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Building IP: JUNO Patent Appl "METHODS FOR T CELL TRANSDUCTION"METHODS FOR T CELL TRANSDUCTIONDOCUMENT IDUS 20230090117 A1 DATE PUBLISHED2023-03-23 INVENTOR INFORMATION NAMECITYSTATEZIP CODECOUNTRYHAIG; Neil Seattle WA N/A US TEOH; Jeffrey Seattle WA N/A US APPLICANT INFORMATION NAME Juno Therapeutics, Inc. CITY Seattle STATE WA ZIP CODE N/A COUNTRY US AUTHORITY N/A TYPE assignee ASSIGNEE INFORMATION NAME Juno Therapeutics, Inc. CITY Seattle STATE WA ZIP CODE N/A COUNTRY US TYPE CODE 02 APPLICATION NO17/796224 DATE FILED2021-01-27 DOMESTIC PRIORITY (CONTINUITY DATA)us-provisional-application US 62967005 20200128 AbstractProvided herein are methods for transduction of T cells. 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 Background/SummaryCROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. provisional application 62/967,005, filed Jan. 28, 2020, entitled “METHODS FOR T CELL TRANSDUCTION,” the contents of which are incorporated by reference in their entirety for all purposes. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing is provided as a file entitled 73504_2023140_SEQLIST.txt, created Jan. 27, 2021, which is 53,345 bytes in size. FIELD [0003] The present disclosure provides methods for transduction of T cells. 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. In some embodiments, such methods result in improving 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, such as one encoding a chimeric receptor such as a chimeric antigen receptor, or other recombinant antigen receptor such as a transgenic T cell receptor, and compositions thereof. In some embodiments, the provided cells and compositions can be used in methods of adoptive immunotherapy. BACKGROUND [0004] Various strategies are available for transducing T cell populations in vitro, including 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, including for research, diagnostic and therapeutic purposes, such that transduction frequency is increased and is more consistent among biological samples. Provided are methods that meet such needs. SUMMARY [0005] Provided herein is a method for increasing 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 enriched in CCR7+ primary T cells; (b) optionally, incubating the input population under stimulatory conditions, thereby generating a stimulated composition, wherein said stimulating 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) a viral vector particle comprising a heterologous polynucleotide encoding a recombinant protein with T cells of the input population of cells, or optionally of the stimulated composition, thereby generating a population of transduced cells. [0006] Also provided herein is a method for increasing 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 enriched in CCR7+ primary T cells; (b) incubating the input population under stimulatory conditions, thereby generating a stimulated composition, wherein said stimulating 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 a viral vector particle comprising a heterologous polynucleotide encoding a recombinant protein with T cells of the stimulated composition, thereby generating a population of transduced cells. [0007] Also provided herein is a method for increasing 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 enriched in CCR7+ primary T cells; and (b) incubating a viral vector particle comprising a heterologous polynucleotide encoding a recombinant protein with T cells of the input population of cells, thereby generating a population of transduced cells. [0008] Also provided herein is a method for increasing transduction frequency of primary T cells, the method comprising: (a) incubating an input population of primary T cells enriched in CCR7+ T cells under stimulatory conditions, thereby generating a stimulated composition, wherein said stimulating 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 a viral vector particle comprising a heterologous polynucleotide encoding a recombinant protein with T cells of the stimulated composition, thereby generating a population of transduced cells. [0009] Also provided herein is a method for increasing transduction frequency of primary T cells, the method comprising incubating a viral vector particle comprising a heterologous polynucleotide encoding a recombinant protein with T cells of an input population of primary T cells enriched in 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 does not comprise selecting for 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 in 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 in CCR7+ and CD45RO+ T cells, optionally wherein less than 85% of the total cells of the input population are CCR7+ and CD45RO+ T cells. In some of any such embodiments, the input population is not enriched in CCR7+ and CD27+ T cells, optionally wherein less than 85% of the total cells of the input population are CCR7+ and CD27+ T cells. In some of any such embodiments, the input population is not enriched in CCR7+ and CD45RA− T cells, optionally wherein less than 85% of the total cells of the input population are CCR7+ and CD45RA− T cells. In some of any such embodiments, the input population is not enriched in CCR7+ and CD62L+ T cells, optionally wherein less than 85% of the total cells of the input population are CCR7+ and CD62L+ T cells. In some of any such embodiments, the input population is not enriched in CCR7+ and CD45RA+ T cells, optionally wherein less than 85% of the total cells of the input population are CCR7+ and CD45RA+ T cells. In some of any such embodiments, the input population is not enriched in CCR7+ and CD62L− T cells, optionally wherein less than 85% of the total cells of 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, are enriched or isolated CD3+ T cells, are enriched or isolated CD4+ T cells, or are 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% 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% 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% 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% cells that are CD4+ T cells and CD8+ T cells. In some of any such embodiments, the ratio of the CD4+ T cells to the CD8+ T cells is or is 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% cells that are CD3+ T cells. [0015] In some of any such embodiments, the input population comprises between 100×10.sup.6 and 500×10.sup.6 total T cells. In some of any such embodiments, the input population comprises between 200×10.sup.6 and 400×10.sup.6 total T cells, optionally at or about 300×10.sup.6 total T cells. 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, TNF-alpha; (iii) are in the G1 or later phase of the cell cycle; and/or (iv) are capable of proliferating. [0017] In some of any such embodiments, the stimulatory reagent comprises a primary agent that specifically binds to a member of a TCR complex, optionally that 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 wherein 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 agents comprise an antibody, optionally wherein the stimulatory reagent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody, or an 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 comprises a bead. In some of any such embodiments, the primary agent and secondary agent are reversibly bound on 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 the streptavidin or streptavidin mutein molecules comprise the amino acid sequence of Val.sup.44-Thr.sup.45-Ala.sup.46-Arg.sup.47 or Ile.sup.44-Gly.sup.45-Ala.sup.46-Arg.sup.47 at sequence positions corresponding to positions 44 to 47 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 34. In some of any such embodiments, each of the plurality of the streptavidin or streptavidin mutein molecules is or comprises: a) the sequence of amino acids set forth in SEQ ID NO: 35 or 56; or b) a sequence of amino acids that exhibit 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 the streptavidin or streptavidin mutein molecules are streptavidin mutein molecules, and wherein each of the plurality of the streptavidin mutein molecules is or comprises: a) the 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 that exhibit 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 of SEQ ID NOS: 36, 41, 48-50, or 53-55 and contain the amino acid sequence corresponding to Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 and/or reversibly bind 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, optionally wherein each of the plurality of the streptavidin mutein molecules is or comprises the amino acid sequence set forth in SEQ ID NO:36 or 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 expressing the recombinant protein is at least 0.5-fold, at least 1-fold, at least 1.5-fold, or at least 2-fold greater as compared to a cell composition that was not enriched for CCR7+ primary T cells through a selection step. [0021] In some of any such embodiments, the incubating the viral vector particle comprises a step of spinoculating the viral vector particles with the input population. In some of any such embodiments, the incubating the viral vector particle comprises a step of spinoculating the viral vector particles with the stimulated composition. [0022] In some of any such embodiments, spinoculating comprises rotating, in an internal cavity of a centrifugal chamber, the viral vector particles and the input population, wherein the rotation is at a relative centrifugal force at an internal surface of the side wall of the cavity that is: between or between about 500 g and 2500 g, 500 g and 2000 g, 500 g and 1600 g, 500 g an 1000 g, 600 g and 1600 g, 600 g and 1000 g, 1000 g and 2000 g, or 1000 g and 1600 g, each inclusive; or at least or at least about 600 g, 800 g, 1000 g, 1200 g, 1600 g, or 2000 g. In some of any such embodiments, spinoculating comprises rotating, in an internal cavity of a centrifugal chamber, the viral vector particles and the stimulated composition, wherein the rotation is at a relative centrifugal force at an internal surface of the side wall of the cavity that is: between or between about 500 g and 2500 g, 500 g and 2000 g, 500 g and 1600 g, 500 g an 1000 g, 600 g and 1600 g, 600 g and 1000 g, 1000 g and 2000 g, or 1000 g and 1600 g, each inclusive; or at least or at least about 600 g, 800 g, 1000 g, 1200 g, 1600 g, or 2000 g. [0023] In some of any such embodiments, spinoculating is for a time 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 or between about 5 minutes and 60 minutes, 10 minutes and 60 minutes, 15 minutes and 60 minutes, 15 minutes and 45 minutes, 30 minutes and 60 minutes, or 45 minutes and 60 minutes, each inclusive. [0024] In some of any such embodiments, the method further comprises, during at least a portion of the incubating, contacting the input population and/or viral vector particles with a transduction adjuvant. In some of any such embodiments, the method further comprises, during at least a portion of the incubating, contacting the stimulated composition and/or viral vector particles with a transduction adjuvant. In some of any such embodiments, the method further comprises, during at least a portion of the incubating, contacting the stimulated composition and/or viral vector particles with a transduction adjuvant. [0025] In some of any such embodiments, the method further comprises, during at least a portion of the incubating, contacting the input population and/or viral vector particles with a transduction adjuvant. [0026] In some of any such embodiments, the contacting is carried out prior to, concomitant with, or after the spinoculating the viral vector particles with the input population. In some of any such embodiments, the contacting is carried out prior to, concomitant with, or after the spinoculating the viral vector particles with the stimulated composition. [0027] In some of any such embodiments, at least a portion of the incubation of the viral vector particle is carried out at or about 37° C.±2° C. In some of any such embodiments, at least a portion of the incubation of the viral vector particle is carried out after the spinoculation. In some of any such embodiments, the at least a portion of the incubation of the viral vector particle is carried out for no more than or no more than about 2 hours, 4 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours, or 72 hours. In some of any such embodiments, the at least a portion of the incubation of the viral vector particle is carried out for or for about 24 hours. In some of any such embodiments, the total duration of the incubation of the viral vector particle is for no more than 12 hours, 24 hours, 36 hours, 48 hours, 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 defective. 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 particle is incubated at a multiplicity of infection of less than or less than about 20.0 or less than or less than about 10.0. In some of any such embodiments, the viral vector particle is incubated at a multiplicity of infection from or from about 1.0 IU/cell to 10 IU/cell, or 2.0 U/cell to 5.0 IU/cell; or the viral vector particle is incubated at a multiplicity of infection of at least 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. [0030] In some of any such embodiments, the stimulated composition comprises at least at or about at least or about 50×10.sup.6 cells, 100×10.sup.6 cells, or 200×10.sup.6 cells. In some of any such embodiments, the stimulated composition comprises between at or about 50×10.sup.6 cells and at or about 300×10.sup.6 cells, inclusive. In some of any such embodiments, the stimulated composition comprises between at or about 100×10.sup.6 cells and at or about 200×10.sup.6 cells, inclusive. [0031] In some of any such embodiments, the input population comprises at least at or about at least or about 50×10.sup.6 cells, 100×10.sup.6 cells, or 200×10.sup.6 cells. In some of any such embodiments, the input population comprises between at or about 50×10.sup.6 cells and at or about 300×10.sup.6 cells, inclusive. In some of any such embodiments, the input population comprises between at or about 100×10.sup.6 cells and at or about 200×10.sup.6 cells, inclusive. [0032] In some of any such embodiments, the T cells incubated with the viral particle comprises at least at or about at least or about 50×10.sup.6 cells, 100×10.sup.6 cells, or 200×10.sup.6 cells. In some of any such embodiments, the T cells incubated with the viral particle comprises between at or about 50×10.sup.6 cells and at or about 300×10.sup.6 cells, inclusive. In some of any such embodiments, the T cells incubated with the viral particle comprises between at or about 100×10.sup.6 cells and at or about 200×10.sup.6 cells, inclusive [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 an intracellular domain of a 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 a 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. In 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. In some of any such embodiments, the disease or condition is a 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 transduced with the 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 the T cells in the population of transduced cells are transduced with the 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 the T cells in the population of transduced cells are transduced with the 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 the T cells transduced with the heterologous polynucleotide are CCR7+. [0037] In some of any such embodiments, the method further comprises recovering or isolating from the population of transduced cells the transduced T cells produced by the method. [0038] In some of any such embodiments, among a plurality of populations of transduced cells, the percentage of T cells in the population of transduced cells that are transduced with the heterologous polynucleotide varies by 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. |
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