. In certain embodiments, the CD4+ T cells are negative for markers of apoptosis, e.g., annexin V or active caspase 3.

[0145] In certain embodiments, 1 x 10 5 Or about 1 x 10 5 ~25,000×10 6 or approximately 25,000 x 10 6 , 1×10 6 Or about 1 x 10 6 ~25,000×10 6 or approximately 25,000 x 10 6 , 10×10 6 Or about 10 x 10 6 ~2,500×10 6 Or about 2,500 x 10 6 , 1×10 6 Or about 1 x 10 6 ~500×10 6 Or about 500 x 10 6 , 50×10 6 Or about 50 x 10 6 ~2,500×10 6 Or about 2,500 x 10 6 , 10×10 6 Or about 10 x 10 6 ~500×10 6 Or about 500 x 10 6 , 100×10 6 Or about 100 x 10 6 ~500×10 6 Or about 500 x 10 6 , 200×10 6 Or about 200 x 10 6 ~400×10 6 Or about 400 x 10 6 , 50×10 6 Or about 50 x 10 6 ~300×10 6 Or about 300 x 10 6CD8+ T cells, e.g., CD8+ T cells of the Input Composition, are incubated, e.g., under stimulatory conditions, e.g., in the presence of a stimulatory reagent. In some embodiments, at least 25 x 10 6 , 50×10 6 , 75×10 6 , 100×10 6 , 125×10 6 , 150×10 6 , 175×10 6 , 200×10 6 , 225×10 6 Or 250 x 10 6 pieces, or 25 x 10 6 , 50×10 6 , 75×10 6 , 100×10 6 , 125×10 6 , 150×10 6 , 175×10 6 , 200×10 6 , 225×10 6 Or 250 x 10 6 pieces, or approximately 25 x 10 6 , 50×10 6 , 75×10 6 , 100×10 6 , 125×10 6 , 150×10 6 , 175×10 6 , 200×10 6 , 225×10 6 Or 250 x 10 6 The CD8+ T cells are incubated, for example, under stimulatory conditions. In some embodiments, the CD8+ T cells are viable CD8+ T cells. In certain embodiments, the CD8+ T cells are negative for markers of apoptosis, such as annexin V or active caspase 3.

[0146] In some embodiments, the conditions for stimulation and / or activation can include one or more of a particular 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.

[0147] In some embodiments, the stimulatory reagent comprises a primary agent, which can be any stimulatory reagent described herein. In some embodiments, the stimulatory reagent comprises a primary agent and a secondary agent. The secondary agent, in some embodiments, can be any stimulatory reagent described herein. In some embodiments, the primary agent specifically binds to a member of the TCR complex, optionally specifically binds to CD3. In some embodiments, the secondary agent specifically binds to a T cell costimulatory molecule, optionally, the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS.

[0148] In some embodiments, the stimulatory conditions or reagents include one or more reagents, e.g., ligands, capable of binding (e.g., specifically binding) to a member of the TCR complex. In some embodiments, the member of the TCR complex is CD3. In some embodiments, the stimulatory conditions or reagents include one or more agents, e.g., ligands, capable of stimulating or activating the intracellular signaling domain of the TCR complex. In some aspects, for example, an agent suitable for delivering a primary signal, e.g., an agent suitable for initiating activation of an ITAM-induced signal, e.g., an agent specific for a TCR component, e.g., anti-CD3, and / or an agent promoting a costimulatory signal, e.g., an agent specific for a T cell costimulatory receptor, e.g., anti-CD28 or anti-4-1BB bound to a solid support such as beads, and / or one or more cytokines, turn on or initiate the TCR / CD3 intracellular signaling cascade in T cells. In some embodiments, the agent that specifically binds to a T cell costimulatory molecule is an agent that specifically binds to CD28, CD137 (4-1BB), OX40, or ICOS. Stimulatory reagents include anti-CD3 / anti-CD28 beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander, and / or ExpACT® beads). Optionally, the expansion method can further comprise adding anti-CD3 and / or anti-CD28 antibodies to the culture medium. In some embodiments, the stimulatory agent comprises a cytokine.

[0149] In certain embodiments, the stimulatory conditions include incubating, culturing, and / or cultivating the cells with a stimulatory reagent. In certain embodiments, the stimulatory reagent is a reagent provided herein, e.g., a reagent described in Section IB-1. In certain embodiments, the stimulatory reagent contains or comprises beads. In certain embodiments, incubation under stimulatory conditions, starting and / or initiating the culturing and / or cultivating of the cells occurs when the cells are contacted and / or incubated with the stimulatory reagent. In certain embodiments, the cells are incubated before, during, and / or after genetic modification of the cells, e.g., introduction of a recombinant polynucleotide into the cells, such as by transduction or transfection.

[0150] In some embodiments, the enriched T cell composition is incubated with a stimulatory reagent and / or bead to cell ratio of at or about 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1 or 0.2:1. In certain embodiments, the ratio of stimulatory reagent and / or beads to cells is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, or between 1.1:1 and 0.9:1. In certain embodiments, the ratio of stimulatory reagent to cells is about 1:1 or is 1:1.

[0151] In certain embodiments, the stimulatory conditions include incubating, culturing, and / or cultivating cells, e.g., cells from the input composition, with and / or in the presence of one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind to and / or are capable of binding to a receptor expressed by and / or endogenous to T cells. In certain embodiments, the one or more cytokines are or include members of the 4-alpha-helix bundle family of cytokines. In some embodiments, members of the four-alpha helix bundle cytokine family include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more cytokines are or include IL-15. In certain embodiments, the one or more cytokines are or include IL-7. In certain embodiments, the one or more cytokines are or include IL-2.

[0152] In certain embodiments, the amount or concentration of one or more cytokines is measured and / or quantified in International Units (IU). International units can be used to quantify vitamins, hormones, cytokines, vaccines, blood products, and similar biologically active substances. In some embodiments, an IU is or includes a unit of measurement for the potency of a biological preparation by comparison to an international standard of a specific weight or potency, such as WHO First International Standard 86 / 504 for human IL-2. International units are the only generally accepted and standardized method for reporting the number of biological activity units and are the result of an international collaborative research effort. In certain embodiments, an IU for a cytokine composition, sample, or source can be obtained by product comparison testing with an analogous WHO standard. For example, in some embodiments, the IU / mg of a composition, sample, or source of human recombinant IL-2, IL-7, or IL-15 is compared to a WHO standard IL-2 preparation (NIBSC code: 86 / 500), a WHO standard IL-17 preparation (NIBSC code: 90 / 530), and a WHO standard IL-15 preparation (NIBSC code: 95 / 554), respectively.

[0153] In some embodiments, biological activity in IU / mg is expressed as the ED 50 ) -1 x10 6 In certain embodiments, the ED of recombinant human IL-2 or IL-15 is equivalent to 50 is equivalent to the concentration required for half-maximal stimulation of cell proliferation (XTT cleavage) using CTLL-2 cells. In certain embodiments, the ED of recombinant human IL-7 50is equivalent to the concentration required for half-maximal stimulation of proliferation of PHA-activated human peripheral blood lymphocytes. Details regarding the assay and calculation of IU for IL-2 are discussed in Wadhwa et al., Journal of Immunological Methods (2013), 379(1-2):1-7 and Gearing and Thorpe, Journal of Immunological Methods (1988), 114(1-2):3-9, and details regarding the assay and calculation of IU for IL-15 are discussed in Soman et al. Journal of Immunological Methods (2009) 348(1-2):83-94.

[0154] In some embodiments, the cells, e.g., input cells, are incubated with a cytokine, e.g., a recombinant human cytokine, at a concentration of from 1 or about 1 IU / mL to 1,000 or about 1,000 IU / mL, from 10 or about 10 IU / mL to 50 or about 50 IU / mL, from 50 or about 50 IU / mL to 100 or about 100 IU / mL, from 100 or about 100 IU / mL to 200 or about 200 IU / mL, from 100 or about 100 IU / mL to 500 or about 500 IU / mL, from 250 or about 250 IU / mL to 500 or about 500 IU / mL, or from 500 or about 500 IU / mL to 1,000 or about 1,000 IU / mL.

[0155] In some embodiments, the cells, e.g., input cells, are incubated with IL-2, e.g., human recombinant IL-2, at a concentration of from 1 or about 1 IU / mL to 500 or about 500 IU / mL, 10 or about 10 IU / mL to 250 or about 250 IU / mL, 50 or about 50 IU / mL to 200 or about 200 IU / mL, 50 or about 50 IU / mL to 150 or about 150 IU / mL, 75 or about 75 IU / mL to 125 or about 125 IU / mL, 100 or about 100 IU / mL to 200 or about 200 IU / mL, or 10 or about 10 IU / mL to 100 or about 100 IU / mL, e.g., in serum-free medium. In certain embodiments, cells, e.g., cells of the input composition, have a cytotoxicity of 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, 150 IU / mL, 160 IU / mL, 170 IU / mL, 180 IU / mL, 190 IU / mL or 100 IU / mL, or about 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, 150 IU / mL, 160 IU / mL, 170 IU / mL, 180 IU / mL, 190 IU / mL or 100 IU / mL. In some embodiments, the cells, e.g., input cells, are incubated with recombinant IL-2 at a concentration of 100 IU / mL, 190 IU / mL, or 100 IU / mL. In some embodiments, the cells, e.g., input cells, are incubated in the presence of 100 IU / mL or about 100 IU / mL of recombinant IL-2, e.g., human recombinant IL-2.

[0156] In some embodiments, the cells, e.g., input cells, are incubated with recombinant IL-7, e.g., human recombinant IL-7, at a concentration of from 100 or about 100 IU / mL to 2,000 or about 2,000 IU / mL, 500 or about 500 IU / mL to 1,000 or about 1,000 IU / mL, 100 or about 100 IU / mL to 500 or about 500 IU / mL, 500 or about 500 IU / mL to 750 or about 750 IU / mL, 750 or about 750 IU / mL to 1,000 or about 1,000 IU / mL, or 550 or about 550 IU / mL to 650 or about 650 IU / mL, e.g., in serum-free medium. In certain embodiments, the cells, e.g., input cells, have a cytotoxicity of at or about 50 IU / mL, 100 IU / mL, 150 IU / mL, 200 IU / mL, 250 IU / mL, 300 IU / mL, 350 IU / mL, 400 IU / mL, 450 IU / mL, 500 IU / mL, 550 IU / mL, 600 IU / mL, 650 IU / mL, 700 IU / mL, 750 IU / mL, 800 IU / mL, 750 IU / mL, 750 IU / mL, or 1,000 IU / mL. In certain embodiments, the cells, e.g., input cells, are incubated with IL-7 at a concentration of 600 IU / mL or about 600 IU / mL, e.g., human recombinant IL-7.

[0157] In some embodiments, the cells, e.g., input cells, are incubated with recombinant IL-15, e.g., human recombinant IL-15, at a concentration of from 1 or about 1 IU / mL to 500 or about 500 IU / mL, from 10 or about 10 IU / mL to 250 or about 250 IU / mL, from 50 or about 50 IU / mL to 200 or about 200 IU / mL, from 50 or about 50 IU / mL to 150 or about 150 IU / mL, from 75 or about 75 IU / mL to 125 or about 125 IU / mL, from 100 or about 100 IU / mL to 200 or about 200 IU / mL, or from 10 or about 10 IU / mL to 100 or about 100 IU / mL, e.g., in serum-free medium. In certain embodiments, cells, e.g., cells of the input composition, have a cytotoxicity of at or about 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, 150 IU / mL, 160 IU / mL, 170 IU / mL, 180 IU / mL, 190 IU / mL, or 200 IU / mL. In some embodiments, the cells, e.g., input cells, are incubated with recombinant IL-15 at a concentration of 100 IU / mL, 190 IU / mL, or 200 IU / mL. In some embodiments, the cells, e.g., input cells, are incubated in the presence of 100 IU / mL or about 100 IU / mL of recombinant IL-15, e.g., human recombinant IL-15.

[0158] In certain embodiments, cells, e.g., cells from the input composition, are incubated under stimulatory conditions, e.g., in serum-free medium, in the presence of IL-2, IL-7, and / or IL-15. In some embodiments, the IL-2, IL-7, and / or IL-15 are recombinant IL-2, IL-7, and / or IL-15. In certain embodiments, the IL-2, IL-7, and / or IL-15 are human IL-2, IL-7, and / or IL-15. In certain embodiments, the one or more cytokines are or comprise human recombinant IL-2, IL-7, and / or IL-15. In certain embodiments, the cells are incubated under stimulatory conditions, e.g., in serum-free medium, in the presence of recombinant IL-2, IL-7, and IL-15.

[0159] The conditions can include one or more of a particular 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.

[0160] 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.

[0161] In some embodiments, the incubation is carried out in a serum-free medium. In some embodiments, the serum-free medium is a defined cell culture medium and / or a chemically defined cell culture medium. In certain embodiments, the serum-free medium is a defined culture medium that has been processed, for example, filtered to remove inhibitors and / or growth factors. In some embodiments, the serum-free medium contains proteins. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and / or attachment factors.

[0162] In some embodiments, at least a portion of the incubation in the presence of one or more stimulating conditions or stimulating reagents is carried out in the internal cavity of a centrifuge chamber, for example, under centrifugal rotation, as described in International Publication No. WO 2016 / 073602. In some embodiments, at least a portion of the incubation carried out in the centrifuge chamber includes mixing with one or more reagents to induce stimulation and / or activation. In some embodiments, cells, for example, selected cells, are mixed with stimulating conditions or stimulating agents in the centrifuge chamber. In some aspects of such processes, a volume of cells is mixed with one or more stimulating conditions or stimulating agents in an amount that is much smaller than the amount used when performing similar stimulation in a cell culture plate or other system.

[0163] In some embodiments, the stimulatory agent is added to the cells in the cavity of the chamber in a substantially reduced amount (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or less) compared to the amount of stimulatory agent that would typically be used or required to achieve selection of the same number of cells or the same volume of cells with about the same or similar efficiency when selection is performed without mixing in the centrifuge chamber, e.g., by intermittent shaking or rotation in a tube or bag. In some embodiments, incubation is, including incubation of reagents, for example, 10 mL to 200 mL, e.g., at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL, or at least about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL, The incubation is performed by adding incubation buffer to the cells and stimulatory agent to achieve a target volume of about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL, or about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL. In some embodiments, the incubation buffer and stimulatory agent are premixed prior to addition to the cells. In some embodiments, the incubation buffer and stimulatory agent are added to the cells separately. In some embodiments, the stimulation incubation is performed under intermittent, gentle mixing conditions, which helps promote energetically favorable interactions, thereby achieving cell stimulation and activation while reducing the total amount of stimulatory agent used.

[0164] In some embodiments, incubation is generally carried out under mixing conditions, e.g., generally with relatively low force or low speed, e.g., a speed less than that used to pellet cells, e.g., at or about 600 rpm to 1700 rpm or about 1700 rpm (e.g., 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or about 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm), e.g., a speed at which the RCF at the sample or wall of the chamber or other container is 80 or about 80 g to 100 g (e.g., 80 g, 85 g, 90 g, 95 g, or 100 g, or about 80 g, 85 g, 90 g, 95 g, or 100 g, or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, rotation is performed using a repeat interval of such slow rotation followed by a rest period, e.g., rotation and / or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, e.g., rotation for approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.

[0165] In some embodiments, the total duration of incubation under stimulatory conditions, e.g., with a stimulatory reagent, is between or about 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours, 12 hours and 24 hours, 18 hours and 30 hours, e.g., at least 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours, e.g., at least about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, the total duration of incubation, e.g., with a stimulatory reagent, is 18 hours or about 18 hours to about 30 hours.

[0166] In some embodiments, cells are cultured, cultivated, and / or incubated under stimulatory conditions prior to and / or during the step of introducing a polynucleotide, e.g., a polynucleotide encoding a recombinant receptor, into the cells, e.g., by transduction and / or transfection, e.g., as described in Section IC. In certain embodiments, cells are cultured, cultivated, and / or incubated under stimulatory conditions for a period of 30 minutes to 2 hours, 1 hour to 8 hours, 6 hours to 12 hours, 12 hours to 18 hours, 16 hours to 24 hours, 18 hours to 30 hours, 24 hours to 48 hours, 24 hours to 72 hours, 42 hours to 54 hours, 60 hours to 120 hours, 96 hours to 120 hours, 90 hours, 1 day to 7 days, 3 days to 8 days, 1 day to 3 days, 4 days to 6 days, or 4 days to 5 days prior to genetic modification. In some embodiments, the cells are incubated under stimulating conditions for at or about 18 to 30 hours. In certain embodiments, the cells are cultured under stimulating conditions for at or about 24 hours.

[0167] In some embodiments, incubating the cells under stimulatory conditions comprises incubating the cells with a stimulatory reagent as described in Section IB-1. In some embodiments, the stimulatory reagent contains or comprises beads, such as paramagnetic beads, and the cells are incubated with the stimulatory reagent at a ratio of less than 3:1 (beads:cells), for example, a ratio of 1:1. In certain embodiments, the cells are incubated with the stimulatory reagent in the presence of one or more cytokines. In some embodiments, the cells are incubated with the stimulatory reagent at a ratio of 1:1 (beads:cells) in the presence of recombinant IL-2, IL-7, and IL-15.

[0168] In certain embodiments, an input composition of cells containing CD4+ and CD8+ T cells is incubated under stimulatory conditions. In certain embodiments, the cells are incubated in serum-free medium. In certain embodiments, the input composition has a ratio of CD4+ T cells to CD8+ T cells of 1:1 or about 1:1. In some embodiments, the input composition has a ratio of CD4+ T cells to CD8+ T cells of 1:1, 1:2, 2:1, 1:3, or 3:1, or about 1:1, 1:2, 2:1, 1:3, or 3:1. In certain embodiments, at least 100 x 10 6 or about 100 x 10 6 cells, e.g., cells from the Input Composition, e.g., 5 x 10 6 Less than or about 5 x 10 cells / mL 6 In certain embodiments, the cells are incubated under stimulatory conditions at a density of at least 50 x 10 cells / mL. 6 or about 50 x 10 6 CD4+ T cells and at least 50 × 10 6 or about 50 x 10 6 CD8+ T cells are incubated under stimulatory conditions. In some embodiments, the cells are incubated for 18 to 30 hours. In certain embodiments, incubating the cells under stimulatory conditions comprises incubating the cells with a stimulatory reagent in the presence of IL-2, IL-7, and / or IL-15. In certain embodiments, the cells are incubated with the stimulatory reagent at a stimulatory reagent to cell ratio of less than 3:1. In some embodiments, the cells are incubated with 50 or about 50 IU / mL to 200 or about 200 IU / mL of IL-2, 400 or about 400 to 1,000 or about 1,000 IU / mL of IL-7, and / or 50 or about 50 IU / mL to 200 or about 200 IU / mL of IL-15.

[0169] In certain embodiments, 100 x 10 of an input composition containing CD4+ and CD8+ T cells at a ratio of 1:1 or about 1:1 6 ~500×10 6In certain embodiments, 200 x 10 cells of an input composition containing CD4+ and CD8+ T cells at a 1:1 or about 1:1 ratio are incubated under stimulatory conditions. 6 ~400×10 6 300×10 cells of the input composition are incubated under stimulatory conditions. In certain embodiments, the cells are viable and negative for apoptotic markers. In some embodiments, ... 6 or approximately 300 x 10 6 In certain embodiments, the cells are incubated in serum-free medium. In certain embodiments, the cells are incubated in a medium containing 3×10 6 cells / mL or approximately 3 x 10 6 In some embodiments, the cells are incubated at a density of 150 x 10 cells / mL. 6 or about 150 x 10 6 CD4+ T cells and 150 × 10 6 or about 150 x 10 6 In certain embodiments, the cells are incubated with a stimulatory reagent at a stimulatory reagent to cell ratio of 1:1 or about 1:1. In certain embodiments, the cells are incubated in the presence of 100 IU / mL or about 100 IU / mL of IL-2, 600 IU / mL or about 600 IU / mL of IL-7, and 50 IU / mL to 200 or about 200 IU / mL of IL-15.

[0170] 1. Irritant Reagents In some embodiments, incubating the enriched cell composition under stimulatory conditions is or includes incubating and / or contacting the enriched cell composition with a stimulatory reagent capable of activating and / or expanding T cells. In some embodiments, the stimulatory reagent can stimulate and / or activate one or more signals within the cell. In some embodiments, the one or more signals are mediated by a receptor. In certain embodiments, the one or more signals are or are associated with changes in signal transduction and / or second messenger levels or amounts, such as cAMP and / or intracellular calcium, changes in the amount, subcellular localization, confirmation, phosphorylation, ubiquitination, and / or cleavage of one or more cellular proteins, and / or changes in cellular activity, such as transcription, translation, proteolysis, cell morphology, activation state, and / or cell division. In certain embodiments, the stimulatory conditions include incubating, culturing, and / or cultivating the cells with a stimulatory reagent. In certain embodiments, the stimulatory reagent contains or includes beads. In certain embodiments, initiation of stimulation occurs when cells are incubated with or contacted with a stimulatory reagent. In certain embodiments, the stimulatory reagent contains or comprises an oligomeric reagent, such as a streptavidin mutein oligomer. In certain embodiments, the stimulatory reagent activates and / or can activate one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules. In some embodiments, both stimulatory reagents are also referred to as primary agents, and / or both stimulatory reagents are also referred to as secondary agents. In some embodiments, the stimulatory reagent comprises a primary agent, e.g., a primary agent that specifically binds to a member of the TCR complex. In some embodiments, the primary agent specifically binds to CD3.In some embodiments, the stimulatory reagent comprises a secondary agent, e.g., a secondary agent that specifically binds to a T cell costimulatory molecule. In some embodiments, the secondary agent specifically binds to CD28, CD137 (4-1-BB), OX40, or ICOS.

[0171] In some embodiments, the stimulatory conditions or stimulatory reagents include one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of the TCR complex. In some embodiments, agents contemplated herein include, but are not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipid lectins, or any other biomolecules with affinity for a desired target. In some embodiments, the desired target is a T cell receptor and / or a component of a T cell receptor. In certain embodiments, the desired target is CD3. In certain embodiments, the desired target is a T cell costimulatory molecule, e.g., CD28, CD137 (4-1-BB), OX40, or ICOS. The one or more agents can be directly or indirectly attached to the beads by a variety of methods known and available in the art. Attachment can be covalent, non-covalent, electrostatic, or hydrophobic and can be achieved by a variety of attachment means, including chemical, mechanical, or enzymatic means. In some embodiments, the agent is an antibody or antigen-binding fragment thereof, e.g., a Fab. In some embodiments, a biomolecule (eg, a biotinylated anti-CD3 antibody) can be attached indirectly via another biomolecule (eg, an anti-biotin antibody) that is directly attached to the bead.

[0172] In some embodiments, the stimulatory reagent contains one or more agents (e.g., antibodies or antigen-binding fragments thereof, e.g., Fabs) that specifically bind to one or more of the following macromolecules on a cell (e.g., a T cell): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHC1, MHCII, CT LA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-gamma R, TNF-alpha R, IL-4R, IL-10R, CD18 / CD11a (LFA-1), CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligands (e.g., Delta-like 1 / 4, Jagged 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3 or fragments thereof, and corresponding ligands for these macromolecules or fragments thereof. In some embodiments, the stimulatory reagent contains one or more agents (e.g., antibodies or antigen-binding fragments thereof, e.g., Fabs) that specifically bind to one or more of the following macromolecules on a cell (e.g., a T cell): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO. In some embodiments, the one or more agents are or can be attached to beads (e.g., paramagnetic beads). In some embodiments, the one or more agents are or can be attached (e.g., reversibly) to an oligomeric reagent, e.g., a streptavidin mutein oligomer.

[0173] In some embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof, e.g., Fab. Antibodies can include polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multiple epitopic specificities, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). In some embodiments, the stimulatory reagent is or comprises an antibody fragment (including an antigen-binding fragment), e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. Antibodies contemplated herein can have constant regions of any isotype, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be derived from any human or animal species (e.g., murine species). It will be understood that a T cell receptor can be obtained. In some embodiments, the agent is or comprises an antibody that binds to and / or recognizes one or more components of the T cell receptor. In particular embodiments, the agent is or comprises an anti-CD3 antibody. In certain embodiments, the agent is or comprises an antibody that binds to and / or recognizes a coreceptor. In some embodiments, the stimulatory reagent is or comprises an anti-CD28 antibody. In some embodiments, the stimulatory reagent comprises a first agent that is or comprises an anti-CD3 antibody or an antigen-binding fragment thereof, and a second agent that is or comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

[0174] In some embodiments, cells, e.g., cells of an input population, are stimulated at a ratio of stimulatory reagent to cells of at or about 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1. In certain embodiments, the ratio of stimulatory reagent to cells is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, or between 1.1:1 and 0.9:1. In certain embodiments, the ratio of stimulatory reagent to cells is about 1:1 or is 1:1.

[0175] In some embodiments, the cells are 6 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg or 10 μg per cell In some embodiments, cells are stimulated in the presence of 0.75 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg, or at least 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg of a stimulatory reagent. 6 In certain embodiments, cells are stimulated in the presence of 10 μg or about 4 μg per cell. 6 In various embodiments, cells are stimulated in the presence of 10 or about 0.8 μg per cell. 6 Stimulation occurs in the presence of 0.8 μg or about 0.8 μg per cell.

[0176] a. Bead Reagent In certain embodiments, the stimulatory reagent contains one or more agents, e.g., particles, e.g., beads, conjugated or linked to biomolecules, that can activate and / or expand cells, e.g., T cells. In some embodiments, the one or more agents are bound to a solid support. In some embodiments, the solid support is or comprises beads. In some embodiments, the one or more agents are bound to beads. In some embodiments, the beads are biocompatible, i.e., the beads are composed of a material suitable for biological use. In some embodiments, the beads are non-toxic to cultured cells, e.g., cultured T cells. In some embodiments, the beads can be any particle to which an agent can be attached in a manner that allows for interaction between the agent and the cell.

[0177] In some embodiments, the stimulatory reagent contains one or more agents capable of activating and / or expanding cells, e.g., T cells, bound to or otherwise attached to the surface of the beads. In certain embodiments, the beads are non-cell particles. In particular embodiments, the beads may comprise colloidal particles, microspheres, nanoparticles, magnetic beads, etc. In some embodiments, the beads are agarose beads. In certain embodiments, the beads are sepharose beads.

[0178] In certain embodiments, the stimulating reagent contains particles that are monodisperse. In certain embodiments, monodisperse beads comprise a size distribution that has a diameter standard deviation of less than 5% from one another.

[0179] In some embodiments, the beads contain one or more agents, e.g., agents coupled, conjugated, or linked (directly or indirectly) to the surface of the beads. In some embodiments, agents contemplated herein may include, but are not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipid lectins, or any other biomolecules with affinity for a desired target. In some embodiments, the desired target is a T cell receptor and / or a component of a T cell receptor. In certain embodiments, the desired target is CD3. In certain embodiments, the desired target is a T cell costimulatory molecule, e.g., CD28, CD137 (4-1-BB), OX40, or ICOS. The one or more agents may be directly or indirectly attached to the beads by a variety of methods known and available in the art. Attachment may be covalent, non-covalent, electrostatic, or hydrophobic and may be achieved by a variety of attachment means, including chemical, mechanical, or enzymatic means. In some embodiments, a biomolecule (eg, a biotinylated anti-CD3 antibody) can be attached indirectly via another biomolecule (eg, an anti-biotin antibody) that is directly attached to the bead.

[0180] In some embodiments, the stimulatory reagent contains beads and one or more agents that directly interact with macromolecules on the cell surface. In certain embodiments, the beads (e.g., paramagnetic beads) interact with cells through one or more agents (e.g., antibodies) specific to one or more macromolecules (e.g., one or more cell surface proteins) on the cells. In certain embodiments, the beads (e.g., paramagnetic beads) are labeled with a first agent described herein, such as a primary antibody (e.g., an anti-biotin antibody) or other biomolecule, and then a second agent, such as a secondary antibody (e.g., a biotinylated anti-CD3 antibody) or other second biomolecule (e.g., streptavidin), is added, whereby the secondary antibody or other second biomolecule specifically binds to the primary antibody or other biomolecule on the particle.

[0181] In some embodiments, the stimulatory reagent contains one or more agents (e.g., antibodies) attached to beads (e.g., paramagnetic beads) and that specifically bind to one or more of the following macromolecules on a cell (e.g., a T cell): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHC1, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-gamma R, TNF-alpha R, IL-4R, IL-10R, CD18 / CD11a (LFA-1, alpha L β2), CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligands (e.g., Delta-like 1 / 4, Jagged 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3 or fragments thereof, or the corresponding ligands for these macromolecules or fragments thereof. In some embodiments, the agent (e.g., an antibody) attached to the bead specifically binds to one or more of the following macromolecules on a cell (e.g., a T cell): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA and / or CD45RO.

[0182] In some embodiments, one or more of the agents attached to the beads is an antibody. Antibodies can include polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multiple epitopic specificities, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). In some embodiments, the stimulatory reagent is an antibody fragment (including an antigen-binding fragment), such as a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. It will be understood that the constant region of any isotype can be used for the antibodies contemplated herein, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from any human or animal species (e.g., murine species). In some embodiments, the agent is an antibody that binds to and / or recognizes one or more components of the T cell receptor. In particular embodiments, the agent is an anti-CD3 antibody. In certain embodiments, the agent binds to and / or recognizes a coreceptor. or a co-receptor-recognizing antibody. In some embodiments, the stimulatory reagent comprises an anti-CD28 antibody. In some embodiments, the beads have a diameter of greater than 0.001 or about 0.001 μm, greater than 0.01 or about 0.01 μm, greater than 0.1 or about 0.1 μm, greater than 1.0 or about 1.0 μm, greater than 10 or about 10 μm, greater than 50 or about 50 μm, greater than 100 or about 100 μm, or greater than 1000 or about 1000 μm, and greater than 1500 or about 1500 μm. In some embodiments, the beads have a diameter of greater than 1.0 or about 1.0 μm, greater than 10 or about 10 μm, greater than 50 or about 50 μm, greater than 100 or about 1000 μm, and greater than 1500 or about 1500 μm. In some embodiments, the beads have a diameter of from about 1.0 μm to 500 or about 500 μm, from 1.0 or about 1.0 μm to 150 or about 150 μm, from 1.0 or about 1.0 μm to 30 or about 30 μm, from 1.0 or about 1.0 μm to 10 or about 10 μm, from 1.0 or about 1.0 μm to 5.0 or about 5.0 μm, from 2.0 or about 2.0 μm to 5.0 or about 5.0 μm, or from 3.0 or about 3.0 μm to 5.0 or about 5.0 μm. In some embodiments, the beads have a diameter of from 3 or about 3 μm to 5 or about 5 μm.In some embodiments, the beads are at least 0.001 μm, 0.01 μm, 0.1 μm, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, 5.0 μm, 5.5 μm, 6.0 μm, 6.5 μm, 7.0 μm, 7.5 μm, 8.0 μm m, 8.5 μm, 9.0 μm, 9.5 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, or 20 μm, or at least about 0.001 μm, 0.01 μm, 0.1 μm, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm , 5.0 μm, 5.5 μm, 6.0 μm, 6.5 μm, 7.0 μm, 7.5 μm, 8.0 μm, 8.5 μm, 9.0 μm, 9.5 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, or 20 μm, or about 0.001 μm, 0.01 μm, 0.1 μm, 0.5 μm, 1.0 μm, 1.5 μm , 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, 5.0 μm, 5.5 μm, 6.0 μm, 6.5 μm, 7.0 μm, 7.5 μm, 8.0 μm, 8.5 μm, 9.0 μm, 9.5 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, or 20 μm. In certain embodiments, the beads have a diameter of 4.5 or about 4.5 μm. In certain embodiments, the beads have a diameter of 2.8 or about 2.8 μm.

[0183] In some embodiments, the beads have a density of 0.001 or about 0.001 g / cm 3 Over 0.01 or approximately 0.01 g / cm 3 Over 0.05 or approximately 0.05 g / cm 3 Over 0.1 or about 0.1 g / cm 3 Over 0.5 or about 0.5 g / cm 3 Over 0.6 or about 0.6 g / cm 3 Over 0.7 or about 0.7 g / cm 3 Over 0.8 or about 0.8 g / cm 3 Over 0.9 or approximately 0.9 g / cm 3 Over 1 or about 1 g / cm 3 Over 1.1 or about 1.1g / cm3 Over 1.2 or about 1.2 g / cm 3 Over 1.3 or about 1.3 g / cm 3 Over 1.4 or about 1.4g / cm 3 Over 1.5 or about 1.5g / cm 3 Over 2 or about 2 g / cm 3 Over 3 or about 3 g / cm 3 Over 4 or about 4g / cm 3 , or 5 or about 5 g / cm 3 In some embodiments, the beads have a density of at or about 0.001 g / cm 3 ~100 or about 100g / cm 3 , 0.01 or approximately 0.01 g / cm 3 ~50 or about 50g / cm 3 , 0.1 or approximately 0.1 g / cm 3 ~10 or about 10g / cm 3 , 0.1 or approximately 0.1 g / cm 3 ~5 or about 5g / cm 3 , 0.5 or about 0.5g / cm 3 ~1 or about 1g / cm 3 , 0.5 or about 0.5g / cm 3 ~1.5 or about 1.5g / cm 3 , 1 or about 1 g / cm 3 ~1.5 or about 1.5g / cm 3 , 1 or about 1 g / cm 3 ~2 or about 2g / cm 3 , or 1 or about 1 g / cm 3 ~5 or about 5g / cm 3 In some embodiments, the beads have a density of 0.5 or about 0.5 g / cm 3 , 0.5 or about 0.5g / cm 3 , 0.6 or approximately 0.6 g / cm 3 , 0.7 or about 0.7g / cm 3 , 0.8 or approximately 0.8g / cm 3 , 0.9 or approximately 0.9 g / cm 3 , 1.0 or about 1.0 g / cm 3, 1.1 or about 1.1g / cm 3 , 1.2 or about 1.2g / cm 3 , 1.3 or about 1.3g / cm 3 , 1.4 or about 1.4g / cm 3 , 1.5 or about 1.5g / cm 3 , 1.6 or about 1.6g / cm 3 , 1.7 or about 1.7g / cm 3 , 1.8 or about 1.8g / cm 3 , 1.9 or approximately 1.9 g / cm 3 , or 2.0 or about 2.0 g / cm 3 In certain embodiments, the beads have a density of at or about 1.6 g / cm 3 In certain embodiments, the beads or particles have a density of at or about 1.5 g / cm 3 In certain embodiments, the particles have a density of at or about 1.3 g / cm 3 It has a density of

[0184] In certain embodiments, the plurality of beads has a uniform density, hi certain embodiments, the uniform density comprises a density standard deviation of less than at or about 10%, less than at or about 5%, or less than at or about 1% of the average bead density.

[0185] In some embodiments, the beads have a particle density of at or about 0.001 m per gram of each particle. 2 (m 2 / g) ~ 1,000 or about 1,000m 2 / g, 0.010 or approximately 0.010m 2 / g~100 or about 100m 2 / g, 0.1 or approximately 0.1m 2 / g~10 or about 10m 2 / g, 0.1 or approximately 0.1m 2 / g~1 or about 1m 2 / g, 1 or about 1m 2 / g~10 or about 10m 2 / g, 10 or approximately 10m 2 / g~100 or about 100m2 / g, 0.5 or approximately 0.5m 2 / g~20 or about 20m 2 / g, 0.5 or approximately 0.5m 2 / g~5 or about 5m 2 / g, or 1 or about 1 m 2 / g~4 or about 4m 2 In some embodiments, the particles or beads have a surface area of ​​1 or about 1 m 2 / g~4 or about 4m 2 / g of surface area.

[0186] In some embodiments, the beads contain at least one material on or near the surface of the beads that can be coupled, linked, or conjugated to an agent. In some embodiments, the beads are surface-functionalized. That is, the beads contain functional groups that can form covalent bonds with binding molecules, such as polynucleotides or polypeptides. In certain embodiments, the beads contain surface-exposed carboxyl, amino, hydroxyl, tosyl, epoxy, and / or chloromethyl groups. In certain embodiments, the beads contain surface-exposed agarose and / or sepharose. In certain embodiments, the bead surface is equipped with a stimulating reagent that can bind or attach to the binding molecule. In certain embodiments, the biomolecule is a polypeptide. In some embodiments, the beads contain surface-exposed protein A, protein G, or biotin.

[0187] In some embodiments, the beads respond in a magnetic field. In some embodiments, the beads are magnetic beads. In some embodiments, the magnetic beads are paramagnetic. In certain embodiments, the magnetic beads are superparamagnetic. In certain embodiments, the beads do not exhibit any magnetic properties unless they are exposed to a magnetic field.

[0188] In certain embodiments, the beads comprise a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the magnetic core contains a metal. In some embodiments, the metal can be, for example, but not limited to, iron, nickel, copper, cobalt, gadolinium, manganese, tantalum, zinc, zirconium, or any combination thereof. In certain embodiments, the magnetic core comprises a metal oxide (e.g., iron oxide), ferrite (e.g., manganese ferrite, cobalt ferrite, nickel ferrite, etc.), hematite, and an alloy (e.g., CoTaZn). In some embodiments, the magnetic core comprises one or more of ferrite, a metal, an alloy, iron oxide, or chromium dioxide. In some embodiments, the magnetic core comprises elemental iron or a compound thereof. In some embodiments, the magnetic core comprises one or more of magnetite (FeO), maghemite (γFeO), or greigite (FeS). In some embodiments, the inner core comprises iron oxide (e.g., FeO).

[0189] In certain embodiments, the beads contain a magnetic, paramagnetic, and / or superparamagnetic core covered with a surface functionalized coat or coating. In some embodiments, the coat can contain a material that can include, for example, but is not limited to, a polymer, a polysaccharide, silica, a fatty acid, a protein, carbon, agarose, sepharose, or a combination thereof. In some embodiments, the polymer can be polyethylene glycol, poly(lactic-co-glycolic acid), polyglutaraldehyde, polyurethane, polystyrene, or polyvinyl alcohol. In certain embodiments, the outer coat or coating comprises polystyrene. In particular embodiments, the outer coating is surface functionalized.

[0190] In some embodiments, the stimulating reagent comprises beads containing a metal oxide core (e.g., an iron oxide core) and a coating, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and the coating comprises at least one polysaccharide (e.g., aminodextran), at least one polymer (e.g., polyurethane), and silica. In some embodiments, the metal oxide core is a colloidal iron oxide core. In certain embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof. In particular embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulating reagent comprises an anti-CD3 antibody, an anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the stimulating reagent comprises an anti-biotin antibody. In some embodiments, the beads have a diameter of about 3 μm to about 10 μm. In some embodiments, the beads have a diameter of about 3 μm to about 5 μm. In certain embodiments, the beads have a diameter of about 3.5 μm.

[0191] In some embodiments, the stimulatory reagent comprises one or more agents attached to beads comprising a metal oxide core (e.g., an iron oxide inner core) and a coat (e.g., a protective coat), wherein the coat comprises polystyrene. In certain embodiments, the beads are monodisperse paramagnetic (e.g., superparamagnetic) beads comprising a paramagnetic (e.g., superparamagnetic) iron core, such as magnetite (Fe3O4) and / or maghemite (γFe2O3)c, and a polystyrene coat or coating. In some embodiments, the beads are non-porous. In some embodiments, the beads contain a functionalized surface to which one or more agents are attached. In certain embodiments, the one or more agents are covalently bound to the beads at their surface. In some embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof. In some embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the one or more agents comprise an anti-CD3 antibody and / or an anti-CD28 antibody and a labeled antibody (e.g., a biotinylated antibody), e.g., an antibody or antigen fragment thereof capable of binding to the labeled anti-CD3 or anti-CD28 antibody. In certain embodiments, the beads have a mass of about 1.5 g / cm 3 density and about 1m 2 / g ~ approx. 4m 2 / g of surface area. In certain embodiments, the beads have a diameter of about 4.5 μm and a surface area of ​​about 1.5 g / cm 3 In some embodiments, the beads are monodisperse superparamagnetic beads having an average diameter of about 2.8 μm and a density of about 1.3 g / cm 3 The superparamagnetic beads are monodisperse and have a density of 0.01 to 0.01.

[0192] In some embodiments, the enriched T cell composition is incubated with a stimulatory reagent at a bead to cell ratio of at or about 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1 or 0.2:1. In certain embodiments, the ratio of beads to cells is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, or between 1.1:1 and 0.9:1. In certain embodiments, the ratio of beads to cells is about 1:1 or is 1:1.

[0193] b. Oligomeric reagents In certain embodiments, the stimulatory reagent contains an oligomeric reagent, e.g., a streptavidin mutein reagent, conjugated, linked, or attached to one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of the TCR complex. In some embodiments, one or more agents are attached with a binding domain or binding partner (e.g., binding partner C) that can bind to a specific binding site (e.g., binding site Z) of the oligomeric reagent. In some embodiments, multiple agents are reversibly bound to the oligomeric reagent. In various embodiments, the oligomeric reagent has multiple specific binding sites, which, in certain embodiments, are reversibly bound to multiple agent binding domains (e.g., binding partner C). In some embodiments, the amount of bound agent is reduced or decreased in the presence of a competing reagent, e.g., a reagent that can also bind to the specific binding site (e.g., binding site Z). Oligomeric stimulatory reagents, including anti-CD3 / anti-CD28 oligomeric streptavidin mutein reagents, are described in International PCT Application No. WO 2018 / 197949.

[0194] In some embodiments, the stimulatory reagent is or includes a reversible system in which at least one agent (e.g., an agent capable of generating a signal in a cell, such as a T cell) is associated, e.g., reversibly associated, with the oligomeric reagent. In some embodiments, the reagent contains multiple binding sites that can bind, e.g., reversibly bind, to the agents. In some embodiments, the stimulatory reagent is reversibly bound to the surface of an oligomeric particle reagent comprising multiple streptavidin molecules or streptavidin mutein molecules. In some embodiments, the stimulatory reagent (e.g., a primary agent and a secondary agent) is reversibly bound to the surface of an oligomeric particle reagent comprising multiple streptavidin molecules or streptavidin mutein molecules. Optionally, the reagent is an oligomeric particle reagent to which at least one agent capable of generating a signal in a cell, such as a T cell, is attached. In some embodiments, the agent contains at least one binding site, e.g., binding site B, that can specifically bind to an epitope or region of the molecule, and also contains a binding partner (also referred to herein as binding partner C) that specifically binds to at least one binding site of the reagent, e.g., binding site Z of the reagent. In some embodiments, the binding interaction between binding partner C and the at least one binding site Z is a non-covalent interaction. Optionally, the binding interaction between binding partner C and the at least one binding site Z is a covalent interaction. In some embodiments, the binding interaction, such as a non-covalent interaction, between binding partner C and the at least one binding site Z is reversible.

[0195] Substances that can be used as oligomeric reagents in such reversible systems are known. See, for example, U.S. Patent Nos. 5,168,049, 5,506,121, 6,103,493, 7,776,562, 7,981,632, 8,298,782, 8,735,540, and 9,023,604, as well as published PCT applications WO 2013 / 124474 and WO 2014 / 076277. Non-limiting examples of reagents and binding partners that can form reversible interactions, as well as substances (e.g., competitive reagents) that can reverse such binding, are described below.

[0196] In some embodiments, the oligomeric reagent is an oligomer of streptavidin, a streptavidin mutein, a streptavidin analog, avidin, an avidin mutein, or an avidin analog (e.g., neutravidin), or a mixture thereof, where such an oligomeric reagent contains one or more binding sites for reversible association with an agent's binding domain (e.g., binding partner C). In some embodiments, the agent's binding domain can be biotin, a biotin derivative, a biotin analog, or a streptavidin-binding peptide, or other molecule capable of specifically binding to streptavidin, a streptavidin mutein, a streptavidin analog, avidin, an avidin mutein, or an avidin analog.

[0197] In certain embodiments, one or more agents (e.g., agents capable of generating a signal in a cell, such as a T cell) associate with (e.g., reversibly bind to) the oligomeric reagent, e.g., via multiple specific binding sites (e.g., binding site Z) present on the oligomeric reagent. In some cases, this allows the agents to be closely spaced relative to one another, such that an avidity effect can occur when the agent is contacted with a target cell bearing (at least two copies of) a cell surface molecule bound by or recognized by the agent.

[0198] In some embodiments, the oligomeric reagent is a streptavidin oligomer, a streptavidin mutein oligomer, a streptavidin analog oligomer, an avidin oligomer, an oligomer composed of an avidin mutein or an avidin analog (e.g., neutravidin), or a mixture thereof. In certain embodiments, the oligomeric reagent contains a specific binding site capable of binding to a binding domain of an agent (e.g., binding partner C). In some embodiments, the binding domain can be biotin, a biotin derivative, a biotin analog, or a streptavidin-binding peptide, or other molecule capable of specifically binding to streptavidin, a streptavidin mutein, a streptavidin analog, avidin, an avidin mutein, or an avidin analog.

[0199] In some embodiments, the streptavidin can be wild-type streptavidin, or a streptavidin mutein or streptavidin analog, such as a streptavidin-like polypeptide. Similarly, the avidin, in some aspects, includes wild-type avidin, or a mutein or analog of avidin, such as neutravidin, which is an arginine-modified, deglycosylated avidin that typically exhibits a more neutral pH and can be used in place of native avidin. Generally, deglycosylated, neutral forms of avidin include commercially available forms such as "Extravidin" available from Sigma-Aldrich or "NeutrAvidin" available from Thermo Scientific or Invitrogen.

[0200] In some embodiments, the reagent is streptavidin or a streptavidin mutein or streptavidin analog. In some embodiments, wild-type streptavidin (wt-streptavidin) has the amino acid sequence (SEQ ID NO:34) disclosed in Argarana et al., Nucleic Acids Res. 14 (1986) 1871-1882. Generally, streptavidin naturally occurs as a tetramer of four identical subunits, i.e., streptavidin is a homotetramer, with each subunit containing a single binding site for biotin, a biotin derivative or biotin analog, or a biotin mimetic. An exemplary sequence of a subunit of streptavidin is the sequence of amino acids set forth in SEQ ID NO:34, although such a sequence may also include sequences present in its homologs from other species of the genus Streptomyces. In particular, each subunit of streptavidin has an equilibrium dissociation constant (K D ) is 10 -14 or about 10 -14 Streptavidin can exhibit strong binding affinity for biotin, on the order of M. In some cases, streptavidin can exist as a monovalent tetramer in which only one of the four binding sites is functional (Howarth et al. (2006) Nat. Methods, 3:267-73; Zhang et al. (2015) Biochem. Biophys. Res. Commun., 63:1059-63), a bivalent tetramer in which two of the four binding sites are functional (Fairhead et al. (2013) J. Mol. Biol., 426:199-214), or as a monomeric or dimeric form (Wu et al. (2005) J. Biol. Chem., 280:23225-31; Lim et al. (2010) Biochemistry, 50:8682-91).

[0201] In some embodiments, the streptavidin can be in any form, such as wild-type or unmodified streptavidin, e.g., streptavidin from a Streptomyces species, or a functionally active fragment thereof, comprising at least one functional subunit containing a binding site for biotin, a biotin derivative, a biotin analog, or a biotin mimetic, e.g., typically comprising at least one functional subunit of wild-type streptavidin from Streptomyces avidinii, or a functionally active fragment thereof, as set forth in SEQ ID NO: 34. For example, in some embodiments, the streptavidin can comprise a fragment of wild-type streptavidin that is N-terminally and / or C-terminally truncated. Such minimal streptavidins include any whose N-terminus begins in the region of amino acid positions 10-16 of SEQ ID NO:34 and whose C-terminus ends in the region of amino acid positions 133-142 of SEQ ID NO:34. In some embodiments, a functionally active fragment of streptavidin contains the sequence of amino acids set forth in SEQ ID NO:35. In some embodiments, a streptavidin, such as that set forth in SEQ ID NO:35, can further contain an N-terminal methionine at the position corresponding to Ala13 in the numbering set forth in SEQ ID NO:34. References to residue positions in streptavidin or streptavidin muteins refer to the residue numbering in SEQ ID NO:34.

[0202] Examples of streptavidin or streptavidin mutein are mentioned in, for example, WO 86 / 02077, DE 19641876 A1, US 6,022,951, WO 98 / 40396 or WO 96 / 24606.Examples of streptavidin mutein are known in the art.See, for example, U.S. Patent No. 5,168,049, U.S. Patent No. 5,506,121, U.S. Patent No. 6,022,951, U.S. Patent No. 6,156,493, U.S. Patent No. 6,165,750, U.S. Patent No. 6,103,493 or U.S. Patent No. 6,368,813, or International PCT Publication No. WO 2014 / 076277.

[0203] In some embodiments, a streptavidin mutein contains amino acids that are not part of unmodified streptavidin or wild-type streptavidin, or can comprise only a portion of wild-type streptavidin or unmodified streptavidin. In some embodiments, a streptavidin mutein contains at least one subunit, which can have one or more amino acid substitutions (replacements) compared to a subunit of unmodified streptavidin or wild-type streptavidin, for example, compared to a subunit of wild-type streptavidin as shown in SEQ ID NO:34 or a functionally active fragment thereof as shown in, for example, SEQ ID NO:35 or SEQ ID NO:56.

[0204] In some embodiments, the binding affinity of the streptavidin or streptavidin mutein to the binding domain, e.g., the dissociation constant (K d ) is 1×10 -4 M, 5 x 10 -4 M, 1 x 10 -5 M, 5 x 10 -5 M, 1 x 10 -6 M, 5 x 10 -6 M or 1 x 10 -7 Less than M or approximately 1 x 10 -4 M, 5 x 10 -4 M, 1 x 10 -5 M, 5 x 10 -5M, 1 x 10 -6 M, 5 x 10 -6 M or 1 x 10 -7 M, but generally, 1 × 10 -13 M, 1 x 10 -12 M, or 1 x 10 -11 For example, peptide sequences (Strep-tags) such as those disclosed in U.S. Pat. No. 5,506,121 can act as biotin mimics, e.g., K D is about 10 -4 ~about 10 -5 The binding affinity of streptavidin is M. In some cases, the binding affinity can be further improved by making mutations in the streptavidin molecule. For example, see U.S. Patent No. 6,103,493 or published PCT application WO 2014 / 076277. In some embodiments, the binding affinity can be determined by a method known in the art, for example, any of the methods described herein.

[0205] In some embodiments, a reagent such as streptavidin or a streptavidin mutein exhibits binding affinity for a peptide ligand binding partner, which can be binding partner C present in an agent (e.g., a receptor-binding agent or a selection agent). In some embodiments, the peptide sequence contains a sequence having the general formula His-Pro-Xaa, where Xaa is glutamine, asparagine, or methionine, e.g., the sequence set forth in SEQ ID NO:51. In some embodiments, the peptide sequence has the general formula set forth in SEQ ID NO:52, e.g., as set forth in SEQ ID NO:42. In one example, the peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also known as Strep-tag® and set forth in SEQ ID NO:43). In one example, the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also known as Strep-tag® II and set forth in SEQ ID NO:37). In some embodiments, the peptide ligand contains a continuous sequence of at least two streptavidin binding modules, the distance between the two modules being at least 0 amino acids and no more than 50 amino acids, one binding module having 3-8 amino acids and containing at least the sequence His-Pro-Xaa, where Xaa is glutamine, asparagine, or methionine, and the other binding module having the same or a different streptavidin peptide ligand, e.g., that set forth in SEQ ID NO:52 (see, e.g., International Published PCT Application WO 02 / 077018; U.S. Patent No. 7,981,632). In some embodiments, the peptide ligand contains a sequence having a formula set forth in either SEQ ID NO:44 or 45. In some embodiments, the peptide ligand has a sequence of amino acids set forth in any of SEQ ID NOs: 38-40, 46, and 47. In most cases, all of these streptavidin-binding peptides bind to the same binding site, namely the biotin-binding site of streptavidin.When one or more such streptavidin-binding peptides are used as binding partners C, e.g., C1 and C2, the multimerization reagent and / or oligomeric particle reagent bound to one or more active substances via binding partner C typically consists of one or more streptavidin muteins.

[0206] In some embodiments, the streptavidin mutein is a variant described in U.S. Patent No. 6,103,493. In some embodiments, the streptavidin mutein contains at least one mutation within the region of amino acid positions 44-53 based on the amino acid sequence of wild-type streptavidin, e.g., as set forth in SEQ ID NO:34. In some embodiments, the streptavidin mutein contains a mutation at one or more residues 44, 45, 46, and / or 47. In some embodiments, the streptavidin mutein contains a replacement of Glu at position 44 of wild-type streptavidin with a hydrophobic aliphatic amino acid, e.g., Val, Ala, Ile, or Leu, a replacement of any amino acid at position 45, an aliphatic amino acid, e.g., a hydrophobic aliphatic amino acid at position 46, and / or Val at position 47 with a basic amino acid, e.g., Arg or Lys, e.g., typically Arg. In some embodiments, Ala is at position 46, and / or Arg is at position 47, and / or Val or Ile is at position 44. In some embodiments, a streptavidin mutant, such as that shown in the exemplary streptavidin mutein containing the sequence of amino acids set forth in SEQ ID NO:48 or SEQ ID NO:49 or 50 (also known as streptavidin mutant 1, SAM1), contains residues Val44-Thr45-Ala46-Arg47. In some embodiments, a streptavidin mutein, such as that shown in the exemplary streptavidin mutein containing the sequence of amino acids set forth in SEQ ID NO:53, 36, or 41 (also known as SAM2), contains residues Ile44-Gly45-Ala46-Arg47. In some cases, such streptavidin muteins are described, for example, in U.S. Patent No. 6,103,493 and are commercially available under the trademark Strep-Tactin®. In some embodiments, the mutein streptavidin contains the sequence of amino acids set forth in SEQ ID NO:54 or SEQ ID NO:55.In certain embodiments, the molecule is a tetramer of streptavidin or streptavidin mutein comprising the sequence set forth in any of SEQ ID NOs: 35, 49, 36, 54, 56, 50 or 41, which as a tetramer contains 20 primary amines, including one N-terminal amine and four lysines per monomer.

[0207] In some embodiments, the streptavidin mutein has a 3.7×10 affinity to the peptide ligand (Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; also known as Strep-tag® and set forth in SEQ ID NO:43). -5 or 3.7×10 -5 Less than or equal to approximately 3.7 x 10 -5 M or less, and / or 7.1 × 10 for the peptide ligand (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys; also known as Strep-tag® II and shown in SEQ ID NO: 37) -5 or 7.1×10 -5 Less than or equal to approximately 7.1 x 10 -5 M or less, and / or 7.0 x 10 for any one of the peptide ligands set forth in any one of SEQ ID NOs: 37, 44-47, 38-40, 42, 43, 51 and 52 -5 M, 5.0 x 10 -5 M, 1.0 × 10 -5 M, 5.0 x 10 -6 M, 1.0 × 10 -6 M, 5.0 x 10 -7 M or 1.0 x 10 -7 M or 7.0 x 10 -5 M, 5.0 x 10 -5 M, 1.0 × 10 -5 M, 5.0 x 10 -6 M, 1.0 × 10 -6 M, 5.0 x 10 -7 M or 1.0 x 10 -7 M or less, or approximately 7.0 x 10 -5 M, 5.0 x 10 -5 M, 1.0 × 10-5 M, 5.0 x 10 -6 M, 1.0 × 10 -6 M, 5.0 x 10 -7 M or 1.0 x 10 -7 M or less, but generally 1×10 -13 M, 1 x 10 -12 M or 1 x 10 -11 m or approximately 1 x 10 -13 M, 1 x 10 -12 M or 1 x 10 -11 The equilibrium dissociation constant (K D ) exhibit binding affinity characterized by

[0208] In some embodiments, the resulting streptavidin mutein has a 2.7×10 affinity to the peptide ligand (Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; also known as Strep-tag® and set forth in SEQ ID NO:43). 4 or 2.7×10 4 or more or approximately 2.7 x 10 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 and set forth in SEQ ID NO: 37) 4 or 1.4×10 4 or more or approximately 1.4 x 10 4 M -1 or greater than 1.43 x 10 for any one of the peptide ligands set forth in any one of SEQ ID NOs: 37, 44-47, 38-40, 42, 43, 51 and 52. 4 M -1 , 1.67×10 4 M -1 , 2 × 10 4 M -1 , 3.33 × 10 4 M -1 , 5×10 4 M -1 , 1×10 5 M -1, 1.11×10 5 M -1 , 1.25×10 5 M -1 , 1.43×10 5 M -1 , 1.67×10 5 M -1 , 2 × 10 5 M -1 , 3.33 × 10 5 M -1 , 5×10 5 M -1 , 1×10 6 M -1 , 1.11×10 6 M -1 , 1.25×10 6 M -1 , 1.43×10 6 M -1 , 1.67×10 6 M -1 , 2 × 10 6 M -1 , 3.33 × 10 6 M -1 , 5×10 6 M -1 , 1×10 7 M -1 or 1.43×10 4 M -1 , 1.67×10 4 M -1 , 2 × 10 4 M -1 , 3.33 × 10 4 M -1 , 5×10 4 M -1 , 1×10 5 M -1 , 1.11×10 5 M -1 , 1.25×10 5 M -1 , 1.43×10 5 M -1 , 1.67×10 5 M -1 , 2 × 10 5 M -1 , 3.33 × 10 5 M -1 , 5×10 5 M -1 , 1×106 M -1 、1.11×10 6 M -1 、1.25×10 6 M -1 、1.43×10 6 M -1 、1.67×10 6 M -1 、2×10 6 M -1 、3.33×10 6 M -1 、5×10 6 M -1 、1×10 7 M -1 以上、もしくは約1.43×10 4 M -1 、1.67×10 4 M -1 、2×10 4 M -1 、3.33×10 4 M -1 、5×10 4 M -1 、1×10 5 M -1 [[ID=:59]]、1.11×10 5 M -1 、1.25×10 5 M -1 、1.43×10 5 M -1 、1.67×10 5 M -1 、2×10 5 M -1 、3.33×10 5 M -1 、5×10 5 M -1 、1×10 6 M -1 、1.11×10 6 M -1 、1.25×10 6 M -1 、1.43×10 6 M -1 、1.67×10 6 M -1 、2×10 6 [[ID=:109]]M -1 、3.33×10 6 M -1 、5×10 6 M-1 , 1×10 7 M -1 That's all, but generally it's 1×10 13 M -1 , 1×10 12 M -1 Or 1 x 10 11 M -1 The equilibrium association constant (K A ) and exhibit a binding affinity characterized by

[0209] In certain embodiments, provided herein are oligomeric particle reagents that are composed of and / or contain a plurality of streptavidin tetramers or streptavidin mutein tetramers. In certain embodiments, the oligomeric particle reagents provided herein reversibly bind to one or more agents, such as stimulatory agents and / or selection agents, or contain a plurality of binding sites that can reversibly bind to one or more agents, such as stimulatory agents and / or selection agents. In some embodiments, the oligomeric particles have a radius of at or about 70 nm to at or about 125 nm, inclusive, e.g., an average or mean radius, 1×10, inclusive. 7 Or about 1 x 10 7 g / mol ~ 1 × 10 9 Or about 1 x 10 9In some embodiments, the oligomeric particle reagent is coupled, e.g., reversibly coupled, to one or more agents, e.g., agents that bind to a molecule, e.g., a receptor, on the surface of a cell. In some embodiments, the one or more agents are or include an antibody or antigen-binding fragment thereof, e.g., Fab. In some embodiments, the one or more agents specifically bind to one or more of the following macromolecules on a cell (e.g., a T cell): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHC1, MHCII, CTLA-4, ICOS, PD-1, OX40 , CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-gamma R, TNF-alpha R, IL-4R, IL-10R, CD18 / CD11a (LFA-1), CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligands (e.g., Delta-like 1 / 4, Jagged 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3 or fragments thereof, and corresponding ligands for these macromolecules or fragments thereof. In some embodiments, the one or more agents specifically bind to one or more of the following macromolecules on a cell (e.g., a T cell): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA and / or CD45RO.In some embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof, e.g., Fab, which can include polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multiple epitopic specificities, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single chain molecules), and antibody fragments (e.g., Fab, F(ab'), and Fv). In some embodiments, the one or more agents are or comprise an antibody fragment (including an antigen-binding fragment), e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab') fragment. It will be understood that constant regions of any isotype can be used for the antibodies contemplated herein, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from any human or animal species (e.g., murine species). In some embodiments, one or more The reagent is or comprises an antibody that binds to and / or recognizes one or more components of the T cell receptor. In certain embodiments, one or more reagents is or comprises an anti-CD3 antibody. In certain embodiments, one or more reagents is or comprises an antibody that binds to and / or recognizes a coreceptor. In some embodiments, one or more reagents is or comprises an anti-CD28 antibody. In some embodiments, one or more reagents is or comprises an anti-CD3 and / or anti-CD28 antibody or antigen-binding fragment thereof, e.g., an antibody or antigen-binding fragment thereof containing a binding partner, e.g., a streptavidin-binding peptide, e.g., Strep-tag® II. In certain embodiments, one or more agents is or comprises a binding partner, e.g., an anti-CD3 Fab and / or anti-CD28 Fab containing a streptavidin-binding peptide, e.g., Strep-tag® II.

[0210] In some embodiments, provided herein are oligomeric particle reagents that are composed of and / or contain a plurality of streptavidin tetramers or streptavidin mutein tetramers. In certain embodiments, provided herein are oligomeric particle reagents that reversibly bind to one or more agents, such as stimulatory agents and / or selection agents, or contain a plurality of binding sites that can reversibly bind to one or more agents, such as stimulatory agents and / or selection agents. In some embodiments, the oligomeric particles have a radius of at or about 80 nm to at or about 120 nm, inclusive, for example, an average radius of 7.5×10 6 Or about 7.5 x 10 6 g / mol~2×10 8 Or about 2 x 10 8 The oligomeric particle reagent has a molecular weight, e.g., an average molecular weight, in g / mol, and / or an amount, e.g., an average amount, of streptavidin or streptavidin mutein tetramers, from at or about 500 to at or about 10,000, inclusive. In some embodiments, the oligomeric particle reagent is bound, e.g., reversibly bound, to one or more agents, e.g., agents that bind to molecules, e.g., receptors, on the surface of cells. In some embodiments, the agents are anti-CD3 Fab and / or anti-CD28 Fab, e.g., Fabs containing a binding partner, e.g., a streptavidin-binding peptide, e.g., Strep-tag® II. In certain embodiments, the one or more agents are anti-CD3 Fab and / or anti-CD28 Fab containing a binding partner, e.g., a streptavidin-binding peptide, e.g., Strep-tag® II.

[0211] In some embodiments, the cells are 6or about 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg or 10 μg per cell, or at least 0.01 μg, In some embodiments, cells are stimulated in the presence of, or at least about, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg of an oligomeric stimulatory reagent. 6 In certain embodiments, cells are stimulated in the presence of 10 μg or about 4 μg per cell. 6 Stimulation occurs in the presence of 0.8 μg or about 0.8 μg per cell. In certain aspects, 4 μg of oligomeric stimulatory reagent is or comprises 3 or about 3 μg of oligomeric particles and 1 or about 1 μg of attached agent, e.g., 0.5 or about 0.5 μg of anti-CD3 Fab and 0.5 or about 0.5 μg of anti-CD28 Fab.

[0212] 2. Removal of irritating reagents from cells In some embodiments, the stimulatory reagent is removed or separated from the cells or cell population before collecting, harvesting, or formulating the cells. In some embodiments, the stimulatory reagent is removed or separated from the cells or cell population after or during incubation, such as the incubation described in Section ID herein. In certain embodiments, the cells or cell population are subjected to a process, procedure, step, or technique for removing the stimulatory reagent after incubation but before the step for collecting, harvesting, or formulating the cells. In certain embodiments, the cells or cell population are subjected to a process, procedure, step, or technique for removing the stimulatory reagent after incubation. In some aspects, if the stimulatory reagent is separated or removed from the cells during incubation, the cells are returned to the same incubation conditions as before separation or removal for the remaining duration of the incubation.

[0213] In certain embodiments, the stimulatory reagent is removed and / or separated from the cells. In particular embodiments, the binding and / or association between the stimulatory reagent and the cells may, in some circumstances, be reduced over time during incubation. In certain embodiments, one or more agents may be added to reduce the binding and / or association between the stimulatory reagent and the cells. In particular embodiments, changes in cell culture conditions, such as the addition of an agent and / or a change in the temperature and / or pH of the medium, may reduce the binding and / or association between the stimulatory reagent and the cells. Thus, in some embodiments, the stimulatory reagent may be removed from the incubation, cell culture system, and / or solution separately from the cells, e.g., rather than removing the cells from the incubation, cell culture system, and / or solution together.

[0214] In certain embodiments, the stimulatory reagent is separated and / or removed from the cells after a certain time. In particular embodiments, the certain time is the time from the start of stimulation. In certain embodiments, the start of incubation is considered to be at or about the time when the cells are contacted with the stimulatory reagent and / or the medium or solution containing the stimulatory reagent. In certain embodiments, the stimulatory reagent is removed or separated from the cells within, or about, 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, or 12 hours from the start of stimulation, inclusive. In certain embodiments, the stimulatory reagent is removed or separated from the cells 48 hours or about 48 hours after stimulation begins. In certain embodiments, the stimulatory reagent is removed or separated from the cells 72 hours or about 72 hours after stimulation begins. In some embodiments, the stimulatory reagent is removed or separated from the cells at or about 96 hours after stimulation is initiated.

[0215] Methods for removing stimulatory reagents (e.g., stimulatory reagents that are or contain bead particles or magnetizable particles) from cells are known. In certain embodiments, bead stimulatory reagents, such as anti-CD3 / anti-CD28 antibody-conjugated paramagnetic beads, are separated or removed from cells or cell populations. In some embodiments, the use of a competing antibody, e.g., an unlabeled antibody, can be utilized, e.g., that binds to the primary antibody of the stimulatory reagent and alters its affinity for its antigen on the cells, thereby allowing for gentle desorption. Optionally, after desorption, the competing antibody remains associated with the particle (e.g., bead particle), while unreacted antibody can be washed away or washed away, leaving cells free of the isolation, selection, enrichment, and / or activation antibody. An example of such a reagent is DETACaBEAD (Friedl et al. 1995, Entschladen et al. 1997). In some embodiments, particles (e.g., bead particles) can be removed in the presence of a cleavable linker (e.g., a DNA linker), in which case the particle-bound antibody is conjugated to the linker (e.g., CELLection, Dynal). Optionally, the linker region is a cleavable site for removing the particles (e.g., bead particles) from the cells after isolation, such as by adding DNase or other release buffers. In some embodiments, other enzymatic methods can also be used to release particles (e.g., bead particles) from cells. In some embodiments, the particles (e.g., bead particles or magnetizable particles) are biodegradable.

[0216] In some embodiments, the stimulatory reagent is magnetic, paramagnetic, and / or superparamagnetic, and / or contains magnetic, paramagnetic, and / or superparamagnetic beads, and the stimulatory reagent can be removed from the cells by exposing the cells to a magnetic field. Examples of suitable equipment containing a magnet for generating a magnetic field include DynaMag CTS (Thermo Fisher), Magnetic Separator (Takara), and EasySep Magnet (Stem Cell Technologies).

[0217] In certain embodiments, the stimulatory reagent is removed or separated from the cells prior to completion of the provided methods, e.g., prior to harvesting, collecting, and / or formulating the modified cells produced by the methods provided herein. In some embodiments, the stimulatory reagent is removed and / or separated from the cells after modifying the cells, e.g., after transduction or transfection. In certain embodiments, the stimulatory reagent is removed after culturing the cells, e.g., prior to culturing the modified, e.g., transfected or transduced, cells under conditions to promote proliferation and / or expression. In certain embodiments, the stimulatory reagent is removed after the cells reach a threshold number, density, and / or expansion during cell culture. In some embodiments, the stimulatory reagent is removed prior to formulating the cells, e.g., before forming a culture of cells, a culture of cells that has reached a threshold number, density, or expansion.

[0218] In some embodiments, the stimulatory bead reagent, for example, the stimulatory magnetic bead reagent, is removed or separated from the cell or cell population before collecting, harvesting, or formulating the cells.In some embodiments, the stimulatory bead reagent, for example, the stimulatory magnetic bead reagent, is removed or separated from the cell or cell population by exposure to a magnetic field during or after incubation, for example, during or after the incubation described in Section ID herein.In certain embodiments, the cell or cell population is exposed to a magnetic field after incubation but before the step of collecting, harvesting, or formulating the cells to remove the stimulatory bead reagent, for example, the stimulatory magnetic bead reagent.In certain embodiments, the cell or cell population is exposed to a magnetic field after incubation to remove the stimulatory bead reagent, for example, the stimulatory magnetic bead reagent.In some aspects, if the stimulatory bead reagent is separated or removed from the cell or cell population during incubation, the remaining duration of incubation returns the cell or cell population to the same incubation conditions as before exposure to the magnetic field.

[0219] In certain embodiments, the stimulatory bead reagent, for example, the stimulatory magnetic bead reagent, is removed or separated from the cells, for example, by exposure to a magnetic field, within 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, or 12 hours, including the end points, from the start of stimulation.In certain embodiments, the stimulatory bead reagent, for example, the stimulatory magnetic bead reagent, is removed or separated from the cells, for example, by exposure to a magnetic field, 72 hours or about 72 hours after stimulation begins.In some embodiments, the stimulatory bead reagent, for example, the stimulatory magnetic bead reagent, is removed or separated from the cells, for example, by exposure to a magnetic field, 96 hours or about 96 hours after stimulation begins.

[0220] In certain embodiments, the stimulatory reagent is separated and / or removed from the cells after a certain time. In particular embodiments, the certain time is the time from the start and / or initiation of incubation under stimulatory conditions. In certain embodiments, the start of incubation is considered to be at or about the time when the cells are contacted with the stimulatory reagent and / or a medium or solution containing the stimulatory reagent. In certain embodiments, the stimulatory reagent is removed or separated from the cells within 28, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 days after the start or initiation of incubation. In some embodiments, the stimulatory reagent is removed or separated from the cells within or about 28, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 days after pooling, combining, and / or mixing the CD4+ T cells and CD8+ T cells into the input composition. In certain embodiments, the stimulatory reagent is removed or separated from the cells within or about 28, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 days after obtaining, isolating, enriching, and / or selecting CD4+ and CD8+ T cells from a biological sample.

[0221] In some embodiments, removal of a stimulatory agent, e.g., removal of a described oligomeric stimulatory reagent, comprises adding a substance, such as a competing agent, to the incubated population of T cells to disrupt, e.g., reduce and / or terminate, signaling of one or more stimulatory agents. In some embodiments, the incubated population of T cells comprises the presence of a substance, such as a competing agent, e.g., biotin or a biotin analog, e.g., D-biotin. In some embodiments, the substance, such as a competing agent, e.g., biotin or a biotin analog, e.g., D-biotin, is present in an amount at least 1.5-fold greater, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 100-fold, or at least 1000-fold greater, or more, than the amount of the substance in a reference population or reference preparation of cultured T cells to which the substance was not exogenously added during incubation. In some embodiments, the amount of a substance, such as a competitive agent, e.g., biotin or a biotin analog, e.g., D-biotin, in a population of cultured T cells is from 10 or about 10 μM to 100 or about 100 μM, 100 or about 100 μM to 1 or about 1 mM, 100 or about 100 μM to 500 or about 500 μM, or 10 or about 10 μM to 100 or about 100 μM. In some embodiments, 10 μM or about 10 μM of biotin or a biotin analog, e.g., D-biotin, is added to a cell or cell population to separate or remove an oligomeric stimulatory reagent from the cell or cell population.

[0222] In certain embodiments, one or more agents (e.g., agents that stimulate or activate TCRs and / or co-receptors) associate (e.g., reversibly bind) with the oligomeric reagent, e.g., via multiple specific binding sites (e.g., binding site Z) present on the oligomeric reagent. In some cases, this allows the agents to be closely spaced relative to one another, such that an avidity effect can occur when a target cell bearing (at least two copies of) a cell surface molecule bound or recognized by the agent is contacted with the agent. In some aspects, the receptor-binding reagent has low affinity for a cellular receptor molecule at binding site B, such that the receptor-binding reagent dissociates from the cell in the presence of a competing reagent. Thus, in some embodiments, the agent is removed from the cell in the presence of a competing reagent.

[0223] In some embodiments, the oligomeric stimulatory reagent is a streptavidin mutein oligomer to which an anti-CD3 Fab and an anti-CD28 Fab are reversibly attached. In some embodiments, the attached Fab contains a streptavidin-binding domain, e.g., one that allows for reversible attachment to the streptavidin mutein oligomer. Optionally, the anti-CD3 Fab and the anti-CD28 Fab are closely spaced such that an avidity effect can occur when CD3- and / or CD28-expressing T cells are contacted with the oligomeric stimulatory reagent bearing the reversibly attached Fab. In some aspects, the Fab has low affinity for CD3 and CD28 such that the Fab dissociates from the cell in the presence of a competing reagent, e.g., biotin or a biotin variant or biotin analog. Thus, in some embodiments, the Fab is removed or dissociated from the cell in the presence of a competing reagent, e.g., D-biotin.

[0224] In some embodiments, the stimulatory oligomeric reagent, e.g., the stimulatory oligomeric streptavidin mutein reagent, is removed or separated from the cell or cell population before collecting, harvesting, or formulating the cells. In some embodiments, the stimulatory oligomeric reagent, e.g., the stimulatory oligomeric streptavidin mutein reagent, is removed or separated from the cell or cell population by contact with or exposure to a competitive reagent, e.g., biotin or a biotin analog, e.g., D-biotin, after or during incubation, e.g., after or during incubation as described in Section ID herein. In certain embodiments, the cell or cell population is contacted with or exposed to a competitive reagent, e.g., biotin or a biotin analog, e.g., D-biotin, after incubation but before the step of collecting, harvesting, or formulating the cells, to remove the stimulatory oligomeric reagent, e.g., the stimulatory oligomeric streptavidin mutein reagent. In certain embodiments, the cells or cell populations are contacted with or exposed to a competitive reagent, e.g., biotin or a biotin analog, e.g., D-biotin, after incubation to remove the stimulatory oligomeric reagent, e.g., the stimulatory oligomeric streptavidin mutein reagent. In some aspects, if the stimulatory oligomeric reagent, e.g., the stimulatory oligomeric streptavidin mutein reagent, is separated or removed from the cells during incubation, such as by contact with or exposure to a competitive reagent, e.g., biotin or a biotin analog, e.g., D-biotin, the cells are returned to the same incubation conditions as before separation or removal for the remainder of the incubation.

[0225] In some embodiments, to remove or separate the oligomeric stimulatory reagent from the cells, the cells are treated with at or about 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 mM, 1 mM, or 10 mM. or 10 mM, or at least 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 mM, 1 mM, or 10 mM, or at least about 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 mM, 1 mM, or 10 mM of competing reagent. To remove or separate from the cells the stimulatory streptavidin mutein oligomers to which the Fab is reversibly attached, the cells are treated with 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 mM, 1 mM, or 10 mM, or about 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 mM, 1 mM, or 10 mM. or 10 mM, or at least 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 mM, 1 mM, or 10 mM, or at least about 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 mM, 1 mM, or 10 mM biotin or a biotin analogue, e.g., D-biotin.

[0226] In certain embodiments, the stimulatory oligomeric reagent, e.g., the stimulatory oligomeric streptavidin mutein reagent, is removed or separated from the cells within, or about, 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, or 12 hours from the start of stimulation, inclusive. In certain embodiments, the stimulatory oligomeric reagent, e.g., the stimulatory oligomeric streptavidin mutein reagent, is removed or separated from the cells 48 hours or about 48 hours after the start of stimulation. In certain embodiments, the stimulatory oligomeric reagent, e.g., the stimulatory oligomeric streptavidin mutein reagent, is removed or separated from the cells 72 hours or about 72 hours after the start of stimulation. In some embodiments, the stimulatory oligomeric reagent, eg, the stimulatory oligomeric streptavidin mutein reagent, is removed or separated from the cells at or about 96 hours after stimulation begins.

[0227] C. Viral Vector Particles In some embodiments, the viral vector particle is a retroviral vector particle, e.g., a lentiviral particle, that contains a nucleic acid encoding a recombinant and / or heterologous molecule, e.g., a recombinant or heterologous protein, e.g., a recombinant and / or heterologous receptor, e.g., a chimeric antigen receptor (CAR) or other antigen receptor, in the genome of the viral vector. The genome of the viral vector particle typically contains sequences in addition to the nucleic acid (e.g., polynucleotide) encoding the recombinant molecule. Such sequences may include sequences that allow the genome to be packaged into viral particles and / or sequences that promote the expression of the nucleic acid encoding the recombinant receptor, e.g., a CAR.

[0228] 1. Viral Vectors In some embodiments, the viral vector particle contains a genome derived from a retroviral genome-based vector, for example, a lentiviral genome-based vector. In some embodiments, the viral vector particle is a lentiviral vector particle. In some aspects of the provided viral vectors, a heterologous nucleic acid (e.g., a polynucleotide) encoding a recombinant protein, for example, an antigen receptor, for example, a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR), is contained and / or located between the 5'LTR sequence and the 3'LTR sequence of the vector genome. In some embodiments, the recombinant protein is an antigen receptor. In some embodiments, the recombinant protein is a T cell receptor (TCR). In some embodiments, the recombinant protein is a chimeric antigen receptor (CAR).

[0229] In some embodiments, the viral vector genome is a lentiviral genome, such as an HIV-1 genome or an SIV genome. In some embodiments, the lentiviral vector particle is replication-deficient. For example, lentiviral vectors are created by multiple attenuation of virulence genes, for example, deleting the genes env, vif, vpu, and nef, making the vector safer for therapeutic purposes. Lentiviral vectors are known. See Naldini et al. (1996 and 1998), Zufferey et al. (1997), Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based and are configured to carry the necessary sequences for integration of foreign nucleic acids, selection, and transfer of nucleic acids (e.g., polynucleotides) into host cells. Known lentiviruses are readily available from depositories or collections such as the American Type Culture Collection ("ATCC," 10801 University Blvd., Manassas, VA 20110-2209) or can be isolated from known sources using publicly available techniques.

[0230] Non-limiting examples of lentiviral vectors include those derived from lentiviruses such as human immunodeficiency virus 1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), human T-lymphotropic virus 1 (HTLV-1), HTLV-2, or equine infectious anemia virus (E1AV). For example, lentiviral vectors have been created by multiple attenuation of HIV virulence genes, such as deletion of the env, vif, vpr, vpu, and nef genes, making the vector safer for therapeutic purposes. Lentiviral vectors are known in the art. See Naldini et al. (1996 and 1998), Zufferey et al. (1997), Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based and are configured to carry the necessary sequences for integration of foreign nucleic acids, selection, and transfer of nucleic acids (e.g., polynucleotides) into host cells. Known lentiviruses are readily available from depositories or collections such as the American Type Culture Collection ("ATCC," 10801 University Blvd., Manassas, VA 20110-2209), or can be isolated from known sources using publicly available techniques.

[0231] In some embodiments, the viral genome vector can contain the 5'LTR and 3'LTR sequences of a retrovirus such as a lentivirus.In some aspects, the viral genome construct can contain the sequences from the 5'LTR and 3'LTR of a lentivirus, and in particular, can contain the R sequence and U5 sequence from the 5'LTR of a lentivirus and the inactivated or self-inactivating 3'LTR of a lentivirus.The LTR sequence can be the LTR sequence from any species of any lentivirus.For example, it can be the LTR sequence from HIV, SIV, FIV or BIV.Typically, the LTR sequence is the HIV LTR sequence.

[0232] In some embodiments, the nucleic acid (e.g., polynucleotide) of a viral vector, such as an HIV viral vector, lacks an additional transcription unit. The vector genome can contain an inactivated or self-inactivating 3' LTR (Zufferey et al. J Virol 72:9873, 1998; Miyoshi et al., J Virol 72:8150, 1998). For example, a deletion in the U3 region of the 3' LTR of the nucleic acid (e.g., polynucleotide) used to produce the viral vector RNA can be used to generate a self-inactivating (SIN) vector. This deletion can then be transferred to the 5' LTR of the proviral DNA during reverse transcription. Self-inactivating vectors generally have a deletion of enhancer and promoter sequences from the 3' long terminal repeat (LTR), which are copied to the 5' LTR upon vector integration. In some embodiments, sufficient sequences can be removed, including the deletion of the TATA box, to abolish the transcriptional activity of the LTR. This prevents the production of full-length vector RNA in transduced cells. In some aspects, the U3 element of the 3' LTR contains deletions of its enhancer sequence, TATA box, Sp1, and NF-kappa B site. As a result of the self-inactivating 3' LTR, the provirus generated following entry and reverse transcription contains an inactivated 5' LTR. This can improve safety by reducing the risk of vector genome mobilization and the effect of the LTR on nearby cellular promoters. The self-inactivating 3' LTR can be constructed by any method known in the art. In some embodiments, this does not affect the vector titer or the vector's properties in vitro or in vivo.

[0233] Optionally, the U3 sequence from the lentivirus 5'LTR can be replaced with a promoter sequence in the viral construct, such as a heterologous promoter sequence. This can increase the titer of the virus recovered from the packaging cell line. Enhancer sequences can also be included. Any enhancer / promoter combination that increases the expression of the viral RNA genome in the packaging cell line can be used. In one example, the CMV enhancer / promoter sequence is used (U.S. Patent No. 5,385,839 and U.S. Patent No. 5,168,062).

[0234] In certain embodiments, the risk of insertional mutagenesis can be minimized by engineering retroviral vector genomes, such as lentiviral vector genomes, to be integration-deficient. Various approaches can be implemented to produce non-integrating vector genomes. In some embodiments, mutations can be incorporated into the integrase enzyme component of the pol gene such that it encodes a protein with an inactive integrase. In some embodiments, the vector genome itself can be modified to prevent integration, for example, by mutating or deleting one or both attachment sites or by rendering the 3' LTR-proximal polypurine tract (PPT) non-functional by deletion or modification. Non-genetic approaches can be utilized in some embodiments, including pharmacological agents that inhibit one or more functions of integrase. These approaches are not mutually exclusive; i.e., two or more of them can be used simultaneously. For example, both the integrase and attachment site can be non-functional, or the integrase and PPT sites can be non-functional, or the attachment site and PPT site can be non-functional, or all of them can be non-functional. Such methods and viral vector genomes are known and available (see Philpott and Thrasher, Human Gene Therapy 18:483, 2007; Engelman et al. J Virol 69:2729, 1995; Brown et al J Virol 73:9011(1999); WO 2009 / 076524; McWilliams et al., J Virol 77:11150, 2003; Powell and Levin J Virol 70:5288, 1996).

[0235] In some embodiments, the vector contains sequences for propagation in a host cell, e.g., a prokaryotic host cell. In some embodiments, the nucleic acid (e.g., polynucleotide) of a viral vector contains one or more origins of replication for propagation in a prokaryotic cell, such as a bacterial cell. In some embodiments, vectors containing a prokaryotic origin of replication may also contain a gene whose expression confers a detectable or selectable marker, such as a drug resistance.

[0236] 2. Nucleic acid encoding a heterologous protein In some embodiments, the viral vector contains a nucleic acid (e.g., polynucleotide) encoding a heterologous recombinant protein. In some embodiments, the heterologous recombinant protein or heterologous recombinant molecule is or comprises a recombinant receptor, such as an antigen receptor, an SB-transposon, such as for gene silencing, a capsid-enclosed transposon, a homologous double-stranded nucleic acid, such as for genome recombination, or a reporter gene (e.g., a fluorescent protein such as GFP or luciferase).

[0237] In some embodiments, the viral vector contains a nucleic acid (e.g., polynucleotide) encoding a recombinant receptor and / or chimeric receptor, such as a heterologous receptor protein.Recombinant receptors, such as heterologous receptors, can include antigen receptors, such as functional non-TCR antigen receptors, including chimeric antigen receptors (CARs), and other antigen-binding receptors, such as transgenic T cell receptors (TCRs).Receptors can also include other receptors, such as other chimeric receptors, for example, receptors that bind to specific ligands and have transmembrane domains and / or intracellular signaling domains similar to those present in CARs.

[0238] In any such example, the nucleic acid (e.g., polynucleotide) is inserted into or located in a region of the viral vector, e.g., generally a nonessential region of the viral genome. In some embodiments, the nucleic acid (e.g., polynucleotide) is inserted into the viral genome in place of certain viral sequences to produce a virus that is replication-deficient.

[0239] In some embodiments, the encoded recombinant antigen receptor, e.g., CAR, is capable of specifically binding to one or more ligands in a targeted cell or disease, e.g., cancer, an infectious disease, an inflammatory disease, an autoimmune disease, or other disease or condition, including those described herein for targeting by the provided methods and compositions.

[0240] In certain embodiments, exemplary antigens are αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (also known as CA9, CAIX, or G250), cancer testis antigen, cancer / testis antigen 1B (also known as CTAG, NY-ESO-1, and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutant (EGFR), vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (Fc receptor like 5: FCRL5, also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand,The antigen may be or include Melan-A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), a pathogen-specific antigen, or an antigen associated with a universal tag, and / or a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens. In some embodiments, the antigen targeted by the receptor includes an antigen associated with a B-cell malignancy, such as any of a number of known B-cell markers. In some embodiments, the antigen is or comprises CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30.

[0241] In some embodiments, exemplary antigens include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3 or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, oncofetal 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, CE7, Wilms' tumor 1 (WT-1), cyclins, e.g., cyclin A1 (CCNA1), and / or biotinylated molecules, and / or molecules expressed by and / or characteristic or specific for HIV, HCV, HBV, HPV and / or other pathogens, and / or oncogenotypes thereof.

[0242] In some embodiments, the antigen is or comprises a pathogen-specific antigen or a pathogen-expressed antigen, hi some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen.

[0243] Antigen receptors, including CARs and recombinant TCRs, and their production and introduction, in some embodiments, are described in, for example, International Patent Application Publication Nos. 200014257, 2013126726, 2012 / 129514, 2014031687, 2013 / 166321, 2013 / 071154, 2013 / 123061, U.S. Patent Application Publication Nos. 2002131960, 2013287748, 20130149337, U.S. Patent Application Nos. those described in Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. 2537416, and / or 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.

[0244] a. Chimeric antigen receptor In some embodiments, the nucleic acid (e.g., polynucleotide) contained in the genome of the viral vector encodes a chimeric antigen receptor (CAR). CARs are generally genetically engineered receptors with an extracellular portion containing an extracellular ligand-binding domain, e.g., an antibody or fragment thereof, linked to one or more intracellular signaling components. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain and / or an intracellular domain connecting the extracellular domain and the intracellular signaling domain. Such molecules typically mimic or approximate the signaling of a natural antigen receptor and / or the signaling of such a receptor in combination with a costimulatory receptor.

[0245] In some embodiments, CARs are constructed with specificity for a particular marker, for example, a marker expressed in a specific cell type targeted for adoptive therapy, such as a cancer marker and / or any of the antigens described. Thus, CARs typically comprise one or more antigen-binding fragments, domains, or portions of an antibody, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, CARs comprise one or more antigen-binding portions of an antibody molecule, such as a variable heavy chain (VH) or an antigen-binding portion thereof, or a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0246] In some embodiments, modified cells, e.g., T cells, are provided that express a CAR with specificity for a particular antigen (or marker or ligand), e.g., an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on disease or condition cells, e.g., tumor cells or pathogenic cells, compared to normal cells or tissues or non-target cells or tissues. In another embodiment, the antigen is expressed on normal cells and / or expressed on modified cells.

[0247] In certain embodiments, the recombinant receptor, e.g., a chimeric receptor, contains an intracellular signaling region, which includes a cytoplasmic signaling domain or region (interchangeably referred to as an intracellular signaling domain or region), e.g., a cytoplasmic (intracellular) region capable of inducing a primary activation signal in T cells, e.g., a cytoplasmic signaling domain or region of a T cell receptor (TCR) component (e.g., the cytoplasmic signaling domain or region of the zeta chain of the CD3-zeta (CD3ζ) chain, or a functional variant or signaling portion thereof), and / or includes an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the CAR includes an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain that includes an ITAM. In some embodiments, the intracellular signaling domain includes the intracellular domain of the CD3-zeta (CD3ζ) chain.

[0248] In some embodiments, the chimeric receptor further comprises an extracellular ligand-binding domain that specifically binds to a ligand (e.g., antigen) antigen. In some embodiments, the chimeric receptor is a CAR that comprises an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the ligand, e.g., antigen, is a protein that is expressed on the surface of a cell. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, e.g., a peptide antigen of an intracellular protein, that is recognized on the cell surface in association with a major histocompatibility complex (MHC) molecule, like a TCR.

[0249] Exemplary antigen receptors, including CARs, and methods for modifying and introducing such receptors into cells are described, for example, in International Patent Application Publication Nos. 200014257, 2013126726, 2012 / 129514, 2014031687, 2013 / 166321, 2013 / 071154, and 2013 / 123061; U.S. Patent Application Publication Nos. 2002131960, 2013287748, and 20130149337; U.S. Patent No. 6,449,514; ,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and those described in European Patent Application No. 2537416, and / or 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. In some aspects, antigen receptors include CARs described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. 2014055668 A1.Examples of CARs include those disclosed in any of the above-mentioned publications, such as WO 2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Patent No. 7,446,190, U.S. Patent No. 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013), Wang et al. (2012) J. Immunother. 35 (9): 689-701, and Brentjens et al., Sci Transl Med. 2013 5 (177). See also WO 2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Pat. No. 7,446,190 and U.S. Pat. No. 8,389,282.

[0250] In some embodiments, CARs are constructed that have specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type targeted for adoptive therapy, e.g., a cancer marker, and / or an antigen against which an attenuated response is intended to be induced, e.g., an antigen expressed on a normal cell type or a non-diseased cell type. Thus, CARs typically comprise one or more antigen-binding molecules, e.g., one or more antigen-binding fragments, antigen-binding domains, or portions thereof, or one or more antibody variable domains, and / or antibody molecules, in their extracellular portion. In some embodiments, CARs comprise one or more antigen-binding portions of an antibody molecule, e.g., a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0251] In some embodiments, the antibody or its antigen-binding portion is expressed on a cell as part of a recombinant receptor, such as an antigen receptor. Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, CARs containing antibodies or antigen-binding fragments that exhibit TCR-like specificity for peptide-MHC complexes can also be referred to as TCR-like CARs. In some embodiments, the extracellular antigen-binding domain of the TCR-like CAR, specific for the MHC-peptide complex, is linked to one or more intracellular signaling components, in some aspects via a linker and / or transmembrane domain. In some embodiments, such molecules can typically mimic or approximate the signaling of a natural antigen receptor, such as a TCR, and optionally the signaling of such a receptor in combination with a costimulatory receptor.

[0252] In some embodiments, the recombinant receptor, e.g., a chimeric receptor (e.g., a CAR), comprises a ligand-binding domain that binds to (e.g., specifically binds to) an antigen (or ligand). Antigens targeted by chimeric receptors include those expressed in association with diseases, conditions, or cell types targeted by adoptive cell therapy. Diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, such as cancers and tumors, e.g., blood cancers, cancers of the immune system, e.g., lymphomas, leukemias, and / or myelomas, e.g., B, T, and myeloid leukemias, lymphomas, and multiple myelomas.

[0253] In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on cells of a disease or condition, such as tumor cells or pathogenic cells, compared to normal cells or tissues or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on modified cells. In some embodiments, the antigen is associated with a disease or condition, such as cancer, an autoimmune disease or disorder, or an infectious disease. In some embodiments, the antigen receptor, e.g., a CAR, specifically binds to the universal tag.

[0254] In some embodiments, the CAR contains an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen, e.g., an intact antigen, expressed on the surface of a cell.

[0255] In some embodiments, the antigen (or ligand) is a tumor antigen or a cancer marker. In some embodiments, the antigen (or ligand) antigen is αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (also known as CA9, CAIX, or G250), a cancer testis antigen, or a cancer / testis antigen. 1B(also known as CTAG, NY-ESO-1, and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutant (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5, also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2) , ganglioside GD3, glycoprotein 100 (gp100), G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha IL-22Ra, IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), nervous system cell adhesion molecule (NCAM), oncofetal antigen, melanoma preferentially expressed antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin,The receptor may be or include trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), a pathogen-specific antigen, or an antigen associated with a universal tag, and / or a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens. In some embodiments, the antigen targeted by the receptor includes an antigen associated with a B-cell malignancy, such as any of a number of known B-cell markers. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30.

[0256] In some embodiments, the antigen or antigen-binding domain is CD19. In some embodiments, the scFv contains VH and VL derived from an antibody or antibody fragment specific to CD19. In some embodiments, the antibody or antibody fragment that binds to CD19 is a mouse-derived antibody such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, for example, as described in U.S. Patent Application Publication No. US 2016 / 0152723.

[0257] In some embodiments, the scFv is derived from FMC63. FMC63 generally refers to a murine monoclonal IgG1 antibody raised against Nalm-1 and Nalm-16 cells expressing CD19 of human origin (Ling, NR, et al. (1987) Leucocyte typing III.302). In some embodiments, the FMC63 antibody comprises a CDRH1 set forth in SEQ ID NO:60, a CDRH2 set forth in SEQ ID NO:61, and a CDRH3 set forth in SEQ ID NO:62 or SEQ ID NO:76, as well as a CDRL1 set forth in SEQ ID NO:57, a CDRL2 set forth in SEQ ID NO:58 or SEQ ID NO:77, and a CDRL3 set forth in SEQ ID NO:59 or SEQ ID NO:78. In some embodiments, the FMC63 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:63. H ) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO:64 L ) is included.

[0258] In some embodiments, the scFv comprises a variable light chain containing the CDRL1 sequence of SEQ ID NO:57, the CDRL2 sequence of SEQ ID NO:58, and the CDRL3 sequence of SEQ ID NO:59, and a variable heavy chain containing the CDRH1 sequence of SEQ ID NO:60, the CDRH2 sequence of SEQ ID NO:61, and the CDRH3 sequence of SEQ ID NO:62. In some embodiments, the scFv comprises a variable light chain containing the CDRL1 sequence of SEQ ID NO:57, the CDRL2 sequence of SEQ ID NO:77, and the CDRL3 sequence of SEQ ID NO:78, and a variable heavy chain containing the CDRH1 sequence of SEQ ID NO:60, the CDRH2 sequence of SEQ ID NO:61, and the CDRH3 sequence of SEQ ID NO:76.

[0259] In some embodiments, the scFv comprises a variable heavy chain region set forth in SEQ ID NO:63 and a variable light chain region set forth in SEQ ID NO:64. In some embodiments, the variable heavy chain and variable light chain are joined by a linker. In some embodiments, the linker is set forth in SEQ ID NO:80. In some embodiments, the scFv comprises, in order, V H , linker, and V L In some embodiments, the scFv comprises, in order, V L , linker, and V H In some embodiments, the scFv is encoded by the sequence of nucleotides set forth in SEQ ID NO:65, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:65. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:65, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:65.

[0260] In some embodiments, the scFv is derived from SJ25C1. SJ25C1 is a murine monoclonal IgG1 antibody raised against Nalm-1 and Nalm-16 cells, which generally express CD19 of human origin (Ling, NR, et al. (1987) Leucocyte typing III.302). In some embodiments, the SJ25C1 antibody comprises CDRH1, H2, and H3 sequences set forth in SEQ ID NOS:69-71, respectively, and CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOS:66-68, respectively. In some embodiments, the SJ25C1 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:72. H ) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO:73 L ) is included.

[0261] In some embodiments, the scFv comprises a variable light chain containing the CDRL1 sequence of SEQ ID NO:66, the CDRL2 sequence of SEQ ID NO:67, and the CDRL3 sequence of SEQ ID NO:68, and a variable heavy chain containing the CDRH1 sequence of SEQ ID NO:69, the CDRH2 sequence of SEQ ID NO:70, and the CDRH3 sequence of SEQ ID NO:71. In some embodiments, the scFv comprises a variable heavy chain region set forth in SEQ ID NO:72 and a variable light chain region set forth in SEQ ID NO:73. In some embodiments, the variable heavy chain and variable light chain are joined by a linker. In some embodiments, the linker is set forth in SEQ ID NO:74. In some embodiments, the scFv comprises, in order, V H , linker, and V L In some embodiments, the scFv comprises, in order, V L , linker, and V H In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:75, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:75.

[0262] In some embodiments, an antibody or antigen-binding fragment (e.g., scFv or V H In some embodiments, the antibody or antigen-binding fragment is derived from or a variant of an antibody or antigen-binding fragment that specifically binds to BCMA.

[0263] In some embodiments, the CAR is an anti-BCMA CAR specific for BCMA, for example, human BCMA. Chimeric antigen receptors containing anti-BCMA antibodies, including mouse anti-human BCMA antibodies and human anti-human antibodies, and cells expressing such chimeric receptors have been described. See Carpenter et al., Clin Cancer Res., 2013,19(8):2048-2060, WO 2016 / 090320, WO 2016090327, WO 2010104949A2, and WO 2017173256. In some embodiments, the antigen or antigen-binding domain is BCMA. In some embodiments, the scFv contains a VH and a VL derived from an antibody or antibody fragment specific for BCMA. In some embodiments, the antibody or antibody fragment that binds BCMA is or contains a VH and VL from the antibodies or antibody fragments set forth in International Patent Application Publication Nos. WO 2016 / 090327 and WO 2016 / 090320.

[0264] In some embodiments, the antigen or antigen-binding domain is GPRC5D. In some embodiments, the scFv contains VH and VL derived from an antibody or antibody fragment specific for GPRC5D. In some embodiments, the antibody or antibody fragment that binds to GPRC5D is or contains VH and VL from the antibody or antibody fragment set forth in International Patent Application Publication Nos. WO 2016 / 090329 and WO 2016 / 090312.

[0265] In some aspects, the CAR contains a ligand (e.g., antigen) binding domain that binds or recognizes (e.g., specifically binds) a universal tag or universal epitope. In some aspects, the binding domain can bind to a molecule, tag, polypeptide, and / or epitope that can be linked to a different binding molecule (e.g., antibody or antigen-binding fragment) that recognizes an antigen associated with a disease or disorder. Exemplary tags or epitopes include dyes (e.g., fluorescein isothiocyanate) or biotin. In some aspects, a binding molecule (e.g., antibody or antigen-binding fragment) linked to a tag that recognizes an antigen associated with a disease or disorder, such as a tumor antigen, together with an engineered cell expressing a CAR specific for that tag, triggers the cytotoxicity or other effector function of the engineered cell. In some aspects, the specificity of the CAR for an antigen associated with a disease or disorder is provided by the tagged binding molecule (e.g., antibody), and different tagged binding molecules can be used to target different antigens. Exemplary CARs specific for universal tags or universal epitopes include those described, for example, in US9,233,125, WO 2016 / 030414, Urbanska et al., (2012) Cancer Res 72:1844-1852, and Tamada et al., (2012). Clin Cancer Res 18:6436-6445.

[0266] In some embodiments, the antigen is or comprises a pathogen-specific antigen or a pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen. In some embodiments, the CAR contains a TCR-like antibody, such as an antibody or antigen-binding fragment (e.g., scFv), that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface as an MHC-peptide complex. In some embodiments, an antibody or its antigen-binding portion that recognizes an MHC-peptide complex can be expressed on a cell as part of a recombinant receptor, such as an antigen receptor. Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). In general, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity for peptide-MHC complexes can also be referred to as a TCR-like CAR.

[0267] "Major histocompatibility complex" (MHC) refers to a protein, typically a glycoprotein, containing a polymorphic peptide-binding site or groove capable of complexing with a polypeptide peptide antigen, such as a peptide antigen processed by the cellular machinery. Optionally, MHC molecules can be displayed or expressed on the cell surface, e.g., as a complex with a peptide, i.e., an MHC-peptide complex, to present the antigen in a conformation that can be recognized by an antigen receptor on a T cell, e.g., a TCR or a TCR-like antibody. Generally, MHC class I molecules are heterodimers containing a membrane-spanning α chain, optionally with three α domains, and noncovalently associated β2-microglobulin. Generally, MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which typically span the membrane. An MHC molecule can comprise an effective portion of an MHC containing one or more antigen-binding sites for binding peptides and the sequences necessary for recognition by the appropriate antigen receptor. In some embodiments, MHC class I molecules deliver peptides generated in the cytosol to the cell surface, where the MHC-peptide complexes are transduced by T cells, e.g., typically CD8 + In some embodiments, MHC class II molecules deliver peptides generated in the vesicle system to the cell surface, where they are typically recognized by CD4+ T cells. + They are recognized by T cells. MHC molecules are generally encoded by a group of linked genetic loci collectively referred to as H-2 in mice and human leukocyte antigens (HLA) in humans. Therefore, human MHC molecules are commonly referred to as human leukocyte antigens (HLA).

[0268] The terms "MHC-peptide complex" or "peptide-MHC complex", or variants thereof, refer to a complex or association between a peptide antigen and an MHC molecule, for example, typically through non-covalent interactions of the peptide in the binding groove or binding cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is present or displayed on the surface of a cell. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor, such as a TCR, a TCR-like CAR, or an antigen-binding portion thereof.

[0269] In some embodiments, peptides of polypeptides, e.g., peptide antigens or peptide epitopes, can associate with MHC molecules, e.g., for recognition by antigen receptors. Generally, peptides are derived from or based on fragments of longer biological molecules, e.g., polypeptides or proteins. In some embodiments, peptides are typically about 8 to about 24 amino acids in length. In some embodiments, peptides have a length of 9 to 22 amino acids or about 9 to 22 amino acids for recognition in an MHC class II complex. In some embodiments, peptides have a length of 8 to 13 amino acids or about 8 to 13 amino acids for recognition in an MHC class I complex. In some embodiments, upon recognition of the peptide in the context of an MHC molecule, e.g., upon recognition of an MHC-peptide complex, an antigen receptor, such as a TCR or TCR-like CAR, generates or triggers an activation signal to a T cell that induces a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response, or other response.

[0270] In some embodiments, the TCR-like antibody or antigen-binding portion is known or can be produced by known methods (see, e.g., US Application Publication Nos. US 2002 / 0150914; US 2003 / 0223994, US 2004 / 0191260, US 2006 / 0034850, US 2007 / 00992530, US 20090226474, US 20090304679, and International PCT Publication No. WO 03 / 068201).

[0271] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to an MHC-peptide complex can be produced by immunizing a host with an effective amount of an immunogen containing a specific MHC-peptide complex. Optionally, the peptide in the MHC-peptide complex is an epitope of an antigen capable of binding to MHC, such as a tumor antigen, e.g., a universal tumor antigen, a myeloma antigen, or other antigens described below. In some embodiments, an effective amount of the immunogen is then administered to the host to elicit an immune response, wherein the immunogen retains its three-dimensional conformation for a period of time sufficient to elicit an immune response to the three-dimensional presentation of the peptide in the binding groove of the MHC molecule. Serum collected from the host is then assayed to determine whether the desired antibodies that recognize the three-dimensional presentation of the peptide in the binding groove of the MHC molecule have been produced. In some embodiments, the produced antibodies can be assayed to confirm that the antibodies can distinguish the MHC-peptide complex from complexes of the MHC molecule alone, the peptide of interest alone, and a peptide unrelated to the MHC. The desired antibodies can then be isolated.

[0272] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to MHC-peptide complexes can be produced by antibody library display methods, such as phage antibody libraries. In some embodiments, phage display libraries of mutant Fab, scFv, or other antibody types can be created, e.g., in which library members are mutated at one or more residues in one or more CDRs. See, e.g., U.S. Application Publication Nos. US 20020150914, US 2014 / 0294841, and Cohen CJ. et al. (2003) J Mol. Recogn. 16:324-332.

[0273] The term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, such as Fab (fragment antigen binding) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, and fragments of variable heavy chains (V) capable of specifically binding to an antigen. H ) regions, single-chain antibody fragments, such as single-chain variable fragments (scFvs), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies, such as bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, such as IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0274] In some embodiments, the antigen-binding proteins, antibodies, and antigen-binding fragments thereof, specifically recognize the antigen of the full-length antibody. In some embodiments, the heavy and light chains of the antibody can be full-length or can be antigen-binding portions (Fab, F(ab')2, Fv, or single-chain Fv fragment (scFv)). In another embodiment, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly selected from, for example, IgG1, IgG2, IgG3, and IgG4, and more particularly selected from IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is selected from, for example, kappa or lambda, particularly kappa.

[0275] The provided antibodies include antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that contains the portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, variable heavy chain (V), and fragments of variable heavy chain (V). H ) regions, single chain antibody molecules, such as scFv and single domain V H These include, but are not limited to, single antibodies and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment, such as an scFv, that contains a variable heavy chain region and / or a variable light chain region.

[0276] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H Domain or V L Even a single V domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be obtained by combining V domains from antibodies that bind to that antigen. H Domain or V L domains to form complementary V L Domain or V H Domains can be isolated by screening libraries (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0277] Single domain antibody is an antibody fragment that comprises all or a part of the heavy chain variable domain or all or a part of the light chain variable domain of an antibody.In certain embodiments, single domain antibody is a human single domain antibody.In some embodiments, CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as a cancer marker, or a cell surface antigen of target cell or disease, such as tumor cell or cancer cell, for example, any of the target antigens described herein or known target antigens.

[0278] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, e.g., a fragment containing a configuration that does not occur in nature, e.g., two or more antibody regions or chains joined by a synthetic linker, e.g., a peptide linker, and / or a fragment containing a configuration that cannot occur by enzymatic digestion of a naturally occurring, intact antibody. In some embodiments, the antibody fragment is an scFv.

[0279] A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FRs. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has been humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0280] Thus, in some embodiments, the chimeric antigen receptor, including the TCR-like CAR, comprises an extracellular portion containing an antibody or antibody fragment. In some embodiments, the antibody or fragment comprises an scFv. In some aspects, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM).

[0281] In some embodiments, the extracellular portion of the CAR, e.g., an antibody portion thereof, further comprises a spacer, e.g., a spacer region between the antigen recognition component, e.g., an scFv, and the transmembrane domain. The spacer can be or comprise at least a portion of an immunoglobulin constant region or a variant or modified form thereof, e.g., a hinge region, e.g., an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In some embodiments, the recombinant receptor further comprises a spacer and / or hinge region. In some embodiments, the constant region or constant portion is that of a human IgG, e.g., IgG4 or IgG1. In some aspects, a portion of the constant region serves as a spacer region between the antigen recognition component, such as an scFv, and the transmembrane domain. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:8 and is encoded by the sequence set forth in SEQ ID NO:9. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:10. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:11.

[0282] In some embodiments, the constant region or constant portion is that of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 12. In some embodiments, the spacer has a sequence of amino acids that exhibits at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 8, 10, 11, and 12.

[0283] In some embodiments, the spacer comprises at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region, e.g., an IgG4 hinge region, and / or a C H 1 / C Land / or Fc region. In some embodiments, the recombinant receptor further comprises a spacer and / or hinge region. In some embodiments, the constant region or constant portion is that of human IgG, e.g., IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen recognition component, such as an scFv, and the transmembrane domain. The spacer can be of a length that increases the responsiveness of the cell after antigen binding compared to when the spacer is absent. In some examples, the spacer is 12 or about 12 amino acids in length, or is 12 amino acids or less in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, including any integer between either endpoint of the recited range. In some embodiments, the spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include an IgG4 hinge only, an IgG4 hinge linked to the CH2 and CH3 domains, or an IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or International Patent Application Publication No. WO 2014 / 031687. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:8 and is encoded by the sequence set forth in SEQ ID NO:9. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:10. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:11.

[0284] In some embodiments, the constant region or constant portion is that of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 12. In some embodiments, the spacer has a sequence of amino acids that exhibits at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 8, 10, 11, and 12.

[0285] The extracellular ligand binding, e.g., antigen recognition domain, is generally linked to one or more intracellular signaling components, e.g., a signaling component that mimics activation by an antigen receptor complex, such as a TCR complex in the case of a CAR, and / or signals from other cell surface receptors. In some embodiments, a transmembrane domain links the extracellular ligand binding domain to the intracellular signaling domain. In some embodiments, the antigen binding component (e.g., an antibody) is linked to one or more transmembrane regions and an intracellular signaling region. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally associates with one of the domains in a receptor, such as a CAR, is used. In some examples, the transmembrane domain is selected or modified by amino acid substitution to avoid binding to the transmembrane domain of the same or different surface membrane protein, in order to minimize interaction with other members of the receptor complex.

[0286] In some embodiments, the transmembrane domain is derived from either a natural or synthetic source. If the source is natural, the domain, in some aspects, is derived from any membrane-bound or transmembrane protein. The transmembrane region includes those derived from (i.e., at least the transmembrane region of) 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, or CD154. Alternatively, the transmembrane domain, in some embodiments, is synthetic. In some aspects, synthetic transmembrane domains contain primarily hydrophobic residues, such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is by a linker, spacer, and / or transmembrane domain.

[0287] In some embodiments, a short oligopeptide or polypeptide linker, e.g., a linker 2-10 amino acids in length, containing glycine and serine, such as a glycine-serine doublet, is present between the transmembrane domain and the cytoplasmic signaling domain of the CAR to form the linkage.

[0288] Recombinant receptors, e.g., CARs, generally comprise at least one intracellular signaling component. In some embodiments, the receptor comprises an intracellular component of the TCR complex, e.g., a TCR CD3 chain, e.g., CD3 zeta chain, which mediates T cell activation and cytotoxicity. Thus, in some aspects, the antigen-binding moiety 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 receptor, e.g., 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, a CAR or other chimeric receptor comprises a chimeric molecule of CD3-zeta (CD3-ζ) or Fc receptor gamma with CD8, CD4, CD25, or CD16.

[0289] In some embodiments, upon ligation of a CAR or other chimeric receptor, the cytoplasmic domain and / or cytoplasmic region or intracellular signaling domain and / or intracellular signaling region of the receptor activates at least one of the normal effector functions or responses of immune cells, such as T cells modified to express the CAR. For example, in some situations, the CAR induces T cell function, such as cytolytic activity or T helper activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of the intact immunostimulatory chain, for example, if it transmits an effector function signal. In some embodiments, the intracellular signaling region, e.g., comprising one or more intracellular domains, comprises the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also includes the cytoplasmic sequence of a coreceptor that acts in concert with such receptor in its natural environment to initiate signal transduction following antigen receptor engagement, and / or the cytoplasmic sequence of any derivative or variant of such molecule, and / or any synthetic sequence having the same functional capability.

[0290] For natural TCR, complete activation generally requires not only TCR signaling but also costimulatory signaling.Therefore, in some embodiments, CAR also comprises the component for generating secondary signaling or costimulatory signaling to promote complete activation.In another embodiment, CAR does not comprise the component for generating costimulatory signaling.In some aspects, additional CAR is expressed in the same cell, and this becomes the component for generating secondary signaling or costimulatory signaling.

[0291] T cell activation, in some aspects, is described as being mediated by at least two types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, a CAR comprises one or both of these signaling components.

[0292] In some aspects, CAR comprises a primary cytoplasmic signaling sequence that regulates the primary activation of TCR complex.The primary cytoplasmic signaling sequence that acts as a stimulator can contain a signaling motif known as immunoreceptor tyrosine-based activation motif (ITAM).Examples of ITAMs that contain primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD8, CD22, CD79a, CD79b and CD66d.In certain embodiments, ITAMs that contain primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma or FcR beta.In some embodiments, the cytoplasmic signaling molecule in CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

[0293] In some embodiments, the CAR comprises a signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, CD27, DAP10, and / or ICOS. In some aspects, the same CAR comprises both an activation region or signaling region and a costimulatory component. In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.

[0294] In some embodiments, the activation domain is contained within one CAR, while the costimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR comprises an activating CAR or stimulatory CAR and a costimulatory CAR, both of which are expressed on the same cell (see WO 2014 / 055668). In some aspects, the CAR is a stimulatory CAR or an activating CAR, and in other aspects, it is a costimulatory CAR. In some embodiments, the cell further comprises an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), e.g., a CAR that recognizes a different antigen, e.g., for the purpose of reducing off-target effects, such that the activation signal delivered by the CAR that recognizes the first antigen is reduced or inhibited by the inhibitory CAR binding to its ligand.

[0295] In certain embodiments, the intracellular signaling domain comprises the transmembrane and signaling domain of CD28 linked to the CD3 intracellular domain, hi some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 costimulatory domain linked to the CD3 intracellular domain.

[0296] In some embodiments, CD8 + The intracellular signaling domain of cytotoxic T cells is CD4 + The intracellular signaling domain is identical to that of a helper T cell. In some embodiments, CD8 + The intracellular signaling domain of cytotoxic T cells is CD4 + It is distinct from the intracellular signaling domain of helper T cells.

[0297] In some embodiments, the CAR comprises one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components CD3 zeta, CD28, and 4-1BB.

[0298] In some embodiments, the recombinant receptor, e.g., CAR, encoded by the nucleic acid (e.g., polynucleotide) in the provided viral vector further comprises one or more markers, for example, for purposes such as verifying cell transduction or cell modification, and / or selecting and / or targeting cells that express the molecule encoded by the polynucleotide. In some aspects, such markers may be encoded by different nucleic acids or polynucleotides, which may be similarly introduced during the genetic modification process, typically by the same method, e.g., by transduction using any of the methods provided herein, e.g., by the same vector or the same type of vector.

[0299] In some aspects, the marker, e.g., a transduction marker, is a protein and / or a cell surface molecule. Exemplary markers are truncated variants of native markers, e.g., endogenous markers, e.g., native cell surface molecules. In some aspects, the variants have reduced immunogenicity, reduced transport function, and / or reduced signaling function compared to native or endogenous cell surface molecules. In some embodiments, the marker is a truncated cell surface receptor, e.g., truncated EGFR (tEGFR). In some aspects, the marker comprises all or a portion (e.g., a truncated form) of CD34, NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a linker sequence, e.g., a cleavable linker sequence, e.g., a polynucleotide encoding T2A P2A, E2A, and / or F2A. See, e.g., WO 2014 / 031687.

[0300] In some embodiments, the marker is a molecule that is not naturally found on or on the surface of a T cell, e.g., a cell surface protein, or portion thereof.

[0301] In some embodiments, the molecule is a non-self molecule, eg, a non-self protein, ie, one that is not recognized as "self" by the immune system of the host into which the cells are to be adoptively transferred.

[0302] In some embodiments, the marker does not serve a therapeutic function and / or has no effect other than being used as a marker for genetic modification, e.g., to select successfully modified cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise exerts some desired effect, such as a ligand for cells encountered in vivo, e.g., a costimulatory molecule or immune checkpoint molecule to enhance and / or attenuate the response of cells when adoptively transferred and encounter the ligand.

[0303] In some cases, the CAR is a first-, second-, and / or third-generation CAR. In some aspects, a first-generation CAR provides only the signal induced by the CD3 chain upon antigen binding, a second-generation CAR provides such a signal plus a costimulatory signal, which includes, for example, an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137, and in some aspects, a third-generation CAR includes multiple costimulatory domains from different costimulatory receptors.

[0304] In some embodiments, the chimeric antigen receptor comprises an extracellular ligand-binding portion, e.g., an antigen-binding portion, e.g., an antibody or fragment thereof, and an intracellular domain. In some embodiments, the antibody or fragment comprises an scFv or single-domain VH antibody, and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain connecting and / or positioned between the extracellular domain and the intracellular signaling region or intracellular signaling domain.

[0305] In some aspects, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any of those described herein. In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule, for example, between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0306] In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains the signaling portion of CD28 or a functional variant thereof and the signaling portion of CD3-zeta or a functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains the signaling portion of 4-1BB or a functional variant thereof and the signaling portion of CD3-zeta or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer containing a portion of an Ig molecule, such as a human Ig molecule, e.g., an Ig hinge, e.g., an IgG4 hinge, e.g., a hinge-only spacer.

[0307] In some embodiments, the transmembrane domain of the receptor, e.g., CAR, is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (Accession Number: P10747.1), or is a transmembrane domain comprising the sequence of amino acids set forth in SEQ ID NO: 15, or a sequence of amino acids exhibiting at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 15; in some embodiments, the transmembrane domain-containing portion of the recombinant receptor is The amino acid sequence set forth in SEQ ID NO:16 or a sequence of amino acids having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity thereto.

[0308] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0309] In some embodiments, the intracellular domain comprises the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as the 41 amino acid domain thereof and / or such a domain with an LL→GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain can comprise a sequence of amino acids set forth in SEQ ID NO:17 or SEQ ID NO:18, or a sequence of amino acids exhibiting at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:17 or SEQ ID NO:18. In some embodiments, the intracellular region and / or intracellular domain comprises the intracellular costimulatory signaling domain of 4-1BB or a functional variant thereof, such as the 42 amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1), or a functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO:19 or a sequence of amino acids exhibiting 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:19.

[0310] In some embodiments, the intracellular signaling region and / or intracellular signaling domain comprises a stimulatory signaling domain of a human CD3 chain, optionally CD3 zeta, or a functional variant thereof, such as the 112 AA cytoplasmic domain of isoform 3 of human CD3ζ (accession number: P20963.2), or a CD3 zeta signaling domain described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993. In some embodiments, the intracellular signaling domain comprises a sequence of amino acids set forth in SEQ ID NO:20, 21 or 22, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:20, 21 or 22.

[0311] In some aspects, the spacer contains only the hinge region of an IgG, e.g., an IgG4 or IgG1 hinge only, e.g., the hinge-only spacer shown in SEQ ID NO:8. In another embodiment, the spacer is an Ig hinge, such as an IgG4 hinge, linked to the CH2 and / or CH3 domains. In some embodiments, the spacer comprises C H 2 and C H In some embodiments, the spacer is a C H An Ig hinge, such as an IgG4 hinge, linked to only three domains, such as that shown in SEQ ID NO: 11. In some embodiments, the spacer is or includes a glycine-serine rich sequence or other flexible linker, such as a known flexible linker.

[0312] For example, in some embodiments, the CAR comprises an extracellular ligand-binding portion, e.g., an antigen-binding portion, e.g., an antibody or fragment thereof, e.g., an sdAb and scFv, that specifically binds to an antigen (e.g., an antigen described herein); a spacer, e.g., any Ig hinge-containing spacer; a transmembrane domain that is a portion of CD28 or a variant thereof; an intracellular signaling domain containing a signaling portion of CD28 or a functional variant thereof; and a signaling portion of a CD3 zeta signaling domain or a functional variant thereof. In some embodiments, the CAR comprises an extracellular ligand-binding portion, e.g., an antigen-binding portion, e.g., an antibody or fragment thereof, sdAb and scFv, that specifically binds to an antigen (e.g., an antigen described herein); a spacer, e.g., any Ig hinge-containing spacer; a transmembrane domain that is a portion of CD28 or a variant thereof; an intracellular signaling domain containing a signaling portion of 4-1BB or a functional variant thereof; and a signaling portion of a CD3 zeta signaling domain or a functional variant thereof.

[0313] In some embodiments, such a CAR construct further comprises a T2A ribosomal skip element and / or a tEGFR sequence, e.g., downstream of the CAR. In some embodiments, the nucleic acid molecule encoding such a CAR construct further comprises a sequence encoding a ribosomal skip element (e.g., T2A) followed by a sequence encoding a tEGFR sequence, e.g., downstream of the sequence encoding the CAR. In some embodiments, T cells expressing an antigen receptor (e.g., a CAR) can also be made to express truncated EGFR (EGFRt) as a non-immunogenic selection epitope (e.g., by introducing a construct encoding CAR and EGFRt separated by a T2A ribosomal switch so that the two proteins are expressed from the same construct), which can then be used as a marker to detect such cells (see, e.g., U.S. Patent No. 8,802,374). In some cases, peptides such as T2A can cause the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element (ribosomal skipping), resulting in separation between the end of the 2A sequence and the next downstream peptide (see, e.g., de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, 2A sequences from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine teschovirus-1 (P2A), as described in U.S. Patent Publication No. 20070116690.

[0314] The recombinant receptor such as CAR that is expressed by the cell administered to the subject generally recognizes or specifically binds to the molecule that is expressed in, associated with, and / or specific to the disease or condition or its cell that is treated.When specifically bound to the molecule, for example, antigen, the receptor generally delivers immunostimulatory signals, such as the signal transmitted by ITAM, into the cell, thereby promoting the immune response that targets disease or condition.For example, in some embodiments, the cell expresses CAR that specifically binds to the antigen that is expressed by the cell or tissue of disease or condition or the antigen that is associated with disease or condition.

[0315] b. T cell receptor (TCR) In some embodiments, the recombinant molecule encoded by the nucleic acid (e.g., polynucleotide) is or comprises a recombinant T cell receptor (TCR). In some embodiments, the recombinant TCR is specific for an antigen, typically 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 from a viral or bacterial pathogen. In some embodiments, modified cells, e.g., T cells, are provided that express a TCR or antigen-binding portion thereof that recognizes a peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, virus, or autoimmune protein. In some embodiments, the TCR specifically binds to an antigen associated with a disease or condition, or specifically binds to a universal tag. In some embodiments, the antigen is associated with a disease or condition, e.g., cancer, an autoimmune disease or disorder, or an infectious disease.

[0316] In some embodiments, a "T cell receptor" or "TCR" is a molecule that contains a variable α chain and a variable β chain (also known as TCRα and TCRβ, respectively), or a variable γ chain and a variable δ chain (also known as TCRα and TCRβ, respectively), or an antigen-binding portion thereof, and is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is of the αβ type. Typically, the structures of TCRs that exist in the αβ and γδ types are generally similar, although the anatomical location or function of the T cells that express them may differ. TCRs may be found on the surface of a cell or in a soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where they are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0317] Unless otherwise stated, the term "TCR" should be understood to encompass complete TCRs as well as antigen-binding portions or fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, including αβ or γδ TCRs. In some embodiments, the TCR is an antigen-binding portion that is shorter than the full-length TCR but binds to a specific peptide bound to an MHC molecule, e.g., binds to an MHC-peptide complex. Optionally, the antigen-binding portion or antigen-binding fragment of a TCR may contain only a portion of the structural domain of a full-length or intact TCR, yet still be capable of binding to a peptide epitope bound by the complete TCR, e.g., an MHC-peptide complex. Optionally, the antigen-binding portion contains sufficient variable domains of the TCR, e.g., the variable α chain and variable β chain of the TCR, to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain the complementarity-determining regions involved in recognizing peptides, MHC, and / or MHC-peptide complexes.

[0318] In some embodiments, the variable domain of a TCR contains hypervariable loops, or complementarity-determining regions (CDRs), which are generally responsible for antigen recognition and binding capacity and specificity. In some embodiments, the CDRs of a TCR or a combination thereof form all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs within the variable region of a TCR chain are generally separated by framework regions (FRs), which generally show less variation between TCR molecules than CDRs (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; also see Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the primary CDR responsible for antigen binding or antigen specificity, or the most important of the three CDRs on a given TCR variable region for antigen recognition and / or for interaction with the processed peptide portion of a peptide-MHC complex. In some circumstances, CDR1 of the alpha chain can interact with the N-terminal portion of a given antigenic peptide. In some circumstances, CDR1 of the beta chain can interact with the C-terminal portion of a peptide. In some circumstances, CDR2 is the primary CDR that most strongly contributes to or is responsible for interaction with or recognition of the MHC portion of an MHC-peptide complex. In some embodiments, the variable region of the beta chain can contain an additional hypervariable region (CDR4 or HVR4), which is generally involved in superantigen binding and not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0319] In some embodiments, the TCR can also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain of the TCR can have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a C-terminal short cytoplasmic tail. In some embodiments, the TCR is associated with the invariant protein of the CD3 complex, which is involved in mediating signal transduction.

[0320] In some embodiments, a TCR chain contains one or more constant domains. For example, the extracellular portion of a given TCR chain (e.g., an α chain or a β chain) comprises two immunoglobulin-like domains, such as a variable domain (e.g., Vα or Vβ; typically, amino acids 1-116 according to the Kabat numbering in Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, 5th ed., 1991), and a constant domain adjacent to the cell membrane (e.g., an α chain constant domain, i.e., Cα, typically positions 117-259 of the α chain according to the Kabat numbering, or a β chain constant region, i.e., C β , typically positions 117-295 of the β chain according to Kabat. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains and two membrane-distal variable domains, each of which contains a CDR. The constant domain of the TCR may contain a short connecting sequence in which cysteine ​​residues form disulfide bonds, thereby linking the two chains of the TCR. In some embodiments, the TCR may have an additional cysteine ​​residue in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domain.

[0321] In some embodiments, the TCR chain contains a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. Optionally, the TCR chain contains a cytoplasmic tail. Optionally, the structure allows the TCR to associate with other molecules, such as CD3 or its subunits. For example, a TCR containing a constant domain and a transmembrane region can anchor the protein to the cell membrane and associate with the CD3 signaling apparatus or an invariant subunit of the CD3 signaling complex. The intracellular tail of the CD3 signaling subunit (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contains one or more immunoreceptor tyrosine-based activation motifs, or ITAMs, that are involved in the signaling ability of the TCR complex.

[0322] In some embodiments, the TCR is a heterodimer of two chains, α and β (or optionally γ and δ), or can be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β or γ and δ chains) linked, such as by one or more disulfide bonds.

[0323] In some embodiments, TCRs can be generated from known TCR sequences, e.g., Vα, β chain sequences, for which substantially full-length coding sequences are readily available. Methods for obtaining full-length TCR sequences, including V chain sequences, from cellular sources are well known. In some embodiments, nucleic acids (e.g., polynucleotides) encoding TCRs can be obtained from a variety of sources, for example, by polymerase chain reaction (PCR) amplification of TCR-encoding nucleic acids (e.g., polynucleotides) within or isolated from one or more given cells, or by synthesis of publicly available TCR DNA sequences.

[0324] In some embodiments, the TCR is obtained from a biological source, e.g., from a cell, e.g., a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, the T cell can be obtained from in vivo isolated cells. In some embodiments, the TCR is a thymus-selected TCR. In some embodiments, the TCR is a neoepitope-restricted TCR. In some embodiments, the T cell can be a cultured T cell hybridoma or a cultured T cell clone. In some embodiments, the TCR, or an antigen-binding portion thereof, or an antigen-binding fragment thereof, can be synthetically generated from knowledge of the sequence of the TCR.

[0325] In some embodiments, TCRs are generated from TCRs identified or selected by screening a library of candidate TCRs against a target polypeptide antigen or its target T cell epitope. TCR libraries can be generated by amplifying Vα and Vβ repertoires from T cells isolated from a subject, including cells present in PBMCs, spleen, or other lymphoid organs. Optionally, T cells can be expanded from tumor-infiltrating lymphocytes (TILs). In some embodiments, TCR libraries can be generated from CD4+ cells or CD8+ cells. In some embodiments, TCRs can be expanded from a T cell source from a normal or healthy subject, i.e., from a normal TCR library. In some embodiments, TCRs can be expanded from a T cell source from a diseased subject, i.e., from a disease TCR library. In some embodiments, degenerate primers are used to amplify Vα and Vβ gene repertoires by RT-PCR in a sample, e.g., T cells obtained from a human. In some embodiments, scTv libraries can be assembled from naive Vα and Vβ libraries, in which amplification products are cloned or assembled so that they are separated by a linker. Depending on the subject and cell source, the library can be HLA allele-specific. Alternatively, in some embodiments, TCR libraries can be generated by mutagenesis and diversification of parent or scaffold TCR molecules. In some aspects, TCRs are subjected to directed evolution, such as by mutagenesis of the α or β chain. In some aspects, specific residues within the CDRs of the TCR are altered. In some embodiments, selected TCRs can be modified by affinity maturation. In some embodiments, antigen-specific T cells can be selected, such as by screening to assess CTL activity against a peptide. In some aspects, TCRs present in antigen-specific T cells can be selected by avidity, such as by a specific affinity or avidity for an antigen.

[0326] In some embodiments, the genetically modified antigen receptor includes a recombinant T cell receptor (TCR) and / or a TCR cloned from a natural T cell. In some embodiments, the TCR is cloned from a natural T cell. In some embodiments, a high affinity T cell clone against a target antigen (e.g., a cancer antigen) is identified and isolated from a patient and introduced into the cell. In some embodiments, a TCR clone against the target antigen is generated in a transgenic mouse modified with human immune system genes (e.g., human leukocyte antigen system, or HLA). See, e.g., tumor antigens (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). In some embodiments, the TCRs, or antigen-binding portions thereof, are modified or altered. In some embodiments, directed evolution methods are used to generate TCRs with altered properties, e.g., higher affinity for specific MHC-peptide complexes. In some embodiments, directed evolution is used to generate TCRs with altered properties, e.g., higher affinity for specific MHC-peptide complexes. In some embodiments, directed evolution is used in a variety of ways, including, but not limited to, yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2004) Nat Immunol, 4, 55-62). al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, the display approach involves alteration or modification of a known parent or reference TCR.For example, in some cases, a wild-type TCR can be used as a template to produce mutant TCRs in which one or more residues in the CDRs are mutated, and mutants are selected that have a desired altered property, such as increased affinity for a desired target antigen.

[0327] In some embodiments, peptides of target polypeptides for use in producing or generating TCRs of interest are known or can be easily identified by those skilled in the art. In some embodiments, peptides suitable for use in generating TCRs or antigen-binding portions can be determined based on the presence of HLA-restricted motifs in target polypeptides of interest, such as the target polypeptides described below. In some embodiments, peptides are identified using available computer prediction models. In some embodiments, when predicting MHC class I binding sites, such models include, but are not limited to, ProPred1 (Singh and Raghava (2001) Bioinformatics 17(12):1236-1237, and SYFPEITHI (Schuler et al. (2007) Immunoinformatics Methods in Molecular Biology, 409(1):75-93 2007). In some embodiments, the MHC-restricted epitope is HLA-A0201, which is expressed in approximately 39-46% of all Caucasians, making it a suitable choice of MHC antigen for use in preparing TCRs or other MHC-peptide binding molecules.

[0328] HLA-A0201 binding motif and proteasome and immunoproteasome cleavage site are known by computer prediction model.When predicting MHC class I binding site, such model includes but is not limited to ProPred1 (described in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237 2001) and SYFPEITHI (referring to Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology, vol. 409(1):75-93 2007).

[0329] In some embodiments, the TCR or its antigen-binding portion can be a recombinantly produced native protein or a variant thereof with one or more modified properties, such as binding characteristics. In some embodiments, the TCR can be derived from one of a variety of animal species, such as human, mouse, rat, or other mammal. The TCR can be cell-associated or soluble. In some embodiments, for the purposes of the provided methods, the TCR is cell-associated and expressed on the surface of a cell.

[0330] In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). In some embodiments, the dTCR or scTCR has the structure described in WO 03 / 020763, WO 04 / 033685 and WO 2011 / 044186.

[0331] In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR contains a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR can form a TCR complex with CD3. In some embodiments, the TCR can be linked to any signaling domain, including dTCR or scTCR, that results in an active TCR on the surface of the T cell. In some embodiments, the TCR is expressed on the surface of the cell.

[0332] In some embodiments, the dTCR contains a first polypeptide in which a sequence corresponding to a TCR α chain variable region sequence is fused to the N-terminus of a sequence corresponding to a TCR α chain constant region extracellular sequence, and a second polypeptide in which a sequence corresponding to a TCR β chain variable region sequence is fused to the N-terminus of a sequence corresponding to a TCR β chain constant region extracellular sequence, the first and second polypeptides being linked by a disulfide bond. In some embodiments, this bond can correspond to the native interchain disulfide bond present in a native dimeric αβ TCR. In some embodiments, the interchain disulfide bond is not present in the native TCR. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequences of the dTCR polypeptide pair. Both native and non-native disulfide bonds may be desirable. In some embodiments, the TCR contains a transmembrane sequence for membrane anchoring.

[0333] In some embodiments, the dTCR comprises a TCR alpha chain comprising a variable alpha domain, a constant alpha domain, and a first dimerization motif attached to the C-terminus of the constant alpha domain, and a TCR beta chain comprising a variable beta domain, a constant beta domain, and a first dimerization motif attached to the C-terminus of the constant beta domain, wherein the first and second dimerization motifs readily interact to form a covalent bond between amino acids in the first and second dimerization motifs that links the TCR alpha and TCR beta chains to one another.

[0334] In some embodiments, the TCR is a scTCR. Typically, scTCRs can be generated using known methods. See, for example, Soo Hoo, WF et al. PNAS(USA)89, 4759(1992), Wuelfing, C. and Plueckthun, A., J. Mol. Biol. 242, 655(1994), Kurucz, I. et al. PNAS(USA)90 3830(1993), PCT International Publication Nos. WO 96 / 13593, WO 96 / 18105, WO 99 / 60120, WO 99 / 18129, WO 03 / 020763, WO 2011 / 044186, and Schlueter, CJ et al. J. Mol. Biol. 256, 859(1996). In some embodiments, the scTCR contains an introduced non-native disulfide interchain bond to enhance TCR chain association (see, e.g., PCT International Publication No. WO 03 / 020763). In some embodiments, the scTCR is a non-disulfide-linked truncated TCR in which a heterologous leucine zipper fused to its C-terminus enhances chain association (see, e.g., PCT International Publication No. WO 99 / 60120). In some embodiments, the scTCR contains a TCR alpha variable domain covalently linked to a TCR beta variable domain via a peptide linker (see, e.g., PCT International Publication No. WO 99 / 18129).

[0335] In some embodiments, the scTCR comprises a first segment composed of an amino acid sequence corresponding to a TCR α chain variable region, a second segment composed of an amino acid sequence corresponding to a TCR β chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR β chain constant domain extracellular sequence, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0336] In some embodiments, the scTCR contains a first segment composed of an alpha chain variable region sequence fused to the N-terminus of an alpha chain extracellular constant domain sequence, a second segment composed of a beta chain variable region fused to the N-terminus of a sequence corresponding to a beta chain extracellular constant and transmembrane sequence, and optionally a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0337] In some embodiments, the scTCR contains a first segment composed of a TCR beta chain variable region sequence fused to the N-terminus of a beta chain extracellular constant domain sequence, a second segment composed of an alpha chain variable region fused to the N-terminus of a sequence corresponding to an alpha chain extracellular constant and transmembrane sequence, and optionally a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0338] In some embodiments, the linker of the scTCR connecting the first and second TCR segments can be any linker capable of forming a single polypeptide chain while maintaining TCR binding specificity. In some embodiments, the linker sequence has the formula -P-AA-P-, for example, where P is proline and AA represents an amino acid sequence containing glycine and serine amino acids. In some embodiments, the first and second segments are paired such that their variable region sequences accommodate such binding. Thus, in some cases, the linker is long enough to span the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but not so long as to prevent or reduce binding of the scTCR to the target ligand. In some embodiments, the linker can contain 10 to 45 or about 10 to 45 amino acids, e.g., 10 to 30 amino acids, or 26 to 41 amino acid residues, e.g., 29, 30, 31, or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P-, where P is proline, G is glycine, and S is serine (SEQ ID NO:29). I have TIFF2025170348000002.tif4128.

[0339] In some embodiments, the scTCR contains a covalent disulfide bond linking residues of the immunoglobulin region of the constant domain of the α chain to residues of the immunoglobulin region of the constant domain of the β chain. In some embodiments, interchain disulfide bonds are absent in native TCRs. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequences of the first and second segments of the scTCR polypeptide. Both native and non-native disulfide bonds may be desirable.

[0340] In some embodiments of dTCRs or scTCRs containing an introduced interchain disulfide bond, there is no native disulfide bond. In some embodiments, one or more native cysteines that form the native interchain disulfide bond are substituted with another residue, such as serine or alanine. In some embodiments, the introduced disulfide bond can be formed by mutating non-cysteine ​​residues on the first and second segments to cysteine. Exemplary non-native disulfide bonds of TCRs are described in PCT International Publication No. WO 2006 / 000830.

[0341] In some embodiments, the TCR or antigen-binding fragment thereof exhibits an affinity for the target antigen with an equilibrium binding constant of 10 to 10 M, or about 10 to about 10 M, and all individual values ​​and ranges therein. In some embodiments, the target antigen is an MHC-peptide complex or an MHC-peptide ligand.

[0342] In some embodiments, one or more nucleic acids (e.g., polynucleotides) encoding TCRs, such as the α and β chains, can be amplified by PCR, cloning, or other suitable means and cloned into one or more suitable expression vectors. The expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for growth and expansion, or for expression, or both, such as plasmids and viruses.

[0343] In some embodiments, the vector can be a pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, La Jolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, CA) vector. In some cases, bacteriophage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. In some embodiments, plant expression vectors can be used, including pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, viral vectors, such as retroviral vectors, are used.

[0344] In some embodiments, recombinant expression vectors can be prepared using standard recombinant DNA techniques. In some embodiments, the vector can contain regulatory sequences, such as transcriptional and translational initiation and termination codons, specific to the type of host into which the vector will be introduced (e.g., bacteria, fungi, plants, or animals), as appropriate, taking into account whether the vector is DNA- or RNA-based. In some embodiments, the vector can contain a non-native promoter operably linked to the nucleotide sequence encoding the TCR or antigen-binding portion (or other MHC-peptide binding molecule). In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of murine stem cell virus. Other known promoters are also contemplated.

[0345] In some embodiments, after obtaining a T cell clone, 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 by a picornavirus 2A ribosomal skipping peptide so that both chains are co-expressed. In some embodiments, the nucleic acid (e.g., polynucleotide) encoding the TCR further comprises a marker to verify transduction or modification of cells to express the receptor. In some embodiments, gene transfer of the TCR is accomplished by retroviral or lentiviral vectors, or by transposons (see, e.g., Baum et al. (2006) Molecular Therapy: The Journal of the American Society of Gene Therapy. 13:1050-1063, Frecha et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:1748-1757, and Hackett et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:674-683).

[0346] In some embodiments, to generate a vector encoding a TCR, the α and β chains are PCR amplified from the total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector. In some embodiments, the α and β chains are cloned into the same vector. In some embodiments, the α and β chains are cloned into different vectors. In some embodiments, the generated α and β chains are incorporated into a retroviral vector, such as a lentiviral vector.

[0347] c. Chimeric Autoantibody Receptor (CAAR) In some embodiments, the recombinant receptor is a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR is specific for an autoantibody. In some embodiments, cells expressing a CAAR, e.g., T cells engineered to express a CAAR, can be used to specifically bind to and kill autoantibody-expressing cells but not normal antibody-expressing cells. In some embodiments, CAAR-expressing cells can be used to treat autoimmune diseases associated with the expression of an autoantigen, e.g., autoimmune disease. In some embodiments, CAAR-expressing cells target B cells that ultimately produce and display autoantibodies on their cell surface, marking these B cells as disease-specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used to efficiently target and kill pathogenic B cells in autoimmune diseases by targeting disease-causing B cells with an antigen-specific chimeric autoantibody receptor. In some embodiments, the recombinant receptor is a CAAR, e.g., any of those described in U.S. Patent Application Publication No. US 2017 / 0051035.

[0348] In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling region comprises a secondary or costimulatory signaling region (secondary intracellular signaling region).

[0349] In some embodiments, the autoantibody binding domain comprises an autoantigen or a fragment thereof. The selection of the autoantigen can depend on the type of autoantibody to be targeted. For example, the autoantigen can be selected because it recognizes an autoantibody on a target cell, e.g., a B cell, that is associated with a particular disease state, e.g., an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoim...

Claims

1. 1. A method for increasing the transduction frequency of primary T cells, comprising: (a) selecting primary T cells that are positive for surface expression of CCR7 from a biological sample containing a population of primary T cells, thereby generating an input population enriched for CCR7+ primary T cells; (b) incubating the input population under stimulatory conditions, thereby generating a stimulated composition, the stimulatory conditions comprising the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and (c) incubating viral vector particles containing a heterologous polynucleotide encoding a recombinant protein with the T cells of the stimulated composition, thereby generating a population of transduced cells. A method comprising:

2. 1. A method for increasing the transduction frequency of primary T cells, comprising: (a) selecting primary T cells that are positive for surface expression of CCR7 from a biological sample containing a population of primary T cells, thereby generating an input population enriched for CCR7+ primary T cells; and (b) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with T cells of the input cell population, thereby generating a population of transduced cells. A method comprising:

3. 1. A method for increasing the transduction frequency of primary T cells, comprising: (a) incubating an input primary T cell population enriched for CCR7+ T cells under stimulatory conditions, thereby generating a stimulated composition, the stimulatory conditions comprising the presence of a stimulatory agent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and (b) incubating viral vector particles containing a heterologous polynucleotide encoding a recombinant protein with the T cells of the stimulated composition, thereby generating a population of transduced cells. A method comprising:

4. 1. A method for increasing the transduction frequency of primary T cells, comprising: incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with T cells of an input primary T cell population enriched for CCR7+ T cells, thereby generating a population of transduced cells. A method comprising:

5. 5. The method of any one of claims 1 to 4, wherein at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the input population are CCR7+ primary T cells.

6. 6. The method of any one of claims 1 to 5, wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the input population are CCR7+ primary T cells.

7. 7. The method of any one of claims 1 to 6, wherein at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the input population are CCR7+ primary T cells.

8. The method of any one of claims 1, 2 and 5 to 7, wherein the biological sample is a blood sample.

9. The method of any one of claims 1, 2 and 5 to 7, wherein the biological sample is a leukapheresis sample.

10. 10. The method of any one of claims 1 to 9, wherein the T cells are unfractionated T cells, enriched or isolated CD3+ T cells, enriched or isolated CD4+ T cells, or enriched or isolated CD8+ T cells.

11. 11. The method of any one of claims 1 to 10, wherein the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells or CD8+ T cells.

12. 12. The method of any one of claims 1-11, wherein the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells.

13. 12. The method of any one of claims 1-11, wherein the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD8+ T cells.

14. 12. The method of any one of claims 1-11, wherein the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells and CD8+ T cells.

15. 15. The method of claim 14, wherein the ratio of CD4+ T cells to CD8+ T cells is or is about 1:1, 1:2, 2:1, 1:3, or 3:

1.

16. 11. The method of any one of claims 1 to 10, wherein the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD3+ T cells.

17. The input group is 100 x 10 6 ~500×10 6 17. The method of any one of claims 1 to 16, comprising a total of T cells.

18. The input group is 200 x 10 6 ~400×10 6 Total T cells, optionally 300 x 10 6 or approximately 300 x 10 6 The method of any one of claims 1 to 17, comprising a total of T cells.

19. 19. The method of claim 17 or claim 18, wherein the total T cells are viable T cells.

20. At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of the cells of the stimulated composition (i) expressing a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB; (ii) comprising intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-gamma, and TNF-alpha; (iii) is in the G1 phase or later of the cell cycle, and / or (iv) having proliferation ability; 10. The method of any one of claims 1, 3, and 519.

21. The irritant agent is a primary agent that specifically binds to a member of the TCR complex, optionally specifically binding to CD3 21. The method of any one of claims 1, 3, and 5-20, comprising:

22. 22. The method of claim 21, wherein the stimulatory reagent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, optionally wherein the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS.

23. 23. The method of claim 21 or claim 22, wherein the primary agent and / or the secondary agent comprises an antibody, and optionally, the stimulatory reagent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody or antigen-binding fragment thereof.

24. 24. The method of any one of claims 21 to 23, wherein the primary agent and / or the secondary agent are present on the surface of a solid support.

25. 25. The method of claim 24, wherein the solid support is or comprises a bead.

26. 24. The method of any one of claims 21-23, wherein the primary agent and the secondary agent are reversibly bound to the surface of an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules.

27. Each of the plurality of streptavidin molecules or streptavidin mutein molecules comprises a Val at a sequence position corresponding to positions 44-47 in relation to their positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO:

34. 44 -Thr 45 -Ala 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47 27. The method of claim 26, comprising the amino acid sequence of:

28. Each of the plurality of streptavidin or streptavidin mutein molecules is a streptavidin mutein molecule, and each of the plurality of streptavidin mutein molecules is (a) a sequence of amino acids set forth in any one of SEQ ID NOs: 36, 41, 48-50, or 53-55; (b) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any one of SEQ ID NOs: 36, 41, 48-50, or 53-55, and contains an amino acid sequence corresponding to Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47, and / or reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide; or (c) a functional fragment of (a) or (b) that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide; is or contains Optionally, each of the plurality of streptavidin mutein molecules is or comprises the amino acid sequence set forth in SEQ ID NO: 36 or SEQ ID NO:

41.

27. The method of claim 26.

29. 29. The method of any one of claims 1-28, wherein the population of transduced cells comprises at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of cells expressing the recombinant protein.

30. 30. The method of any one of claims 1-29, wherein the population of transduced cells comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of cells that express the recombinant protein.

31. 31. The method of any one of claims 1-30, wherein the percentage of cells in the population of transduced cells that express the recombinant protein is at least 0.5-fold, at least 1-fold, at least 1.5-fold, or at least 2-fold greater compared to a cell composition that has not been enriched for CCR7+ primary T cells by the selection step.

32. 32. The method of any one of claims 1 to 31, wherein the step of incubating the viral vector particles comprises subjecting the viral vector particles to spinoculation with an input population or a stimulated composition.

33. subjecting to spinoculation includes spinning the viral vector particles and the input population or stimulated composition in an inner cavity of a centrifugation chamber; The rotation, 500g to 2500g, 500g to 2000g, 500g to 1600g, 500g to 1000g, 600g to 1600g, 600g to 1000g, 1000g to 2000g, or 1000g to 1600g, inclusive; or about 500g to 2500g, 500g to 2000g, 500g to 1600g, 500g to 1000g, 600g to 1600g, 600g to 1000g, 1000g to 2000g, or 1000g to 1600g, inclusive; At least 600g, 800g, 1000g, 1200g, 1600g, or 2000g, or at least about 600g, 800g, 1000g, 1200g, 1600g, or 2000g is the relative centrifugal force at the inner surface of the cavity side wall, 33. The method of claim 32.

34. To subject to spinoculation, greater than or about 5 minutes, greater than or about 10 minutes, greater than or about 15 minutes, greater than or about 20 minutes, greater than or about 30 minutes, greater than or about 45 minutes, greater than or about 60 minutes, greater than or about 90 minutes, or greater than or about 120 minutes, or 5 to 60 minutes, 10 to 60 minutes, 15 to 60 minutes, 15 to 45 minutes, 30 to 60 minutes, or 45 to 60 minutes, inclusive; or approximately 5 to 60 minutes, 10 to 60 minutes, 15 to 60 minutes, 15 to 45 minutes, 30 to 60 minutes, or 45 to 60 minutes 34. The method of claim 32 or claim 33, wherein the method is carried out for a period of time that is

35. 35. The method of any one of claims 1, 3 and 5-34, further comprising contacting the primed composition and / or viral vector particles with a transduction adjuvant during at least a portion of the incubating step.

36. 35. The method of any one of claims 2, 4-34, further comprising contacting the input population and / or viral vector particles with a transduction adjuvant during at least a portion of the incubating step.

37. 37. The method of claim 35 or claim 36, wherein the contacting step is performed before, simultaneously with, or after subjecting the viral vector particles to spinoculation with the input population or stimulated composition.

38. 38. The method of any one of claims 1 to 37, wherein at least a portion of the incubation of the viral vector particles is carried out at or about 37°C±2°C.

39. 39. The method of any one of claims 1 to 38, wherein at least a portion of the incubation of the viral vector particles is carried out after spinoculation.

40. 40. The method of claim 38 or claim 39, wherein at least a portion of the incubation of the viral vector particles is carried out for less than or equal to about 2, 4, 12, 18, 24, 30, 36, 48, 60, or 72 hours.

41. 41. The method of any one of claims 38-40, wherein at least a portion of the incubation of the viral vector particles is carried out for 24 hours or about 24 hours.

42. 42. The method of any one of claims 1 to 41, wherein the total duration of incubation of the viral vector particles is no more than 12 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours.

43. 43. The method of any one of claims 1 to 42, wherein the viral vector particle is a lentiviral vector particle.

44. 44. The method of claim 43, wherein the lentiviral vector particle is replication-deficient.

45. 45. The method of any one of claims 1 to 44, wherein the viral vector particle is pseudotyped with a viral envelope glycoprotein.

46. 46. ​​The method of claim 45, wherein the viral envelope glycoprotein is VSV-G.

47. 47. The method of any one of claims 1 to 46, wherein the viral vector particles are incubated at a multiplicity of infection of less than or about 20.0 or less than or about 10.

0.

48. viral vector particles are incubated at a multiplicity of infection of 1.0 IU / cell to 10 IU / cell or 2.0 U / cell to 5.0 IU / cell, or about 1.0 IU / cell to 10 IU / cell or 2.0 U / cell to 5.0 IU / cell; or the viral vector particles are incubated at a multiplicity of infection of at least 1.6 IU / cell, 1.8 IU / cell, 2.0 IU / cell, 2.4 IU / cell, 2.8 IU / cell, 3.2 IU / cell, 3.6 IU / cell, 4.0 IU / cell, 5.0 IU / cell, 6.0 IU / cell, 7.0 IU / cell, 8.0 IU / cell, 9.0 IU / cell, or 10.0 IU / cell, or at least about 1.6 IU / cell, 1.8 IU / cell, 2.0 IU / cell, 2.4 IU / cell, 2.8 IU / cell, 3.2 IU / cell, 3.6 IU / cell, 4.0 IU / cell, 5.0 IU / cell, 6.0 IU / cell, 7.0 IU / cell, 8.0 IU / cell, 9.0 IU / cell, or 10.0 IU / cell; 48. The method of any one of claims 1 to 47.

49. The stimulated composition is at least 50 x 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately at least 50 × 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately 50 x 10 6 cells, 100×10 6 cells or 200 x 10 6 The method of any one of claims 1, 3 and 5 to 48, comprising a cell.

50. The stimulated composition is 50 x 10 inclusive. 6 or about 50 x 10 6 Cell ~300×10 6 or approximately 300 x 10 6 Cells, optionally 100 x 10 inclusive 6 or about 100 x 10 6 Cell ~200×10 6 or approximately 200 x 10 6 50. The method of any one of claims 1, 3 and 5-49, comprising a cell.

51. The input population is at least 50 × 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately at least 50 × 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately 50 x 10 6 cells, 100×10 6 cells or 200 x 10 6 The method of any one of claims 2 and 4 to 48, comprising a cell.

52. The input population is 50 × 10, including both extreme values. 6 or about 50 x 10 6 Cell ~300×10 6 or approximately 300 x 10 6 Cells, optionally 100 x 10 inclusive 6 or about 100 x 10 6 Cell ~200×10 6 or approximately 200 x 10 6 50. The method of any one of claims 2 and 4 to 49, comprising a cell.

53. T cells incubated with viral particles must be at least 50 × 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately at least 50 × 10 6 cells, 100×10 6 cells or 200 x 10 6 cells, or approximately 50 x 10 6 cells, 100×10 6 cells or 200 x 10 6 53. The method of any one of claims 1 to 52, comprising a cell.

54. T cells incubated with viral particles were 50 × 10 inclusive. 6 or about 50 x 10 6 Cell ~300×10 6 or approximately 300 x 10 6 Cells, optionally 100 x 10 inclusive 6 or about 100 x 10 6 Cell ~200×10 6 or approximately 200 x 10 6 54. The method of any one of claims 1 to 53, comprising a cell.

55. 55. The method of any one of claims 1 to 54, wherein the recombinant protein is an antigen receptor.

56. 56. The method of claim 55, wherein the antigen receptor is a transgenic T cell receptor (TCR).

57. 56. The method of claim 55, wherein the antigen receptor is a chimeric antigen receptor (CAR).

58. 58. The method of claim 57, wherein the CAR comprises an extracellular antigen recognition domain that specifically binds to a target antigen, an intracellular signaling domain comprising an ITAM, and a transmembrane domain that links the extracellular domain and the intracellular signaling domain.

59. The method of claim 58, wherein the intracellular signaling domain comprises the intracellular domain of the CD3 zeta (CD3ζ) chain.

60. 60. The method of claim 58 or claim 59, wherein the transmembrane domain comprises the transmembrane portion of CD28.

61. The method of any one of claims 58 to 60, wherein the intracellular signaling domain further comprises an intracellular signaling domain of a T cell costimulatory molecule.

62. 62. The method of claim 61, wherein the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.

63. 63. The method of any one of claims 55-62, wherein the antigen receptor specifically binds to an antigen associated with the disease or condition or specifically binds to a universal tag.

64. 64. The method of claim 63, wherein the disease or condition is cancer, an autoimmune disease or disorder, or an infectious disease.

65. 65. The method of any one of claims 1 to 64, wherein the population of transduced cells comprises T cells that have been transduced with a heterologous polynucleotide.

66. 66. The method of claim 65, wherein at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the T cells in the population of transduced cells are transduced with the heterologous polynucleotide.

67. 67. The method of claim 65 or claim 66, wherein at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the T cells in the population of transduced cells are transduced with the heterologous polynucleotide.

68. 68. The method of any one of claims 65-67, wherein at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the T cells transduced with the heterologous polynucleotide are CCR7+.

69. 67. The method of claim 65 or claim 66, further comprising the step of recovering or isolating the transduced T cells produced by the method from the population of transduced cells.

70. 70. The method of any one of claims 1-69, wherein the variation in the percentage of T cells in a population of transduced cells that are transduced with the heterologous polynucleotide among the plurality of populations of transduced cells is no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%.

71. The method of any one of claims 1 to 70, which is carried out in vitro or ex vivo.

72. 72. A composition comprising a population of transduced cells produced by the method of any one of claims 1 to 71.

73. 73. The composition of claim 72, further comprising a cryopreservation material.

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