RU2780156C2 - Production of constructed cells for adoptive cell therapy - Google Patents

Abstract

FIELD: biotechnology; cell biology.

SUBSTANCE: present invention relates to biotechnology and cell biology, in particular to methods for transduction of T-cells and methods for adoptive cell therapy. For the implementation of methods for transduction of T-cells, first, a retroviral vector particle, for example of a lentiviral vector, is incubated, containing recombinant nucleic acid encoding a heterologous molecule, with a composition containing T-cells. Then, the composition obtained at the first stage is incubated ex vivo in the presence of stimulating agents capable of activating T-cells. After that, a signal is induced by means of TCR complex, or T-cell proliferation is induced. At the same time, the first stage is started not late than 24 hours after taking a sample in an individual. In this case, the initial composition was not subjected to ex vivo stimulation, including incubation in the presence of agents capable of activating T-cells. The incubation at the final stage is carried out during not late than 10 days after the first incubation stage.

EFFECT: present invention allows for improvement strategies of transduction of cell populations in vitro for a possibility of their use in researches, diagnostics, and therapy.

90 cl, 3 dwg, 1 tbl, 2 ex

Description

Cross-reference to related applications

[0001] The present application claims priority of U.S. Provisional Application No. 62/430349, filed December 5, 2016 and entitled "Production of Engineered Cells for Adoptive Cell Therapy", the contents of which are incorporated herein in their entirety by reference.

Including a sequence listing via a link

[0002] The contents of the following, presented in an ASCII text file, are incorporated herein in their entirety by reference, namely, as a sequence listing in computer format (CRF) (file name 735042005040SeqList.txt, creation date: November 28, 2017, size : 50233 bytes).

Field of invention

[0003] The present invention relates to methods for genetically engineering cells, including cells for use in combination with adoptive cell therapy. In some embodiments of the invention, the described methods include transducing cells by incubation with a retroviral vector particle, for example, with a lentiviral vector, where, prior to incubation, the cells were not incubated with an activating or stimulating agent, for example, they were not incubated with anti-CD3/ anti-CD28 antibodies and/or with one or more recombinant cytokines. In some embodiments of the invention, such methods provide features associated with a reduction or improvement in the genetic engineering of cells. The present invention also relates to resulting cells that have been transduced with a recombinant or heterologous gene, such as a gene encoding a chimeric receptor, such as a chimeric antigen receptor, or another recombinant antigen receptor, such as a transgenic T-cell receptor, and compositions thereof. In some embodiments of the invention, the described cells and compositions can be used in methods of adoptive immunotherapy.

State of the art

[0004] There are various strategies for in vitro transduction of T cell populations, including in vitro transduction of antigen-specific T cells for use in adoptive cell immunotherapy or in cancer therapy. For the transduction of cell populations in vitro , including their applications in research, diagnostics and therapy, improved strategies need to be developed. The present invention relates to reagents, methods, articles of manufacture and kits that meet such requirements.

Essence

[0005] The present invention relates to a method for transducing T cells, comprising incubation of a viral vector particle containing a recombinant nucleic acid, and an initial composition containing a plurality of T cells, where the specified plurality of T cells was obtained from a sample containing cells taken from an individual, where the incubation is started no more than 24 hours after the sample is taken from the individual; 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, or 24 hours after sampling from an individual; and/or prior to incubation, T cells were not brought to a temperature equal to, greater than, or approximately greater than 37°C ± 2.0°C for a period of time greater than 15 minutes, 30 minutes, 1 hours or 2 hours after sampling from an individual. In some embodiments of the invention, incubation begins no more than or no more than about 1 hour, 3 hours, 6 hours, 12 hours, or 18 hours after the sample is taken from the individual.

[0006] In some of any such embodiments of the invention, prior to said incubation, the method does not include stimulation of T cells under conditions that promote cell activation. In some of any such embodiments of the invention, prior to said incubation, the parent composition has not been subjected to ex vivo stimulation, including incubation at a temperature greater than or approximately greater than 37° C. ± 2.0° C. and/or incubation in the presence of an agent or agents capable of activating T cells, CD4 ⁺ -T cells and/or CD8 ⁺ -T cells, incubation in the presence of an agent or agents capable of inducing a signal through the TCR complex and/or incubation in the presence of an agent or agents capable of inducing proliferation of T cells, CD4 ⁺ -T cells and/or CD8 ⁺ -T cells; CD3 binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7.

[0007] In some of any such embodiments of the invention, prior to said incubation, no more than 5%, 10%, 20%, 30%, or 40% of T cells that are activated cells express a surface marker selected from the group , consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB; include an expressed intracellular cytokine selected from the group consisting of IL-2, IFN-gamma, TNF-alpha; are present in the G1 phase or in a later phase of the cell cycle and/or are capable of proliferating.

[0008] In some of any such embodiments of the invention, the method includes the incubation of a viral vector particle containing a recombinant nucleic acid, and the original composition containing T cells, where these T cells were obtained from a sample taken from an individual, where before the specified by incubation, the T cells or parent composition have not been subjected to ex vivo stimulation, including incubation at a temperature greater than or approximately greater than 37° C. ± 2.0° C. and/or incubation in the presence of an agent or agents capable of activating T- cells, CD4 ⁺ -T cells and/or CD8 ⁺ -T cells, incubation in the presence of an agent or agents capable of inducing a signal through the TCR complex and/or incubation in the presence of an agent or agents capable of inducing T cell proliferation, CD4 ⁺ -T-cells and/or CD8 ⁺ -T-cells; CD3 binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7.

[0009] In some of any such embodiments of the invention, one or more agents comprise an anti-CD3 antibody and/or an anti-CD28 antibody.

[0010] The present invention also relates to a method for transducing T cells, including incubation of a viral vector particle containing a recombinant nucleic acid, and the initial composition containing T cells, where these T cells were obtained from a sample taken from an individual, where before of said incubation, no more than 5%, 10%, 20%, 30%, or 40% of activated T cells express a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB; include an expressed intracellular cytokine selected from the group consisting of IL-2, IFN-gamma, TNF-alpha; and/or are present in the G1 phase or in a later phase of the cell cycle.

[0011] In some of any such embodiments of the invention, immediately prior to incubation, no more than 10% of the T cells in the initial composition contain a T cell activation marker selected from the group consisting of HLA-DR, CD25, CD69, CD71 , CD40L and 4-1BB. In some of any such embodiments of the invention, prior to said incubation, more than 5%, 10%, 20%, 30%, or 40% of the T cells express the low density lipid receptor (LDL-R).

[0012] In some of any such embodiments of the invention, the individual is a human.

[0013] In some of any such embodiments of the invention, T cells were not brought to a temperature of 2°C to 8°C and/or maintained at this temperature for more than 48 hours prior to incubation.

[0014] In some of any such embodiments of the invention, the sample is a blood sample. In some of any such embodiments of the invention, the sample is a sample taken after leukapheresis.

[0015] In some of any such embodiments, 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. In some of any such embodiments of the invention, T cells have been selected or enriched from a sample taken from an individual, and in some aspects of the invention, these cells produce an enriched composition and/or the original composition.

[0016] In some of any such embodiments of the invention, the method also includes, prior to incubation, taking a sample from the individual and, optionally, selecting or enriching T cells from the sample, where these cells, in some aspects of the invention, produce an enriched composition and/ or original composition. In some cases, the percentage of T cells in the original composition is greater than or approximately greater than 75%, 80%, 85%, 90%, 95% T cells.

[0017] In some of any such embodiments of the invention, T cells contain CD4 ⁺ or CD8 ⁺ cells. In some embodiments of the invention, T-cells contain CD4 ⁺ - and CD8 ⁺ -cells. In some cases, the ratio of CD4 ⁺ cells to CD8 ⁺ cells is or approximately 1:1, 1:2, 2:1, 1:3 or 3:1.

[0018] In some of any such embodiments, the sample contains serum or plasma at a concentration of at least or at least about 10% (v/v), at least or at least about 15% (v/v) , at least or at least about 20% (v/v), at least or at least about 25% (v/v), at least or at least about 30% (v/v), at at least or at least about 33% (v/v), at least or at least about 35% (v/v), or at least or at least about 40% (v/v); and/or, prior to incubation, the sample is contacted ex vivo with serum or plasma at a concentration of at least or at least about 10% (v/v), at least or at least about 15% (v/v), at least or at least about 20% (v/v), at least or at least about 25% (v/v), at least or at least about 30% (v/v), at least at least or at least about 33% (v/v), at least or at least about 35% (v/v), or at least or at least about 40% (v/v).

[0019] In some of any such embodiments of the invention, the sample contains serum or plasma at a concentration of at least or at least about 30% (v/v); and/or, prior to incubation, the sample is contacted ex vivo with serum or plasma at a concentration of at least or at least about 30% (v/v). In some embodiments, the serum or plasma is human. In some cases, serum or plasma is autologous to an individual.

[0020] In some of any such embodiments of the invention, the sample contains an anticoagulant and/or the anticoagulant was added to the sample prior to incubation. In some cases, the anticoagulant contains a free citrate ion.

[0021] In some of any such embodiments of the invention, the method includes, prior to incubation, cryopreservation of T cells, optional T cells of the sample or enriched composition in the presence of a cryoprotectant to obtain a cryopreserved composition. In some aspects of the invention, the method includes, prior to incubation, washing the cryopreserved composition under conditions that reduce or remove the cryoprotectant and/or form the original composition.

[0022] In some of any such embodiments of the invention, the original composition contains N-acetylcysteine (NAC); serum, optionally human serum; recombinant interleukin-2 (IL-2), recombinant interleukin-15 (IL-15), and/or recombinant interleukin-7 (IL-7).

[0023] In some of any such embodiments of the invention, the original composition contains N-acetylcysteine at a concentration of from or about 0.4 mg/ml to 4 mg/ml, from 0.8 mg/ml to 3.6 mg/ml or from 1.6 mg/ml to 2.4 mg/ml, inclusive; or the original composition contains N-acetylcysteine at a concentration of at least or at least about or about 0.4 mg/ml, 0.8 mg/ml, 1.2 mg/ml, 1.6 mg/ml, 2.0 mg/ml, 2.4 mg/ml, 2.8 mg/ml, 3.2 mg/ml, 3.6 mg/ml or 4.0 mg/ml. In some of any such embodiments of the invention, the initial composition contains serum, optionally human serum, at a concentration of from or about 0.5% to 25% (v/v), from 1.0% to 10% (v/v) or from 2.5% to 5.0% (v/v), inclusive; or the starting composition contains serum, optionally human serum, at a concentration of at least or at least about or about 0.5%, 1.%, 2.5%, 5% (v/v), or 10%.

[0024] In some of any such embodiments of the invention, the initial composition contains recombinant IL-2, optionally recombinant human IL-2 at a concentration of from or about 10 IU/ml to 500 IU/ml, from 50 IU/ml to 250 IU /ml or from 100 IU/ml to 200 IU/ml, inclusive; or at a concentration of at least or at least about 10 IU/ml, 50 IU/ml, 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, or 500 IU/ml; and/or the parent composition contains recombinant IL-15, optionally recombinant human IL-15, at a concentration of from or about 1 IU/mL to 100 IU/mL, from 2 IU/mL to 50 IU/mL, or from 5 IU/mL up to 10 IU / ml, inclusive; or at a concentration of at least or at least about 1 IU/ml, 2 IU/ml, 5 IU/ml, 10 IU/ml, 25 IU/ml, or 50 IU/ml; and/or the original composition contains recombinant IL-7, optionally recombinant human IL-7, at a concentration of from or about 50 IU/ml to 1500 IU/ml, from 100 IU/ml to 1000 IU/ml, from 200 IU/ml ml up to 600 IU / ml, inclusive; or at a concentration of at least or at least about 50 IU/ml, 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml , 800 IU/ml, 900 IU/ml or 1000 IU/ml.

[0025] In some of any such embodiments of the invention, the incubation includes the step of centrifugally inoculating the viral vector particles with the parent composition. In some cases, centrifugal inoculation involves rotating, in the inner compartment of the centrifuge chamber, particles of the viral vector and the original composition, where the rotation is carried out with a relative centrifugal force applied to the inner surface of the side wall of the compartment, and ranging from or approximately from 500 g to 2500 g, from 500 g to 2000 g, from 500 g to 1600 g, from 500 g to 1000 g, 600 g and 1600 g, from 600 g to 1000 g, from 1000 g to 2000 g or 1000 g and 1600 g inclusive; or at least or at least about 600 g, 800 g, 1000 g, 1200 g, 1600 g or 2000 g.

[0026] In some embodiments of the invention, centrifuge inoculation is carried out for a period of time greater than or about 5 minutes, more than or about 10 minutes, more than or about 15 minutes, more than or about 20 minutes, more, more than or about 30 minutes, more than or about 45 minutes, more than or about 60 minutes, more than or about 90 minutes, more than or about 120 minutes; or from or about 5 minutes to 60 minutes, 10 minutes to 60 minutes, 15 minutes to 60 minutes, 15 minutes to 45 minutes, 30 minutes to 60 minutes, or 45 minutes to 60 minutes, inclusive.

[0027] In some of any such embodiments, the method also includes contacting the parent composition and/or viral vector particles with a transduction adjuvant. In some cases, contact is made before, during, or after centrifugal inoculation of the viral vector particles with the parent composition.

[0028] In some of any such embodiments of the invention, at least part of the incubation is carried out at or approximately at 37°C ± 2°C. In some aspects of the invention, at least part of the incubation is carried out after centrifugal inoculation. In some cases, at least part of the incubation is carried out for a period of time 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 embodiments of the invention, at least part of the incubation is carried out within or approximately within 24 hours. In some of any such embodiments of the invention, the entire incubation period is no more than 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours.

[0029] In some of any such embodiments of the invention, the viral vector particle is a lentiviral vector particle. In some cases, the lentiviral vector particle is derived from HIV-1. In some of any such embodiments of the invention, the viral vector particle is pseudotyped with a viral envelope glycoprotein. In some cases, the viral envelope glycoprotein is VSV-G.

[0030] In some of any such embodiments of the invention, the viral vector particle contains a lentiviral protein having SAMHD1 inhibitory activity, where the specified protein is packaged in a viral particle. In some instances, the SAMHD1 inhibitory protein is a wild-type Vpx protein, a wild-type Vpr protein, or a variant or portion of a wild-type Vpx or Vpr protein having SAMHD1 inhibitory activity. In some cases, the SAMHD1 inhibitory protein is heterologous to the retroviral vector particle. In some embodiments, the SAMHD1 inhibitory protein is a wild-type Vpx protein or a variant or portion of a wild-type Vpx protein having SAMHD1 inhibitory activity.

[0031] In some of any such embodiments, the viral vector particle is incubated at an MOI of less than or less than about 20.0, or less than or less than about 10.0. In some of any such embodiments of the invention, the viral vector particle is incubated at a moi of infection from or about 1.0 IU/cell to 10 IU/cell, or from 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/cage or 3.6 IU/cage, 4.0 IU/cage, 5.0 IU/cage, 6.0 IU/cage, 7.0 IU/cage, 8.0 IU/cage, 9.0 IU/cage or 10.0 IU/cage.

[0032] In some of any such embodiments, the starting composition contains at least or about at least or about 50 x 10 ⁶ cells, 100 x 10 ⁶ cells, or 200 x 10 ⁶ cells.

[0033] In some of any such embodiments of the invention, the recombinant nucleic acid encodes an antigen receptor. In some cases, the antigen receptor is a transgenic T cell receptor (TCR). In some embodiments, the antigen receptor is a chimeric antigen receptor (CAR). In some cases, the chimeric antigen receptor (CAR) contains an extracellular antigen recognition domain that specifically binds to the target antigen and to an intracellular signal transduction domain containing ITAM. In some aspects of the invention, the intracellular signal-transmitting domain contains an intracellular domain of the CD3-zeta chain (CD3ζ). In some of any such embodiments of the invention, the CAR also contains a transmembrane domain that binds an extracellular domain and an intracellular signal-transmitting domain. In some cases, the transmembrane domain contains the transmembrane portion of CD28.

[0034] In some of any such embodiments of the invention, the intracellular signal-transmitting domain also contains the intracellular signal-transmitting domain of the T-cell costimulatory molecule. In some instances, the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.

[0035] In some of any such embodiments of the invention, the antigen receptor specifically binds to an antigen associated with a disease or condition, or specifically binds to a universal label. In some cases, the disease or condition is cancer, an autoimmune disease or disorder, or an infectious disease.

[0036] In some of any such embodiments of the invention, the method includes obtaining a final composition containing T cells transduced with a recombinant nucleic acid. In some instances, at least 30%, or at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the T cells in the final composition are transduced with the recombinant nucleic acid. acid. In some aspects of the invention, the method also includes obtaining or isolating from the final composition transduced T cells obtained by this method. In some cases, the method also includes activating or expanding the cells of the final composition or cells transduced by this method. In some cases, activation and/or propagation is carried out ex vivo .

[0037] In some of any such embodiments of the invention, after incubation, the cells in the final composition are also incubated in the presence of one or more stimulatory agents capable of activating T cells, inducing signaling through the TCR complex, and/or inducing T cell proliferation. In some aspects of the invention, one or more stimulant agents are selected from the group consisting of CD3 binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7. In some embodiments, the one or more stimulatory agents comprise an anti-CD3 antibody and/or an anti-CD28 antibody.

[0038] In some cases, activation and/or propagation is carried out in vivo . In some embodiments of the invention, activation and/or propagation occurs in the presence of an antigen that specifically binds to an antigen receptor, and/or activation and/or propagation is transgene-specific.

[0039] In some of any such embodiments of the invention, after incubation, the cells in the final composition are not further incubated ex vivo in the presence of one or more stimulatory agents, optionally consisting of CD3-binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7 and/or cells in the final composition is not additionally incubated at a temperature of more than 30°C for more than 24 hours.

[0040] the Present invention also relates to genetically engineered T-cells obtained by any of the methods described here. The present invention also relates to compositions containing genetically engineered T cells described in this application, and a pharmaceutically acceptable carrier.

[0041] The present invention also provides a method of treatment comprising administering to an individual suffering from a disease or disorder a composition as described above. In some cases, the composition is or will be administered to an individual and/or the composition is prepared for analysis within no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, or no more than 5 days after sampling from an individual. In some cases, the composition is or will be administered to an individual, and/or the composition is prepared for analysis within no more than 1 day, 2 days, 3 days, or 4 days after the sample is taken from the individual. In some cases, the resulting composition may be administered to the individual for no more than 21 days, no more than 20 days, no more than 19 days, no more than 15 days, no more than 14 days, no more, than 13 days, not more than 12 days, not more than 10 days, not more than 9 days, not more than 8 days, not more than 7 days, not more than 6 days, or not more than 5 days after taking the sample from the individual.

[0042] The present invention also relates to a method of adoptive cell therapy, including the enrichment or isolation of T cells from a sample taken from an individual suffering from a disease or disorder; transducing an initial composition containing enriched or isolated T cells with a viral vector particle by any of the methods described herein, to obtain a final composition containing transduced cells, where the viral vector particle contains a recombinant nucleic acid encoding an antigen receptor that specifically binds to an antigen associated with disease or disorder; and administering the final composition containing the transduced cells to an individual for the treatment of the disease or condition.

[0043] The present invention also provides a method of treatment that includes administering a final composition comprising T cells transduced with a recombinant nucleic acid to an individual for the treatment of a disease or condition, wherein the final composition is obtained by any of the cell transduction methods described herein.

[0044] In one embodiment of any such methods, the composition is or will be administered to the individual, or the composition is prepared for analysis for no more than 11 days, no more than 9 days, no more than 8 days, no more, than 7 days, not more than 6 days, or not more than 5 days after the sample is taken from the individual. In some embodiments of the invention, the composition is administered or will be administered to the individual for no more than 1 day, 2 days, 3 days, 4 days, or 5 days after the sample is taken from the individual. In some cases, prior to administration of the composition, the transduced cells or the cells of the final composition are prepared in a pharmaceutically acceptable buffer.

[0045] In some of any such embodiments of the invention, before administration of the final composition containing transduced cells, these cells are cultured ex vivo for a period of time up to 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after transduction, where said cultivation is carried out at a temperature of more than 30°C. In some embodiments of the invention, after transduction, the final composition or cells containing the transduced cells are cultured in the presence of one or more stimulatory agents capable of activating T cells, inducing signal transduction through the TCR complex, and/or inducing T cell proliferation to obtain a composition containing transduced cells.

[0046] In some of any such embodiments of the invention, before the introduction of the transduced cells, the final composition or cells containing the transduced cells are not additionally incubated ex vivo in the presence of one or more stimulating agents, and/or are not additionally incubated at a temperature of more than 30°C for more than 24 hours.

[0047] In some of any such embodiments of the invention, one or more stimulant agents are selected from the group consisting of CD3-binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7; a vaccine containing an antigen specifically recognized by an antigen receptor; and an anti-idiotypic antibody that specifically binds to an antigen receptor.

[0048] In some instances, one or more stimulatory agents comprise an anti-CD3 antibody and/or an anti-CD28 antibody. In some embodiments of the invention, the cells of the final composition or transduced cells are administered at a suboptimal dose.

[0049] In some of any such embodiments, the method also includes administering to the individual one or more agents to induce or enhance stimulation and/or expand the transduced T cells in vivo . In some cases, one or more agents are transgene-specific and/or stimulate or activate cells through an expressed transgene, which is or optionally contains an antigen receptor. In some aspects of the invention, one or more agents are selected from a vaccine containing an antigen specifically recognized by an antigen receptor; an anti-idiotypic antibody that specifically binds to an antigen receptor, or an agent capable of chemically inducing antigen receptor dimerization. In some embodiments of the invention, one or more agents are an immunomodulatory agent; immune checkpoint inhibitor; an inhibitor of extracellular adenosine or an adenosine receptor, optionally an A2aR receptor; a kynurenine pathway modulator; and signaling pathway modulators such as kinase inhibitors.

[0050] The present invention also provides a composition comprising a population of primary human T cells genetically engineered to express a chimeric antigen receptor (CAR) or transgenic TCR that specifically binds to a target antigen, wherein such population contains a plurality of resting T cells, and wherein the plurality of resting T cells contains at least 7.5% of the genetically engineered cells in the composition. In some cases, genetically engineered resting T cells contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the genetically engineered cells in the composition.

[0051] In some embodiments, resting T cells are negative for a T cell activation surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L (CD154) and 4-1BB (CD137) ; do not express an intracellular cytokine selected from the group consisting of IL-2, IFN-gamma and TNF-alpha; are present in the G0 or G ₀ G _1a phase of the cell cycle; and/or contain active SAMHD1. In some cases, resting T cells are negative for surface CD25 and CD69 (CD25 ^- /CD69 ^- ). In some aspects of the invention, resting T cells contain CD4 ⁺ - and/or CD8 ⁺ -T cells.

[0052] In some of any such embodiments of the invention, the target antigen is associated with a disease or disorder. In some cases, the disease or disorder is an infectious disease or condition, an autoimmune disease, an inflammatory disease, or cancer.

[0053] In some of any such embodiments, the target antigen is selected from the group consisting of ROR1, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL- 13R-alpha2, kdr, kappa light chain, Lewis Y antigen, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1 ligands, MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, GD-2, MAGE A3, CE7, antigen Wilms tumor 1 (WT-1) and cyclin A1 (CCNA1). In some embodiments of the compositions described herein, primary human T cells are genetically engineered to express a CAR containing an extracellular antigen recognition domain that specifically binds to a target antigen and to an intracellular signal transduction domain containing ITAM. In some cases, the intracellular signal-transmitting domain contains an intracellular domain of the CD3-zeta chain (CD3ζ). In some cases, CAR also contains a transmembrane domain linking an extracellular domain and an intracellular signal-transmitting domain. In some aspects of the invention, the transmembrane domain contains the transmembrane portion of CD28.

[0054] In some of any such embodiments of the invention, the intracellular signal-transmitting domain of CAR also contains the intracellular signal-transmitting domain of a T-cell co-stimulatory molecule. In some instances, the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.

[0055] In some of any such embodiments of the invention, the composition contains a pharmaceutically acceptable carrier.

Brief description of the drawings

[0056] FIG. 1A-1B are dotted curves for side scatter (SSC; y-axis) and EGFRt surface marker expression (x-axis), namely an indicator of T cell transgene expression with or without activation prior to transduction by transgene-expressing lentiviral vector particles. In FIG. 1A shows dotted curves for EGFRt expression after transduction with lentiviral vector particles, in CD4 ⁺ and CD8 ⁺ T cells activated with anti-CD3/anti-CD28 antibody bead reagent 1 or anti-CD3 bead reagent 2. /anti-CD28 antibodies prior to transduction. In FIG. 1B shows dotted curves for EGFRt expression after transduction with lentiviral vector particles at various virus concentrations (in 2-fold serial dilutions from baseline) in CD4 ⁺ and CD8 ⁺ T cells that were not activated prior to transduction. The percentage of cells that are EGFRt ⁺ cells is also shown in the box.

[0057] FIG. 2 illustrates the frequency of expression of surrogate surface markers (indicative of the frequency of transduction) for CD4 ⁺ - and CD8 ⁺ -T cells on the indicated days after selection, transduction and processing under various conditions.

Detailed description

I. Overview

[0058] The present invention relates to methods for transferring viral vectors into cells (e.g., T cells), where these methods include the transduction of cells, such as immune cells, for example, T cells, without first activating the cells and/or for a period of time no more than 24 hours after sampling from an individual, and/or where these cells have not been brought to a temperature of more than 15°C-25°C, for example, more than or more than about 37°C ± 2 0°C, for more than a few hours (and no more than 24 hours) after taking the cells from the individual and prior to transduction. In some embodiments, the methods described include incubating and/or contacting a retroviral vector particle, such as a lentiviral vector, with a population of cells, such as immune cells, e.g., T cells with a retroviral vector particle, such as lentiviral particles, without initial activation. and/or stimulation, ie prior to transduction, of T cells with an ex vivo stimulating reagent (eg, anti-CD3/anti-CD28 reagent) prior to and/or simultaneously with contact or incubation of cells with viral particles.

[0059] In some embodiments of the invention, the described methods are used to genetically engineer such cells with a heterologous molecule, such as a recombinant receptor, for example, an antigen receptor, such as a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR). The resulting genetically engineered cells can be used in adoptive immunotherapy. In some such embodiments, the methods described can be used to generate immune cells, such as T cells for adoptive therapy, where said methods do not include the step of activating and/or stimulating T cells. In some aspects of the invention, due to the absence of the need to activate or stimulate cells, the described methods allow to reduce the procedure for constructing and/or obtaining cells for adoptive cell therapy.

[0060] Generally speaking, retroviral vectors can be used to stably integrate genes of interest into cells. Although lentiviral vectors pseudotyped with the G protein of vesicular stomatitis virus (VSV-g) can be transferred into non-dividing cells, however, resting cells do not provide sufficient transduction. Therefore, with respect to existing retroviral vectors, it is not always possible to efficiently genetically engineer stable quiescent and/or quiescent cells, such as quiescent myeloid cells or quiescent T cells, using a retroviral vector in an amount sufficient for their subsequent use, for example, in cell therapy. . In some cases, transduction into T cells may require activation of T cells through their binding to the T cell receptor (TCR) or cytokine stimulation. In some cases, one observation showed a low level of expression of the LDL receptor, a binding partner for VSV-G, in resting T cells. In some cases, it has been shown that activation leads to an increase in the level of expression of the LDL receptor and, thereby, to an increase in the degree of uptake of lentiviral vectors. Typically, T cells are activated at least one day (and sometimes 3 days or more) prior to transduction in order to conduct adoptive T cell therapy. For example, protocols for transducing T cells with lentiviruses typically involve activation at least 24 hours prior to transduction (Amirache et al. (2014) Blood, 123:1422-1424).

[0061] In some cases, current methods of obtaining genetically engineered T-cells for adoptive immunotherapy may include sequential selection, activation, transduction, and ex vivo expansion. However, ex vivo stimulation or activation of immune cells, such as T cells, may not always be desirable in order to prepare cells for use in some adoptive immunotherapy methods. Thus, for example, carrying out the step(s) of activation and/or stimulation, for example, prior to transduction, may increase the time, cost, number of reagents and/or complicate the processing of the resulting cells for adoptive cell therapy. Such steps may increase the risk of variability between different methods and/or cells taken from different individuals. Thus, in some embodiments of the invention, the described methods are preferred because they do not include the stage of activation and/or stimulation prior to treatment with a retroviral vector particle compared to other methods.

[0062] In addition, in some aspects, the persistence and/or depletion of T cells after adoptive cell therapy may be associated with stimulation and/or activation of T cells prior to administration, for example, before or during genetic engineering (for example, the introduction of a nucleic acid encoding a genetically engineered molecule such as a receptor, such as an antigen receptor, such as CAR). Thus, for example, activation of T cells to facilitate transduction may result in a change in the differentiation or activation status of T cells, which in turn may result in and/or reduce in vivo persistence when genetically engineered cells are administered to an individual. Changes in differentiation status can be, in some cases, loss of native phenotype, loss of memory T cell phenotype, and/or generation of effector cells with an attenuated T cell phenotype. Depletion of T cells can lead to progressive loss of T cell functions and/or cell depletion (Yi et al . (2010) Immunology , 129:474-481).

[0063] The described methods are based on the observation that sufficient transduction of primary cells taken from an individual can be achieved by incubating and/or contacting a population of cells, such as a population of T cells, with retroviral vector particles, such as lentiviral vector particles immediately after selection and/or enrichment of cells taken from the individual. In some embodiments of the invention, it was found that the process of transduction in accordance with the methods described here is not limited to the need to stimulate cells by binding to CD3 and/or CD28 proteins and/or activate LDL receptors by preactivating cells. Without wishing to be bound by any particular theory, the authors merely note that the above-described processing of selected and/or enriched cells, including collection by apheresis, as well as T-cell selection and/or enrichment processes, may already promote activation of LDL receptor expression and/or any other message to T-cells of the ability to penetrate the virus into them. Accordingly, it has been found that it is possible to transduce T cells immediately after selection without having to activate them for at least 24 hours, thus shortening the process. In some cases related to the methods described, enriched and/or selected cells may be cryopreserved prior to transduction, provided that such enriched and/or selected cells are not subjected to ex vivo stimulation with a stimulating agent or agents prior to incubation and/or contact.

[0064] In some embodiments of the invention, the described methods include incubation and/or contact of the original composition containing transducible cells, where before incubation, the cells of the original composition were not subjected to ex vivo stimulation, including incubation with an agent that starts or initiates a cascade of transfer reactions an intracellular TCR/CD3 signal in a T cell, such as a CD4 ⁺ T cell and/or a CD8 ⁺ T cell. Such agents are binding molecules or antibodies, such as antibodies specific for a TCR component and/or a costimulatory receptor, e.g. with one or more cytokines, for example recombinant cytokines such as IL-2 and/or IL-15 and/or IL-7. In some embodiments of the invention, the described methods, prior to the introduction of the described retroviral vector, do not include activation and/or stimulation of a population of cells containing T cells, one or more components, such as an anti-CD3 antibody, anti-CD28 antibody and/or recombinant cytokines IL-2, IL-15 or IL-7. In some embodiments of the invention, the term "recombinant" refers to cytokines that were not obtained in the usual way from a sample taken from an individual, but instead, they were obtained by recombinant DNA methods, such as methods involving expression in a cell from DNA, in which has been artificially or exogenously introduced a gene sequence encoding a protein. Therefore, the term "recombinant cytokines" does not include cytokines that may be present in a sample of an individual, for example, in a sample taken by apheresis, and/or present in the serum obtained from such an individual. In some embodiments of the invention, the original composition contains a population of primary cells that were obtained from a sample taken from the individual, and/or enriched with a specific subpopulation of cells, such as T cells.

[0065] In some embodiments of the invention, the population of cells, for example, the original composition may be a population of cells that has been previously subjected to cryopreservation. In some such embodiments, prior to cell cryopreservation, the cell population has not been treated and/or exposed and/or incubated under any conditions that induce cell activation or stimulation, such as T cell activation or stimulation. In some embodiments, prior to cell cryopreservation, the cell population has not been treated and/or exposed and/or incubated in the presence of one or more agents capable of activating the intracellular signal transduction domain of the TCR complex, such as anti-CD3 and/or anti-CD28 an antibody and/or one or more cytokines such as IL-2 and/or IL-15 and/or IL-7.

[0066] In some embodiments of the invention, the population of cells, for example, the original composition, which was incubated with the described retroviral vector, is a composition that, prior to incubation with the retroviral vector particle, has not been exposed and/or brought to a temperature that exceeds 0 °C, for example, is more than 4°C, 15°C, 20°C, or more than 25°C for more than about 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours , 48 hours or 72 hours.

[0067] In some embodiments, incubation and/or contact begins no more than or no more than about 1 hour, 3 hours, 6 hours, 12 hours, 18 hours, or 24 hours after receiving the sample, e.g., sample obtained by apheresis from an individual containing primary cells. In some embodiments of the invention, prior to incubation, T cells have not been brought to a temperature of more than or more than about 15°C, 18°C, 22°C, or 25°C for more than 1 hour, 2 hours , 4 hours, 6 hours, 8 hours, 12 hours, or 24 hours after the individual is sampled, e.g., they have not been brought to temperatures greater than or greater than approximately 37°C ± 2.0°C for more than, than 15 minutes, 30 minutes, 1 hour, or 2 hours after sampling from an individual.

[0068] In some embodiments of the invention, the described methods include obtaining the final composition containing the transduced cells, where such activation of the cells was not carried out prior to transduction. In some embodiments of the invention, these methods can be used to transduce a population of T cells in which at least 40%, 50%, 60%, 70%, 80%, 90% or more of the T cells in the population are resting T cells, such as T cells that do not contain a T cell activation marker, such as a surface marker or an intracellular cytokine or other marker, and/or T cells that are present in the G ₀ or G ₀ G _1a phase of the cell cycle.

[0069] In some embodiments, the methods include obtaining a final composition in which at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of all cells (or target cells of a particular type, such as T cells) have been transduced with said viral vector and/or expressed the recombinant gene product encoded by the vector.

[0070] In some embodiments of the invention, the described methods allow relatively efficient transduction of immune cells, such as silent and/or resting cells, such as non-activated or unstimulated immune cells, for example, resting T cells. In some embodiments of the invention, at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95 % of silent or resting cells, such as resting T cells in the cell population, for example, in the final composition, were transduced with retroviral vector particles in accordance with the methods described here.

[0071] In some embodiments of the invention, no more than 5%, 10%, 20%, 30%, or 40% of T cells in the initial composition and / or in the final composition, which are activated cells, express the surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB; contain an intracellularly expressed cytokine selected from the group consisting of IL-2, IFN-gamma, TNF-alpha, are present in the G1 phase or in a later phase of the cell cycle, and/or are capable of proliferating. Thus, for example, in some aspects of the invention, the cell population of the initial composition and/or the final composition is a population in which at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% are negative for superficial CD25 and/or CD69, eg ^CD25- and CD69.

[0072] Methods and technologies for assessing and/or analyzing cell cycle status, for example, resting T cells, are known (Tumeh et al. (2010) J Immunother., 33:759-768). In some embodiments, resting T cells are smaller than proliferating T cells, such as activated T cells. Therefore, in some aspects of the invention, cells that have been sized on an automated cell counter in the presence or absence of a viability indicator dye such as trypan blue can be used. In some embodiments, most or substantially all of the T cells in a population are cells having a diameter of less than 4 µm, e.g., generally less than 3 µm, e.g., from or about 1 µm. up to 3 microns, for example, mostly about or about 2 microns. In some embodiments, metabolic substrate uptake can be assessed, for example, using tritium-labeled 2'-deoxy-D-glucose ([ ³ H])([ ³ H]-2'DDG), ³ H-labeled 3 '-deoxy-3'-fluorothymidine ([ ³ H]-3'FLT) and/or ³ H-labeled 3'-deoxy-3'-fluoroarabinofluranosylcytosine ([ ³ H]-FAC), which are markers taken up by activated or stimulated cells. In some embodiments of the invention, the majority of cells or, in general, all T cells in the population are not able to absorb and/or accumulate the metabolic substrate marker. In some embodiments of the invention, the cell cycle can be assessed or analyzed directly, for example, by quantifying DNA content, for example, using propidium iodide (PI), 7-aminoactinomycin-D (7-AAD), Hoechst 33342, 33258 and S769121 , TO-PRO-3, 4'6'-diamidino-2-phenylindole (DAPI), DRAQ5™ or DRAQ7™.

[0073] Methods and technologies for assessing the expression and/or levels of a T cell activation marker are known to those skilled in the art. Antibodies and reagents for detecting such markers are well known to those skilled in the art and are readily available. Assays and methods for detecting such markers include, but are not limited to, flow cytometry, including intracellular flow cytometry, ELISA, ELISPOT, sphere cytometry or other multiplex methods, Western blot analysis, and other immunoaffinity-based methods.

[0074] In some embodiments of the invention, these methods allow you to achieve at least a certain efficiency of transduction under certain conditions. Thus, for example, in some embodiments of the invention, if the initial composition includes virus and cells in a ratio equal to or approximately from 1 infectious unit (IU) per cell to 10 IU per cell, for example, at least or equal to or approximately equal to 1 infectious unit (IU) per cell, or at least or equal to or approximately equal to 2 IU per cell, at least or equal to or approximately equal to 5 IU per cell, or at least or equal to or approximately equal to 10 IU per cell, this means that this method allows to obtain the final composition, in which at least 10%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the cells in the composition obtained in this way contain a recombinant viral vector, for example, these cells are transduced with a recombinant viral vector.

[0075] In some embodiments, the efficiency of cell transduction by a retroviral vector particle can be monitored and/or evaluated by measuring the expression level of a recombinant molecule or protein, such as a heterologous molecule or protein encoded by a nucleic acid contained in the genome of the retroviral vector, after transduction or other form of transfer of the vector into a cell, such as a resting host cell, for example, a resting T cell, or a population of these cells. A number of well known methods for assessing expression levels of recombinant molecules can be used, such as affinity detection methods, eg immunoaffinity detection, and in the case of cell surface proteins, flow cytometry methods. In some examples, the level of expression is measured by detecting a transduction marker and/or a reporter construct. In some embodiments of the invention, a nucleic acid encoding a truncated surface protein is included in the vector and is used as a marker of expression and/or increase in its level.

[0076] In some embodiments of the invention, the described methods may include a cryopreservation step carried out before or after incubation, for example, transduction of cells with viral particles. In some embodiments of the invention, such a stage in this method is an intermediate stage of the introduction of materials, samples of materials or "maintenance" of the patient's condition prior to therapy.

[0077] Although in some aspects of the invention the described method does not include the stage of pre-activation and/or stimulation of cells, however, this method may include activation or stimulation of cells simultaneously with transduction and/or after transduction. Activation or stimulation may be performed ex vivo or in vivo . In some embodiments of the invention, after incubation, for example, transduction of cells with viral particles, these cells can be introduced into the patient by infusion for activation and propagation in vivo .

[0078] In some embodiments of the invention, during or after incubation, the methods described herein may also include culturing the initial composition, the final composition and/or transduced cells ex vivo , for example, under conditions of cell stimulation, for example, to induce their proliferation and/ or activation. In some embodiments of the invention, the cultivation is carried out at a temperature of more than 30°C, for example, more than or more than about 32°C, 35°C or 37°C, for more than 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or more. In some embodiments of the invention, the stimulation is carried out in the presence of one or more stimulating agents. In some embodiments, the one or more stimulatory agents are a CD3 binding molecule, a CD28 binding molecule, or a cytokine such as recombinant IL-2, recombinant IL-15, or recombinant IL-7. In some embodiments, the binding molecule is an antibody, or an antigen-binding fragment thereof, such as an anti-CD3 antibody and/or an anti-CD28 antibody. In some embodiments of the invention, additional cultivation is carried out under conditions effective for propagation of cells, with obtaining a therapeutically effective dose of cells for their introduction to the individual using adoptive cell therapy.

[0079] In some embodiments of the invention, the final composition or transduced cells are not subjected to additional ex vivo culture at a temperature of more than 30°C and/or in the presence of one or more stimulating agents.

[0080] In some embodiments of the invention, the described methods avoid significant changes and / or minimize changes in the differentiation status of ex vivo T cells during the introduction, transfer to T cells of a nucleic acid encoding a recombinant receptor, such as CAR, and / or transduction of T cells with this nucleic acid. In some embodiments, nucleic acid introduced cells produced according to the methods described herein have a less depleted phenotype.

[0081] In some embodiments of the invention, the initial composition and/or the final composition, for example, a composition containing T cells, includes a smaller number or a lower percentage of effector cells, such as effector cells with a depleted T-cell phenotype, than cells in the composition in which the cells of the original composition were activated and/or stimulated prior to incubation, for example, activated and/or stimulated with an anti-CD3/anti-CD28 antibody for at least or at least about 24 hours. In some embodiments of the invention, the T cells in the initial composition and/or in the final composition contain a smaller number or a smaller percentage of memory effector T cells (TEM) or effector T cells (TEFF), such as CD45RO ⁺ CD62L ^- CCR7 ^- - cells than cells in a composition in which the cells of the original composition were activated and/or stimulated prior to incubation, e.g., activated and/or stimulated with an anti-CD3/anti-CD28 antibody for at least or at least about 24 hours. In some embodiments of the invention, the number or percentage is reduced by at least or at least about 1.5, 2, 3, 4 or 5 times.

[0082] In some embodiments of the invention, the initial composition and/or the final composition, for example, a composition containing T cells, includes a greater number or a greater percentage of T cells with a memory T cell phenotype than cells in a composition in which cells the original composition were activated and/or stimulated prior to incubation, for example, activated and/or stimulated with anti-CD3/anti-CD28 antibody for at least or at least about 24 hours. Thus, for example, in some embodiments of the invention, memory T cells are cells having a central memory T cell (TCM) phenotype, for example, CD45RO ⁺ CCR7 ⁺ CD62L ⁺ -T cells and/or CD45RO ⁺ CCR7 ⁺ CD27 ⁺ CD28 ⁺ CD62L ⁺ -T cells. In some embodiments of the invention, the number or percentage is reduced by at least or at least about 1.5, 2, 3, 4 or 5 times.

[0083] In some embodiments of the invention, the described methods are carried out so that one, several or all of the steps for obtaining cells for their clinical use, for example, in adoptive cell therapy, are carried out without processing the cells in non-sterile conditions and without storing them in a sterile chamber or in a sterile environment. In some embodiments of such a method, cells are isolated, separated or selected, transduced, washed, optionally activated or stimulated, and prepared in a closed system. In some embodiments of the invention, the closed system is or includes a device having a centrifuge chamber. In some embodiments of the invention, these methods are carried out automatically. In some embodiments of the invention, one or more of the stages is carried out outside of a closed system or closed device, for example, outside of a system with a centrifuge chamber.

[0084] In some embodiments of the invention, the described methods are an optimized or improved process in which cells are treated ex vivo for a shorter period of time, which saves time and reduces the cost of the procedure. In some aspects of the invention, the described methods may also include obtaining transduced cells with a better or more desirable phenotype, for example, cells with a less depleted phenotype and/or with more central memory cells compared to effector memory cells, for administration to an individual.

[0085] In some embodiments of the invention, such cells obtained by the specified method, or a composition containing such cells, is administered to an individual for the treatment of a disease or disorder.

[0086] In some embodiments, the described methods include administering a suboptimal dose of cells to an individual. In some embodiments, the cell dose is less than or about 1.5, 2, 3, 4, 5, or 10 times less than the therapeutically effective cell dose for treating the disease or disorder. In such an example, expansion of cells to obtain a therapeutically effective number of cells can be carried out in vivo after administration of the cells to an individual.

[0087] In some embodiments, the methods described herein include cell propagation in vivo . In some aspects of the invention, in vivo cell expansion can be carried out in vivo by transgene-specific activation or stimulation of the transduced cells. In some embodiments, the antigen receptor (eg, CAR) is stimulated, eg, activated or amplified after antigen recognition. In some embodiments of the invention, one or more agents are administered to an individual to enhance, increase or increase the level of stimulation, activation or expansion of cells in vivo in the individual.

[0088] In some embodiments of the invention, the described methods include obtaining genetically engineered T cells, which, when administered to an individual, increase the persistence of T cells and/or reduce the degree of their depletion. In some embodiments of the invention, genetically engineered cells with increased persistence and/or less depletion are more active in the individual to which they are administered. In some embodiments, the described retroviral vector particles and methods can reduce the variability in treatment outcomes in adoptive immunotherapy methods, for example, by minimizing and/or reducing the number of ex vivo manipulations of T cells prior to their administration to an individual. In some embodiments of the invention, the absence of the need for ex vivo activation of T cells allows an improvement in the method of producing or obtaining genetically engineered T cells for adoptive immunotherapy by reducing the time and number of reagents required for ex vivo manipulations.

[0089] In some embodiments of the invention, the selection of transduced or engineered T cells is carried out after their construction by genetic engineering. In some embodiments, engineered nondividing and/or quiescent or quiescent cells, such as quiescent T cells, can be enriched for use in adoptive immunotherapy.

[0090] In addition, in some of its embodiments, the present invention relates to obtained genetically engineered cells, such as cells expressing a genetically engineered receptor, such as a recombinant antigen receptor, such as CAR and/or other recombinant receptor, as well as to methods and the use of such genetically engineered cells for adoptive immunotherapy.

II. Transduction methods

[0091] The present invention relates to a method for incubating or contacting cells of an initial composition with a retroviral vector particle, for example, lentiviral vector particles. In some aspects of the invention, the original composition is a composition of primary cells taken from an individual, where, in some cases, a subpopulation or subset of cells were selected and/or enriched. The present invention relates to the original composition with distinctive features.

[0092] In some embodiments of the invention, cells include one or more nucleic acids introduced by the methods of genetic engineering according to the invention, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, nucleic acids are heterologous, that is, they are not normally present in a cell or in a sample derived from cells, such as cells derived from another organism or cell, i.e., such nucleic acids are not normally present in the engineered cell and/or in the organism from which such a cell is isolated. In some embodiments, the nucleic acids are non-natural nucleic acids that are not naturally present, including nucleic acids containing chimeric combinations of nucleic acids encoding different domains derived from many different cell types.

[0093] The processing steps may include any one or more of the various cell processing steps, alone or in combination. In specific embodiments, the processing steps include transducing the cells with viral vector particles containing a retroviral vector, such as a vector encoding a recombinant product for expression in cells. These methods may additionally or alternatively include other processing steps such as the steps of isolating, separating, selecting, washing, suspending, diluting, concentrating and/or obtaining cells. In some instances, these methods may also include an ex vivo culture step (eg, stimulating the cells, eg, to induce their proliferation and/or activation). In other instances, the step of stimulating or activating cells is carried out in vivo after administering the cells to the individual via antigen recognition and/or after administering one or more agents to the individual to enhance or increase the rate of proliferation, activation and/or proliferation of cells in the individual. In some embodiments of the invention, these methods include isolating cells from an individual, as well as obtaining, processing, culturing and/or constructing them, and then introducing them to the same individual before or after cryopreservation.

[0094] In some embodiments of the invention, this method includes processing steps carried out in the following order: cells, for example, primary cells, are first isolated, for example, selected or separated from a biological sample, and then the selected cells are incubated with viral vector particles for transduction , and the transduced cells are formulated into a composition. In some cases, the transduced cells are activated, expanded, or cultured ex vivo , for example, by stimulation in the presence of a stimulating reagent. In some embodiments of the invention, this method may include one or more processing steps, namely washing, suspending, diluting and/or concentrating steps, which can be performed before, during or after one or more isolation steps, such as separating or selection, as well as transduction, stimulation and/or preparation steps.

[0095] In some embodiments, one or more or all of the processing steps, e.g., isolation, selection and/or enrichment, processing, incubation in combination with transduction and engineering, and preparation steps are performed using a system, device, or equipment in an integrated or a self-limiting system and/or in an automated or programmable manner. In some aspects of the invention, the system or equipment includes a computer and/or a computer program associated with the system or equipment that allows the user to program, adjust, evaluate the result and/or correct various aspects of the stages of processing, isolation, construction and preparation. In one example, such a system is the system described in International Patent Application Publication No. WO2009/072003 or US 20110003380 A1. In one example, such a system is the system described in International Application Publication No. WO2016/073602.

[0096] In some embodiments of the invention, one or more stages of processing cells in combination with preparation, processing and / or incubation of cells in combination with the transduction method described here can be carried out in the internal compartment of a centrifuge chamber, such as a generally rigid chamber , which is essentially cylindrical and rotates around its own axis, which may provide some advantages over other methods used. In some embodiments of the invention, all processing steps are carried out in the same centrifuge chamber. In some embodiments of the invention, one or more processing steps are carried out in different centrifuge chambers, such as a plurality of centrifuge chambers of the same type. Such methods include any of the methods described in International Application Publication WO2016/073602.

[0097] Representative centrifuge chambers are those manufactured and supplied by Biosafe SA, including chambers with the Sepax® and Sepax® 2 system, including the A-200/F and A-200 centrifuge chambers, and various kits for using such systems. Representative chambers, processing and room systems and equipment are described, for example, in US Patent No. 6123655, in US patent no. 6,733,433, in US Published Application No.: US 2008/0171951 and in Published International Patent Application No. WO 00/38762, the contents of which are incorporated herein in their entirety by reference. Depending on the particular method (eg, dilution, washing, transduction, preparation), the person skilled in the art can independently choose a particular kit suitable for implementing such a method. Representative kits for use with such systems include, but are not limited to, disposable kits available from BioSafe SA under the trademarks CS-430.1, CS-490.1, CS-600.1, or CS-900.2.

[0098] In some embodiments of the invention, this system is introduced into other equipment and / or connected to this equipment, including equipment for performing, automated execution, regulation and / or monitoring of the processing steps in various aspects of the invention carried out in this system. In some embodiments of the invention, this equipment is indoors. In some embodiments of the invention, the equipment includes a chamber that has a housing containing control circuits, a centrifuge, a lid, motors, pumps, sensors, displays, and a user interface. An example of such a device is described in US Patent No. 6123655, in US patent no. 6,733,433 and in US application 2008/0171951.

[0099] In some embodiments of the invention, the system includes a series of containers, such as bags, tubes, stopcocks, clips, connectors, and a centrifuge chamber. In some embodiments, containers, such as pouches, include one or more containers, such as pouches, containing transduced cells and viral vector particles in the same or different containers, e.g., in the same pouch or in separate packages. In some embodiments of the invention, the system for carrying out these methods also includes one or more containers, such as bags containing a medium, such as a solution for dilution and/or washing, which are placed in a chamber, and/or other components for dilution, resuspension and /or for washing, and/or composition. The containers may be connected to the system in one or more positions, for example, in a position corresponding to an inlet line, a dilution line, a flush line, a waste line, and/or an outlet line.

[0100] In some embodiments of the invention, the system, such as a closed system, is sterile. In some embodiments of the invention, all connecting parts of the components of the system, for example, located between the tubing line and the container, connected by a connector, are manufactured under sterile conditions. In some embodiments of the invention, connectors are made in laminar flow. In some embodiments of the invention, the connectors are made using a sterile device, which allows you to get sterile connectors, for example, through sterile welding, between the tube system and the container. In some embodiments of the invention, the sterile connector device allows contact at a temperature that is high enough to maintain sterility, for example, at a temperature of at least 200°C, such as a temperature of at least 260°C or 300°C.

[0101] In some embodiments, the system may be disposable, such as a disposable kit. In some embodiments, a disposable kit may be used to run multiple cycles of a process or processes, such as at least 2, 3, 4, 5 or more times, such as processes that are performed in a continuous or semi-continuous manner. In some embodiments of the invention, a system, such as a disposable kit, is used to process cells taken from a single patient.

[0102] The centrifuge chamber usually rotates around its axis, and its compartment is usually coaxial with respect to this chamber. In some embodiments of the invention, the centrifuge chamber also includes a movable element, such as a piston, which can typically move (eg, axially) through the chamber and change the volume of the compartment. Thus, in specific embodiments of the invention, the inner compartment is connected to the side wall, to the end wall of the chamber and to the movable element and has a varying volume, which can be adjusted due to the movement of the movable element. The movable element may be made of rigid, substantially or mostly rigid or flexible materials, or combinations thereof.

[0103] The chamber also typically includes one or more openings, such as one or more inlets, one or more outlets, and/or one or more inlets/outlets, which can absorb and introduce liquid and/or gas into the compartment and from the compartment. In some instances, the orifice may be an inlet/outlet that may allow absorption and entry of liquid and/or gas. In some cases, one or more inlets may be separate from or spaced apart from one or more outlets. The hole or holes may be on one of the end walls. In some embodiments of the invention, liquid or gas enters the compartment and/or leaves the compartment through the movement of the movable element, which increases and/or reduces the volume of the compartment. In other embodiments of the invention, liquid and/or gas may enter and/or exit the compartment via a piping or other conduit that is connected to the opening, such as by connecting a line or conduit to a pump, syringe, or other mechanism that can controlled automatically.

[0104] In some embodiments, the chamber is part of a closed system, such as a sterile system, having various additional components such as tubing lines, connectors, and caps, and processing steps are performed in this system. Thus, in some embodiments of the invention, the described methods or steps are carried out in a fully enclosed or semi-enclosed space, such as a closed or semi-enclosed sterile system, facilitating the production of cells for therapeutic administration to an individual, without the need for a separate sterile space, such as as a biological safety chamber or room. In some embodiments of the invention, the methods are carried out in an automatic or partially automatic manner.

[0105] In some embodiments of the invention, the chamber is connected to a centrifuge, which rotates the chamber, for example, around its axis. Rotation may be performed before, during and/or after incubation in one or more processing steps. Thus, in some embodiments of the invention, one or more of the various stages of processing is carried out with rotation, for example, with a specific force. Typically, the chamber rotates vertically or substantially vertically, and therefore, during centrifugation, the chamber is set to a vertical position, the side wall and shaft are set to a vertical or nearly vertical position, and the end wall(s) are set to horizontal or almost horizontal position.

[0106] In some aspects of these methods, these processes are not necessarily carried out in the same closed system, such as the same centrifuge chamber, and they may be carried out in a different closed system, such as a different centrifuge chamber, and in some embodiments implementation of the invention, such different centrifuge chambers, when carrying out these methods, are placed in appropriate positions with respect to the same system, for example, they are placed on the same centrifuge. In some embodiments of the invention, all processing steps are carried out in a closed system, where all steps or part of one or more separate processing steps are carried out in the same or in different centrifuge chambers.

A. Sample and cell preparation

[0107] The cells are usually eukaryotic cells, such as mammalian cells, and usually human cells. In some embodiments of the invention, these cells are derived from blood, bone marrow, lymph, or lymphoid organs, and such cells are cells of the immune system, such as cells of the innate or adaptive immune system, for example, myeloid or lymphoid cells, including lymphocytes, and usually, T cells and/or NK cells. Other representative cells are stem cells such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).

[0108] Cells are typically primary cells, eg, cells isolated directly from an individual and/or isolated from an individual and frozen. In some embodiments, the cells are one or more subpopulations of T cells or other cell types, such as populations of whole T cells, CD4 ⁺ cells, CD8 ⁺ cells, and subpopulations thereof, such as subpopulations with specific functions, such as activation status, maturation, differentiation, multiplication, recycling, localization and/or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With regard to the individual being treated, such cells may be allogeneic and/or autologous. These methods include commercially available methods. In some aspects, such as those using commercially available technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments of the invention, the methods include isolating cells from an individual, as well as preparing, processing, culturing and/or constructing them, and reintroducing them to the same individual before or after cryopreservation.

[0109] The subtypes and subpopulations of T cells and/or CD4 ⁺ - and/or CD8 ⁺ -T cells are "naive" T(T _N ) cells, effector T cells (T _EFF ), memory T cells, and their subtypes such as memory stem T cells (T _SCM ), central memory T cells (T _CM ), effector memory T cells (T _EM ) or end-stage memory effector T cells, tumor-infiltrating lymphocytes ( TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T cells (MAIT), natural and adaptive regulatory T cells (Treg), 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.

[0110] 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.

[0111] In some embodiments of the invention, the cells are derived from cell lines, for example, T-cell lines. In some embodiments, the cells are obtained from a xenogeneic source, such as from mice, rats, non-human primates, and pigs.

[0112] In some embodiments of the invention, cells can be isolated from a sample, such as a biological sample, for example, a sample obtained from or taken from an individual. In some embodiments of the invention, the individual from whom cells are isolated is an individual suffering from a disease or disorder or in need of cell therapy, or an individual who will receive cell therapy. In some embodiments, the subject is a human in need of a particular therapeutic treatment, such as adoptive cell therapy, for which cells have been isolated, processed, and/or engineered.

[0113] Accordingly, in some embodiments, the cells are primary cells, such as primary human cells. Samples are tissue, fluid, and other samples taken directly from the individual, as well as samples obtained in one or more processing steps such as separation, centrifugation, genetic engineering (eg, transduction with a viral vector), washing, and/or incubation. The biological sample may be a sample obtained directly from a biological source, or a sample that has been processed. Biological samples include, but are not limited to, physiological fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived from these samples.

[0114] In some aspects of the invention, the sample from which the cells are derived or from which the cells have been isolated is blood, or a blood-derived sample, or a product of apheresis or leukapheresis, or a product derived therefrom. Representative specimens are whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemic cells, lymphoma, lymph node, intestinal lymphoid tissue, mucosal lymphoid tissue, spleen, other lymphoid tissues, liver, lungs , stomach, small intestine, large intestine, kidney, pancreas, breast, bone, prostate, cervix, testes, ovary, tonsils, or other organs and/or cells derived from these organs. For cell therapy, samples are samples for adoptive cell therapy, for example, samples derived from autologous and allogeneic sources.

[0115] In some examples, cells taken from an individual's bloodstream are obtained, for example, by apheresis or leukapheresis. In some aspects of the invention, the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other germline leukocytes, erythrocytes and/or platelets, and in some aspects, they contain cells that are not erythrocytes and platelets.

[0116] In some embodiments of the invention, blood cells taken from an individual are washed, for example, to remove plasma fractions and to introduce the cells into an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution is free of calcium and/or magnesium and/or many or all of the divalent cations. In some aspects of the invention, the washing step is performed on a semi-automated "through-flow" centrifuge (eg, a Cobe 2991 cell centrifuge, Baxter) according to the manufacturer's instructions. In some aspects of the invention, the washing step is carried out by tangential flow filtration (TFF) in accordance with the manufacturer's instructions. In some embodiments of the invention, the cells are resuspended in various biocompatible buffers after washing, such as, for example, PBS without Ca ⁺⁺ /Mg ⁺⁺ . In some embodiments of the invention, the blood cell sample components are removed and the cells directly resuspended in culture medium.

[0117] In some embodiments, prior to cell enrichment and/or cell selection, the sample is contacted with serum or plasma and/or the sample contains serum or plasma, such as human serum or plasma. In some embodiments, the serum or plasma is autologous to the individual from whom the cells are taken. In some embodiments, serum or plasma is present in the sample at a concentration of at least or at least about 10% (v/v), at least or at least about 15% (v/v), at least or at least about 20% (v/v), at least or at least about 25% (v/v), at least or at least about 30% (v/v), at least or at least at least about 35% (v/v) or at least or at least about 40% (v/v). In some embodiments of the invention, before the selection and/or transduction of cells, the sample containing the primary cells is contacted with an anticoagulant, or this sample contains an anticoagulant. In some embodiments of the invention, the anticoagulant is or contains a free citrate ion, for example, a solution of the anticoagulant, citrate and dextrose, Solution A (ACD-A).

[0118] In some embodiments of the invention, prior to enrichment and/or cell selection, the cells taken from the sample are transferred to or suspended in serum-free medium. In some embodiments of the invention, the serum-free medium is a medium for culturing cells with a defined and/or well-defined chemical composition. In some embodiments, the serum-free medium is a controlled cell medium that has been treated, such as filtered, to remove inhibitors and/or growth factors. In some embodiments of the invention, the serum-free medium contains proteins. In some embodiments of the invention, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins and/or binding factors. In some embodiments, the serum-free medium contains proteins, for example, albumin, such as bovine serum albumin, human serum albumin, and/or recombinant albumin. In some embodiments, the serum-free medium contains a basal medium, such as DMEM or RPMI 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources. In some embodiments of the invention, components such as, but not limited to, albumin, lipids of a certain chemical composition, growth factors, insulin, cytokines and/or antioxidants are added to the serum-free medium. In some embodiments of the invention, the serum-free medium is prepared to support the growth, proliferation, health, homeostasis of certain types of cells, such as immune cells, T cells and/or CD4 ⁺ - and / or CD8 ⁺ -T cells.

[0119] In some embodiments of the invention, before the selection and/or enrichment of cells, the sample or cells in the sample can be left or saved until the next stages of processing. In some embodiments, the sample is maintained or stored at or about 2°C to 8°C for 48 hours, such as 12 hours, 24 hours, or 36 hours.

[0120] In some embodiments of the invention, methods of preparation include the steps of freezing, for example, cryopreservation of cells before or after isolation, selection and/or enrichment and/or incubation for transduction and construction. In some embodiments of the invention, freezing and subsequent thawing steps are performed to remove granulocytes, and to a certain extent, monocytes, from the cell population. In some embodiments of the invention, the cells are suspended in a freezing solution, for example, after a washing step, to remove plasma and platelets. However, in some aspects of the invention, any of the various and known freezing solutions and their parameters can be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing media. Then, these solutions are diluted 1:1 with medium so that the final concentration of DMSO and HSA is 10% and 4%, respectively. The cells are then typically frozen to -80° C. at a rate of 1° per minute in the vapor phase of a liquid nitrogen storage tank.

[0121] In some embodiments, cell isolation includes one or more steps of preparing and/or separating cells by non-affinity methods. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich with desired components, lyse, or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more properties, such as density, adhesive properties, size, sensitivity, and/or resistance to particular components.

[0122] In some embodiments, the methods include methods for separating cells by their density, such as methods for preparing leukocytes from peripheral blood by lysing erythrocytes and centrifuging in a Percoll or Ficoll gradient.

[0123] In some embodiments, isolation methods include separating different types of cells based on the expression or presence of one or more specific molecules in the cells, such as surface markers, such as surface proteins, intracellular markers, or nucleic acid. In some embodiments of the invention, any known method of separation based on such markers can be applied. In some embodiments of the invention, separation is carried out by affinity or immunoaffinity methods. Thus, for example, in some aspects of the invention, isolation involves separating cells and cell populations based on the expression or level of expression by the cells of one or more markers, and usually cell surface markers, for example, by incubation with an antibody or binding partner that specifically bind to such markers, followed by the steps of washing and separating cells having bound antibody or its binding partner from cells lacking bound antibody or its binding partner.

[0124] Such separation steps may be based on positive selection, where cells having bound reagents are retained for later use, and/or negative selection, where cells lacking bound antibody or its binding partner are retained. In some examples, both fractions are retained for later use. In some aspects of the invention, negative selection may be particularly useful if there is no antibody that can specifically identify a cell type in a heterogeneous population, and therefore separation is best done on the basis of markers expressed by cells other than those of the target population.

[0125] Separation will not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. Thus, for example, positive selection or enrichment for a particular cell type, such as marker-expressing cells, means an increase in the number or percentage of such cells, but not necessarily the complete absence of cells that do not express the marker. Similarly, negative selection, removal or depletion of cells of a particular type, such as cells expressing a marker, means a decrease in the number or percentage of such cells, but not necessarily the complete removal of all these cells.

[0126] In some examples, multiple rounds of separation steps are performed, where a positively or negatively selected fraction of one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can contribute to the simultaneous depletion of cells expressing multiple markers at once, for example, by incubating cells with multiple antibodies or their binding partners, each of which is specific for a negative selection marker. Similarly, many cell types can be positively and simultaneously selected by incubating the cells with a variety of antibodies or their binding partners expressed on different cell types.

[0127] Thus, for example, in some aspects, specific subpopulations of T cells, such as cells positive for one or more surface markers or expressing high levels of these markers, for example, CD28 ⁺ -, CD62L ⁺ -, CCR7 ⁺ -, CD27 ⁺ -, CD127 ⁺ -, CD4 ⁺ -, CD8 ⁺ -, CD45RA ⁺ - and/or CD45RO ⁺ -T cells isolated by positive or negative selection methods.

[0128] For example, CD3 ⁺ -, CD28 ⁺ -T cells can be positively selected using magnetic spheres conjugated with anti-CD3 / anti-CD28 antibodies (for example, on the DYNABEADS® M-450 CD3 / CD28 device T Cell Expander).

[0129] In some embodiments of the invention, the selection is carried out by enrichment in specific cell populations through positive selection or depletion of a specific cell population through negative selection. In some embodiments of the invention, positive or negative selection is performed by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker ⁺ ) at a relatively higher level (marker ^high ) on positively or negatively selected cells, respectively.

[0130] In some embodiments, T cells are separated from a PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, such as CD14 cells. In some aspects, the step of selecting CD4 ⁺ or CD8 ⁺ cells is performed to separate helper CD4 ⁺ and CD8 ⁺ cells and cytotoxic CD8 ⁺ T cells. Such CD4 ⁺ and CD8 ⁺ populations can be further sorted into subpopulations by positive or negative selection for expressed markers, or markers expressed relatively higher, on one or more subpopulations of naive T cells, memory T cells, and /or effector T cells.

[0131] In some embodiments, CD8 ⁺ cells are also enriched or depleted in or depleted in naive cells, central memory cells, memory effector cells, and/or central memory stem cells by positive or negative selection on surface antigens, associated with the respective subpopulation. In some embodiments of the invention, enrichment in central memory T cells (T _CM ) is carried out to increase efficiency, for example, to increase lifespan, increase reproduction and/or improve graft engraftment after administration, where in some aspects, such enrichment is especially suitable for such subpopulations. See Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J. Immunother 35(9):689-701. In some embodiments of the invention, the association of T _CM -rich in CD4 ⁺ - and/or CD8 ⁺ -T cells also improves efficiency.

[0132] In some embodiments, memory T cells are present in both CD62L ⁺ - and CD62L ^- subpopulations of CD8 ⁺ peripheral blood lymphocytes. PBMCs can be enriched or depleted in CD62L ^- CD8 ⁺ and/or CD62L ⁺ CD8 ⁺ fractions, for example using anti-CD8 and anti-CD62L antibodies.

[0133] In some embodiments, enrichment in central memory T cells (T _CM ) is based on positive surface expression or high expression of CD45RO, CD62L, CCR7, CD28, CD3 and/or CD127; and in some aspects, based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8 ⁺ population enriched in T _CM cells is accomplished by depletion of cells expressing CD4, CD14, CD45RA , and positive selection or enrichment for cells expressing CD62L. In one aspect of the invention, central memory T cells ( _TCM ) are enriched based on a negative fraction of cells selected for CD4 expression, where said cells are negatively selected based on CD14 and CD45RA expression and positively selected based on CD62L expression. In some aspects, such selection is carried out simultaneously, and in other aspects, sequentially, in any order. In some aspects, the same selection step for CD4 expression carried out to prepare a population or subset of CD8 ⁺ cells is also performed to prepare a population or subset of CD4 ⁺ cells such that positive and negative CD4 ⁺ separation fractions are retained. and used in subsequent steps of these methods, optionally after one or more additional positive or negative selection steps.

[0134] In a specific example, a sample of PBMC or a sample of other leukocytes is subjected to selection for CD4 ⁺ cells, where the negative and positive selection fractions were retained. The negative fraction is then subjected to negative selection based on CD14 and CD45RA or CD19 expression, and positive selection based on markers specific to central memory T cells such as CD62L or CCR7, where positive and negative selection is done in any order.

[0135] Helper CD4 ⁺ T cells are sorted into naive cells, central memory cells, and effector cells by identifying cell populations having cell surface antigens. CD4 ⁺ -lymphocytes can be obtained by standard methods. In some embodiments of the invention, "naive" CD4 ⁺ -T-lymphocytes are CD45RO ^- , CD45RA ⁺ -, CD62L ⁺ -, CD4 ⁺ -T cells. In some embodiments, the central CD4 ⁺ memory cells are CD62L ⁺ and CD45RO ⁺ . In some embodiments, the effector CD4 ⁺ cells are CD62L ^- and CD45RO ^- .

[0136] In one example, for enrichment in CD4 ⁺ cells by negative selection, the mixture of monoclonal antibodies typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments of the invention, the antibody or its binding partner is attached to a solid carrier or to a matrix, such as a magnetic sphere or a paramagnetic sphere, in order to separate cells by positive and/or negative selection. Thus, for example, in some embodiments, cells and cell populations are separated or isolated by immunomagnetic (or affinity magnetic) separation methods (described in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: SA Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[0137] In some aspects, a sample or composition of cells to be separated is incubated with a small magnetized or magnetically susceptible material, such as magnetically susceptible particles or microparticles, such as paramagnetic spheres (for example, such as Dynalbeads or MACS spheres). The magnetically receptive material, e.g., particles, is generally attached directly or indirectly to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on the cell, cells, or cell population required for separation. , for example, cells required for negative or positive selection.

[0138] In some embodiments, the magnetic particle or sphere contains a magnetically susceptible material associated with a specific binding partner, such as an antibody or other binding partner. There are many well known magnetically susceptible materials used in magnetic separation techniques. Suitable magnetic particles are described by Molday in US Patent No. 4452773 and in the description of the European patent application EP 452342 B, which is introduced into the present description by reference. Other examples are colloidal particles of a certain size, such as those described by Owen in U.S. Patent No. 4795698 m Liberti et al., in US patent no. 5200084.

[0139] Incubation is typically carried out under conditions where antibodies or binding partners or molecules, such as second antibodies or other reagents, that specifically bind to such antibodies or binding partners, which are in turn attached to magnetic particles or spheres. , bind specifically to cell surface molecules if they are present on the cells in the sample.

[0140] In some aspects, the sample is placed in a magnetic field, and cells having associated magnetically susceptible or magnetized particles will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells that are attracted to the magnet are retained, and for negative selection, cells that are not attracted to the magnet (unlabeled cells) are retained. In some aspects, the combination of negative and positive selection is carried out in the same selection stage, where the positive and negative fractions are retained and then processed or additional separation steps are carried out.

[0141] In some embodiments, the magnetically susceptible particles are coated with "first" antibodies or other binding partners, "second" antibodies, lectins, enzymes, or streptavidin. In some embodiments of the invention, the magnetic particles are attached to cells by coating with first antibodies specific for one or more markers. In some embodiments, instead of spheres, cells are labeled with a first antibody or its binding partner, and then magnetic particles coated with a second antibody or other binding partner (e.g., streptavidin) specific for a particular cell type are added. In some embodiments of the invention, streptavidin-coated magnetic particles are used in combination with biotinylated first or second antibodies.

[0142] In some embodiments, the magnetically susceptible particles remain associated with cells, which are then incubated, cultured, and/or engineered, and in some aspects, the particles remain associated with cells for administration to a patient. In some embodiments of the invention, magnetized or magnetically susceptible particles are removed from the cells. Methods for removing magnetized particles from cells are known and include, for example, the use of competing unlabeled antibodies and magnetized particles or antibodies conjugated to cleavable linkers. In some embodiments of the invention, the magnetized particles are biodegradable.

[0143] In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetically Activated Cell Sorting Systems (MACS) allow selection of high purity cells having magnetized particles associated with these cells. In some embodiments of the invention, MACS is carried out in a mode in which non-target molecules and target molecules are sequentially eluted after the application of an external magnetic field. That is, cells bound to the magnetized particles are preserved, while unbound cells are eluted. Then, after this first elution step is completed, the molecules that have been captured by the magnetic field and that have not been eluted are released by a certain mechanism so that they can be eluted and can be isolated. In some embodiments, non-target cells are labeled and isolated from a heterogeneous population of cells.

[0144] In some embodiments, isolation or separation is performed using a system, device, or equipment that performs one or more isolation, cell preparation, separation, processing, incubation, culture, and/or composition steps. In some aspects, this system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, improve user experience, and/or reduce contamination. In one example, such a system is the system described in International Patent Application Publication No. WO2009/072003 or US 20110003380 A1. In one example, such a system is the system described in International Application Publication No. WO2016/073602.

[0145] In some embodiments of the invention, the methods include cell selection, where all or part of the selection procedure is carried out in the internal compartment of the centrifuge chamber, for example, with centrifugal rotation. In some embodiments, cells are incubated with selection reagents, such as immunoaffinity selection reagents, in a centrifuge chamber.

[0146] For example, selection based on immunoaffinity may depend on a favorable energetic interaction between the cells being separated and a molecule that specifically binds to a marker on the cell, such as an antibody or other binding partner on a solid surface, such as a particle. . In some known affinity separation methods using particles such as spheres, particles and cells are incubated in a container such as a tube or bag with shaking or agitation at a constant ratio of cell density to particle (eg sphere) to facilitate stimulation of energetically favorable interactions. . Such methods may not be ideal for use in large scale production, for example, because they may require large volumes to maintain an optimal or desired cell-to-particle ratio while maintaining the desired number of cells. Accordingly, such methods may require batch processing or processing in a batch format, which can be time consuming, require more steps and more careful processing, increase cost, and increase the risk of user error.

[0147] In some embodiments of the invention, when conducting such stages of selection or parts thereof (for example, incubation with particles coated with antibodies, for example, magnetic spheres) in the compartment of the centrifuge chamber, the user can adjust some parameters, such as the volume of various solutions, the addition solution during processing, as well as their synchronization, which can provide advantages over other available methods. For example, the ability to reduce the volume of liquid in the compartment during incubation allows you to increase the concentration of particles (eg, reagent spheres) used in the selection, and thus the chemical potential of the solution, without affecting the total number of cells in the compartment. This, in turn, can enhance pairwise interactions between treated cells and particles used for selection. In some embodiments of the invention, conducting the incubation step in the chamber, for example, in association with systems, regulatory schemes and controls, as described in this application, allows the user to mix the solution at the right time during the incubation, which can also improve interaction.

[0148] In some embodiments, at least part of the selection step is performed in a centrifuge chamber, and this step includes incubating the cells with a selection reagent. In some aspects of such methods, the volume of cells is mixed with an amount of the desired affinity selection reagent, which is much less than would normally be used when performing such selection in a tube or container to select the same number of cells and/or cell volume according to manufacturer's instructions. In some embodiments of the invention, the amount of reagent or reagents for selection is no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 50%, not more than 60%, not more than 70%, or not more than 80% of the amount of the same selection reagents used to collect cells in a tube or container for incubation of the same number cells and/or cell volume according to the manufacturer's instructions.

[0149] Incubation with a reagent or selection reagents, for example, as part of the selection methods that can be performed in the compartment of the camera, includes the use of one or more reagents to select cells of one or more different types based on expression or presence in the cell or on the cell of one or more specific molecules, such as surface markers, such as surface proteins, intracellular markers, or nucleic acid. In some embodiments of the invention, any known method of using a selection reagent or reagents for separation based on such markers can be used. In some embodiments, the selection reagent or reagents allow separation based on affinity or immunoaffinity. Thus, for example, in some aspects of the invention, selection includes incubation with a reagent or reagents to separate cells and cell populations based on the expression or level of expression by the cells of one or more markers, and usually cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed by the steps of washing and separating cells having bound antibody or binding partner from cells that are not bound to the antibody or binding partner.

[0150] In some embodiments of the invention, for selection, for example, immunoaffinity selection of cells, these cells are incubated in the chamber compartment in a composition that also contains a selection buffer along with a selection reagent, such as a molecule that specifically binds to a surface marker on the cell , which is desired to be enriched and/or depleted, but not on other cells in the composition, e.g., an antibody that is optionally attached to a scaffold, such as a polymer or a surface, e.g., a sphere, e.g. antibodies specific for CD4 and CD8. In some embodiments of the invention, as described above, the selection reagent is added to the cells in the chamber compartment in an amount that is substantially less (for example, no more than 5%, 10%, 20%, 30%, 40%, 50% , 60%, 70%, or 80%) of the amount of selection reagent that is normally used, or that must be used to achieve approximately the same or a similar effect on the selection of the same number of cells or the same volume of cells, if such selection is carried out in a tube with shaking or with vortex mixing. In some embodiments of the invention, the incubation is carried out with the addition of cell selection buffer and selection reagent to achieve the desired volume when the reagent is incubated, for example, from 10 ml to 200 ml, for example, at least or at least about or about or 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 150 ml or 200 ml. In some embodiments of the invention, the selection buffer and the selection reagent are premixed before they are added to the cells. In some embodiments, the selection buffer and the selection reagent are separately added to the cells. In some embodiments, the selection incubation is performed with occasional gentle agitation, which can facilitate the stimulation of energetically favorable interactions and thereby allow the use of fewer total selection reagents while achieving high selection efficiency.

[0151] In some embodiments of the invention, the total duration of incubation with the selection reagent is from or about 5 minutes to 6 hours, for example, from 30 minutes to 3 hours, for example, at least or at least about 30 minutes, 60 minutes, 120 minutes or 180 minutes.

[0152] In some embodiments of the invention, the incubation is usually carried out under mixing conditions, for example, by centrifugation, usually at a relatively low force or speed, such as a speed lower than the speed used to pellet the cells, for example, from or about 600 rpm /min up to 1700 rpm (for example, at or approximately or at least at 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), for example, with RCF acting on the sample or wall chamber or other container and between or about 80 g to 100 g (eg, at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments of the invention, centrifugation is performed at repetitive intervals at low speed, followed by breaks, where centrifugation and / or breaks follow after 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 seconds, for example, after centrifugation for about 1 or 2 seconds, a break of about 5, 6, 7 or 8 seconds is taken.

[0153] In some embodiments of the invention, such a method is carried out in a completely closed system, in which the camera is an integral part of it. In some embodiments of the invention, this method (and in some aspects, also one or more additional steps, such as a preliminary step of washing the sample containing cells, such as a sample after apheresis) is carried out in an automatic mode, in which cells, reagent and other components are taken into the chamber and ejected from the chamber at the appropriate times during centrifugation so that the washing and binding step is carried out in one closed system by an automated program.

[0154] In some embodiments of the invention, after incubation and/or mixing of cells and reagent and/or reagents for selection, the incubated cells are subjected to separation for cell selection, based on the presence or absence of a particular reagent or reagents. In some embodiments of the invention, further selection is carried out outside the centrifuge chamber. In some embodiments of the invention, the separation is carried out in the same closed system in which the centrifuge chamber is present and in which the cells are incubated with the selection reagent. In some embodiments of the invention, after incubation with selection reagents, the incubated cells, including cells in which the selection reagent is bound, are removed from the centrifuge chamber, that is, transferred from the centrifuge chamber to an immunoaffinity cell separation system. In some embodiments, the immunoaffinity separation system is or includes a magnetic separation column. In some embodiments of the invention, prior to separation, one or more other processing steps, such as washing, may be performed in the chamber.

[0155] In some aspects, separation and/or other steps are performed using the CliniMACS system (Miltenyi Biotic), for example, to automatically separate cells at a clinically scalable level in a closed and sterile system. Components may include an embedded microcomputer, a magnetic separation device, a peristaltic pump, and various pressure valves. In some aspects of the invention, an embedded computer regulates all components of the device and directs the system to perform repetitive procedures in a standardized sequence. In some aspects of the invention, the magnetic separation device includes a movable permanent magnet and a sampling column holder. The peristaltic pump regulates the flow rate through the entire row of tubes, along with pressure valves, which ensures controlled buffer flow through the system and continuous cell suspension.

[0156] In some aspects of the invention, the CliniMACS system uses magnetized particles associated with the antibody, which are fed into a sterile, pyrogen-free solution. In some embodiments of the invention, after labeling the cells with magnetic particles, the cells are washed to remove excess particles. Thereafter, the cell preparation bag is connected to a tubing system, which in turn is connected to the bag containing the buffer and to the cell collection bag. The tubing system consists of pre-assembled sterile tubing, including pre-column and separation column, and is for single use only. After starting the separation program, the system automatically feeds the cell sample to the separation column. Labeled cells are retained on the column while unlabeled cells are removed through a series of washes. In some embodiments of the invention, the cell populations used in the methods described here are unlabeled and are not stored in the column. In some embodiments of the invention, the cell populations used in the methods described here are labeled and stored in a column. In some embodiments, the cell populations used in the methods described herein are eluted on the column after the magnetic field has been removed and collected in a cell collection bag.

[0157] In some embodiments, the separation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some aspects of the invention, the CliniMACS Prodigy system is equipped with a cell processor that provides automatic washing and cell fractionation by centrifugation. The CliniMACS Prodigy system can also include an integrated camera and an image recognition computer program that determines the optimal cell fractionation end point, allowing differentiation of macroscopic layers of the cell source product. For example, peripheral blood is automatically divided into erythrocytes, leukocytes and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber that performs cell culture protocols such as, for example, cell differentiation and expansion, antigen loading, and long-term cell culture. Inlets can be used for sterile removal and replenishment of media, and cell monitoring can be done with an integrated microscope. See, for example, Klebanoff et al. (2012) J. Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J. Immunother. 35(9):689-701.

[0158] In some embodiments, the cell population described herein is harvested and enriched (or depleted) by flow cytometry, where cells stained for a plurality of cell surface markers are captured in a fluid stream. In some embodiments of the invention, the cell population described herein is collected and enriched (or depleted) using preparative scalable (FACS) sorting. In some embodiments, the cell population described herein is harvested and enriched (or depleted) using microelectromechanical systems (MEMS) chips in combination with a FACS 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 a variety of markers, allowing the isolation of well-defined subpopulations of T cells with high purity.

[0159] In some embodiments, the antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on the binding of antibodies or other binding partners specific for one or more cell surface markers is carried out in a fluid stream, for example, using fluorescence-activated cell sorting (FACS), including preparative scalable (FACS) - sorting, and/or use of microelectromechanical systems (MEMS) chips, for example in combination with a flow cytometric detection system. Such methods allow simultaneous positive and negative selection based on multiple markers.

[0160] In some embodiments, the cells to transduce the retroviral vector particles described herein are, for example, monocytes, monocyte-derived macrophages, monocyte-derived dendritic cells, or resting T cells. In specific embodiments of the invention, the described retroviral particles can infect resting T cells. In some embodiments of the invention, the initial composition includes a plurality of cells, such as immune cells, for example, T cells, which are non-dividing and/or silent and/or resting, and/or cells in which a plurality of cells are present, for example, more than 50%, 60%, 70%, 80%, 80% or more of cells that have been transduced and are non-dividing and/or silent and/or quiescent. In some embodiments of the invention, the initial composition contains a population of T cells in which at least 40%, 50%, 60%, 70%, 80%, 90% or more of the T cells in the population are resting T cells , such as T cells lacking a T cell activation marker such as a surface marker or an intracellular cytokine or other marker and/or T cells that are at the G ₀ or G ₀ G _1a stage of the cell cycle. In some embodiments of the invention, the cell contains active SAMHD1, such as unphosphorylated SAMHD1. In some embodiments of the invention, the cells are at the G ₀ , G ₀ /G _1a or G1 stage of the cell cycle.

[0161] In some embodiments, the methods include transducing T cells in which no more than 5%, 10%, 20%, 30%, or 40% of the T cells express a T cell activation marker. In some embodiments, no more than 5%, 10%, 20%, 30%, or 40% of T cells are positive for one or more surface markers of T cell activation HLA-DR, CD25, CD69, CD71, CD40L (CD154) and/or 4-1BB (CD137). In some embodiments, no more than 5%, 10%, 20%, 30%, or 40% of T cells express an intracellular cytokine that is IL-2, IFN-gamma, and/or TNF-alpha.

B. Viral vector particles

[0162] In some embodiments, the viral vector particles are retroviral vector particles, such as lentiviral particles, containing a nucleic acid encoding a recombinant and/or heterologous molecule, for example, a recombinant or heterologous protein, such as a recombinant and/or heterologous receptor, for example, a chimeric antigen receptor (CAR) or other antigen receptor in the genome of a viral vector. The genome of a viral vector particle typically includes sequences other than the nucleic acid encoding the recombinant molecule. Such sequences may include sequences that allow the genome to be packaged into a viral particle and/or sequences that stimulate the expression of a nucleic acid encoding a recombinant receptor, such as CAR.

1. Viral vector

[0163] In some embodiments, the viral vector particles comprise a genome derived from a vector based on a retroviral genome, such as a vector based on a lentiviral genome. In some aspects of the viral vectors described herein, a heterologous nucleic acid encoding a recombinant receptor, such as an antigen receptor, such as CAR, is contained and/or located between the 5'-LTR and 3'-LTR sequences of the vector genome.

[0164] In some embodiments, the viral vector genome is a lentiviral genome, such as the HIV-1 genome or the SIV genome. For example, lentiviral vectors have been generated by attenuating multiple virulence genes, for example, the env, vif, vpu and nef genes can be deleted, making the vector safer for therapeutic use. Lentiviral vectors are known. See, Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, US Pat. Nos. 6013516 and 5994136). In some embodiments, these viral vectors are derived from a plasmid or a virus and are configured to carry base sequences for incorporating a foreign nucleic acid in order to select and transfer the nucleic acid into a host cell. Known lentiviruses can be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC"; 10801 University Blvd., Manassas, Va.20110-2209), or isolated from known sources using publicly available methods.

[0165] Non-limiting examples of lentiviral vectors are vectors derived from a lentivirus 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 generated by attenuating multiple HIV virulence genes, eg the env, vif, vpu and nef genes have been deleted, making the vector safer for therapeutic use. Lentiviral vectors are known to those skilled in the art, see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, US Pat. Nos. 6013516 and 5994136). In some embodiments, these viral vectors have been derived from a plasmid or virus and are configured to carry base sequences for incorporating a foreign nucleic acid in order to select and transfer the nucleic acid into a host cell. Known lentiviruses can be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC"; 10801 University Blvd., Manassas, Va.20110-2209), or isolated from known sources using publicly available methods.

[0166] In some embodiments, the viral genome vector may contain the 5'- and 3'-LTR sequences of a retrovirus, such as a lentivirus. In some aspects of the invention, the viral genome construct may contain lentivirus 5'- and 3'-LTR sequences, and in particular, it may contain R and U5 sequences from lentivirus 5'-LTR and inactivated or self-inactivating lentivirus 3'-LTR. The LTR sequences may be the LTR sequences of any lentivirus derived from any species. For example, they may be the LTR sequences from HIV, SIV, FIV or BIV. Typically, the LTR sequences are HIV LTR sequences.

[0167] In some embodiments, the viral vector nucleic acid, such as the HIV viral vector, does not contain additional transcription units. The vector genome may contain inactivated or self-inactivating 3'-LTRs (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 nucleic acid used to produce viral vector RNA can be introduced to produce self-inactivating (SIN) vectors. This deletion can then be transferred to the 5'-LTR of the proviral DNA by reverse transcription. A self-inactivating vector typically has a deletion in the enhancer and promoter sequences from the 3' long terminal repeat (LTR) that will be copied to the 5'-LTR during vector integration. In some embodiments of the invention, a sufficient portion of the sequence may be removed, including the TATA box, in order to abolish the transcriptional activity of the LTR. This prevents the production of full length vector RNA in the transduced cells. In some aspects, the 3′-LTR U3 element contains a deletion in the enhancer sequence, in the TATA box, and in the Sp1 and NF-kappa B sites. contains inactivated 5'-LTR. This may improve safety by reducing the risk of mobilization of the vector genome and the effect of LTR on neighboring cellular promoters. The self-inactivating 3'-LTR may be constructed by any method known to those skilled in the art. In some embodiments of the invention, this does not affect the titers of the vector or the in vitro or in vivo properties of the vector.

[0168] The U3 sequence of the 5'-LTR of a lentivirus may optionally be replaced by a promoter sequence in the viral construct, such as a heterologous promoter sequence. This may increase the titer of the virus isolated from the packaging cell line. An enhancer sequence may also be included. Any enhancer/promoter combination that increases the level of expression of the viral RNA genome in the packaging cell line can also be used. In one example, the CMV enhancer/promoter sequence (US Pat. No. 5,385,839 and US Pat. No. 5,168,062) is used.

[0169] In some embodiments, the risk of insertional mutagenesis can be minimized by constructing a retroviral vector genome, such as an integration-defective lentiviral vector genome. A number of methods can be applied to obtain a non-integrating vector genome. In some embodiments of the invention, mutation(s) may be introduced(s) into a gene component of the Pol integrase enzyme such that it encodes a protein with an inactive integrase. In some embodiments of the invention, the vector genome itself can be modified to prevent integration, for example, by mutation or deletion of one or both binding sites, obtaining a 3'-LTR-proximal polypurine cascade (PPT) that has been rendered non-functional by the deletion or modification. . In some embodiments, non-genetic methods may also be used; these methods involve the use of pharmacological agents that inhibit one or more integrase functions. These approaches are not mutually exclusive, that is, more than one of these approaches can be applied at the same time. For example, the integrase and the binding sites may be non-functional, or the integrase and the PPT site may be non-functional, or the binding sites and the PPT site may be non-functional, or all of them may 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).

[0170] In some embodiments, the vector contains sequences for propagation in a host cell, such as a prokaryotic host cell. In some embodiments of the invention, the nucleic acid of the viral vector contains one or more origins of replication for propagation in a prokaryotic cell, such as a bacterial cell. In some embodiments, vectors comprising a prokaryotic origin of replication may also contain a gene that expresses a detectable or selectable marker, such as a drug resistance marker.

2. SAMHD1 inhibitory accessory protein

[0171] In one embodiment, the present invention provides retroviral vector particles comprising a SAMHD1 inhibitory accessory protein encapsulated in a virion. In some aspects, such particles allow efficient transduction of non-dividing cells, such as non-dividing immune cells, such as resting T cells. In some aspects, such particles allow efficient transduction of monocytes, monocyte-derived macrophages, or monocyte-derived dendritic cells. In some aspects of the invention, the described retroviral vector particles minimize and/or reduce the need for ex vivo manipulations for cell transduction, such as cell activation and/or stimulation.

[0172] SAMHD1 is a protein present in some silent and/or resting immune cells and has dGTP-dependent deoxynucleotide triphosphohydrolase activity, and such a protein can dephosphorylate cellular deoxynucleotide triphosphates (dNTPs). The presence of active SAMHD1 in cells that has antiviral activity, such as unphosphorylated SAMHD1, can reduce the nucleotide pool. Such a reduction can thereby limit retroviral transduction by preventing the viral vector from interacting with enough nucleotides to sufficiently facilitate the reverse transcription reaction of the viral RNA, eg the viral genome. Although in some cases resting cells, such as activated or proliferating T cells, can express SAMHD1, however, in some aspects, the antiviral activity of SAMHD1 is regulated by phosphorylation of SAMHD1 in a cell cycle dependent manner (Cribier et al. (2013) Cell Reports, 3: 1036-1043). For example, in dividing cells, SAMHD1 usually interacts with proteins associated with the cell cycle, and in some aspects, is phosphorylated by cyclin-dependent kinase 1 (CDK1). A representative sequence of human SAMHD1 is shown in SEQ ID NO: 19 (see, for example, UniProt No. Q9Y3Z3). Thus, for example, in some embodiments of the invention, SAMHD1 can be phosphorylated at a position corresponding to position T592 of the representative SAMHD1 sequence shown in SEQ ID NO: 19. Phosphorylated SAMHD1 has reduced and/or minimal activity, indicating no restriction on dividing T- virus cells.

[0173] In some embodiments of the vector particles and methods described herein, by incorporating the SAMHD1 inhibitory accessory protein into the virion, SAMHD1 is inhibited, eg, degraded, in cells into which the described retroviral vector particle has been introduced. This leads to an increase in the nucleotide pool, and thereby allows the transduction of silent and/or non-dividing cells that express SAMHD1. In some embodiments, the described retroviral particles contain a SAMHD1 inhibitory protein and can transduce cells containing active SAMHD1, such as a non-phosphorylated SAMHD1 protein. In some embodiments, transduction of the described retroviral vector particles occurs in cells in which SAMHD1 is not phosphorylated at the position corresponding to position T592 in the representative SAMHD1 sequence shown in SEQ ID NO: 19. Methods for assessing SAMHD1 expression and/or phosphorylation can be performed using any known technique, e.g. Western blot analysis using a SAMHD1-specific antibody after SDS-PAGE and/or after SDS-PAGE based on phosphate affinity using acrylamide technology using phosphate labels or other similar methods (see for example Cribier et al.).

[0174] In specific embodiments, the described retroviral vector particles modified to contain a SAMHD1 inhibitory accessory protein in their virion are any viral vector that is based on host nucleotides for transduction. Such viral vector particles may include retroviral vector particles, for example, lentiviral or gamma-retroviral vector particles, which use cellular dGTPs to synthesize viral cDNA from viral RNA via viral reverse transcriptase.

[0175] In some embodiments, the SAMHD1 inhibitory protein is a host cell viral protein that has SAMHD1 inhibitory activity. In some embodiments, the SAMHD1 inhibitory protein is a lentiviral protein, such as the lentiviral Vpx protein or Vpr protein, or a variant or portion thereof, that has SAMHD1 inhibitory activity. Vpx and Vpr, which are expressed in various lentiviruses, share common sequences and structural homology, and are virion-associated proteins that are packaged into the virion during assembly. Once introduced into a cell, Vpx, and in some aspects Vpr, can block the ubiquitinization of SAMHD1 through a ubiquitin ligase complex in the cell derived from host cell proteins CUL4A-DDB1-DCAF1, which will lead to proteasomal degradation of SAMHD1 (Schwefel et al . (2014) Nature 505:234-238). In some embodiments, inhibition, such as degradation of SAMHD1 by a Vpr or Vpx protein, is mediated by binding of a lentiviral Vpr or Vpx protein to SAMHD1 and/or one or more DDB1 or DCAF1 in a ubiquitin ligase complex.

[0176] In some embodiments, the SAMHD1 inhibitory protein is an antibody or fragment thereof that specifically binds to SAMHD1 and/or inhibits SAMHD1.

[0177] In some embodiments, the disclosed retroviral vector particles have the activity to degrade SAMHD1 in host cells, such as resting T cells, into which the viral vector particle has been introduced. In some aspects, SAMHD1 can be reduced by 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 35x, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more compared to the expression or level of SAMHD1 in the same cells that were not injected with retroviral particles vector. In some such embodiments, degradation of SAMHD1 can be monitored or observed following transduction or other form of transfer of the disclosed retroviral vector particle containing the SAMHD1 inhibitory protein into silent host cells, such as resting T cells. Degradation can be assessed by determining the relative level of SAMHD1 expression compared to SAMHD1 expression in the same cells in which no retroviral vector particles have been introduced. A number of well known methods for assessing the level of protein expression can be applied. In some embodiments of the invention, SAMHD1 can be detected by immunoblotting using a SAMHD1-specific antibody, for example, in a sample of lysed cells. The degree or level of expression can also be assessed and/or quantified using standard procedures such as densitometry. In some embodiments of the invention, levels and/or expression of SAMHD1 may be monitored over a period of time and/or may be monitored for changes in the original virus.

[0178] In some aspects, described retroviral vector particles containing SAMHD1 inhibitory proteins, such as Vpx or Vpr protein, render nondividing and/or silent or quiescent immune cells even more receptive to transduction and administration of a genetically engineered molecule encoded by such a vector, for example, a vector encoding genetically engineered receptors, such as recombinant antigen receptors, such as CARs and/or other recombinant receptors, such as receptors containing an extracellular ligand-binding or recognition domain and an intracellular signal transduction domain, or domains capable of transmitting the T stimulatory signal cell, such as ITAM-containing domains and/or T-cell co-stimulatory domains. In some embodiments, the present invention relates to methods of using the described retroviral vector particles containing a SAMHD1 inhibitory protein, such as a Vpx protein or a Vpr protein, to transduce and/or introduce a genetically engineered receptor, such as recombinant antigen receptors, such as CAR, and /or other recombinant receptors, into monocyte-derived macrophages, monocyte-derived dendritic cells, or resting T cells.

[0179] In some embodiments, the SAMHD1 inhibitor is a Vpx protein, a Vpr protein, or a functional variant or portion of a Vpx or Vpr protein having SAMHD1 inhibitory activity. In some embodiments, Vpx is an 18 kDa 112 amino acid (a.a.) protein found in primate lentiviruses. Vpx from SIV and HIV-2 are 83% identical at the amino acid level (Goujon et al., J Virol , 82:12335-12345 (2008) and have SAMHD1 binding and degradation activity (Laguette et al., Nature , 474 : 654-657 (2011) Vpx is present only in some lentiviruses and has not been found in HIV-1 The closely related Vpr gene, which has a high degree of structural similarity and sequence homology to Vpx, is encoded by all primate lentiviruses and, in some cases, it also has SAMHD1 activity Both Vpx and Vpr are packaged into the virion through its interaction with the p6 region of the p55gag precursor.

[0180] Any suitable Vpx and/or Vpr protein having SAMHD1 inhibitory activity can be used in the contemplated particles, compositions and methods. A protein has SAMHD1 inhibitory activity if it is able to inhibit, for example, degrade SAMHD1 in the cell. In some cases, inhibition can be monitored by assessing the degradation of SAMHD1 in quiescent cells, such as resting T cells, following administration of a viral vector particle containing such a protein, compared to a viral vector lacking such a protein, using the methods described. in the literature or in this application. In some embodiments, Vpx, Vpr, and variants thereof are tested for the ability to inhibit SAMHD1 activity according to one or more different known methods. See Lim et al., Cell Host & Microbe , 11, 194-204 (2012) and Lahouassa et al., Nature Immunol , 13:3, 223-229 (2012). In some cases, monitoring of inhibition can be done by assessing the efficiency of transduction in silent cells, such as resting T cells, after administration of a viral vector particle containing such a protein, compared to a viral vector that is comparable to or is the same. , but does not contain Vpx or Vpr protein, using the methods described in the literature or in this application.

[0181] In some embodiments of the invention, Vpx and/or Vpr or a variant or part thereof are intended for packaging and introduction into a virion. In some embodiments, a gene encoding a Vpx protein, a Vpr protein, or a variant or portion of a Vpx or Vpr protein is incorporated into and expressed in the lentiviral genome if the viral particle infects a target cell.

[0182] In some embodiments, the SAMHD1 inhibitory protein is a Vpx protein that is encoded by a wild-type lentivirus, such as a wild-type primate lentivirus, or a variant or portion thereof, having SAMHD1 inhibitory activity (see, e.g., Fregoso (2013 ) PLoS Pathogens, 9:e1003496). Primate lentiviruses that encode Vpx are HIV-2/SIVsmc-related and SIVrcm/SIVmnd2-related lineage viruses (Lim et al. (2012) Cell Host & Microbe, 11:194-204), such as HIV-2, SIV black mangabey (SIVsm), SIV red-headed mangabey (SIVrcm) and SIV macaque (SIVmac), SIV mandrill (SIVmnd2), SIV drill (SIVdrl), SIV pig-tailed macaque (SIVmne).

[0183] In some embodiments, retroviral vector particles, such as lentiviral vector particles, comprise a Vpx protein encoded by HIV-2, SIVsm, SIVrcm, SIVmac, SIVmnd2, SIVdrl, SIVmne, or a variant or portion thereof, which has SAMHD1- inhibitory activity. In some embodiments, the Vpx protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 ( SIVmac Vpx) or is a functional fragment or part of such a protein. In some embodiments, the Vpx protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 ( SIVsm Vpx) or is a functional fragment or part of such a protein. In some embodiments, the Vpx protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 (SIVrcm Vpx) or is a functional fragment or part of such a protein. In some embodiments, the Vpx protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4 (HIV-2) or is a functional fragment or part of such a protein. In some embodiments, the Vpx protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16 (SIVmnd2) or is a functional fragment or part of such a protein. In some embodiments, the Vpx protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17 (SIVdrl) or is a functional fragment or part of such a protein. In some embodiments, the Vpx protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18 (SIVmne) or is a functional fragment or part of such a protein.

[0184] In some embodiments, the SAMHD1 inhibitory protein is a Vpr protein that has SAMHD1 inhibitory activity. Vpr is usually encoded by natural lentiviruses. But not all Vpr proteins have SAMHD1 inhibitory activity. For example, Vpr from SIVdeb, SIVmus, SIVagm, SIVden, SIVtal, SIVgsn, SIVmon and SIVsyk have been reported to have SAMHD1 inhibitory activity (Lim et al. (2012) Cell Host & Microbe, 11:194-204).

[0185] In some embodiments, the SAMHD1 inhibitory protein is SIVdeb Vpr, SIVmus Vpr, SIVagm Vpr, SIVden Vpr, SIVtal Vpr, SIVgsn Vpr, SIVmon Vpr, or SIVsyk Vpr, or a variant or portion thereof having SAMHD1 inhibitory activity. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5 ( SIVdeb) or is a functional fragment or part of it. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6 ( SIVmus) or is a functional fragment or part of it. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 ( SIVagm Vpr) or is a functional fragment or part of it. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8 ( SIVagm Vpr) or is a functional fragment or part of it. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 ( SIVdeb Vpr) or is a functional fragment or part of it. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10 ( SIVden Vpr) or is a functional fragment or part of it. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11 ( SIVtal Vpr) or is a functional fragment or part of it. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12 ( SIVgsn Vpr) or is a functional fragment or part of it. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 ( SIVgsn Vpr) or is a functional fragment or part of it. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14 ( SIVmon Vpr) or is a functional fragment or part of it. In some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 ( SIVsyk Vpr) or is a functional fragment or part of it.

[0186] In some embodiments, the SAMHD1 inhibitory protein, such as the Vpx or Vpr protein, is heterologous to the virus. In this context, the term "heterologous" means that the protein comes from a different viral family, class or species than the family of the class or virus species into which it is introduced. Thus, for example, in some aspects, the Vpx protein from SIVmac can be introduced into an HIV-1 vector particle. In other aspects, a Vpx protein derived from any lentivirus, such as SIVmac, can be introduced into a gamma retrovirus, such as MLV.

[0187] In some embodiments, a SAMHD1 inhibitory protein, such as a Vpx or Vpr protein, inhibits, e.g., degrades human SAMHD1, e.g., as shown in SEQ ID NO:19 (Lim et al. (2012) Cell Host & Microbe , 11:194-204). In some embodiments, the Vpx protein that degrades human SAMHD1 is encoded by HIV-2 or SIVmac, or is a variant or functional fragment or portion of a protein that degrades human SAMHD1. For example, in some embodiments, the Vpx protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 4 or is a functional fragment or portion of a human SAMHD1 degrading protein. In some embodiments, the Vpr protein that degrades human SAMHD1 may be encoded by SIVdeb or SIVmus, or is a variant or functional fragment or portion of a protein that degrades human SAMHD1. For example, in some embodiments, the Vpr protein has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5 or SEQ ID NO:6 or is a functional fragment or portion of a human SAMHD1 degrading protein.

[0188] In some embodiments of the invention, variants are proteins containing one or more amino acid deletions, insertions and/or substitutions compared to the sequence of the wild-type protein or reference protein Vpx or Vpr. In some cases, the options are either non-conservative or conservative substitutions. In some cases, sequence alignment information for Vpx proteins and/or Vpr proteins can be used to generate functional variants and variants of functional Vpx or Vpr fragments. Thus, for example, one or more conservative or non-conservative substitutions can be made at positions that differ between viruses encoding a Vpx or Vpr protein.

[0189] Thus, for example, in some embodiments of the invention, residues that are identical between the Vpx proteins encoded by SIV and HIV-2 do not vary or do not change in the variant of the Vpx protein described here. For example, the following mutations have been shown to reduce or abolish Vpx activity: deletion of the proline-rich C-terminal 11 residue, Ser13Ala, Lys84Ala/Lys85Ala, Thr17Ala, Thr28Ala, Gly86Ala/Cys87Ala, Ser13Ala/Thr17Ala/Thr28Ala, His39Ala, Tyr66Ala/Tyr69Ala /Tyr71Ala, Trp49Ala/Trp53Ala/Trp56Ala, Lys68Ala/Lys77Ala, Gln76Ala, Pro9Gly, Asn12Ala, Glu15Ala, Glu16Ala, Phe80Ala, where each residue is compared to the residues in SEQ ID NO: 1. See Goujon et al., Gramberg et al. . (2010) Journal of Virology , 84:1387-1396., Laguette et al. (2011). Therefore, in some embodiments of the invention, the Vpx protein variant does not contain a substitution at the residue position corresponding to any of the above residues in SEQ ID NO:1, which have been shown to be necessary to communicate activity. In some embodiments, the Vpx protein variant contains a conservative substitution, but not a non-conservative substitution, at a residue corresponding to any of the above residues in SEQ ID NO:1.

[0190] In some embodiments, the Vpx or Vpr protein variants may include a mutation that is not known to affect SAMHD1 inhibitory activity (see Goujon et al., J Virol , 82:12335-12345 (2008)) . Thus, for example, in some embodiments, mutations such as amino acid substitutions corresponding to one or more of the Asn26Ala, Ser52Ala, and Ser63Ala/Ser65Ala mutations in SEQ ID NO: 1 do not affect Vpx function.

[0191] In some embodiments, inhibition and/or degradation of SAMHD1 requires nuclear localization of a SAMHD1 inhibitory protein, such as a Vpx protein, a Vpr protein, or a variant or portion thereof. Vpx induces SAMHD1 ubichitinization and nuclear degradation such that both SAMHD1 and Vpx localize to the nucleus. Thus, in some embodiments of the invention, the Vpx protein, the Vpr protein, or a variant or portion thereof, contains a nuclear localization signal. In some embodiments, the Vpx protein, or variant or portion thereof, contains a nuclear localization signal corresponding to residues 62-80 or 65-72 in SEQ ID NO:4.

[0192] In some embodiments, inhibition and/or degradation of SAMHD1 includes binding to and/or interaction with DCAF. Thus, in some embodiments of the invention, the Vpx protein, the Vpr protein, or a variant or portion thereof, binds to DCAF. In some embodiments, the Vpx protein, the Vpr protein, or a variant or portion thereof, comprises a Wx4Φx2Φx3AΦxH motif (represented in SEQ ID NO: 26; Wei et al. (2012) Cell Microbiol., 14:1745-56). Thus, for example, in some embodiments, the Vpx protein, the Vpr protein, or a variant or portion thereof, contains the Wx4Φx2Φx3AΦxH motif at residues corresponding to residues 24-39 of SEQ ID NO:1 or SEQ ID NO:4. In some embodiments of the invention, the Vpx protein, the Vpr protein, or a variant or part thereof, contains the Gln (Q) motif at a position corresponding to position 76 of SEQ ID NO:1.

[0193] In some embodiments, the Vpx protein, the Vpr protein, or a variant or portion thereof, contain residues corresponding to residues corresponding to the N-terminal, helix 1, helix 2, and/or helix 3 residues of the protein (Gramberg et al. (2010) Journal of Virology, 84:1387-1396). Thus, for example, in some embodiments of the invention, the Vpx protein, the Vpr protein, or a variant or portion thereof, comprise an amino acid sequence corresponding to residues 1-86 of the amino acid sequence shown in SEQ ID NO:1, or a variant thereof with amino acid substitutions that is at least least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to residues 1-86 of SEQ ID NO: 1 and has SAMHD1 inhibitory activity.

[0194] In some embodiments, the Vpx protein, Vpr protein, or functional portion or variant thereof is packaged in a virion, allowing the proteins to inhibit, for example, degrade SAMHD1 after host cell infection prior to provirus integration. In some embodiments, the retroviral vector particle contains a p6 domain of the Gag protein that stimulates the packaging of the SAMHD1 inhibitory lentiviral protein into the retroviral vector. Vpx and Vpr are packaged through interactions with an amino acid packaging motif in the p6 region of the Gag precursor polypeptide (p6gag). In some embodiments of the invention, a sufficient level of introduction is achieved using endogenous viral p6 Gag. In some embodiments of the invention, the introduction is carried out and/or facilitated using a chimeric p6 Gag containing a packaging motif that stimulates packaging.

[0195] In some embodiments, the packaging motif corresponds to a motif that is present in the N-terminal region of P6 containing amino acids corresponding to amino acid residues 17-23 of SEQ ID NO:21 (Accola et al. (1999) J. Virol., 73:9992-9). Thus, for example, in some embodiments of the invention, the packaging motif contains the leucine-containing motif DXAXXLL (SEQ ID NO:22).

[0196] In some embodiments, the retroviral vector particle is modified by introducing a packaging motif that mediates and/or stimulates the packaging of Vpx or Vpr into the virion. In some embodiments of the invention, the packaging motif is inserted into the appropriate position of the endogenous p6 viral region. In some embodiments, the packaging motif comprises the amino acid sequences DPAVDLL (SEQ ID NO:23) or DPAVDLLKNY (SEQ ID NO:24) that correspond to amino acids 17-23 or amino acids 17-26, respectively, of SIVmac p6 shown in SEQ ID NO:21. In some embodiments, the packaging motif is any of the packaging motifs described in U.S. Published Application No. US2013/0183334. In some embodiments of the invention, the transfer of the motif to the appropriate position of HIV-1 p6, presented in SEQ ID NO:20, results in the packaging of Vpx into HIV-1 virions.

[0197] In some embodiments, the Vpx protein, Vpr protein, or variant or portion thereof is packaged without modifying the p6 region of the Gag protein. In some such embodiments, the retroviral vector is derived from HIV-1 and a sufficient level of administration is achieved using p6 Gag of HIV-1. In certain such embodiments, the SAMHD1 inhibitory protein is SIVmac Vpx, or a variant or portion thereof, that has SAMHD1 inhibitory activity.

[0198] In some embodiments, the retroviral vector is derived from HIV-1 and contains a chimeric or fusion p6 domain containing HIV-1 p6 into which a p6 SIVmac packaging motif has been inserted. Thus, for example, in some embodiments of the invention, the retroviral vector contains a chimeric or hybrid p6 domain containing, in the following order: amino acid residues corresponding to amino acids 1-14 of HIV-P6, presented in SEQ ID NO: packaging motif shown as residues 17-26 of SEQ ID NO:21 and the C-terminal portion of HIV-1 p6 corresponding to amino acid residues 21-52 of SEQ ID NO:20 (designated SIV 17-23b; see US2013/0183334; Sunseri et al (2011) J. Virol., 85:6263-6274). Thus, for example, in some embodiments of the invention, the hybrid or chimeric p6 domain contains the amino acid sequence shown in SEQ ID NO:25.

3. Nucleic acid encoding a heterologous protein

[0199] In some embodiments, the viral vector contains a nucleic acid that encodes a heterologous recombinant protein. In some embodiments, the heterologous recombinant molecule is a recombinant receptor, e.g., an antigen receptor, SB transposons, e.g., for gene silencing, transposons enclosed in a capsid, a homologous double-stranded nucleic acid, e.g., for genomic recombination, or reporter genes ( e.g. fluorescent proteins such as GFP) or luciferase).

[0200] In some embodiments, the viral vector contains a nucleic acid that encodes a recombinant receptor and/or a chimeric receptor, such as a heterologous receptor protein. A recombinant receptor, such as a heterologous receptor, may 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 may also include other receptors, such as other chimeric receptors, for example, receptors that bind to specific ligands and have transmembrane and/or intracellular signal transduction domains similar to those present in CAR.

[0201] In any such example, the nucleic acid is inserted or introduced into a region of a viral vector, such as a vector present primarily in a non-core region of the viral genome. In some embodiments, the nucleic acid is inserted into the viral genome in place of certain viral sequences to produce a replication-deficient virus.

[0202] In some embodiments, the encoded recombinant antigen receptor, e.g., CAR, is a receptor capable of specifically binding to one or more ligands on a cell or site affected by a disease, such as a cancer, infectious disease, inflammatory, or autoimmune disease or other disease or disorder, including the diseases described herein, being treated using the methods and compositions described herein.

[0203] In some embodiments, the representative antigen is or includes an ανβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic acid anhydrase 9 (CA9, also known as CAIX or G250), testicular cancer antigen, testicular cancer antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, chemokine ligand C-C motif 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 ), mutated epidermal growth factor receptor type III (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, receptor 5, like Fc receptor (FCRL5; also known as the Fc receptor homologue 5 or FCRH5), the fetal acetylcholine receptor (fetal th 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 (tyrosine kinase receptor erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, 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), L1-CAM CE7 epitope, leucine-rich repeat containing A8 family member (LRRC8A), Lewis Y antigen, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE- A6, mesothelin, c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer cell group 2 D member antigen ligands (NKG2D), melan A (MART-1), cle neural cell precise adhesion molecule (NCAM), oncofetal antigen, predominantly expressed melanoma antigen (PRAME), progesterone receptor, prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA) , orphan receptor 1 like tyrosine kinase receptor (ROR1), survivin, trophoblastic 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 antigen 1 (WT-1), a pathogen-specific antigen or an antigen associated with a universal tag and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV or other pathogens. In some embodiments, the antigens that are targeted by the receptors include antigens associated with B cell malignancy, such as any of the known B cell marker antigens. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig-kappa, Ig-lambda, CD79a, CD79b, or CD30.

[0204] In some embodiments, representative antigens are orphan tyrosine kinase receptor ROR1, tEGFR, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3 or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL -13R-alpha2, kdr, kappa light chain, Lewis Y antigen, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1 ligands, MUC16, PSCA, NKG2D, 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 antigen 1 (WT-1), cyclin such as cyclin A1 (CCNA1), and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV, HPV, and/or other pathogens and/or their oncogenic variants, and/or characteristic or specific for these pathogens.

[0205] In some embodiments, the antigen is or includes a pathogen-specific antigen or an antigen expressed by a pathogen. In some embodiments, the antigen is a viral antigen (such as a viral antigen derived from HIV, HCV, HBV, and the like), bacterial antigens, and/or parasitic antigens.

[0206] In some embodiments, antigen receptors, including CARs and recombinant TCRs, and their production and administration are described, for example, in International Patent Application Publication Nos. /071154, WO2013/123061, в публикациях заявок на патент США US2002131960, US2013287748, US20130149337, в патентах США NN: 6451995, 7446190, 8252592, 8339645, 8398282, 7446179, 6410319, 7070995, 7265209, 7354762, 7446191, 8324353, и 8479118 , and European Patent Application No. EP2537416, and/or Sadelain et al., Cancer Discov. April 2013; 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.

a. Chimeric antigen receptors

[0207] In some embodiments, the nucleic acid contained in the genome of the viral vector encodes a chimeric antigen receptor (CAR). A CAR is essentially a genetically engineered receptor with an extracellular ligand-binding domain, such as an extracellular portion containing an antibody or fragment thereof linked to one or more intracellular signal-transmitting components. In some embodiments of the invention, the chimeric antigen receptor includes a transmembrane domain and/or an intracellular domain connecting the extracellular domain and the intracellular signal-transmitting domain. Such molecules typically mimic or approximate the signal transmitted by the natural antigen receptor and/or the signal transmitted by such a receptor in combination with a co-stimulatory receptor.

[0208] In some embodiments, CARs are designed to be specific for a particular marker, such as a marker expressed in a particular cell type that is targeted for adoptive therapy, such as a cancer marker and/or any of the described antigens. Thus, a CAR typically includes one or more antigen-binding fragments, domains, or portions of an antibody, or one or more variable domains of antibodies and/or antibody molecules. In some embodiments, a CAR comprises an antigen-binding portion or portions of an antibody molecule, such as a heavy chain variable region (VH) or an antigen-binding portion thereof, or a single chain antibody fragment (ScFv) derived from a heavy chain variable region (VH) and a variable the light chain (VL) region of a monoclonal antibody (mAb).

[0209] In some embodiments, the present invention relates to engineered cells, such as T cells, that express CAR with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments of the invention, the antigen is a carbohydrate or other molecule. In some embodiments of the invention, the antigen is selectively expressed or overexpressed on cells that cause a disease or disorder, such as a tumor, or on pathogenic cells, compared to normal cells or tissues, or non-target cells or tissues. In other embodiments of the invention, the antigen is expressed on normal cells and/or is expressed on engineered cells.

[0210] In specific embodiments, the recombinant receptor, such as a chimeric receptor, comprises an intracellular signal-transmitting region that includes a cytoplasmic signal-transmitting domain or a region (also referred to as an intracellular signal-transmitting domain or region), such as a cytoplasmic (intracellular ) a region capable of inducing a primary activation signal in a T cell, e.g., a cytoplasmic signal transduction domain or a T cell receptor (TCR) component region (e.g., a cytoplasmic signal transduction domain or zeta chain region, CD3-zeta chain (CD3ζ) or a functional variant or signal-transmitting part thereof) and/or comprising an immunoreceptor tyrosine (ITAM) activation motif.

[0211] In some embodiments, the chimeric receptor also contains an extracellular ligand-binding domain that specifically binds to a ligand antigen (eg, antigen). In some embodiments, the chimeric receptor is a CAR containing an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments of the invention, the ligand, such as an antigen, is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a truncated peptide antigen, such as an intracellular protein peptide antigen that, like TCR, is recognized on the cell surface surrounded by a major histocompatibility complex (MHC) molecule.

[0212] Representative antigen receptors, including CARs and methods for constructing and introducing such receptors into cells, are described, for example, in International Patent Application Publication Nos. 123061, в публикациях заявок на патент США US2002131960, US2013287748, US20130149337, в патентах США NN: 6451995, 7446190, 8252592, 8339645, 8398282, 7446179, 6410319, 7070995, 7265209, 7354762, 7446191, 8324353, и 8479118, и в Европейской патентной application no. EP2537416 and/or described in Sadelain et al., Cancer Discov. April 2013; 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 of the invention, the antigen receptors are CARs as described in US Patent No. 7446190 and International Patent Application Publication No.: WO/2014055668 A1. Examples of CARs are those described in any of the above publications such as WO2014031687, US 8339645, US 7446179, US 2013/0149337, US Patent No.: 7446190, US Patent No.: 8389282, 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 WO2014031687, US 8339645, US 7446179, US 2013/0149337, US Patent No: 7446190 and US Patent No.: 8389282.

[0213] In some embodiments, the CAR is designed to be specific for a particular antigen (marker or ligand), such as an antigen expressed in a particular cell type that is targeted for adoptive therapy, such as a cancer marker and/or an antigen that is expected to induce a decrease in response, such as an antigen expressed on normal or unaffected cells. Thus, a CAR typically includes one or more antigen-binding molecules of an extracellular portion, such as one or more antigen-binding fragments, domains, or moieties, or one or more variable domains of antibodies and/or antibody molecules. In some embodiments, a CAR comprises an antigen-binding portion or portions of an antibody molecule, such as a single chain antibody fragment (ScFv) derived from a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody (mAb).

[0214] In some embodiments, the antibody, or antigen-binding portion thereof, is expressed on cells as part of a recombinant receptor, such as an antigen receptor. Antigen receptors are, among others, functional non-TCR antigen receptors such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment thereof having a TCR-like specificity directed against peptide-MHC complexes may also be abbreviated herein as a TCR-like CAR. In some embodiments, an extracellular antigen-binding domain specific for the MHC-peptide complex of a TCR-like CAR is associated with one or more intracellular signal-transmitting components, and in some aspects, via linkers and/or transmembrane domain(s). ov). In some embodiments of the invention, such molecules usually mimic or approximate the signal transmitted by a natural angiogenic receptor, such as TCR, and optionally, the signal transmitted by such a receptor in combination with a co-stimulatory receptor.

[0215] In some embodiments, the recombinant receptor, such as a chimeric receptor (eg, CAR), includes a ligand-binding domain that binds, eg, specifically binds, to an antigen (or ligand). Chimeric receptor target antigens are antigens that are expressed in certain diseases, disorders, or in certain types of cells that are targets for adoptive cell therapy. Such diseases and disorders are proliferative, neoplastic and malignant diseases and disorders, including cancer and tumors, including hematopoietic system malignancies, immune system malignancies such as lymphomas, leukemias and/or myelomas such as B-, T- and myeloid leukemias. , lymphomas and multiple myelomas.

[0216] In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, the antigen is a carbohydrate or other molecule. In some embodiments of the invention, the antigen (or ligand) is selectively expressed or overexpressed on cells that cause a disease or disorder, such as a tumor, or on 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 is expressed on engineered cells.

[0217] In some embodiments, the CAR comprises an antibody or an antigen-binding fragment thereof (eg, ScFv) that specifically recognizes an antigen, such as an intact antigen expressed on a cell surface.

[0218] In some embodiments, the antigen (or ligand) is a tumor antigen or cancer marker. In some embodiments, the antigen (or ligand) is or includes an ανβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic acid anhydrase 9 (CA9, also known as CAIX or G250), testicular cancer antigen, testicular cancer antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, chemokine ligand C-C motif 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 ), mutated epidermal growth factor receptor type III (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, receptor 5, like Fc receptor (FCRL5; also known as the Fc receptor homologue 5 or FCRH5), a fetal acetylcholine receptor (fetal AchR), without folate-binding block (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), G protein-coupled receptor 5D (GPCR5D), Her2/neu (tyrosine kinase erb-B2 receptor), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, human melanoma-associated high molecular weight 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, cell-adhesive L1 molecule (L1-CAM), L1-CAM CE7 epitope, leucine-rich repeat containing A8 family member (LRRC8A), Lewis Y antigen, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer cell group 2 D member antigen ligands (NKG2D), melan A (MART-1), cell-adhesion neuronal cell molecule (NCAM), oncofetal antigen, predominantly expressed melanoma antigen (PRAME), progesterone receptor, prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor orphan 1, tyrosine kinase receptor-like (ROR1), survivin, trophoblastic 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 antigen 1 (WT-1), a pathogen-specific antigen or universal tag-associated antigen, and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV, or other pathogens. In some embodiments, the antigens that are targeted by the receptors include antigens associated with B cell malignancy, such as any of the known B cell marker antigens. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig-kappa, Ig-lambda, CD79a, CD79b, or CD30.

[0219] In some embodiments, the antigen is or includes a pathogen-specific antigen or an antigen expressed by a pathogen. In some embodiments, the antigen is a viral antigen (such as a viral antigen derived from HIV, HCV, HBV, and the like), bacterial antigens, and/or parasitic antigens. In some embodiments, the CAR comprises a TCR-like antibody, such as an antibody or an antigen-binding fragment thereof (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, the antibody, or antigen-binding portion thereof, that recognizes the MHC-peptide complex may be expressed on cells as part of a recombinant receptor, such as an antigen receptor. Antigen receptors are, among others, functional non-TCR antigen receptors such as chimeric antigen receptors (CARs). Generally speaking, a CAR containing an antibody or antigen-binding fragment thereof that has a TCR-like specificity directed against peptide-MHC complexes may also be referred to as a TCR-like CAR.

[0220] The term "major histocompatibility complex" (MHC) means a protein, usually a glycoprotein, that contains a polymorphic peptide-binding site or binding groove that can, in some cases, complex with peptide antigens of polypeptides, including peptide antigens processed by cellular mechanisms. In some cases, MHC molecules may be presented or expressed on the cell surface, including a complex with a peptide, i.e., an MHC-peptide complex to present an antigen in a conformation recognized by an antigen receptor on T cells, such as a TCR or a TCR-like antibody. . Typically, class I MHC molecules are heterodimers having a transmembrane alpha chain, and in some cases three alpha domains, and a non-covalently bound β2-microglobulin. Typically, class II MHC molecules consist of two transmembrane glycoproteins, α and β, which both typically span the entire membrane. The MHC molecule may include an active part of the MHC, which contains the antigen-binding site or sites for binding to the peptide and sequences necessary for recognition by the appropriate antigen receptor. In some embodiments, MHC class I molecules deliver peptides located in the cytosol to the cell surface, where the MHC-peptide complex is recognized by T cells, such as CD8 ⁺ -T cells, and in some cases, CD4 ⁺ -T- cells. In some embodiments of the invention, MHC class II molecules deliver peptides located in the vesicular system to the cell surface, where they are usually recognized by CD4 ⁺ -T cells. Typically, MHC molecules are encoded by a group of linked loci, collectively referred to as H-2 in mice and referred to as human leukocyte antigen (HLA) in humans. Therefore, in general, the human MHC may also be referred to as the human leukocyte antigen (HLA).

[0221] The term "MHC-peptide complex" or "peptide-MHC complex", or variants thereof, means the complex or association of a peptide antigen and an MHC molecule, for example, typically through non-covalent interactions of the peptide in the binding groove or cleft of the MHC molecule. In some embodiments of the invention, the MHC-peptide complex is presented or expressed on the surface of cells. In some embodiments, the MHC-peptide complex may be specifically recognized by an antigen receptor, such as a TCR, a TCR-like CAR, or antigen-binding portions thereof.

[0222] In some embodiments, a peptide, such as a peptide antigen or a polypeptide epitope, can be associated with an MHC molecule, for example, for recognition by an antigen receptor. Typically, a peptide is derived from or derived from a fragment of a larger biological molecule, such as a polypeptide or protein. In some embodiments, the peptide is typically between about 8 and about 24 amino acids in length. In some embodiments of the invention, the peptide is from or about 9 to 22 amino acids in length and is recognized in the MHC class II complex. In some embodiments, the peptide is from or about 8 to about 13 amino acids in length and is recognized in an MHC class I complex. In some embodiments, upon recognition of the peptide in the environment of an MHC molecule, such as a TCR or TCR-like CAR, produces or triggers an activation signal to T cells to induce a T cell response such as T cell proliferation, cytokine production, cytotoxic T cell response, or other response.

[0223] In some embodiments, the TCR-like antibody, or antigen-binding portion thereof, is known or can be prepared by known methods (see, for example, US Published Applications 2002/0150914; US 2003/0223994; US 2004/0191260; US 2006/0034850; US 2007/00992530; US20090226474; US20090304679; and PCT International Application Publication WO 03/068201).

[0224] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to the MHC-peptide complex can be obtained by immunizing the host with an effective amount of an immunogen containing the specific MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of an antigen capable of binding to the MHC, such as a tumor antigen, for example, a universal tumor antigen, myeloma antigen, or other antigen 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 shape for a period of time sufficient to elicit an immune response against the three-dimensional presentation of the peptide in the binding groove of the MHC molecule. Thereafter, serum taken from the host is analyzed to determine if the desired antibodies are being produced that recognize the three-dimensional presentation of the peptide in the binding groove of the MHC molecule. In some embodiments of the invention, the obtained antibodies can be evaluated to confirm whether the antibody can distinguish the complex "MHC-peptide" from only one MHC molecule, one peptide of interest and the MHC complex and the wrong peptide. The desired antibodies can then be isolated.

[0225] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to an MHC-peptide complex can be generated using antibody library presentation techniques, such as antibody phage libraries. In some embodiments, phage display libraries of mutant Fab, ScFv, or other forms of antibodies can be obtained, for example, by introducing mutations into library members at positions of one or more CDR residues or multiple CDRs. See, for example, U.S. Published Application No. US20020150914, US2014/0294841; and Cohen CJ et al. (2003) J. Mol. Recognize. 16:324-332.

[0226] The term "antibody" is used herein in its broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including antigen-binding (Fab) fragments, F(ab') ₂ fragments , Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable regions (V _H ) capable of specifically binding to an antigen, single chain antibody fragments, including single chain variable fragments (ScFv) and single domain antibody fragments (e.g., sdAb, sdFv, nanoantibodies). This term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, multispecific antibodies such as bispecific antibodies, diantibodies, tribodies and tetrabodies, tandem di-ScFv, tandem tri-ScFv. Unless otherwise stated, the term "antibody" is intended to encompass functional fragments of an antibody. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and subclasses thereof, IgM, IgE, IgA, and IgD.

[0227] In some embodiments, antigen-binding proteins, antibodies, and antigen-binding fragments thereof specifically recognize an antigen for a full-length antibody. In some embodiments, the antibody heavy and light chains may be full length or may be an antigen-binding portion (Fab, F(ab') ₂ , Fv, or single chain Fv fragment (ScFv)). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, and in particular, for example, from IgG1, IgG2, IgG3, and IgG4, and more specifically, IgG1 (eg, human IgG1). In another embodiment, the antibody light chain constant region is selected from, for example, kappa or lambda chains, and in particular kappa.

[0228] Considered antibodies are, among others, fragments of antibodies. The term "antibody fragment" means a molecule that is not an intact antibody, but includes a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diantibodies; linear antibodies; heavy chain variable regions (V _H ), single chain antibody molecules such as scFv, and single domain single chain V _H antibodies; and multispecific antibodies formed by antibody fragments. In specific embodiments, the antibodies are single chain antibody fragments containing a heavy chain variable region and/or a light chain variable region, such as scFv.

[0229] The term "variable region" or "variable domain" means the domain of the heavy and light chains of an antibody that is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (V _H and V _L , respectively) of a native antibody typically 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., WH Freeman & Co., page 91 (2007). A single V _H or V _L domain may be sufficient to communicate antigen binding specificity. In addition , antibodies that bind to a particular antigen can be isolated using the V _H or V _L domain of an antibody that binds to the antigen to screen for a library of complementary V _L or V _H domains, respectively.See, for example, Portolano et al., J. Immunol 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0230] Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domain, or all or part of the light chain variable domain of an antibody. In some embodiments, the single domain antibody is a human single domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as a cancer marker or cell surface antigen on a target cell or lesion, such as tumor or cancer cells, e.g., any of the target antigens. described in this application or known to experts.

[0231] Antibody fragments can be obtained by various methods, including, but not limited to, proteolytic cleavage of intact antibodies, as well as production of antibodies by recombinant host cells. In some embodiments, antibodies are recombinantly produced fragments, such as fragments containing rearrangements that do not occur naturally, such as fragments in which two or more regions or chains of an antibody are joined by synthetic linkers, e.g., peptide linkers, and/or fragments that cannot be obtained by enzymatic cleavage of a natural intact antibody. In some embodiments, the antibody fragments are scFv.

[0232] A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are from non-human CDRs and all or substantially all of the FR amino acid residues are from human FRs. A humanized antibody may optionally include at least an antibody portion of an antibody constant region derived from a human antibody. The term "humanized form" of a non-human antibody means a variant of a non-human antibody that is humanized, typically to reduce immunogenicity in humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments of the invention, some FR residues in a humanized antibody are replaced with the corresponding residues of a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to restore or increase the specificity or affinity of the antibody.

[0233] Thus, in some embodiments, the chimeric antigen receptor, including TCR-like CARs, includes an extracellular portion containing an antibody or antibody fragment. In some embodiments, the antibody or fragment comprises a ScFv. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment thereof and an intracellular signal-transmitting region. In some embodiments of the invention, the intracellular signal-transmitting region contains an intracellular signal-transmitting domain. In some embodiments, the intracellular signal transduction domain is or comprises a primary signal transduction domain, a signal transduction domain capable of inducing a primary activation signal in T cells, a T cell receptor (TCR) component signal transduction domain, and/or a signal-transmitting domain containing an immunoreceptor tyrosine-based activation motif (ITAM).

[0234] In some embodiments, the extracellular portion of the CAR, such as part of an antibody, also includes a spacer, such as a spacer region between the antigen recognition component, such as ScFv, and the transmembrane domain. The spacer may be or include at least a portion of an immunoglobulin constant region, or a variant or modified variant thereof, such as a hinge region, eg an IgG4 hinge region, and/or a CH1/CL and/or an Fc region. In some embodiments of the invention, the recombinant receptor further comprises a spacer and/or a hinge region. In some embodiments, the constant region or portion thereof is a constant region or portion of a human IgG, such as IgG4 or IgG1. In some aspects of the invention, a portion of the constant region serves as a spacer region between the antigen recognition component, eg, ScFv, and the transmembrane domain. In some embodiments, the spacer has the sequence given in SEQ ID NO: 27 and is encoded by the sequence given in SEQ ID NO: 28. In some embodiments, the spacer has the sequence given in SEQ ID NO: 29. In some embodiments, of the invention, the spacer has the sequence shown in SEQ ID NO: 30.

[0235] In some embodiments of the invention, the constant region or part of it is a constant region or part of IgD. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 31. In some embodiments, the spacer has an amino acid sequence that is at least, or at least about 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of any of SEQ ID NO: 27, 29, 30 and 31.

[0236] In some embodiments, the spacer may be or include at least a portion of an immunoglobulin constant region, or a variant or modified variant, such as a hinge region, e.g., an IgG4 hinge region, and/or C _H 1/C _L and /or Fc region. In some embodiments of the invention, the recombinant receptor further comprises a spacer and/or a hinge region. In some embodiments, the constant region or portion thereof is a constant region or portion of a human IgG, such as IgG4 or IgG1. In some aspects of the invention, a portion of the constant region serves as a spacer region between the antigen recognition component, eg, ScFv, and the transmembrane domain. The spacer may be of a length that provides increased sensitivity of cells after antigen binding compared to sensitivity in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids long, or no more than 12 amino acids long. Representative spacers are spacers having a length of 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. 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 whole number between specified boundary values in any of the listed intervals. In some embodiments, the spacer region is about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Representative spacers are only the IgG4 hinge region, the IgG4 hinge region associated with the CH2 and CH3 domains, or the IgG4 hinge region associated with the CH3 domain. Representative spacers include, but are not limited to, those described in Hudeček et al., (2013) Clin., Cancer Res, 19:3153 or International Patent Application Publication WO2014/031687. In some embodiments, the spacer has the sequence given in SEQ ID NO: 27 and is encoded by the sequence given in SEQ ID NO: 28. In some embodiments, the spacer has the sequence given in SEQ ID NO: 29. In some embodiments, of the invention, the spacer has the sequence shown in SEQ ID NO: 30.

[0237] In some embodiments of the invention, the constant region or part of it is a constant region or part of IgD. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 31. In some embodiments, the spacer has an amino acid sequence that is at least or at least about 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identical to any of SEQ ID NOs: 27, 29, 30 and 31 .

[0238] A domain that binds to an extracellular ligand, such as an antigen recognition domain, is typically associated with one or more intracellular signal-transmitting components, such as signal-transmitting components that mimic activation via an antigen-receptor complex, such as the TCR, in the case of CAR, and/or through signaling through another cell surface receptor. In some embodiments of the invention, the transmembrane domain connects extracellular ligand binding domains and intracellular signal-transmitting domains. In some embodiments of the invention, the antigen-binding component (eg, antibody) is associated with one or more transmembrane and intracellular signal-transmitting regions. In some embodiments of the invention, CAR includes a transmembrane domain attached to an extracellular domain. In one embodiment of the invention, a transmembrane domain is used that is inherently associated with one of the domains in the receptor, such as CAR. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same protein or different membrane surface proteins in order to minimize interactions with other members of the receptor complex.

[0239] In some embodiments, the transmembrane domain is from a natural or synthetic source. In some aspects, if the source is natural, then the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions are regions derived from (i.e. containing at least the transmembrane region(s)) of the T cell receptor alpha, beta or zeta chain, CD28, CD3-epsilon, CD45, CD4, CD5, CD8, CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. Alternatively, in some embodiments of the invention, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain contains predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be located at each end of the synthetic transmembrane domain. In some embodiments of the invention, the binding is via linkers, spacers and/or transmembrane(s) domain(s).

[0240] In some embodiments, a short oligo- or polypeptide linker, e.g., a 2 to 10 amino acid linker, such as a linker containing glycines and serines, e.g., a glycine-serine doublet, is present and forms a link between the transmembrane domain and the cytoplasmic signal-transmitting domain of CAR.

[0241] A recombinant receptor, for example, CAR, typically includes at least one intracellular signal-transmitting component or signal-transmitting components. In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as the TCR-CD3 chain, which mediates T cell activation and T cell cytotoxicity, such as the CD3-zeta chain. Thus, in some aspects, the antigen-binding portion is associated with one or more modules that transmit a signal to cells. In some embodiments, cell signaling modules include the CD3 transmembrane domain, CD3 intracellular signal transduction domains, and/or other CD transmembrane domains. In some embodiments of the invention, the receptor, for example, CAR, also includes part of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. Thus, for example, in some aspects, the CAR or other chimeric receptor comprises a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0242] In some embodiments, upon ligation of a CAR or other chimeric receptor, the cytoplasmic domain and/or region or intracellular signal-transmitting domain and/or region of the receptor activates at least one normal effector function or response in immune cells, e.g., in a T cell engineered to express CAR. Thus, for example, in some aspects, 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 signal-transmitting domain of an antigen receptor component or co-stimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it signals an effector function. In some embodiments of the invention, intracellular signal-transmitting regions, for example, containing an intracellular domain or domains, include cytoplasmic sequences of the T-cell receptor (TCR), and in some aspects, they are also co-receptors that in their natural environment act together with such receptors to initiate signal transduction upon binding to an antigen receptor, and/or they include any derivative or variant of such molecules and/or any synthetic sequence that has the same functional activity.

[0243] In the case of natural TCR, full activation usually requires signaling not only through the TCR, but also through a co-stimulatory signal. Thus, in some embodiments of the invention, in order to stimulate full activation, a component for transmitting a secondary or costimulatory signal is also included in the CAR. In other embodiments of the invention, the CAR does not include a component for generating a costimulatory signal. In some aspects, the additional CAR is expressed in the same cell and is the component that generates the secondary or co-stimulatory signal.

[0244] In some aspects, T cell activation is described as activation mediated by at least two classes of cytoplasmic signal transduction sequences: sequences that initiate antigen-dependent primary activation via TCR (primary cytoplasmic signal transduction sequences), and sequences that which act by an antigen-independent mechanism and generate a secondary or costimulatory signal (secondary cytoplasmic signal-transmitting sequences). In some aspects, the CAR includes one or both of these signal-transmitting components.

[0245] In some aspects, CAR includes a primary cytoplasmic signal transduction sequence that regulates the primary activation of the TCR complex. Primary cytoplasmic signal transduction sequences that act in a stimulatory manner may contain signal transduction motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signal transducing sequences are sequences derived from TCR or CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD8, CD22, CD79a, CD79b and CD66d. In some embodiments, the ITAM-containing primary cytoplasmic signal transducing sequences are sequences derived from TCR or CD3 zeta, FcR gamma, or FcR beta. In some embodiments of the invention, the cytoplasmic(s) signal-transmitting(s) molecule(s) in CAR contains(at) a cytoplasmic signal-transmitting domain, part thereof, or a sequence derived from CD3 zeta.

[0246] In some embodiments, the CAR includes a signal transduction domain and/or a transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, CD27, DAP10, and ICOS. In some aspects, the same CAR includes an activating or signaling region and costimulatory components.

[0247] In some embodiments, the activating domain is included in one CAR while the co-stimulatory component is delivered by a different antigen that recognizes the other CAR. In some embodiments, CARs include activating or stimulatory CARs and costimulatory CARs that are both expressed on the same cell (see WO2014/055668). In some aspects, the CAR is a stimulating or activating CAR; and in other aspects, it is a costimulatory CAR. In some embodiments, the cells also include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine , 5(215) (December, 2013), such as a CAR that recognizes a different antigen, resulting in an activating signal delivered by a CAR recognizing the first antigen is attenuated or inhibited by the binding of the inhibitory CAR to its ligand, which will result in, for example, a reduction in undesirable effects.

[0248] In some embodiments of the invention, the intracellular signal-transmitting domain contains a transmembrane and signal-transmitting domain of CD28 associated with the intracellular domain of CD3. In some embodiments of the invention, the intracellular signal-transmitting domain contains chimeric co-stimulatory domains of CD28 and CD137 associated with the intracellular domain of CD3.

[0249] In some embodiments, the intracellular signal-transmitting domain of cytotoxic CD8 ⁺ T cells is the same as the intracellular signal-transmitting domain of CD4 ⁺ helper T cells. In some embodiments of the invention, the intracellular signal-transmitting domain of cytotoxic CD8 ⁺ -T cells is different from the intracellular signal-transmitting domain of helper CD4 ⁺ -T cells.

[0250] In some embodiments, the CAR includes one or more, eg, two or more, co-stimulatory domains and an activation domain, eg, a primary activation domain in the cytoplasmic portion. Representative CARs are the intracellular components of CD3-zeta, CD28 and 4-1BB.

[0251] In some embodiments, the recombinant(s) receptor(s), e.g., CAR, encoded by the nucleic acid(s) in the viral vectors provided herein also includes(s) one or more markers, e.g., for confirmation of transduction or construction of cells expressing the receptor, and/or selection and/or targeting of cells expressing the molecule(s)encoded(s) by the polynucleotide. In some aspects, such a marker may be encoded by another nucleic acid or other polynucleotide, which can also be introduced by genetic engineering, and usually by the same method, for example, by transduction using any of the methods described here, for example, using the same vector or a vector of the same type.

[0252] In some aspects, the marker, for example, a marker for transduction, is a protein and/or a cell surface molecule. Representative markers are truncated versions of natural, eg, endogenous markers, such as natural cell surface molecules. In some aspects, the variants have reduced immunogenicity, reduced transport function, and/or reduced signal transduction function compared to a native or endogenous cell surface molecule. In some embodiments, the marker is a truncated cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (eg, a truncated form) of CD34, NGFR, or an epidermal growth factor receptor (eg, tEGFR). In some embodiments, a nucleic acid encoding a marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, for example, T2A P2A, E2A and/or F2A. See, for example, WO2014/031687.

[0253] In some embodiments, the marker is a molecule, such as a cell surface protein, that does not naturally occur on T cells, or does not occur naturally on the surface of T cells, or a portion thereof.

[0254] In some embodiments of the invention, the molecule is a foreign molecule, for example, a foreign protein, that is, a protein that is not recognized as "self" by the immune system of the host into whose cells it is adoptively transferred.

[0255] In some embodiments, the marker has no therapeutic function and/or no effect other than being used as a marker for genetic engineering, such as the selection of successfully engineered cells. In other embodiments of the invention, the marker may be a therapeutic molecule or a molecule that provides a slightly different desired effect, for example, a ligand for interaction with a cell in vivo , such as a co-stimulatory molecule or an immune checkpoint molecule that enhances and/or attenuates cell responses after adoptive transfer and interaction with the ligand.

[0256] In some instances, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is a CAR that produces only a signal induced by the CD3 chain upon antigen binding; in some aspects, a second generation CAR is a CAR that produces just such a signal and a costimulatory signal, eg, a CAR comprising an intracellular signal transduction domain from a costimulatory receptor such as CD28 or CD137; and in some aspects, a third generation CAR is a CAR that includes multiple costimulatory domains of various costimulatory receptors.

[0257] In some embodiments, the chimeric antigen receptor includes an extracellular ligand-binding portion, such as an antigen-binding portion, such as an antibody or fragment thereof, and an intracellular domain. In some embodiments, the antibody or fragment thereof comprises a ScFv or VH of a single domain antibody and the intracellular domain comprises ITAM. In some aspects, the intracellular signal-transmitting domain includes the signal-transmitting domain of the CD3-zeta chain (CD3ζ). In some embodiments of the invention, the chimeric antigen receptor includes a transmembrane domain connecting an extracellular domain and an intracellular signal-transmitting region or domain, and/or a transmembrane domain located between them.

[0258] 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 via a spacer, such as any spacer described herein. In some embodiments of the invention, the chimeric antigen receptor contains the intracellular domain of a T-cell co-stimulatory molecule, for example, between the transmembrane domain and the intracellular signal-transmitting domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0259] In some embodiments, the CAR comprises 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 signal-transmitting domain containing the signal-transmitting portion of CD28 or a functional variant thereof and the signal-transmitting part of the CD3 zeta or its functional variant. In some embodiments, the CAR comprises 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 signal-transmitting domain containing the signal-transmitting portion of CD28 or a functional variant thereof, and the signal-transmitting part 4-1BB or its functional variant, and the signal-transmitting part CD3 zeta or its functional variant. In some such embodiments, the receptor also includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, eg, an Ig hinge, eg, an IgG4 hinge, namely, a hinge connected only by the spacer.

[0260] In some embodiments, a receptor transmembrane domain, e.g., CAR, is a transmembrane domain of human CD28 or a variant thereof, e.g., a 27 amino acid human CD28 transmembrane domain (Reg. No. P10747.1) or a transmembrane domain that contains an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence that is at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO: 34; and in some embodiments of the invention, the transmembrane domain containing part of the recombinant receptor contains the amino acid sequence shown in SEQ ID NO: 35 or an amino acid sequence that is at least or at least about 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to this sequence.

[0261] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T-cell costimulatory molecule. In some aspects of the invention, the T cell costimulatory molecule is CD28 or 4-1BB.

[0262] In some embodiments, the intracellular domain comprises an intracellular costimulatory signal-transmitting domain of human CD28, or a functional variant or portion thereof, e.g., a 41 amino acid domain and/or a domain with an LL to GG substitution at positions 186-187 of the native protein CD28. In some embodiments of the invention, the intracellular signal-transmitting region and/or domain may contain the amino acid sequence shown in SEQ ID NO: 36 or 37 or an amino acid sequence that is at least or at least about 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 36 or 37. B In some embodiments, the intracellular region and/or intracellular domain comprises an intracellular co-stimulatory signal-transmitting domain 4-1BB or a functional variant thereof, e.g., the 42 amino acid cytoplasmic domain of human 4-1BB (Reg. No. Q07011.1) or a functional variant thereof. variant or part, for example, the amino acid sequence shown in SEQ ID NO: 38 or an amino acid sequence that is at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO: 38.

[0263] In some embodiments, the intracellular signal-transmitting region and/or signal-transmitting domain comprises a human CD3 chain, optionally a CD3-zeta stimulatory signal-transmitting domain or a functional variant thereof, such as the human CD3ζ isoform 3 cytoplasmic domain of 112 amino acids (reg. No. P20963.2) and/or signal-transmitting domain of CD3 zeta, as described in US patent No. 7446190 or in US patent No. 8911993. In some embodiments of the invention, the intracellular signal-transmitting region contains the amino acid sequence presented in SEQ ID NO: 39, 40 or 41 or an amino acid sequence that is at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of SEQ ID NO: 39, 40 or 41.

[0264] In some aspects, the spacer contains only the hinge region of IgG, for example, only the hinge region of IgG4 or IgG1, for example, the spacer only from the hinge region, presented in SEQ ID NO: 27. In other embodiments of the invention, the spacer is a hinge region Ig, for example, an IgG4 hinge region associated with CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge associated with the C _H 2 and C _H 3 domains shown in SEQ ID NO: 29. In some embodiments, the spacer is an Ig hinge. , for example, an IgG4 hinge region associated only with a C _H 3 domain, such as the hinge region shown in SEQ ID NO: 30. In some embodiments, the spacer is or contains a glycine-serine rich sequence or other flexible linker, such as known flexible linkers.

[0265] For example, in some embodiments, a CAR includes: an extracellular ligand-binding portion, such as an antigen-binding portion, such as an antibody or fragment thereof, including sdAb and scFv, that specifically binds to an antigen, such as those described herein. antigen; a spacer, such as any spacer containing an Ig hinge region; a transmembrane domain, which is part of CD28 or variants thereof; an intracellular signal-transmitting domain containing the signal-transmitting portion of CD28 or a functional variant thereof; and the signal-transmitting portion of the signal-transmitting domain of CD3 zeta, or a functional variant thereof. In some embodiments, a CAR includes: an extracellular ligand-binding portion, such as an antigen-binding portion, such as an antibody or fragment thereof, including sdAb and scFv, that specifically binds to an antigen, such as the antigen described herein; a spacer, such as any spacer containing an Ig hinge region; a transmembrane domain, which is part of CD28 or a variant thereof; an intracellular signal-transmitting domain containing the signal-transmitting portion of 4-1BB or a functional variant thereof; and the signal-transmitting portion of the signal-transmitting domain of CD3 zeta, or a functional variant thereof. In some embodiments, such CAR constructs also include a T2A ribosome excision and/or a tEGFR sequence, such as downstream of the CAR.

b. T cell receptors (TCR)

[0266] In some embodiments, the recombinant molecule(s) encoded by the nucleic acid(s) is or includes a recombinant T cell receptor (TCR). In some embodiments, the recombinant TCR is specific for an antigen, and in general for an antigen present on a target cell, such as a tumor-specific antigen, an antigen expressed on specific cell types associated with an autoimmune or inflammatory disease, or an antigen derived from from a viral pathogen or a bacterial pathogen. In some embodiments, the present invention provides engineered cells, such as T cells, expressing a TCR or an antigen-binding portion thereof, that recognize a peptide epitope or T-cell epitope of a target polypeptide, such as a tumor, viral, or autoimmune protein antigen.

[0267] In some embodiments, a “T cell receptor” or “TCR” is a molecule containing α- and β-variable chains (also known as TCRα and TCRβ, respectively) or γ- and δ-variable chains ( also known as TCRα and TCRβ, respectively), or antigen-binding portions thereof, and capable of specifically binding to a peptide associated with an MHC molecule. In some embodiments of the invention, the TCR is present in the form of αβ. Typically, TCRs present in the αβ and γδ forms are essentially structurally similar, but T cells expressing these forms may have different anatomical positions or functions. The TCR may be present on the surface of the cell, or it may be present in a soluble form. Generally speaking, the TCR is present on the surface of T cells (or T lymphocytes), where it is normally responsible for recognizing antigens associated with major histocompatibility complex (MHC) molecules.

[0268] Unless otherwise stated, it should be noted that the term "TCR" encompasses full-length TCRs, as well as their antigen-binding portions or antigen-binding fragments. In some embodiments of the invention, the TCR is an intact or full-length TCR, including TCR in the form of αβ or γδ. In some embodiments, the TCR is an antigen-binding portion that is less than the length of a full-length TCR but that binds to a specific peptide associated with an MHC molecule, such as an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR may contain only a subset of the structural domains of a full-length or intact TCR, but is still able to bind to a peptide epitope, such as an MHC-peptide complex, to which the full-length TCR binds. In some instances, the antigen binding portion contains TCR variable domains, such as the TCR variable α chain and the TCR variable β chain, sufficient to form a binding site for a specific MHC-peptide complex. Generally speaking, TCR variable chains contain hypervariable regions involved in peptide, MHC and/or MHC-peptide recognition.

[0269] In some embodiments, the TCR variable domains contain hypervariable loops or hypervariable regions (CDRs), which typically make a major contribution to antigen recognition, binding capacity, and specificity. In some embodiments, the TCR CDRs, or a combination thereof, form all or almost all of the antigen-binding site of a given TCR molecule. The various CDRs found in the variable region of the TCR chain are usually separated by framework regions (FRs), which generally exhibit less inter-TCR variability compared to CDRs (see, e.g., Jores et al., Proc. Nat'l Acad. Sci U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, the CDR3 is the primary CDR responsible for antigen binding or antigen specificity, or plays the most important role of all three CDRs on a given TCR variable region in antigen recognition and/or interaction with the truncated peptide portion of the peptide complex. - MNS. In some cases, the alpha chain CDR1 may interact with the N-terminal portion of some antigenic peptides. In some cases, the CDR1 of the beta chain may interact with the C-terminal portion of the peptide. In some aspects, CDR2 makes a major contribution to, or participates in, rendering the primary CDR capable of interacting with or recognizing the MHC portion of the MHC-peptide complex. In some embodiments, the β-chain variable region may contain another hypervariable region (CDR4 or HVR4) that is primarily involved in superantigen binding and does not recognize antigen (Kotb (1995) Clinical Microbiology Reviews, 8:411-426 ).

[0270] In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each TCR chain may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments of the invention, the TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction.

[0271] In some embodiments, the TCR chain contains one or more constant domains. Thus, for example, the extracellular portion of a given TCR chain (eg, α-chain or β-chain) may contain two immunoglobulin-like domains, such as a variable domain (eg, Vα or Vβ; usually amino acids 1-116, numbered according to Kabat, Kabat et al., ʺSequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and a constant domain (e.g., α-chain constant domain or Cα, usually at positions 117-259 of the Kabat chain or β-chain constant domain or Cβ, usually at positions 117-295 of the Kabat chain) adjacent to the cell membrane. For example, in some cases, the extracellular part of the TCR, formed by two chains, contains two constant domains located near the membrane, and two variable domains located at a distance from the membrane, where each variable domain contains a CDR. The constant domain of the TCR may contain short bridging sequences in which the cysteine residue forms a disulfide bond, which will facilitate the linking of the two TCR chains. In some embodiments of the invention, the TCR may have an additional cysteine residue in each of the α- and β-chains, resulting in the TCR will contain two disulfide bonds in the constant domains.

[0272] In some embodiments, the TCR chains contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, this structure allows the TCR to associate with other molecules such as CD3 and their subunits. For example, a TCR containing constant domains with a transmembrane region can anchor a protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of the CD3 signal-transmitting subunits (eg, CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more tyrosine- or ITAM-based immunoreceptor activation motifs that confer signal transduction capability on the TCR complex.

[0273] In some embodiments, the TCR may be a two-chain heterodimer of α and β (or optionally γ and δ), or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) linked, for example, by a disulfide bond or disulfide bonds.

[0274] In some embodiments, the TCR may be derived from known TCR sequence(s), such as V α,β chain sequences, for which a substantially full-length coding sequence is readily available. Methods for obtaining full length TCR sequences, including V chain sequences, from cellular sources are well known. In some embodiments of the invention, TCR-encoding nucleic acids can be obtained from various sources, for example, by amplifying TCR-encoding nucleic acids by polymerase chain reaction (PCR) in a given cell or in cells or outside such cells, or by synthesis of publicly available TCR DNA sequences.

[0275] In some embodiments, the TCR is obtained from a biological source, for example, from cells such as T cells (eg, cytotoxic T cells), T cell hybridomas, or other commonly available sources. In some embodiments of the invention, T cells can be obtained from cells isolated in vivo . In some embodiments of the invention, the TCR is a TCR isolated from the thymus. In some embodiments, the TCR is a neoepitope-restricted TCR. In some embodiments, the T cells may be cultured T cell hybridomas or clones. In some embodiments, the TCR, or an antigen-binding portion or antigen-binding fragment thereof, can be synthesized from a known TCR sequence.

[0276] In some embodiments, the TCR is derived from a TCR identified or selected by screening a TCR candidate library against a target polypeptide antigen or target T cell epitope. TCR libraries can be obtained by amplifying the Vα and Vβ repertoire from T cells isolated from an individual, including cells present in PBMC, in the spleen, or in another lymphoid organ. In some cases, T cells can be amplified from tumor infiltrating lymphocytes (TILs). In some embodiments of the invention, TCR libraries can be obtained from CD4 ⁺ or CD8 ⁺ cells. In some embodiments of the invention, TCRs can be amplified from a healthy individual's T-cell source, ie, from libraries of normal TCRs. In some embodiments of the invention, TCRs can be amplified from a T-cell source from an individual with a disease, ie, from pathological TCR libraries. In some embodiments, the degenerate primers are used to amplify the Vα and Vβ gene repertoire, for example by RT-PCR, in samples such as human T cells. In some embodiments, scTv libraries can be constructed from native Vα and Vβ libraries in which the amplified products have been cloned or assembled to be separated by a linker. Depending on the individual and the source of the cells, the libraries may be specific for the HLA allele. Alternatively, in some embodiments, TCR libraries can be obtained by mutagenesis or diversification of the parent or backbone TCR molecule. In some aspects, TCRs undergo directed evolution, eg, via mutagenesis, eg, from the α or β chain. In some aspects, specific residues in the TCR CDRs are modified. In some embodiments, selected TCRs can be modified by affinity maturation. In some embodiments of the invention, antigen-specific T cells can be selected, for example, by screening to assess the activity of the TCR against the peptide. In some aspects, TCRs present, for example, on antigen-specific T cells, can be selected, for example, by binding activity, for example, by specific affinity or avidity for the antigen.

[0277] In some embodiments, the genetically engineered antigen receptors are recombinant T cell receptors (TCRs), and/or TCRs cloned from natural T cells. In some embodiments, the TCR is a TCR cloned from natural T cells. In some embodiments, a T cell clone having high affinity for a target antigen (eg, a cancer antigen) is identified and isolated from a patient and then introduced into cells. In some embodiments, the TCR clone for the target antigen has been generated from transgenic mice that have been introduced with human immune system genes (eg, human leukocyte antigen or HLA systems). See, for example, tumor antigens (see, for example, 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 the TCR against the target antigen (see, for example, Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349- 354). In some embodiments, the TCR or antigen-binding portion thereof has been modified or engineered. In some embodiments, directed evolution techniques have been applied to produce TCRs with altered properties, such as increased affinity for a specific MHC-peptide complex. In some embodiments, directed evolution is achieved by presentation methods, including but not limited to yeast presentation (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et 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 of the invention, the representation methods include constructing or modifying a known, parental, or reference TCR. For example, in some cases, wild-type TCRs can be used as templates for the production of mutagenic TCRs in which one or more CDR residues have been mutated, followed by selection for mutants that have the desired modified properties, such as higher affinity for the desired antigen. targets.

[0278] In some embodiments, the target polypeptide peptides for use in the production or production of the TCR of interest are known or can be easily identified by those skilled in the art. In some embodiments, suitable peptides for use in the production of TCRs or antigen-binding portions can be determined based on the presence of an HLA-restricted motif in a target polypeptide of interest, such as the target polypeptide described below. In some embodiments of the invention, peptides are identified using available computer prediction models. In some embodiments, for predicting class I MHC binding sites, such models are, but are not limited to, ProPred1 (Singh and Raghava (2001), Bioinformatics 17(12):1236-1237 2001), and SYFPEITHI (see 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 Indo-Europeans and therefore represents a suitable choice of MHC antigen for use in the production of a TCR or other molecule that binds to MHC peptide.

[0279] HLA-A0201 binding motifs and cleavage sites for proteasomes and immune proteasomes, obtained using predictive computer models, are known. For class I MHC binding site prediction, such models include, but are not limited to, ProPred1 (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237, 2001) , and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007).

[0280] In some embodiments, the TCR, or an antigen-binding portion thereof, may be a recombinantly produced natural protein, or a mutant form thereof, in which one or more properties, such as binding properties, have been modified. In some embodiments, the TCR may be from one of various animal species such as humans, mice, rats, or other mammals. The TCR may be present in a cell-bound form or in a soluble form. In some embodiments of the invention, to implement the methods described here, the TCR is obtained in a cell-bound form expressed on the cell surface.

[0281] In some embodiments, the TCR is a full length TCR. In some embodiments of the invention, the TCR is an antigen-binding portion. In some embodiments of the invention, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single chain TCR (sc-TCR). In some embodiments of the invention, dTCR or scTCR have the structures described in WO 03/020763, WO 04/033685, WO2011/044186.

[0282] In some embodiments of the invention, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments of the invention, the TCR does not contain a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR is capable of complexing the TCR with CD3. In some embodiments of the invention, any TCR, including dTCR or scTCR, can be associated with signal-transmitting domains that form an active TCR on the surface of a T cell. In some embodiments of the invention, TCR is expressed on the surface of cells.

[0283] In some embodiments, the dTCR comprises a first polypeptide wherein a sequence corresponding to the TCR α chain variable region sequence is fused to the N-terminus of a sequence corresponding to the TCR α chain extracellular constant region sequence, and a second polypeptide wherein the sequence , corresponding to the sequence of the TCR β-chain variable region, is attached to the N-terminus of the sequence corresponding to the sequence of the TCR β-chain extracellular constant region, where the first and second polypeptides are linked by a disulfide bond. In some embodiments, the bond may correspond to a native interchain disulfide bond present in native αβ dimeric TCRs. In some embodiments of the invention, interchain disulfide bonds are not present in native TCR. For example, in some embodiments, one or more cysteines can be included in the extracellular sequences of the dTCR constant region polypeptide pair. In some cases, both native and non-native disulfide bonds may be desirable. In some embodiments of the invention, the TCR contains a transmembrane sequence anchored to the membrane.

[0284] In some embodiments, the dTCR comprises a TCR α-chain comprising an α variable domain, an α constant domain and a first dimerization motif attached to the C-terminus of the α constant domain, and a TCR β-chain comprising a β variable domain, constant a β domain and a first dimerization motif attached to the C-terminus of the constant β domain, where the first and second dimerization motifs readily interact to form a covalent bond between the amino acid in the first dimerization motif and the amino acid in the second dimerization motif connecting the TCR α-chain and the β-chain TCR.

[0285] In some embodiments, the TCR is an scTCR. In general, scTCRs can be prepared by known methods, see, for example, Soo Hoo, W. F. et al. PNAS (USA) 89, 4759 (1992); Wülfing, C. and Plückthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); publication of PCT International Application No. WO 96/13593, WO 96/18105, WO99/60120, WO99/18129, WO 03/020763, WO2011/044186; and Schlueter, C. J. et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, the scTCR contains an introduced non-native interchain disulfide bond to facilitate association of TCR chains (see, for example, PCT International Application Publication No. WO 03/020763). In some embodiments, the scTCR is a non-disulfide truncated TCR in which heterologous C-terminal leucine zippers facilitate chain association (see, for example, PCT International Application Publication No. WO99/60120). In some embodiments, the scTCR comprises a TCRα variable domain covalently linked to a TCRβ variable domain via a peptide linker (see, for example, PCT International Application Publication No. WO99/18129).

[0286] In some embodiments, the scTCR comprises a first segment formed by an amino acid sequence corresponding to the variable region of the TCR α chain; second segment formed by an amino acid sequence corresponding to the TCR β-chain variable region sequence attached to the N-terminus of the amino acid sequence corresponding to the extracellular sequence of the TCR β-chain constant domain, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment .

[0287] In some embodiments, the scTCR comprises a first segment formed by an α-chain variable region sequence fused to the N-terminus of an α-chain extracellular constant domain sequence and a second segment formed by a β-chain variable region sequence fused to N - to the end of the extracellular constant and transmembrane sequence of the β-chain and, optionally, a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0288] In some embodiments, the scTCR comprises a first segment formed by a TCR β chain variable region sequence fused to the N-terminus of a β chain extracellular constant domain sequence and a second segment formed by an α chain variable region sequence fused to the N-terminus of the extracellular constant and transmembrane α-chain sequence and, optionally, a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0289] In some embodiments, the scTCR linker that binds the first and second segments of the TCR can be any linker capable of forming a single polypeptide chain while retaining TCR binding specificity. In some embodiments of the invention, the linker sequence may have, for example, the formula -P-AA-P-, where P means proline, and AA means an amino acid sequence in which the amino acids are glycine and serine. In some embodiments of the invention, the first and second segments form pairs, and therefore, the variable region sequences are targeted for such binding. Therefore, in some cases, the linker is long enough to bridge the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but not too long to block or reduce scTCR binding to the target ligand. In some embodiments of the invention, the linker may contain from or about 10 to 45 amino acids, for example, from 10 to 30 amino acids, or from 26 to 41 amino acid residues, for example, 29, 30, 31 or 32 amino acids. In some embodiments of the invention, such a linker has the formula -PGGG-(SGGGG) ₅ -P-, where P is proline, G is glycine, and S is serine (SEQ ID NO: 48). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 49).

[0290] In some embodiments, the scTCR comprises a covalent disulfide bond linking an immunoglobulin region α-chain constant domain residue to an immunoglobulin region β-chain constant domain residue. In some embodiments, there is no interchain disulfide bond in the native TCR. For example, in some embodiments of the invention, one or more cysteines can be included in the extracellular sequences of the first and second segments of the constant region scTCR polypeptide. In some cases, both native and non-native disulfide bonds may be desirable.

[0291] In some variants of dTCR or scTCR containing introduced interchain disulfide bonds, native disulfide bonds are absent. In some embodiments of the invention, one or more native cysteines forming native interchain disulfide bonds are replaced by another residue, such as serine or alanine. In some embodiments of the invention, the introduced disulfide bond can be formed by replacing non-cysteine residues in the first and second segments with cysteine. Representative non-native disulfide bonds in TCR are described in PCT International Application Publication No. WO2006/000830.

[0292] In some embodiments, the TCR, or an antigen-binding fragment thereof, has an affinity for an equilibrium binding constant for a target antigen that is within or about 10 to 5 and 10 to 12 M and all individual values and ranges between them. In some embodiments, the target antigen is an MHC-peptide complex or a ligand.

[0293] In some embodiments, the TCR-encoding nucleic acid or nucleic acids, such as α-strands and β-strands, can be amplified by PCR, cloning, or other suitable methods, and cloned into a suitable expression vector or vectors. The expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors are vectors that have been designed for amplification and propagation or for expression or both, such as plasmids and viruses.

[0294] In some embodiments, the vector may be pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala , Sweden), or the pEX series (Clontech, Palo Alto, Calif.). In some cases, bacteriophage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 may also be used. In some embodiments, plant expression vectors can be used and are pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In some embodiments, the animal expression vectors are pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments of the invention, a viral vector is used, such as a retroviral vector.

[0295] In some embodiments of the invention, recombinant expression vectors can be obtained using standard methods of recombinant DNA. In some embodiments, these vectors may contain regulatory sequences, such as transcription and translation initiation and termination codons, that are specific to the particular type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is inserted, if desired. , considering whether the given vector is a DNA or RNA vector. In some embodiments, the vector may contain a non-native promoter operably linked to a nucleotide sequence encoding a TCR or an antigen-binding moiety (or other molecule that binds to an MHC peptide). In some embodiments, the promoter may be a non-viral promoter or a viral promoter such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and a promoter present in the long terminal repeat of mouse stem cell virus. Other known promoters are also contemplated.

[0296] In some embodiments of the invention, after obtaining a T-cell clone, the alpha and beta chains of the TCR are isolated and cloned into a gene expression vector. In some embodiments, the alpha and beta TCR genes are fused via a picornavirus 2A ribosome cleavage site peptide such that both strands are co-expressed. In some embodiments of the invention, the nucleic acid encoding the TCR further includes a marker that confirms the transduction or construction of cells expressing the receptor. In some embodiments of the invention, the genetic transfer of the TCR is via retroviral or lentiviral vectors or via transposons (see, for example, 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).

[0297] In some embodiments of the invention, to obtain a vector encoding a TCR, α-chains and β-chains are subjected to PCR amplification from a total cDNA isolated from a T-cell clone expressing a TCR of interest, and cloned into an expression vector. In some embodiments of the present invention, the α chains and β chains are cloned into the same vector. In some embodiments of the invention, α chains and β chains are cloned into different vectors. In some embodiments, the resulting α-chains and β-chains are introduced into a retroviral, eg, lentiviral, vector.

With. Chimeric autoantibody receptor (CAAR)

[0298] 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, a CAAR expressing cell, such as a T cell engineered to express CAAR, can be used to specifically bind to and kill cells expressing the autoantibody, but not normal antibody expressing cells. In some embodiments of the invention, CAAR-expressing cells can be used to treat an autoimmune disease associated with the expression of autoantigens, such as autoimmune diseases. In some embodiments of the invention, CAAR-expressing cells can be targeted to B cells that will eventually produce autoantibodies and present autoantibodies on cell surfaces that are markers of these B cells as disease-specific targets in order to therapeutic treatment. In some embodiments, CAAR-expressing cells can be used to effectively target and kill pathogenic B cells in autoimmune diseases by targeting disease-causing B cells using an antigen-specific chimeric autoantibody receptor. In some embodiments, the recombinant receptor is a CAAR, such as any CAAR described in US Patent Application Publication No. 2017/0051035.

[0299] In some embodiments, the CAAR contains an autoantibody-binding domain, a transmembrane domain, and an intracellular signal-transmitting region. In some embodiments of the invention, the intracellular signal-transmitting region contains an intracellular signal-transmitting domain. In some embodiments, the intracellular signal-transmitting domain is or comprises a primary signal-transmitting domain, a signal-transmitting domain capable of inducing a primary activation signal in a T cell, a T-cell receptor (TCR) component signal-transmitting domain, and /or a signal-transmitting domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments of the invention, the intracellular signal-transmitting region contains a secondary or co-stimulatory signal-transmitting region (secondary intracellular signal-transmitting regions).

[0300] In some embodiments, the autoantibody-binding domain comprises a self-antigen or a fragment thereof. The choice of autoantigen may depend on the type of target autoantibody. Thus, for example, an autoantigen may be selected because it recognizes an autoantibody on a target cell, such as a B cell associated with a particular pathological condition, such as an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease includes pemphigus vulgaris (VP). Examples of self antigens are desmoglein 1 (Dsg1) and Dsg3.

d. Multitargeting

[0301] s. In some embodiments of the invention, the use of cells and methods includes multi-targeting strategies, such as the expression of two or more genetically engineered receptors per cell, each of which distinguishes one antigen from another antigen, and, as a rule, each of them includes different intracellular signal-transmitting Components. Such multi-targeting strategies are described, for example, in International Patent Application Publication No: WO 2014055668 A1 (which describes combinations of activating and costimulatory CAARs, e.g. targeting two different antigens present separately on non-target cells, e.g. normal cells, but present together only on the cells responsible for the disease or condition being treated) and in Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013) (which describes cells expressing both an activating and an inhibitory CAR, such as a CAR wherein the activating CAR binds to a single antigen expressed on normal or unaffected cells and on cells responsible for a disease or condition treated, and the inhibitory CAR binds to another antigen expressed only on normal cells or cells that should not be killed).

[0302] Thus, for example, in some embodiments, the cell includes a receptor that expresses a first genetically engineered antigen receptor (e.g., CAR or TCR) that is capable of inducing an activating or stimulatory signal in the cell, usually after specific binding to the first recognized antigen. receptor, for example, with the first antigen. In some embodiments, the cell further comprises a second genetically engineered antigen receptor (e.g., CAR or TCR), e.g., a chimeric costimulatory receptor, that is capable of inducing a costimulatory signal in an immune cell, typically after specific binding to a second antigen recognized by the second receptor. In some embodiments of the invention, the first antigen and the second antigen are the same. In some embodiments of the invention, the first antigen and the second antigen are different.

[0303] In some embodiments, the first and/or second genetically engineered antigen receptor (eg, CAR or TCR) is capable of inducing an activating or stimulatory signal in a cell. In some embodiments of the invention, the receptor includes an intracellular signal-transmitting component containing ITAM motifs or ITAM-like motifs. In some embodiments of the invention, the activation induced by the first receptor includes signal transduction or a change in the level of protein expression in the cell, which will lead to the initiation of an immune response, such as ITAM phosphorylation and/or the initiation of an ITAM-mediated signal transduction cascade, to the formation of an immunological synapse. and/or clustering a molecule in the vicinity of the associated receptor (e.g. CD4 or CD8, etc.), to activate one or more transcription factors such as NF-kB and/or AP-1 and/or to induce gene expression of the factors, such as cytokines, to proliferation and/or to increased survival.

[0304] In some embodiments, the first and/or second receptor includes intracellular signal-transmitting domains of co-stimulatory receptors such as CD28, CD137 (4-1BB), OX40, and/or ICOS. In some embodiments of the invention, the first and second receptors include the intracellular signal-transmitting domain of the co-stimulatory receptor, and these receptors are different from each other. In one embodiment of the invention, the first receptor contains the co-stimulatory signal-transmitting region of CD28, and the second receptor contains the co-stimulatory signal-transmitting region 4-1BB, or vice versa.

[0305] In some embodiments, the first and/or second receptor comprises an intracellular signal-transmitting domain containing ITAM or ITAM-like motifs and an intracellular signal-transmitting domain of a co-stimulatory receptor.

[0306] In some embodiments of the invention, the first receptor contains an intracellular signal-transmitting domain containing ITAM motifs or ITAM-like motifs, and the second receptor contains an intracellular signal-transmitting domain of a co-stimulatory receptor. A costimulatory signal, in combination with an activating or stimulating signal induced in the same cell, is a signal that produces an immune response, such as a reliable and sustained immune response, such as increased gene expression, secretion of cytokines and other factors, and effector T-cell mediated functions such as cell killing.

[0307] In some embodiments of the invention, neither ligation of only one first receptor, nor ligation of only one second receptor induces a reliable immune response. In some aspects, if only one receptor is ligated, then the cell becomes resistant or unresponsive to the antigen, or is inhibited and/or does not induce proliferation or secretion of factors, or does not perform effector functions. However, in some such embodiments, if a plurality of receptors are ligated, for example, after interaction with a cell expressing the first and second antigens, then the desired response is achieved, such as complete immune activation or stimulation, indicated, for example, by the secretion of one or more cytokines. , proliferation, persistence, and/or performance of an immune effector function, such as cytotoxic killing of a target cell.

[0308] In some embodiments, two receptors induce, respectively, an activating and inhibitory signal in a cell, whereby binding of one receptor to the antigen activates the cell or induces a response, and binding of the second inhibitory receptor to the antigen induces a signal that suppresses or quenches this reaction. Examples are a combination of activating CARs and inhibitory CARs or iCARs. Such a strategy can be used, for example, if an activating CAR binds to an antigen that is expressed in the event of a disease or disorder, but is also expressed on normal cells, and an inhibitory receptor binds to a separate antigen that is expressed on normal cells, but not on cells that cause disease or disorder.

[0309] In some embodiments of the invention, multi-targeting strategies are applied when an antigen associated with a particular disease or condition is expressed on an unaffected cell and/or is expressed on the engineered cell itself, or transiently (for example, after stimulation in combination with a gene engineering), or permanently. In such cases, specificity, selectivity and/or potency can be increased, and this requires ligation of two separate and individually specific antigen receptors.

[0310] In some embodiments, a plurality of antigens, such as the first and second antigens, are expressed on cells, tissues, or target sites associated with a disease or condition, such as cancer cells. In some aspects, cells, tissues, diseases or conditions refer to multiple myeloma or multiple myeloma cells. In some embodiments, one or more of the plurality of antigens is typically also expressed on cells that are undesirable as a target for cell therapy, such as normal or unaffected cells or tissues and/or engineered cells themselves. In such embodiments of the invention, ligation of multiple receptors is necessary to achieve the desired cellular response, specificity and/or efficacy.

e. Other regulatory elements

[0311] In some embodiments of the methods and compositions discussed herein, a nucleic acid sequence contained in the genome of a viral vector encoding a recombinant receptor, such as an antigen receptor, e.g., CAR, is operably linked via other genetic elements, e.g., transcription regulation sequences, including promoters or enhancers, to regulate the expression of a sequence of interest in a specific manner. In some cases, such transcriptional control sequences are sequences whose activity is temporally and/or spatially regulated. Expression control elements that can be used to regulate the expression of components are known to those skilled in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, enhancers, and other regulatory elements. In some embodiments of the invention, the nucleic acid sequence contained in the genome of the viral vector includes a plurality of expression regulation elements that regulate various encoded components, for example, various receptor components and/or signal-transmitting components, resulting in expression, function and/or the activity of the recombinant receptor and/or the engineered cell, eg, a cell expressing the engineered receptor, may be regulated, eg, inducible, inhibited, regulated and/or controlled by the user. In some embodiments of the invention, one or more vectors may contain one or more nucleic acid sequences that contain one or more expression control elements and/or one or more encoded components, whereby the nucleic acid sequences taken together can regulate expression, the activity and/or function of the encoded components, for example, a recombinant receptor or an engineered cell.

[0312] In some embodiments, a nucleic acid sequence encoding a recombinant receptor, such as an antigen receptor, such as CAR, is operably linked to internal promoter/enhancer regulatory sequences. The promoters used may be constitutive, tissue-specific, inducible and/or suitable, under appropriate conditions, to provide a high level of expression of the introduced DNA segment. The promoter may be heterologous or endogenous. In some embodiments of the invention, the promoter and/or enhancer is obtained by synthesis. In some embodiments, the promoter and/or enhancer is obtained by recombinant cloning and/or nucleic acid amplification technology.

[0313] In some cases, the nucleic acid sequence encoding the recombinant receptor contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide, such as a representative signal peptide of the GMCSFR alpha chain shown in SEQ ID NO: 51 and encoded by the nucleotide sequence shown in SEQ ID NO: 50. In some cases, the nucleotide sequence an acid encoding a recombinant receptor, such as a chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. Non-limiting representative examples of signal peptides are, for example, the GMCSFR alpha chain signal peptide shown in SEQ ID NO: 51 and encoded by the nucleotide sequence shown in SEQ ID NO: 50, or the CD8 alpha signal peptide shown in SEQ ID NO: 52 .

[0314] In some embodiments, the polynucleotide encoding the recombinant receptor contains at least one promoter that is operably linked to regulate expression of the recombinant receptor. In some embodiments of the invention, the polynucleotide contains two, three or more promoters operably linked to regulate the expression of the recombinant receptor.

[0315] In some cases, when nucleic acid molecules encode two or more different polypeptide chains, for example, a recombinant receptor and a marker, each polypeptide chain may be encoded by a separate nucleic acid molecule. Thus, for example, two separate nucleic acids are considered, each of which can be separately transferred or introduced into a cell for expression in the cell. In some embodiments, a nucleic acid encoding a recombinant receptor and a nucleic acid encoding a marker are operably linked to the same promoter and separated, but not necessarily, by an internal ribosome binding site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes cleavage of the ribosome, and which is, but not necessarily, T2A, but P2A, but E2A or F2a. In some embodiments, the nucleic acids encoding the marker and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters. In some embodiments of the invention, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are present at or inserted into different positions in the cellular genome. In some embodiments of the invention, a polynucleotide encoding a recombinant receptor is introduced into a composition containing cultured cells, for example, by retroviral transduction, transfection, or transformation.

[0316] In some embodiments, for example, where the polynucleotide comprises a first and a second nucleic acid sequence, the sequences encoding each of the different polypeptide chains may be operably linked to a promoter and the chains may be the same or different. In some embodiments of the invention, the nucleic acid molecule may contain a promoter that drives the expression of two or more different polypeptide chains. In some embodiments of the invention, such nucleic acid molecules can be multicistronic (bicistronic or tricistronic, see, for example, US patent No. 6060273). In some embodiments of the invention, the transcription units can be designed as a bicistronic unit containing an IRES (internal ribosome binding site), which allows co-expression of gene products (for example, encoding a marker and a recombinant receptor) through a single messenger promoter. Alternatively, in some cases, a single promoter may regulate the expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (eg, encoding a marker and a recombinant receptor) separated from each other by sequences encoding a self-cleaving peptide (eg , sequence 2A), or a protease recognition site (eg, furin). Thus, the ORF encodes a single polypeptide, which, either during (in the case of 2A) or after translation, is processed into separate proteins. In some cases, a peptide such as T2A can confer on the ribosome the ability to block (ribosomal excision) the synthesis of a peptide bond at the C-terminus of the 2A element, resulting in separation of 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)). Various elements of 2A are known. Examples of 2A sequences that can be used in the methods and systems disclosed herein are, but are not limited to, 2A sequences of foot and mouth disease virus (F2A, e.g. SEQ ID NO: 47), equine rhinitis virus A (E2A, e.g. SEQ ID NO : 46), Thosea asigna virus (T2A, e.g., SEQ ID NO: 32 or 43), and porcine techovirus-1 (P2A, e.g., SEQ ID NO: 44 or 45) as described in US Patent Publication No. 20070116690.

[0317] Any of the recombinant receptors described herein may be encoded by polynucleotides containing one or more nucleic acid sequences encoding recombinant receptors, in any combination or rearrangement. Thus, for example, one, two, three or more polynucleotides may encode one, two, three or more different polypeptides, such as recombinant receptors. In some embodiments of the invention, one vector or one construct contains a sequence encoding a marker, and a separate vector or a separate construct contains a nucleic acid sequence encoding a recombinant receptor, for example, CAR. In some embodiments, a nucleic acid encoding a marker and a nucleic acid encoding a recombinant receptor are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the recombinant receptor is located downstream of the nucleic acid encoding the marker.

[0318] In some embodiments, the vector backbone contains a nucleic acid sequence encoding one or more markers. In some embodiments, the one or more markers are a transduction marker, a surrogate marker, and/or a selection marker.

[0319] In some embodiments, the marker is a transduction marker or a surrogate marker. A transduction marker or surrogate marker can be used to detect cells into which a polynucleotide has been introduced, for example a polynucleotide encoding a recombinant receptor. In some embodiments of the invention, the transduction marker can identify or confirm cell modification. In some embodiments, the surrogate marker is a protein that has been designed to be co-expressed on the cell surface along with a recombinant receptor, such as CAR. In specific embodiments of the invention, such a surrogate marker is a surface protein that has been modified to have little or no activity. In some embodiments, the surrogate marker is encoded by the same polynucleotide that encodes the recombinant receptor. In some embodiments, a nucleic acid sequence encoding a recombinant receptor is operably linked to a nucleic acid sequence encoding a marker and is separated, but not necessarily, by an internal ribosome binding site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes cutting the ribosome, and which is a 2A sequence such as T2A, P2A, E2A or F2A. In some cases, external marker genes can be used in combination with the engineered cell to detect or select cells, and in some cases also to induce cell suicide.

[0320] Representative surrogate markers can be truncated cell surface polypeptides such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (EGFRt, representative sequence of EGFRt shown in SEQ ID NO: 33 or 42), or membrane prostate specific antigen (PSMA) or a modified form thereof. EGFRt may contain an epitope recognized by the cetuximab antibody (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule that can be used to identify or select cells that have been engineered using the EGFRt construct and a recombinant receptor such as a chimeric antigen receptor ( CAR), and/or to remove or separate cells expressing the receptor. See US Patent No. 8802374 and Liu et al., Nature Biotech. April 2016; 34(4): 430-434). In some aspects, the marker, eg, a surrogate marker, includes all or part (eg, a truncated form) of CD34, NGFR, or an epidermal growth factor receptor (eg, tEGFR). In some embodiments, a nucleic acid encoding a marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, such as T2A. Thus, for example, the marker and optionally the linker sequence may be any sequence described in PCT Application Publication No. WO2014031687. Thus, for example, the marker may be a truncated EGFR (tEGFR) which is optionally attached to a linker sequence, such as a cleavable T2A linker sequence. A representative truncated EGFR polypeptide (e.g., tEGFR) contains the amino acid sequence shown in SEQ ID NO: 33 or 42, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 33 or 42.

[0321] In some embodiments, the marker is or comprises a fluorescent protein, such as a green fluorescent protein (GFP), an enhanced fluorescence green fluorescence protein (EGFP), such as supercoiled GFP, red fluorescent protein (RFP) such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, blue fluorescent protein (CFP), blue fluorescent protein (BFP), fluorescent protein in the blue spectral range with increased fluorescence intensity (EBFP), and protein, fluorescent in the yellow range of the spectrum (YFP), and their variants, including species variants, monomeric variants and codon-optimized and/or activated variants of fluorescent proteins. In some embodiments, the marker is or includes an enzyme such as luciferase, E. coli LacZ gene, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Representative light-emitting reporter genes are luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0322] In some embodiments of the invention, the marker is a selection marker. In some embodiments, the selection marker is or includes a polypeptide conferring resistance to exogenous agents or drugs. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene conferring antibiotic resistance to the mammalian cell. In some embodiments, the selection marker is or includes a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or a modified form thereof.

[0323] Additional nucleic acids, for example, genes for introduction, are genes that increase the effectiveness of therapy, for example, by stimulating the viability and/or function of the transferred cells; genes encoding a genetic marker for selection and/or evaluation of cells, for example, for evaluation of survival or localization in vivo ; genes to improve safety, for example, by obtaining cells susceptible to negative selection in vivo , as described by Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also PCT/US91/08442 and PCT/US94/05601, Lupton et al., which describe the use of bifunctional selective hybrid genes resulting from the addition of a dominant positive selectable marker to a negative selectable marker. See, for example, Riddell et al., U.S. Patent No. 6040177, columns 14-17.

[0324] In some embodiments, the promoter and/or enhancer may be a promoter and/or enhancer that is inherently associated with a nucleic acid sequence, as they can be obtained by isolating 5' non-coding sequences located upstream coding segment and/or exon. Alternatively, in some embodiments, the nucleic acid coding segment may be under the control of a recombinant and/or heterologous promoter and/or enhancer that is not normally associated with the nucleic acid coding sequence in nature. For example, representative promoters used to construct recombinant DNA include, but are not limited to, β-lactamase (penicillinase), lactose, tryptophan (trp), RNA polymerase (Pol) III promoters, including human and mouse U6 Pol promoters. III, as well as human and mouse H1 Pol III RNA promoters; RNA polymerase (pol) II promoters; the cytomegalovirus immediate early promoter (pCMV), the elongation factor 1 alpha (EF-1 alpha) promoter, and the Rous sarcoma virus (pRSV) long terminal repeat promoter systems. In some embodiments, the promoter may be derived from, for example, the genomes of viruses such as polyoma virus, fowl pox virus, adenovirus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40 ). The promoter may also be, for example, a heterologous mammalian promoter, such as an actin or immunoglobulin promoter, a heat shock protein promoter, or a promoter normally associated with a native sequence, provided such promoters are compatible with the target cell. In one embodiment of the invention, the promoter is a natural viral promoter in a viral expression system.

[0325] In some embodiments of the invention, the promoter may be constitutively active. Non-limiting examples of constitutive promoters that can be used are the ubiquitin promoter (US Patent No. 5510474; WO 98/32869), CMV (Thomsen et al., PNAS 81:659, 1984; US Patent No. 5168062), beta-actin (Gunning et al. 1989 Proc. Natl. Acad. Sci. USA 84:4831-4835) and pgk (see e.g. Adra et al. 1987 Gene 60:65-74; Singer-Sam et al. 1984 Gene 32 :409-417 and Dobson et al 1982 Nucleic Acids Res 10:2635-2637).

[0326] In some embodiments, the promoter may be a tissue-specific promoter and/or a target cell-specific promoter. In some embodiments of the invention, the promoters can be selected so that they induce the expression of the sequence of interest. A number of systems are known to induce expression, including a tetracycline responsive system, a lac repressor operator system, and promoters sensitive to various environmental or physiological changes, including heat shock promoters, metal ions such as metallothionein, interferons, hypoxia promoters. , steroids such as the progesterone receptor or glucocorticoid promoter, and a radiation sensitive promoter such as the VEGF promoter. In some embodiments, a tetracycline (tet)-regulated system that relies on the inhibitory effect of Escherichia coli 's tet (tetr) repressor on the tet operator sequence (TECO) can be modified for use in mammalian systems and as an expression regulation element in expression clusters. These systems are well known. (See Goshen and Badgered, Proc. Natl. Acad. Sci. USA 89: 5547-51 (1992), Shockett et al., Proc. Natl. Acad. Sci. USA 92:6522-26 (1996), Lindemann et al., Mol. Med. 3:466-76 (1997)).

[0327] A combination of promoters can also be used to achieve the desired expression of a gene of interest. A person skilled in the art can select a promoter based on the desired expression pattern of the gene in the organism or target cell of interest.

[0328] In some embodiments, an enhancer may also be present in the viral construct to increase the expression level of the gene of interest. Enhancers are typically cis-acting nucleic acid elements, generally about 10 to 300 bp in length, that act on a promoter to increase its level of transcription. Many enhancers in viral genomes such as HIV or CMV are known. For example, the CMV enhancer (Boshart et al. Cell, 41:521, 1985) can be used. Other examples are, for example, the SV40 enhancer downstream of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer downstream of the replication origin, and adenovirus enhancers. In some cases, the enhancer is derived from a mammalian gene, such as an enhancer for globin, elastase, albumin, alpha-fetoprotein, or insulin. An enhancer may be used in combination with a heterologous promoter. The enhancer may be spliced into the vector at position 5' or 3' in the polynucleotide sequence encoding the gene of interest, but is typically located 5' from the promoter. The person skilled in the art can select the appropriate enhancer based on the desired expression pattern.

[0329] The viral vector genome may also contain additional genetic elements. The types of elements that can be included in the designs are not limited in any way and can be chosen by the person skilled in the art.

[0330] For example, a signal can be included to facilitate entry of the viral genome into the nucleus of the target cell. An example of such a signal is the HIV-1 capture sequence (in some cases, said sequence is called the capture sequence). In addition, the vector genome may contain one or more genetic elements designed to enhance the expression of a gene of interest. In some embodiments, the genome contains a post-transcriptional regulatory element (PRE) or a modified form thereof that has post-transcriptional activity. For example, in some embodiments, the woodchuck hepatitis virus post-transcriptional sensing element (WPRE) can be introduced into the construct (Zufferey et al. 1999 . J. Virol. 74:3668-3681; Deglon et al. 2000 . Hum. Gene Ther 11:179-190). In some embodiments of the invention, the vector genome does not contain sequences for capture and/or WPRE. In some embodiments, the vector genome contains a mutated or defective capture and/or WPRE sequence.

[0331] In some cases, more than one open reading frame encoding individual heterologous proteins may be included. For example, in some embodiments of the invention, if a reporter and/or detectable and/or selective gene is included in the expression construct, then an internal ribosome binding site (IRES) sequence may be included. Typically, additional genetic elements are operably linked to and independently regulated by the promoter/enhancer. The additional genetic element may be a reporter gene, selectable marker, or other desired gene.

[0332] In some embodiments of the invention, various other regulatory elements may include a region of initiation and/or termination of transcription. Expression vectors may also contain sequences for terminating transcription and for stabilizing the mRNA. Such sequences are known and often occur naturally in the 5' and sometimes in the 3' untranslated regions of eukaryotic or viral DNA or cDNA. Examples of transcription termination regions include, but are not limited to, polyadenylation signal sequences. Examples of polyadenylation signal sequences include, but are not limited to, poly(A) bovine growth hormone (BGH), poly(A) late SV40, poly(A) rabbit beta-globin (RBG), poly(A) thymidine kinase (TK) and any of their options.

[0333] In some embodiments of the invention, regulatory elements may include regulatory elements and/or systems that provide controlled expression and/or activity of a recombinant receptor, for example, CAR. In some embodiments of the invention, regulated expression and/or activity is achieved through the configuration of the recombinant receptor containing or under the control of specific regulatory elements and/or systems. In some embodiments of the invention, one or more additional receptors can be used in expression regulation systems. In some embodiments of the invention, systems for regulating expression may include systems that require exposure to a specific ligand or binding to a specific ligand that can regulate the expression and/or activity of the recombinant receptor. In some embodiments, regulated expression of a recombinant receptor, such as CAR, is achieved by a regulated transcription factor release system, such as a modified Notch signal transduction system (see, for example, Roybal et al., Cell (2016) 164:770- 779; Morsut et al., Cell (2016) 164:780-791). In some embodiments of the invention, the regulation of the activity of the recombinant receptor is achieved by introducing an additional agent that can induce conformational changes and/or multimerization of polypeptides, for example, the recombinant receptor. In some embodiments, the additional agent is a chemical inducer (see, for example, US Patent Publication No. 2016/0046700, Clackson et al. (1998) Proc. Natl. Acad. Sci. USA. 95(18):10437 -42 Spencer et al (1993) Science 262(5136):1019-24 Farrar et al (1996) Nature 383(6596):178-81 Miyamoto et al (2012) Nature Chemical Biology 8(5 ): 465-70 Erhart et al (2013) Chemistry and Biology 20(4): 549-57).

4. Preparation of viral vector particles

[0334] The viral vector genome is typically constructed in the form of a plasmid that can be transferred to a packaging or producer cell line. A number of different known methods can be used to produce retroviral particles whose genome contains an RNA copy of the viral vector genome. In some embodiments of the invention, at least two components are involved in the creation of a virus-based gene delivery system, one of which is packaging plasmids containing structural proteins, as well as enzymes necessary for the formation of a viral vector particle, and the second is the viral vector itself, that is, the transferred genetic material. In the design of one or both of these components, biosafety agents may be included.

[0335] In some embodiments, the packaging plasmid may contain all retroviral proteins, such as HIV-1, that are not envelope proteins (Naldini et al., 1998). In other embodiments of the invention, viral vectors may not contain additional viral genes, such as genes that are associated with virulence, such as vpr, vif, vpu and nef and/or Tat, i.e., the primary transactivator of HIV. In some embodiments of the invention, lentiviral vectors, such as HIV-based lentiviral vectors, contain only three genes of the parent virus: gag, pol and rev, which reduce or eliminate the possibility of recombination of the wild-type virus.

[0336] In some embodiments, the viral vector genome is introduced into a packaging cell line containing all components necessary to package the viral genomic RNA transcribed from the viral vector genome into viral particles. Alternatively, the viral vector genome may include one or more genes encoding viral components in addition to one or more sequences, such as recombinant nucleic acids of interest. However, in some aspects, to prevent genome replication in the target cell, endogenous viral genes required for replication are removed and introduced separately into the packaging cell line.

[0337] In some embodiments of the invention, the packaging cell line is transfected with one or more plasmid vectors containing components necessary for the formation of particles. In some embodiments, the packaging cell line is transfected with a plasmid containing the viral vector genome, including an LTR, a cis-acting packaging sequence, and a sequence of interest, i.e., a nucleic acid encoding an antigen receptor, such as CAR, and one or more helper plasmids encoding enzymatic and/or structural components of the virus, such as gag, pol and/or rev. In some embodiments of the invention, multiple vectors are used to separate the various genetic components that generate retroviral vector particles. In some such embodiments, the introduction of individual vectors into the packaging cell reduces the likelihood of recombination events that may otherwise generate replication-competent viruses. In some embodiments, a single plasmid vector having all retroviral components can be used.

[0338] In some embodiments, a retroviral vector particle, such as a lentiviral vector particle, is pseudotyped to improve the transduction efficiency of host cells. Thus, for example, in some embodiments, a retroviral vector particle, such as a lentiviral vector particle, is pseudotyped with VSV-G glycoprotein, which provides a wider range of host cells that can be transduced. In some embodiments, the packaging cell line is transfected with a plasmid or polynucleotide encoding a non-native envelope glycoprotein such that it includes xenotropic, polytropic, or amphotropic envelopes such as Sindbis virus envelope, GALV, or VSV-G.

[0339] In some embodiments, the packaging cell line contains components, including viral regulatory and structural proteins, that are required in trans to package the viral genomic RNA into lentiviral vector particles. In some embodiments, the packaging cell line can be any cell line capable of expressing lentiviral proteins and producing functional lentiviral vector particles. In some aspects, suitable packaging cell lines are 293 (ATCC CCL X), 293T, HeLA (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th ( ATCC CRL 1430).

[0340] In some embodiments, the packaging cell line stably expresses the viral(e) protein(s). Thus, for example, in some aspects of the invention, a packaging cell line can be constructed that contains gag, pol, rev, and/or other structural genes, but not LTR and packaging components. In some embodiments, the packaging cell line can be transiently transfected with nucleic acid molecules encoding one or more viral proteins, together with a viral vector genome containing a nucleic acid molecule encoding a heterologous protein and/or a nucleic acid encoding an envelope glycoprotein.

[0341] In some embodiments, viral vectors and packaging and/or helper plasmids are introduced by transfection or infection of a packaging cell line. The packaging cell line produces viral vector particles containing the viral vector genome. Methods for transfection or infection are well known. Non-limiting examples are methods using calcium phosphate, DEAE-dextran and methods of lipofection, electroporation and microinjection.

[0342] By introducing the recombinant plasmid and the retroviral LTRs and packaging sequences into a dedicated cell line (e.g., by calcium phosphate precipitation), the packaging sequences can package the recombinant plasmid RNA transcript into viral particles, which can then be secreted into the culture medium. Then, in some embodiments of the invention, the medium containing the recombinant retroviruses is collected, optionally concentrated and used for gene transfer. For example, in some aspects, after co-transfection of the packaging plasmids and transfer of the vector to the packaging cell line, the viral vector particles are isolated from the culture medium and titrated by standard methods used by those skilled in the art.

[0343] In some embodiments, a retroviral vector, such as a lentiviral vector, can be produced in a packaging cell line, such as a representative HEK 293T cell line, by introducing plasmids to generate lentiviral particles. In some embodiments, the packaging cell is transfected with a polynucleotide encoding gag and pol and a polynucleotide encoding a recombinant receptor, such as an antigen receptor, for example, CAR, and/or the cell contains these components. In some embodiments, the packaging cell line is optionally and/or additionally transfected with a polynucleotide encoding the rev protein and/or the line contains such a polynucleotide. In some embodiments, the packaging cell line is optionally and/or additionally transfected with a polynucleotide encoding a non-native envelope glycoprotein, such as VSV-G, and/or the line contains such a polynucleotide. In some such embodiments, approximately two days after transfection of cells, such as HEK293T cells, the cell supernatant contains recombinant lentiviral vectors that can be isolated and titrated.

[0344] Reconstituted and/or derived retroviral vector particles can be used to transduce target cells by the methods described. The viral RNA is then reverse transcribed in the target cells, imported into the nucleus, and stably integrated into the host genome. One or two days after integration of the viral RNA, expression of a recombinant protein, for example an antigen receptor such as CAR, can be detected.

C. Incubation

[0345] In some embodiments, contemplated methods include methods for transducing cells by contacting, for example, incubating, a cell composition comprising a plurality of cells (hereinafter also referred to as the "original composition") with (1) a viral particle. In some embodiments of the invention, the original composition is or includes primary cells taken from the individual, for example, enriched cells and/or cells selected from the individual.

[0346] In some embodiments of the invention, the original composition contains primary cells taken from the individual. In some aspects, the sample is a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukapheresis product.

[0347] In some embodiments, prior to cell selection and/or transduction, a sample containing primary cells is contacted ex vivo with serum or plasma, and/or the sample contains serum or plasma at a concentration of at least or at least at least about 10% (v/v), at least or at least about 15% (v/v), at least or at least about 20% (v/v), at least or at least about 25% (v/v), at least or at least about 30% (v/v), at least or at least about 35% (v/v), at least or at least about 40% (v/v) or at least or at least about 50%. In some embodiments, the sample contains serum or plasma at a concentration that is or is about or at least about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33% , 34%, or 35% (v/v). In some embodiments, the serum or plasma is human. In some embodiments, the serum or plasma is autologous to the individual. In some embodiments of the invention, before the selection and/or transduction of cells, the sample containing the primary cells is contacted with an anticoagulant, or this sample contains an anticoagulant. In some embodiments, the anticoagulant is or contains a free cytarate ion, such as anticoagulant citrate and dextrose solution A (ACD-A).

[0348] In some embodiments, prior to cell collection and/or transduction, the sample is maintained at or about 2°C to 8°C for 48 hours, such as 12 hours, 24 hours, or 36 hours.

[0349] In some embodiments of the invention, the original composition contains T cells and/or enriched in T cells, including CD4 ⁺ - and/or CD8 ⁺ -T cells. In some aspects, enrichment can be accomplished by affinity selection by incubating the primary cells with one or more affinity selection or maturation reagents that specifically bind to a cell surface molecule expressed on a subset of the primary cells that will promote enrichment of the primary cells based on their binding to the selection reagent. In some embodiments of the invention, enrichment can be accomplished by incubating the cells with antibody-coated particles, such as magnetic spheres.

[0350] In some embodiments of the invention, the initial composition contains more than or more than about 75%, 80%, 85%, 90%, 95% or more T cells obtained from a sample taken from an individual. In some aspects, prior to incubation, no more than 5%, 10%, 20%, 30%, or 40% of the T cells of the original composition are activated cells, express a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB; contain an intracellularly expressed cytokine selected from the group consisting of IL-2, IFN-gamma, TNF-alpha, are present in the G1 phase or in a later phase of the cell cycle, and/or are capable of proliferating.

[0351] In some embodiments, the initial composition containing such cells, for example, cells that have not been subjected to ex vivo stimulation with a stimulating agent or agents prior to incubation and / or contact, is a composition in which more than 20%, 30%, 40%, 50%, 60%, 70% or more of the cells express the low density lipid receptor (LDL-R). In some embodiments of the invention, the initial composition is enriched with T cells and/or selected for T cells, such as CD4 ⁺ - and / or CD8 ⁺ - T cells and before the specified incubation, more than 20%, 30%, 40 %, 50%, 60%, 70% or more of T cells express low density lipid receptor (LDL-R).

[0352] In some embodiments of the invention, during or during at least part of the process of incubation and/or contact, the original composition may include one or more cytokines. In some embodiments, the cytokine is selected from IL-2, IL-7, or IL-15. In some embodiments of the invention, the cytokine is a recombinant cytokine. In some embodiments of the invention, the concentration of the cytokine in the original composition is independently from or about 1 IU/ml to 1500 IU/ml, for example, from or about 1 IU/ml to 100 IU/ml, from 2 IU/ml to 50 IU/ml, 5 IU/ml to 10 IU/ml, 10 IU/ml to 500 IU/ml, 50 IU/ml to 250 IU/ml or 100 IU/ml to 200 IU/ml, from 50 IU/ml to 1500 IU/ml, 100 IU/ml to 1000 IU/ml or 200 IU/ml to 600 IU/ml. In some embodiments of the invention, the concentration of the cytokine in the original composition is independently at least or at least about 1 IU/ml, 5 IU/ml, 10 IU/ml, 50 IU/ml, 100 IU/ml, 200 IU/ml , 500 IU/ml, 1000 IU/ml or 1500 IU/ml. In some aspects, an agent capable of activating an intracellular signal-transmitting domain of the TCR complex, such as an anti-CD3 and/or anti-CD28 antibody domain, may also be included during or during at least part of the incubation process or after incubation.

[0353] In some embodiments of the invention, during or during at least part of the process of incubation and/or contact, the original composition may contain serum. In some embodiments, the serum is fetal bovine serum. In some embodiments of the invention, the serum is human serum. In some embodiments, the serum is present in the original composition at a concentration of from or about 0.5% to 25% (v/v), from 1.0% to 10% (v/v), or from 2.5% to 5.0% (v/v), inclusive. In some embodiments, whey is present in the original composition at a concentration of at least or at least about 0.5% (v/v), 1.0% (v/v), 2.5% (v/v) , 5% (V/V) or 10% (V/V).

[0354] In some embodiments of the invention, during or during at least part of the process of incubation and / or contact, the original composition does not contain and / or essentially does not contain serum. In some embodiments of the invention, during or during at least part of the process of incubation and/or contact, the original composition is incubated and/or enter into contact in the absence of serum. In specific embodiments of the invention, during or during at least part of the process of incubation and/or contact, the original composition is incubated and/or entered into contact in serum-free medium. In some embodiments of the invention, the serum-free medium is a cell culture medium having a defined and/or well-defined composition. In some embodiments, the serum free medium is a controlled culture medium that has been treated, eg filtered, to remove inhibitors and/or growth factors. In some embodiments of the invention, the serum-free medium contains proteins. In some embodiments of the invention, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins and/or binding factors. In some embodiments, the serum-free medium contains proteins, for example, albumin, such as bovine serum albumin, human serum albumin, and/or recombinant albumin. In some embodiments, the serum-free medium contains a basal medium, such as DMEM or RPMI 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources. In some embodiments, the serum-free medium is supplemented with components such as, but not limited to, albumin, lipids of certain chemical composition, growth factors, insulin, cytokines, and/or antioxidants. In some embodiments of the invention, the serum-free medium is prepared so that it supports the growth, proliferation, health, homeostasis of cells of a certain type, such as cells of the immune system, T cells, and/or CD4 ⁺ - and CD8 ⁺ -T cells.

[0355] In some embodiments of the invention, during or during at least part of the process of incubation and/or contact, the original composition contains N-acetylcysteine. In some embodiments of the invention, the concentration of N-acetylcysteine in the original composition is from or about 0.4 mg/ml to 4 mg/ml, from 0.8 mg/ml to 3.6 mg/ml, or from 1.6 mg /ml up to 2.4 mg/ml, inclusive. In some embodiments of the invention, the concentration of N-acetylcysteine in the original composition is at least or at least approximately or approximately 0.4 mg / ml, 0.8 mg / ml, 1.2 mg / ml, 1.6 mg / ml, 2.0 mg/ml, 2.4 mg/ml, 2.8 mg/ml, 3.2 mg/ml, 3.6 mg/ml or 4.0 mg/ml.

[0356] In some embodiments of the invention, the concentration of cells of the original composition is from or about 1.0 x 10 ⁵ cells/ml to 1.0 x 10 ⁸ cells/ml, for example, at least or at least about or about 1.0 x 10 ⁵ cells/ml, 5 x 10 ⁵ cells/ml, 1 x 10 ⁶ cells/ml, 5 x 10 ⁶ cells/ml, 1 x 10 ⁷ cells/ml, 5 x 10 ⁷ cells/ml or 1 x 10 ⁸ cells/ml.

[0357] In some embodiments of the invention, viral particles are obtained at a certain ratio of copies of viral vector particles or infectious units (IU) per total number of cells (IU/cell) in the original composition or total number of transduced cells. Thus, for example, in some embodiments of the invention, viral particles are present at the time of contact in an amount that is either about or at least about or about, or at least or about 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 or 60 IU viral vector particles per cell.

[0358] In some embodiments of the invention, the titer of viral vector particles is in the range or approximately in the range from 1 × 10 ⁶ IU/ml to 1 × 10 ⁸ IU/ml, for example, within or approximately within the range of 5 × 10 ⁶ IU/ml up to 5 x 10 ⁷ IU/ml, e.g. at least 6 x 10 ⁶ IU/ml, x 10 ⁶ IU/ml, 8 x 10 ⁶ IU/ml, 9 x 10 ⁶ IU/ml, 1 x 10 ⁷ IU / ml, 2 × 10 ⁷ IU / ml, 3 × 10 ⁷ IU / ml, 4 × 10 ⁷ IU / ml or 5 × 10 ⁷ IU / ml.

[0359] In some embodiments, transduction can be achieved with a MOI of less than 100, eg, typically less than 60, 50, 40, 30, 20, 10, 5, or less.

[0360] In some embodiments of the invention, the method includes contact or incubation, for example, mixing cells with viral particles. In some embodiments of the invention, the contact is carried out for a period of time from 30 minutes to 72 hours, for example, from 30 minutes to 48 hours, from 30 minutes to 24 hours, or from 1 hour to 24 hours, for example, at least or at least least for approximately 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours or more.

[0361] In some embodiments of the invention, the contact is carried out in solution. In some embodiments of the invention, cells and viral particles are brought into contact in a volume of from or about 0.5 ml to 500 ml, for example, from or about 0.5 ml to 200 ml, from 0.5 ml to 100 ml, 0.5 ml to 50 ml 0.5 ml to 10 ml 0.5 ml to 5 ml 5 ml to 500 ml 5 ml to 200 ml 5 ml to 100 ml 5 ml to 50 ml, 5 ml to 10 ml, 10 ml to 500 ml, 10 ml to 200 ml, 10 ml to 100 ml, 10 ml to 50 ml, 50 ml to 500 ml, 50 ml or more up to 200 ml, from 50 ml to 100 ml, from 100 ml to 500 ml, from 100 ml to 200 ml or from 200 ml to 500 ml.

[0362] In some embodiments of the invention, the contact can be carried out by centrifugation, such as centrifugal inoculation (eg, inoculation in a centrifuge). In some embodiments of the invention, the composition containing cells, viral particles and reagent can be subjected to rotation, usually at a relatively low force or speed, such as a speed lower than the cell sedimentation rate, for example, from or about 600 rpm up to 1700 rpm (eg, at or about or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm). In some embodiments of the invention, the rotation is carried out at a force, for example, a relative centrifugal force from or about 100 g to 3200 g (for example, at or about at, or at least at or about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g) as measured, for example, on the inside or outside wall of the chamber or compartment. The term "relative centrifugal force" or RCF usually means the effective force imparted to an object or substance (such as a cell, sample or sediment and/or position in a chamber or other rotating container) relative to the force of gravity at a particular point in space with respect to the axis of rotation. This value can be determined from well-known formulas, taking into account the force of gravity, the speed of rotation and the radius of rotation (distance from the axis of rotation and the object, substance or particle on which the RCF is measured).

[0363] In some embodiments of the invention, incubation of cells with viral vector particles allows to obtain or produce the final composition containing cells transduced with viral vector particles.

D. Other processing steps

[0364] In some embodiments, the transduction processing steps, for example, in combination with cell engineering, may further include culturing, growing, stimulating, activating, expanding, and/or preparing cells. In some embodiments of the invention, the final compositions or cells are incubated under stimulating conditions or in the presence of a stimulating agent. Such conditions include those necessary to induce proliferation, expansion, activation and/or survival of cells in a population and/or to mimic antigen exposure. Stimulation can be carried out ex vivo or in vivo after administration to the individual.

1. Activation after transduction and/or cell expansion

[0365] In some embodiments of the invention, the cells, for example, the final composition is additionally incubated and / or additionally cultured by genetic engineering methods. The incubation steps may include culturing, growing, stimulating, activating and/or propagating. In some such embodiments, the additional incubation is carried out under conditions that promote integration of the viral vector into the genome of one or more host cells. The incubation and/or construction may be carried out in a culture vessel such as a device, chamber, well, column, tube, tubing, valve, vial, culture dish, bag or other cell growth or culture container. In some embodiments of the invention, compositions or cells are incubated under stimulation conditions or in the presence of a stimulating agent. Such conditions are conditions conducive to the induction of proliferation, reproduction, activation and/or increase in the lifespan of cells in a population; mimicking antigen exposure; and/or initiating genetic engineering of cells, for example, to introduce a recombinant antigen receptor.

[0366] In some embodiments of the invention, additional incubation is carried out at a temperature above room temperature, for example, more than or more than about 25°C, for example, usually more than or more than about 32°C, 35° C or 37°C. In some embodiments of the invention, additional incubation is carried out at a temperature equal to or approximately equal to 37°C ± 2°C, for example, at a temperature equal to or approximately equal to 37°C.

[0367] In some embodiments of the invention, additional incubation is carried out under conditions conducive to stimulation and / or activation of cells, and such conditions may include the use of one or more specific environments, temperature, oxygen content, carbon dioxide content, time, use of specific agents, for example, 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.

[0368] In some embodiments, the stimulation conditions or agents are one or more agents (eg, stimulants and/or adjuncts), for example, a ligand capable of activating the intracellular signal-transmitting domain of the TCR complex. In some aspects, the agent starts or initiates the intracellular TCR/CD3 signaling cascade in the T cell, and such agents are suitable for primary signaling, for example, to initiate the activation of an ITAM-induced signal, such as a signal specific to a TCR component, and /or suitable for stimulating a costimulatory signal, such as a signal specific for a T cell costimulatory receptor, e.g., an anti-CD3 antibody, an anti-CD28 antibody, or an anti-41-BB antibody, e.g., optionally associated with a solid carrier such as spheres , and/or with one or more cytokines. Stimulating agents are, among others, spheres with anti-CD3/anti-CD28 antibodies (for example, spheres containing anti-CD3/anti-CD28 antibody DYNABEADS® M-450 for expansion of T cells and/or ExpACT® spheres). The propagation method may also optionally include the step of adding an anti-CD3 and/or anti-CD28 antibody to the culture medium. In some embodiments, the stimulatory agents are IL-2 and/or IL-15, such as IL-2 at a concentration of at least about 10 units/mL.

[0369] In some embodiments of the invention, the stimulation conditions or agents are one or more agents, for example, a ligand capable of activating the intracellular signal-transmitting domain of the TCR complex. In some aspects, the agent starts or initiates a TCR/CD3 intracellular signaling cascade in a T cell. Such agents may be antibodies, such as antibodies specific for a TCR component and/or a costimulatory receptor, e.g., an anti-CD3 antibody, an anti-CD28 antibody, e.g. cytokines. The propagation method may also optionally include the step of adding anti-CD3 and/or anti-CD28 antibodies to the culture medium (eg, at a concentration of at least about 0.5 ng/mL). In some embodiments, the stimulatory agents are IL-2 and/or IL-15, e.g., IL-2 at a concentration of at least about 10 units/mL, at least about 50 units/mL, at least about 100 units /ml or at least about 200 units/ml.

[0370] The conditions may include the use of one or more specific media, temperature, oxygen content, carbon dioxide content, time, the use of specific agents, for example, 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 intended to activate cells.

[0371] In some aspects, the incubation is carried out by methods such as those described in US patent No. 6040177, 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.

[0372] In some embodiments of the invention, additional incubation is carried out in the same container or device in which the contact is made. In some embodiments of the invention, additional incubation is carried out without rotation or centrifugation, which is usually carried out after at least part of the incubation process during rotation, for example, in combination with centrifugation or centrifugal inoculation. In some embodiments of the invention, additional incubation is carried out outside the stationary phase, for example, outside the chromatographic matrix, for example, in solution.

[0373] In some embodiments of the invention, additional incubation is carried out in another container or device in which contact is made, for example, by transfer, for example, by automatically transferring the cell composition to another container or device after contact with viral particles and reagent.

[0374] In some embodiments, T cells are propagated by adding them to a culture initiating feeder cell composition, such as non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., such that the final cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T-lymphocyte in the original breeding population); and incubating the culture (eg, for a period of time sufficient to expand a certain number of T cells). In some aspects, non-dividing feeder cells may contain gamma-irradiated PBMC feeder cells. In some embodiments of the invention, PBMCs are irradiated with gamma rays in the range of approximately 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the T cell populations.

[0375] In some embodiments of the invention, stimulating conditions include a temperature suitable for the growth of human T-lymphocytes, for example, at least about 25 degrees Celsius, usually at least about 30 degrees Celsius, and most often 37 degrees Celsius or about 37 degrees Celsius. Incubation may also include, but not necessarily, the addition of nondividing virus-transformed lymphoblastoid cells (LCL) as feeder cells, where, for example, the virus may be EBV, CMV, or influenza virus, and the transformed LCL present the viral antigen on the surface of the cells, optionally in the presence of MHC. In some embodiments, the T cells express a TCR that recognizes a viral antigen. In some embodiments, the viral antigen may be an EBV, CMV, or influenza virus antigen. The LCL can be irradiated with gamma rays in the range of approximately 6000 to 10000 rads. In some aspects, LCL feeder cells are produced in any suitable amount such that the ratio of LCL feeder cells to original T lymphocytes is at least about 10:1.

[0376] In some embodiments of the invention, additional cultivation or incubation, carried out, for example, to facilitate ex vivo propagation, is carried out for more than or approximately more than 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. In some embodiments of the invention, additional cultivation or incubation is carried out for no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, or no more than 24 hours.

[0377] In some embodiments of the invention, the total duration of incubation, for example, with a stimulating agent is in the range or approximately in the range from 1 hour to 96 hours, from 1 hour to 72 hours, from 1 hour to 48 hours, from 4 hours to 36 hours, 8 hours to 30 hours, or 12 hours to 24 hours, such as at least or at least about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments of the invention, additional incubation is carried out for a period of time from or about 1 hour to 48 hours, 4 hours to 36 hours, 8 hours to 30 hours, or 12 hours to 24 hours, inclusive.

[0378] In some embodiments of the invention, the methods contemplated here do not include additional cultivation or incubation, for example, do not include ex vivo propagation steps or include a generally shorter ex vivo propagation step.

[0379] In some embodiments, the entire process of constructing cells, for example, selection and / or enrichment, incubation along with transduction and / or additional cultivation or propagation is carried out for a period of time no more than 9 days, no more than 8 days , no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, or no more than 1 day after taking a sample from an individual. Obviously, such a period of time does not include any period of time during which the cells are subjected to cryopreservation.

[0380] In some embodiments of the methods contemplated here, the engineered cells, eg, the final composition or the prepared composition, are administered to the individual immediately or immediately after transduction, without any significant ex vivo expansion. In some embodiments of the invention, engineered cells can be introduced immediately after the stage of transduction. In some embodiments, the engineered cells can be introduced immediately after the transduction step, e.g., without any significant ex vivo expansion, or generally in a shorter ex vivo expansion step than in standard methods where significant activation may be required. , propagation and/or enrichment in vitro . For example, in some embodiments of the methods described herein, engineered cells can be introduced three, two, or one day after transduction. In some embodiments, the engineered cells can be administered 48, 36, 24, 20, 16, 12, 8, 4, 2, 1 or less hours after the transduction step. In some embodiments, the engineered cells are subjected to a substantially shorter in vitro propagation step than standard methods, such as 48, 36, 24, 20, 16, 12, 8, 4, 2, 1 or less hours. .

[0381] In any of such embodiments, expansion and/or activation of cells may occur in vivo following antigen treatment, eg, expansion of the engineered cells in an individual may occur following administration of the cells. In some embodiments of the invention, the level, extent or amount of in vivo reproduction can also be increased, enhanced or increased by various methods, allowing modulation, for example, to increase the level of reproduction, proliferation, survival and/or efficiency of the injected cells, for example, cells expressing the recombinant receptor.

[0382] In some embodiments of the invention, such methods include the introduction of engineered cells that are further modified with an agent, for example, a nucleic acid to change (e.g., increase or decrease) the level of expression or activity of the molecule, where such a change in the level of expression or activity increases, enhancing or increasing the level of multiplication, proliferation, survival and/or efficiency of the injected cells. In some embodiments of the invention, the expression of an agent, for example, a nucleic acid, is induced, suppressed, regulated and/or controlled by the user, for example, by introducing an inducer or other modulating molecule.

[0383] In some embodiments of the invention, such methods include methods of combined administration, for example, simultaneous or sequential administration together with a drug or agent that increases, enhances or increases the level of reproduction, proliferation, survival and / or efficiency of the administered cells, for example, cells expressing the recombinant receptor. Examples of such drugs and agents are described in Section III.

2. Preparation of the composition

[0384] In some embodiments of the invention, after additional incubation, the method of obtaining cells may also include washing or preparing cells. Thus, the processing steps may include, among others, the preparation of such compositions.

[0385] The present invention also relates to pharmaceutical compositions or preparations for their use in such methods, where in some embodiments, such methods are carried out in combination with the processing methods discussed here, for example, in a closed system in which other processing steps are carried out, for example, in automatic or semi-automatic mode.

[0386] In some embodiments, the cells and compositions are administered to the individual in the form of a pharmaceutical composition or preparation, such as a composition containing cells or cell populations and a pharmaceutically acceptable carrier or excipient.

[0387] The term "pharmaceutical composition" means a preparation that is prepared in such a form that imparts to the active ingredient contained in this composition effective biological activity, and which does not contain any additional components that are unacceptably toxic to the individual who is administered this composition.

[0388] In some embodiments, the pharmaceutical compositions further comprise other pharmaceutically active agents or drugs such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, these agents are administered in the form of a salt, such as a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts are salts of mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids and organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic , succinic and arylsulfonic acids, for example, p-toluenesulfonic acid.

[0389] The term "pharmaceutically acceptable carrier" means an ingredient in a pharmaceutical composition that is not an active ingredient and that is non-toxic to an individual. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0390] In some aspects, the choice of carrier is determined in part by the particular cell and/or route of administration. Accordingly, there are various suitable compositions. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may be, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of from about 0.0001% to about 2% by weight of the entire composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (approximately less than 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

[0391] In some aspects, the compositions include buffering agents. Suitable buffering agents are, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of from about 0.001% to about 4% by weight of the entire composition. Methods for preparing injectable pharmaceutical compositions are known. Representative methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).

[0392] Compositions may include aqueous solutions. The preparation or composition may also contain more than one active ingredient suitable for the treatment of the particular disease, disorder or condition being treated using cells, and preferably cells whose activity corresponds to the activity of cells, where the respective activities do not adversely affect each other. . Such active ingredients are preferably present in combination in amounts effective to achieve the desired objectives. Thus, in some embodiments of the invention, the pharmaceutical composition also includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine and/or vincristine.

[0393] In some embodiments of the invention, the pharmaceutical composition contains cells in an amount effective for the treatment or prevention of a disease or condition, for example, a therapeutically effective or prophylactically effective amount. In some embodiments of the invention, individuals undergoing treatment are periodically examined to evaluate therapeutic or prophylactic efficacy. The desired dose can be delivered by a single bolus of cells, multiple boluses of cells, or by continuous cell infusion.

[0394] Compositions are compositions for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments of the invention, cell populations are administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments of the invention, the cells are administered to the individual via peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0395] In some embodiments of the invention, the compositions are prepared as sterile liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which, in some aspects, can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous formulations, and solid formulations. In addition, liquid formulations are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions may be formulated within an appropriate viscosity range so as to provide a longer period of contact with certain tissues. Liquid or viscous compositions may contain carriers, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0396] Sterile injectable solutions can be prepared by incorporating the cells into a solvent, for example, admixed with a suitable carrier, diluent, or excipient such as sterile water, saline, glucose, dextrose, or the like. The compositions may contain excipients such as wetting, dispersing or emulsifying agents (e.g. methylcellulose), pH buffering agents, gelling agents or viscosity enhancers, preservatives, flavorings and/or coloring agents, depending on the desired route of administration and formulation. . In some aspects of the invention, methods for preparing suitable preparations can be found in standard manuals.

[0397] Various additives may be added to the compositions to improve the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be provided using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol and sorbic acid. Prolonged absorption of an injectable pharmaceutical form can be achieved using agents that delay absorption, such as aluminum monostearate and gelatin.

[0398] Compositions used for in vivo administration are usually sterile. Sterility can be easily achieved, for example, by filtration through sterile filter membranes.

III. Therapeutic methods and compositions for administration

[0399] In some aspects, the results of the methods are used in methods of treatment, for example, in therapy, namely, for the introduction of cells and compositions to an individual undergoing adoptive cell therapy. The present invention also relates to such methods and uses of cells treated and obtained by such methods, and to pharmaceutical compositions and preparations for their use in these purposes. Contemplated methods typically involve administering to individuals the cells or compositions, eg, the final composition and/or the prepared compositions.

[0400] In some embodiments, the cell expresses recombinant receptors, such as CARs, or other antigen receptors, such as transgenic TCRs, such as the TCRs used in the transduction methods described here. Such cells are typically administered to patients suffering from a disease or condition that is associated with a ligand specifically recognized by the receptor. In one embodiment, the cells express a recombinant receptor or chimeric receptor, such as an antigen receptor, such as CAR or TCR, that specifically binds to a ligand associated with a disease or disorder or expressed in cells or tissues. Thus, for example, in some embodiments of the invention, the receptor is an antigen receptor, and the ligand is an antigen specific to a disease or disorder and/or associated with such a disease or disorder. The administration will generally aid in the alleviation of one or more symptoms of a disease or condition and/or the treatment or prevention of such disease or condition or symptom thereof.

[0401] Diseases, conditions and disorders are, among others, tumors, including solid tumors, hematological malignancies and melanomas, including localized and metastatic tumors, infectious diseases, such as infections caused by a virus or other pathogen, such as HIV , HCV, HBV, CMV, and parasitic diseases, as well as autoimmune and inflammatory diseases. In some embodiments, the diseases or conditions are tumors, cancer, malignancies, neoplasms, or other proliferative diseases or disorders. Such diseases include, but are not limited to, leukemia, lymphoma, such as chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, intractable follicular lymphoma, cerebral cortex lymphoma, mild B-cell lymphoma, B -cell malignancies, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone and brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, adenocarcinoma pancreas, Hodgkin's lymphoma, cervical carcinoma, rectal and colon cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma and/or mesothelioma.

[0402] In some embodiments of the invention, such diseases are, but are not limited to, leukemia, lymphoma, for example, acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), reticuloendotheliosis (RE), small cell lymphocytic lymphoma (MLL), cerebral cortex cell lymphoma (LCCC), marginal zone lymphoma, Burkitt's lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), follicular lymphoma, intractable follicular lymphoma, diffuse large B-cell lymphoma (DLCL), and multiple myeloma (MM). In some embodiments, the diseases or disorders are B-cell malignancies selected from acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLCL) . In some embodiments, the diseases or disorders are NHL, wherein the NHL is selected from the group consisting of aggressive NHL, diffuse large B-cell lymphoma (DLCL), NOS (De Novo lymphoma and lymphoma transformed from mild lymphoma), primary large B-cell mediastinal α-cell lymphoma (MCCL), T-cell/histocyte-rich large B-cell lymphoma (TCCL), Burkitt's lymphoma, cerebral cortex cell lymphoma (CCCL), and/or follicular lymphoma (FL) and optionally follicular lymphoma of the class 3B (FL-3B).

[0403] In some embodiments, the diseases or conditions are infectious diseases or conditions such as, but not limited to, viral, retroviral, bacterial and protozoal infections, immunodeficiency, cytomegalovirus (CMV) infections, Epstein-Barr virus infections (EBV), adenovirus, and polyomavirus BK. In some embodiments, the diseases or conditions are an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), type I diabetes mellitus, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and/or a disease or condition associated with transplantation.

[0404] In some embodiments, the antigen associated with the disease or disorder is or includes an ανβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic acid -anhydrase 9 (CA9, also known as CAIX or G250), testicular cancer antigen, testicular cancer antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, chemokine ligand 1 (CCL-1) C-C motif, 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), mutated epidermal growth factor receptor type III (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), receptor estrogen, Fc receptor-like receptor 5 (FCRL5; also known as the Fc receptor 5 homologue or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alfa, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), G protein-coupled receptor 5D ( GPCR5D), Her2/neu (erb-B2 tyrosine kinase receptor), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, 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), L1-CAM CE7 epitope, leucine-rich repeat containing A8 family member (LRRC8A), Lewis Y antigen, melanoma-associated antigen (MAGE)-A1, MAGE- A3, MAGE-A6, mesothelin, c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural kille cell group 2 member antigen ligands ditch (NKG2D), melan A (MART-1), neuronal cell adhesion molecule (NCAM), oncofetal antigen, predominantly expressed melanoma antigen (PRAME), progesterone receptor, prostate specific antigen, prostate stem cell antigen ( PSCA), prostate-specific membrane antigen (PSMA), orphan receptor 1 like tyrosine kinase receptor (ROR1), survivin, trophoblastic 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 antigen 1 (WT-1), pathogen-specific or universal tag-associated antigen and/or biotinylated molecules and/or molecules expressed by HIV , HCV, HBV or other pathogens. In some embodiments, the antigens that are targeted by the receptors include antigens associated with B cell malignancy, such as any of the known B cell marker antigens. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig-kappa, Ig-lambda, CD79a, CD79b, or CD30.

[0405] In some embodiments, the antigen associated with the disease or disorder being treated with cells or compositions is selected from the group consisting of orphan tyrosine kinase receptor ROR1, tEGFR, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP , fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y antigen, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1 ligands, MUC16, PSCA, NKG2D, 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 antigen 1 (WT-1), cyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.

[0406] In some embodiments, the antigen is or includes a pathogen-specific antigen or an antigen expressed by a pathogen. In some embodiments, the antigen is a viral antigen (eg, a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

[0407] As used herein, the term "treatment" (and its grammatical variations such as "treat" or "therapy") refers to the total or partial amelioration or reduction of a disease, or condition, or disorder, or symptom, adverse effect, or result, or phenotype associated with them. Desirable effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequence of the disease, prevention of the occurrence of metastases, reduction in the rate of disease progression, improvement or temporary relief of the disease state, and remission or improved prognosis. This term does not include the complete cure of a disease or the complete elimination of any symptom or effect(s) affecting all symptoms or treatment outcome.

[0408] As used herein, the term "slowing the development of a disease" means delaying, delaying, preventing, stabilizing, suppressing, and/or inhibiting the development of a disease (eg, cancer). This delay may vary in time, depending on the history of the disease and/or the particular individual being treated. It will be apparent to one skilled in the art that a sufficient or significant delay may essentially encompass prophylaxis, that is, the situation where the individual does not develop a disease. For example, there may be a delay in the onset of a late stage cancer, such as the development of metastases.

[0409] As used herein, the term "prevention" includes preventing the onset or recurrence of a disease in an individual who may be predisposed to the disease but who has not yet been diagnosed with the disease. In some embodiments of the invention, considered cells and compositions are used to slow down the development of the disease or slow down the progression of the disease.

[0410] As used herein, the term "suppression" of function or activity means a decrease in function or activity compared to function or activity in other conditions, excluding the condition or parameter of interest, or alternatively compared to another condition. Thus, for example, cells that inhibit tumor growth reduce the rate of tumor growth compared to the rate of tumor growth in the absence of these cells.

[0411] Methods for introducing cells for adoptive cell therapy are known to those skilled in the art and can be used in combination with the methods and compositions discussed herein. For example, adoptive T cell therapy methods are described, for example, in U.S. Patent Application Publication No. 2003/0170238 Gruenberg et al; in US Patent No. 4690915 Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

[0412] The disease or condition being treated can be any disease or condition in which antigen expression is associated with the etiology of the condition or disorder and/or is involved in the development of such condition or disorder, such as causing, exacerbating, or otherwise otherwise involved in the development of such a disease, condition or disorder. Representative diseases and conditions can be diseases or conditions associated with malignancy or transformation of cells (eg, cancer), autoimmune or inflammatory diseases, or an infectious disease, for example, caused by a bacterial, viral, or other pathogen. Examples of antigens, which include antigens associated with various diseases and conditions that can be treated, are described above. In specific embodiments, the chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with a disease or condition.

[0413] Cells and compositions can be administered by standard methods of administration using drugs and/or devices. The cells administered may be autologous or heterologous, eg allogeneic. Thus, for example, immunoresponsive or progenitor cells may be taken from one individual and administered to the same individual or another compatible individual. Immunoresponsive cells or their progeny derived from peripheral blood (for example, obtained in vivo , ex vivo or in vitro ) can be administered by local injection, including introduction by catheter, systemic injection, localized injection, intravenous injection or parenteral administration. When a therapeutic composition is administered (eg, a pharmaceutical composition containing genetically modified immune sensitive cells), such a composition is usually prepared in the form of an injectable unit dosage form (solution, suspension, emulsion).

[0414] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is performed by autologous transfer, where T cells are isolated and/or otherwise taken from the individual receiving the cell therapy or isolated from a sample taken from such an individual. Thus, in some aspects of the invention, cells are taken from an individual, such as a patient in need of therapy and cell administration, and after isolation and processing, the cells are administered to the same individual.

[0415] In some embodiments of the invention, cell therapy, for example, adoptive cell therapy, for example, adoptive T-cell therapy is carried out by allogeneic transfer, where cells are isolated and / or otherwise taken from an individual that is not an individual who is being treated cell therapy or who ultimately receives such cell therapy, for example, in the first individual. In such embodiments, the cells are then administered to another individual, such as a second individual of the same species. In some embodiments of the invention, the first and second individuals are genetically identical. In some embodiments of the invention, the first and second individuals are genetically similar. In some embodiments, the second individual expresses an HLA of the same class or supertype as the first individual.

[0416] Cells can be administered by any suitable method, for example, by bolus infusion, by injection, for example, by intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, vitreous injection, trans-septal injection, injection into subscleral area, intrachoroidal space injection, intra-chamber injection, subconjunctival injection, sub-Tenon's capsule injection, retrobulbar injection, peribulbar injection, or posterior injection near the sclera. In some embodiments of the invention, these injections are administered parenterally, intrapulmonary and intranasally, and, if necessary, locally by injection into the affected area. Parenteral infusions are intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus of cells, by multiple bolus of cells, eg, over a time period of no more than 3 days, or by a continuous infusion of cells.

A. Doses and Administration

[0417] Cells or compositions are administered at a dose containing cells expressing a therapeutically effective amount of the recombinant receptor (eg, CAR) in vivo to treat a disease or condition. For the prevention or treatment of a disease, the appropriate dose may depend on the type of disease being treated, the type of cells or recombinant receptors, the administration of other drugs or agents in combination, to increase, increase or increase the rate of cell reproduction, the severity and course of the disease, regardless of whether these cells are administered prophylactically or therapeutically, from previous treatments, the patient's medical history, and response to the cells, as well as at the discretion of the attending physician. In some embodiments of the invention, the compositions and cells are suitable for their introduction to the individual immediately or as a course of treatment.

[0418] The term "effective amount" of an agent, e.g., pharmaceutical, cells, or composition, when referring to administration, means an amount effective at specific doses/levels and for a period of time necessary to achieve the desired result, such as therapeutic or preventive effect.

[0419] The term "therapeutically effective amount" of an agent, such as a pharmaceutical composition or cells, means an amount effective at certain doses and for a period of time necessary to achieve the desired therapeutic result, for example, to treat a disease, condition or disorder, and / or the pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary depending on such factors as the pathological condition, age, sex and weight of the individual, and on the population of cells administered to the individual, as well as on other drugs or agents administered in combination, for example, simultaneously.

[0420] The term "prophylactically effective amount" means an amount effective at the indicated doses and for periods of time necessary to achieve the desired prophylactic result. Since a prophylactic dose is administered to individuals prior to the development of the disease or early in its development, usually, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount.

[0421] In some embodiments, the cells or compositions are administered in an amount that is effective for treating or preventing a disease or condition, such as a therapeutically effective or prophylactically effective amount. Thus, in some embodiments of the invention, methods of administration include the introduction of cells and compositions in effective amounts. In some embodiments of the invention, patients undergoing treatment are periodically examined to evaluate therapeutic or prophylactic efficacy. In the case of repeated administrations over several days or more, depending on the condition, the treatment is repeated until the desired suppression of the symptoms of the disease. However, other dosing regimens may be used and selected.

[0422] In some embodiments of the invention, a therapeutically effective amount of cells is administered to the individual. In some embodiments of the invention, a suboptimal dose of cells is administered to the individual, for example, in some cases, when the cells are administered under conditions suitable for cell propagation in vivo .

[0423] In some embodiments, cells or distinct populations of cell subtypes are administered to an individual in the range of from about 0.1 million to about 100 billion cells and/or in volume per kilogram of the individual's body weight, for example, from 0.1 million to about 50 billion cells (e.g., less than 0.5 million cells, less than 1 million cells, approximately 0.1 million cells, approximately 0.2 million cells, approximately 0.3 million cells, approximately 0.4 million cells, approximately 0.5 million cells approximately 1 million cells approximately 5 million cells approximately 25 million cells approximately 500 million cells approximately 1 billion cells approximately 5 billion cells approximately 20 billion cells approximately 30 billion cells approximately 40 billion cells, or within the range of any two of the above), from 1 million to about 50 billion cells (e.g., approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or within, defined by any two of the above), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells , approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or or within the range of any two of the above), and in some cases, from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells , approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells) or in any number within these intervals and/or in the amount per kilogram of body weight of the individual. Doses may vary depending on the particular signs of the disease or disorder and/or the patient and/or other treatments. In some embodiments of the invention, such values mean the number of cells expressing the recombinant receptor, and in other embodiments of the invention, these values mean the number of administered T cells or PBMCs or all cells.

[0424] In some embodiments of the invention, cell therapy includes the introduction of a dose containing a number of cells that is at least or at least approximately or exactly or approximately 0.1 × 10 ⁶ cells / kg of body weight of the individual, 0.2 × 10 ⁶ cells/kg, 0.3 × 10 ⁶ cells/kg, 0.4 × 10 ⁶ cells/kg, 0.5 × 10 ⁶ cells/kg, 1 × 10 ⁶ cells/kg, 2.0 × 10 ⁶ cells/kg, 3 x 10 ⁶ cells/kg or 5 x 10 ⁶ cells/kg.

[0425] In some embodiments, the cell therapy comprises administering a dose containing a cell number in the range or approximately in the range of 0.1 x 10 ⁶ cells/kg of an individual's body weight to 1.0 x 10 ⁷ cells/kg, in the range or approximately within the range of 0.5 × 10 ⁶ cells/kg to 5 × 10 ⁶ cells/kg, within or approximately within the range of 0.5 × 10 ⁶ cells/kg to 3 × 10 ⁶ cells/kg, within or approximately within the range of 0.5 × 10 ⁶ cells/kg to 2 × 10 ⁶ cells/kg, within or approximately within the range of 0.5 × 10 ⁶ cells/kg to 1 × 10 ⁶ cells/kg, within or approximately within the range of 1.0 × 10 ⁶ cells/kg of body weight to 5 × 10 ⁶ cells/kg, within or approximately within the range of 1.0 × 10 ⁶ cells/kg to 3 × 10 ⁶ cells/kg, within or approximately within 1.0 × 10 ⁶ cells/kg to 2 × 10 ⁶ cells/kg, within or approximately within 2.0 × 10 ⁶ cells/kg body weight to 5 × 10 ⁶ cells/ kg, within or approximately within x from 2.0 × 10 ⁶ cells/kg to 3 × 10 ⁶ cells/kg, or within or approximately within the range from 3.0 × 10 ⁶ cells/kg of body weight to 5 × 10 ⁶ cells/kg, inclusive.

[0426] In some embodiments of the invention, the dose of cells is from or about 2 x 10 ⁵ cells/kg to or about 2 x 10 ⁶ cells/kg, for example, from or about 4 x 10 ⁵ cells/kg to or up to about 1 x 10 ⁶ cells/kg, or from or about 6 x 10 ⁵ cells/kg to or up to about 8 x 10 ⁵ cells/kg. In some embodiments of the invention, the dose of cells is no more than 2 x 10 ⁵ cells (eg, antigen-expressing, such as CAR-expressing cells) per kilogram of body weight of the individual (cells/kg), for example, no more than or about 3 x 10 ⁵ cells/kg, no more than or about 4 x 10 ⁵ cells/kg, no more than or about 5 x 10 ⁵ cells/kg, no more than or about 6 x 10 ⁵ cells/kg , no more than or approximately 7 × 10 ⁵ cells/kg, no more than or approximately 8 × 10 ⁵ cells/kg, no more than or approximately 9 × 10 ⁵ cells/kg, no more than or approximately 1 × 10 ⁶ cells/kg, or no more than or about 2 × 10 ⁶ cells/kg.

[0427] In some embodiments of the invention, the dose of cells is at least or at least about or exactly or about 2 x 10 ⁵ cells (for example, antigen-expressing, such as CAR-expressing cells) per kilogram of individual body weight (cells /kg), for example, at least or at least approximately or exactly or approximately 3 × 10 ⁵ cells/kg, is at least or at least approximately or exactly or approximately 4 × 10 ⁵ cells/kg, is at least at least or at least about or exactly or about 5 x 10 ⁵ cells/kg is at least or at least about or exactly or about 6 x 10 ⁵ cells/kg is at least or at least about or exactly or approximately 7 × 10 ⁵ cells/kg, is at least or at least approximately or exactly or approximately 8 × 10 ⁵ cells/kg, is at least or varies more than about or exactly or about 9 x 10 ⁵ cells/kg, is at least or at least about or exactly or about 1 x 10 ⁶ cells/kg, or is at least or at least about or exactly or approximately 2 x 10 ⁶ cells/kg.

[0428] In some embodiments, the cell dose is a flat cell dose or a fixed cell dose, i.e., a cell dose that is independent of or calculated from body surface area or weight of the individual.

[0429] In some embodiments, cells or distinct populations of cell subtypes are administered to an individual in the range of about one million to about 100 billion cells and/or in volume per kilogram of body weight, e.g., about 1 million to about 50 billion cells ( for example, approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or as defined by any two of the above. values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells approx. 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or or within the range of any two of the above), and in some cases, approximately 100 million cells to approximately 50 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately cells, approximately 30 billion cells, approximately 45 billion cells) or in any amount within these intervals and/or in the amount per kilogram of body weight of the individual. Doses may vary depending on the particular signs of the disease or disorder and/or the patient and/or other treatments.

[0430] In some embodiments of the invention, for example, if the individual is a human, then the dose includes less than about 5 x 10 ^{8 of} all cells expressing the recombinant receptor (for example, CAR), T cells, or peripheral blood mononuclear cells (PBMC) , for example, ranging from approximately 1 x 10 ⁶ to 5 x 10 ⁸ such cells, for example, a total of 2 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 5 x 10 ⁷ , 1 x 10 ⁸ or 5 x 10 ⁸ such cells, or within any two of the above values.

[0431] In some embodiments, cell therapy comprises administering a dose containing a cell number of from or about 1 x 10 ⁵ to 5 x 10 ^{8 of} all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells ( PBMC), from or about 5 x 10 ⁵ to 1 x 10 ^{7 of} all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMC), or from or about 1 x 10 ⁶ to 1 x 10 ⁷ all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMCs), inclusive. In some embodiments, cell therapy comprises administering a dose of cells containing a cell count of at least or at least about 1 x 10 ^{5 of} all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMCs), e.g. , at least or at least about 1 x 10 ⁶ , at least or at least about 1 x 10 ⁷ , at least or at least about 1 x 10 ⁸ such cells. In some embodiments of the invention, the specified number means the number of all CD4 ⁺ - and/or CD8 ⁺ -cells, and in some cases, also cells expressing the recombinant receptor (eg, CAR ⁺ ). In some embodiments of the invention, cell therapy includes the introduction of a dose containing a number of cells from or approximately from 1 x 10 ⁵ to 5 x 10 ^{8 of} all CD3 ⁺ - and / or CD8 ⁺ -T cells or CD3 ⁺ - and / or CD8 ⁺ - cells expressing the recombinant receptor, from or about 5 x 10 ⁵ to 1 x 10 ^{7 of} all CD3 ⁺ - and / or CD8 ^{+ -} T cells or CD3 ⁺ - and / or CD8 ⁺ cells expressing the recombinant receptor, or from or about 1 x 10 ⁶ to 1 x 10 ^{7 of} all CD3 ⁺ and/or CD8 ⁺ T cells or CD3 ⁺ and/or CD8 ⁺ cells expressing the recombinant receptor, inclusive. In some embodiments of the invention, cell therapy includes the introduction of a dose containing a number of cells from or about 1 x 10 ⁵ to 5 x 10 ^{8 of} all CD3 ⁺ /CAR ⁺ or CD8 ⁺ /CAR ⁺ cells, from or about 5 x 10 ⁵ to 1 x 10 ^{7 of} all CD3 ⁺ /CAR ⁺ or CD8 ⁺ /CAR ⁺ cells, or from or about 1 x 10 ⁶ to 1 x 10 ^{7 of} all CD3 ⁺ /CAR ⁺ or CD8 ⁺ /CAR ⁺ cells, inclusive.

[0432] In some embodiments, the dose T cells are CD4 ⁺ T cells, CD8 ⁺ T cells, or CD4 ⁺ and/or CD8 ⁺ T cells.

[0433] In some embodiments of the invention, for example, if the individual is a human, then the CD8 ⁺ -T cells of the dose, including the dose that includes CD4 ⁺ - and CD8 ⁺ -T cells, contains from about 1 x 10 ⁶ to 5 x 10 ^{8 of} all CD8 ⁺ cells expressing a recombinant receptor (eg, CAR), for example, in the range from approximately 5 × 10 ⁶ to 1 × 10 ⁸ such cells, namely, 1 × 10 ⁷ , 2.5 × 10 ⁷ , 5 x 10 ⁷ , 7.5 x 10 ⁷ , 1 x 10 ⁸ , or 5 x 10 ⁸ of all cells, or between any two of the above. In some embodiments of the invention, fractional doses are administered to the patient, and each of these doses or total doses can be in the range of any two of the above values. In some embodiments of the invention, the introduction of a dose of cells includes the introduction of from or about 1 x 10 ⁷ to 0.75 x 10 ^{8 of} all CD8 ⁺ -T cells expressing the recombinant receptor, from 1 x 10 ⁷ to 2.5 x 10 ⁷ of all CD8 ⁺ -T cells expressing the recombinant receptor, from or about 1 x 10 ⁷ to 0.75 x 10 ^{8 of} all CD8 ⁺ -T cells expressing the recombinant receptor, inclusive. In some embodiments, administering a dose of cells comprises administering a dose of cells that is or approximately 1 x 10 ⁷ , 2.5 x 10 ⁷ , 5 x 10 ⁷ , 7.5 x 10 ⁷ , 1 x 10 ⁸ , or 5 x 10 ^{8 of} all CD8 ⁺ -T cells expressing the recombinant receptor.

[0434] In some embodiments of the invention, the dose of cells, for example, T cells expressing the recombinant receptor, is administered to the individual as a single dose, or is administered only once over a period of time for two weeks, one month, three months, six months , 1 year or more.

[0435] In the case of adoptive cell therapy, administering a given "dose" of cells includes administering a given number or number of cells in a single composition, or a single continuous administration, such as a single injection or continuous infusion, as well as administering a given number or number of cells in the form of a divided dose or in the form of multiple compositions, presented as several separate compositions or infusions, over a certain period of time, for example, for no more than 3 days. Thus, in some cases, the dose is administered by a single or continuous administration of a certain number of cells taken or initiated at a certain point in time. However, in some cases, the dose is administered by multiple injections or infusions over no more than three days, for example, once a day for three days or two days, or by multiple infusions over one day.

[0436] Thus, in some aspects, the dose cells are administered as a single pharmaceutical composition. In some embodiments of the invention, the cells of the dose are administered as a plurality of compositions collectively containing the cells of the given dose.

[0437] The term "fractional dose" means a dose that is divided so that it can be administered over more than one day. This type of dosing is within the scope of the methods of the invention and is considered to be a single dose. In some embodiments, the fractional dose cells are administered as a plurality of compositions that collectively comprise the dose cells over a period of no more than three days.

[0438] Thus, the dose of cells can be administered as a fractional dose, such as a fractional dose administered over a period of time. So, for example, in some embodiments of the invention, the dose may be administered to the individual within 2 days or 3 days. Representative methods for administering divided doses include administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments, 33% of the dose may be administered on the first day and the remaining 67% administered on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the divided dose is administered over no more than 3 days.

[0439] In some embodiments, dose cells may be administered by administering multiple compositions or solutions, such as first and second compositions and solutions, and possibly more, each containing some dose cells. In some aspects, multiple compositions, each containing cells of different populations and/or subtypes, are administered separately or independently, optionally over a period of time. Thus, for example, the cell population or subtypes may include CD8 ⁺ - and CD4 ⁺ -T cells, respectively, and/or populations enriched in CD8 ⁺ - and/or CD4 ⁺ -T cells, respectively, for example, CD4 ⁺ - and /or CD8 ⁺ -T cells, each of which separately includes cells genetically engineered to express the recombinant receptor. In some embodiments of the invention, the introduction of the dose includes the introduction of a first composition containing a dose of CD8 ⁺ -T cells or a dose of CD4 ⁺ -T cells, and the introduction of a second composition containing a different dose of CD4 ⁺ -T cells and CD8 ⁺ -T- cells.

[0440] In some embodiments of the invention, the introduction of a composition or dose, for example, the introduction of multiple cell compositions, includes the introduction of cell compositions separately. In some aspects, the individual introductions are carried out simultaneously or sequentially in any order. In some embodiments, the dose comprises a first composition and a second composition, wherein the first composition and second composition are administered at intervals of 0 to 12 hours, 0 to 6 hours, or 0 to 2 hours. In some embodiments, the start of administration of the first composition and the start of administration of the second composition are at intervals of no more than 2 hours, no more than 1 hour, or no more than 30 minutes, no more than 15 minutes, no more than 10 minutes or no more than 5 minutes. In some embodiments of the invention, the beginning and/or completion of the introduction of the first composition and the completion and/or beginning of the introduction of the second composition is carried out at intervals of no more than 2 hours, no more than 1 hour, or no more than 30 minutes, not more than 15 minutes, no more than 10 minutes or no more than 5 minutes.

[0441] In some embodiments of the composition, the first composition, for example, the first composition of the dose, includes CD4 ⁺ -T cells. In some embodiments of the composition, the first composition, for example, the first composition of the dose, includes CD8 ⁺ -T cells. In some embodiments of the invention, the first composition is administered prior to the introduction of the second composition.

[0442] In some embodiments of the invention, the dose or composition of cells includes a specific or target ratio of CD4 ⁺ cells expressing the recombinant receptor to CD8 ⁺ cells expressing the recombinant receptor, and/or CD4 ⁺ cells to CD8 ⁺ cells, where such a ratio is, but not necessarily, about 1:1 or in the range from about 1:3 to about 3:1, for example, about 1:1. In some aspects, administering a composition or dose at a target or desired ratio of different cell populations (such as a ratio of CD4 ⁺ cells:CD8 ⁺ cells or a ratio of CAR ⁺ CD4 ⁺ cells:CAR ⁺ CD8 ⁺ cells, for example, 1 :1) involves administering a cell composition containing one of the populations, and then administering a separate cell composition containing cells from another population, wherein the administration is in or about the target or desired ratio. In some aspects, the introduction of a dose or composition of cells in a certain respect increases the degree of reproduction, persistence and/or antitumor activity of T cells used in T cell therapy.

[0443] In some embodiments of the invention, multiple doses are administered to the individual, for example, two or more doses or several consecutive doses of cells. In some embodiments of the invention, two doses are administered to the individual. In some embodiments, the individual is dosed sequentially, for example, the second dose is administered approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose. In some embodiments of the invention, several consecutive doses are administered after the first dose, so that an additional dose or additional doses are administered after the subsequent dose is administered. In some aspects, the number of cells administered to an individual in an additional dose is the same or similar to the number of cells in the first dose and/or in a subsequent dose. In some embodiments of the invention, the additional dose or additional doses exceed the previous doses.

[0444] In some aspects of the invention, the size of the first and / or subsequent dose is determined based on one or more criteria, such as the response of the individual to previous treatment, for example, chemotherapy, the severity of the disease in the individual, for example, tumor burden, volume, size, or the degree, level, or type of metastasis, stage, and/or likelihood or possibility of an individual developing toxic effects, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or host immune response to administration of cells and/or recombinant receptors.

[0445] In some aspects, the time between the first dose and the next dose is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some embodiments, the next dose is administered more than about 14 days and less than about 28 days after the first dose. In some aspects, the time between the first dose and the next dose is approximately 21 days. In some embodiments of the invention, an additional dose or doses, for example, subsequent doses, is administered after the administration of the subsequent dose. In some aspects, the additional subsequent dose or doses are administered at least about 14 and less than about 28 days after the previous dose. In some embodiments, an additional dose is administered less than about 14 days after the previous dose, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the previous dose. In some embodiments, less than about 14 days after the previous dose, the dose is not administered, and/or the dose is not administered more than about 28 days after the previous dose.

[0446] In some embodiments, the dose of cells, e.g., cells expressing the recombinant receptor, comprises two doses (e.g., a double dose) containing a first dose of T cells and a subsequent dose of T cells, where administration of one or both of the first dose and the second dose comprises administering a fractional dose of T cells.

[0447] In some embodiments of the invention, the dose of cells should usually be large enough to be effective in reducing the severity of the disease.

[0448] In some embodiments of the invention, the cells are administered at the desired dose, which, in some aspects, includes the desired dose or number of cells or cells of certain type(s) and/or the desired ratio of cells of certain types. Thus, in some embodiments of the invention, the dose of cells is calculated based on the total number of cells (or number per kg of body weight) and the desired ratio of individual populations or subtypes, such as the ratio of CD4 ⁺ cells to CD8 ⁺ cells. In some embodiments of the invention, the dose of cells is calculated based on the desired total number (or number per kg of body weight) of cells in individual cell populations or cell types. In some embodiments of the invention, the dose of cells is calculated based on a combination of such features as the desired number of total cells, the desired ratio, and the desired total number of cells in individual populations.

[0449] In some embodiments of the invention, cells of certain populations or subtypes, such as CD8 ⁺ - and CD4 ⁺ - T cells, are administered within or within acceptable ranges of desired doses of all cells, such as the desired dose of T cells. In some aspects, the desired dose is the desired number of cells or the desired number of cells per unit body weight of the individual to which the cells are administered, eg, cells/kg. In some aspects, the desired dose is equal to or greater than the minimum number of cells or the minimum number of cells per unit of body weight. In some aspects, of all cells administered at the desired dose, cells of certain populations or subtypes are present in a ratio or approximately in the desired final ratio (such as the ratio of CD4 ⁺ cells to CD8 ⁺ cells), for example, within certain acceptable differences or errors of this kind.

[0450] In some embodiments, the cells are administered within or within acceptable ranges of the desired dose of one or more distinct cell populations or subtypes, such as the desired dose of CD4 ⁺ cells and/or the desired dose of CD8 ⁺ cells. In some aspects, the desired dose is the desired number of cells of a certain subtype or population, or the desired number of such cells per unit body weight of the individual to whom these cells are administered, for example, cells/kg. In some aspects, the desired dose is equal to or greater than the minimum number of cells of a certain population and a certain subtype, or the minimum number of cells of a population or subtype per unit of body weight.

[0451] Thus, in some embodiments of the invention, the dose is calculated from the desired fixed dose of all cells and the desired ratio and/or from the desired fixed dose of one or more cells, for example, each cell of certain subtypes or subpopulations. Thus, in some embodiments of the invention, the dose is calculated based on the desired fixed or minimum dose of T cells and the desired ratio of CD4 ⁺ cells to CD8 ⁺ cells and / or based on the desired fixed or minimum dose of CD4 ⁺ cells and / or CD8 ⁺ -cells.

[0452] In some embodiments, the cells are administered within or within an acceptable range of the desired final ratio of multiple cell populations or subtypes, such as CD4 ⁺ and CD8 ⁺ cells or subtypes thereof. In some aspects, the desired ratio may be a specific ratio or range of such ratios. Thus, for example, in some embodiments of the invention, the desired ratio (for example, the ratio of CD4 ⁺ cells to CD8 ⁺ cells) is in the range from or about 5:1 to or about 5:1 (or more than about 1 :5 and less than about 5:1), or ranging from or about 1:3 to or up to about 3:1 (or greater than about 1:3 and less than about 3:1), for example, within from or about 2:1 to or up to about 1:5 (or more than about 1:5 and less than about 2:1, e.g., equal to or about equal to 5:1, 4.5:1, 4: 1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5: 1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1: 1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1: 3.5, 1:4, 1:4.5, or 1:5 In some aspects, the allowable difference is within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in these ranges.

[0453] In specific embodiments of the invention, the number and/or concentration of cells means the number of cells expressing the recombinant receptor (eg, CAR). In other embodiments of the invention, the number and/or concentration of cells means the number or concentration of all administered cells, T cells or peripheral blood mononuclear cells (PBMC).

[0454] In some aspects, the dose size is determined based on one or more criteria, such as the response of the individual to prior treatment, such as chemotherapy, the severity of the disease in the individual, such as tumor burden, volume, size or extent, level or type of metastasis , stage, and/or likelihood or possibility of an individual developing toxic effects, eg, CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or host immune response to administration of cells and/or recombinant receptors.

[0455] In some embodiments, the methods also include administering one or more additional doses of cells expressing a chimeric antigen receptor (CAR) and/or lymph depletion therapy, and/or repeating one or more of the steps of these methods. In some embodiments of the invention, one or more additional doses are the same as the initial dose. In some embodiments of the invention, one or more additional doses differ from the initial dose, and, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times or more the initial dose, or are, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times or less than the initial dose. In some embodiments of the invention, the administration of one or more additional doses depends on the response of the individual to the first treatment or previous treatment, the severity of the disease in the individual, for example, tumor burden, volume, size or extent, level or type of metastasis, stage and / or likelihood or the potential for an individual to develop toxic effects, eg, CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or host immune response to administration of cells and/or recombinant receptors.

[0456] In some embodiments, a relatively lower cell dose may be administered, such as a suboptimal cell dose or a cell dose less than a therapeutically effective amount, and that dose, after in vivo stimulation (e.g., with an endogenous antigen or exogenous agent) may contribute to an increase in the level, for example, an increase in the number or degree of reproduction of the engineered cells present in the individual. In any of such embodiments of the invention, expansion and/or activation of cells can occur under the influence of antigen in vivo , for example, expansion of the engineered cells in the body of the individual after the introduction of cells. In some embodiments of the invention, the level, extent, or magnitude of in vivo proliferation can be further increased, enhanced, or increased in various ways, allowing modulation, for example, increase the level, increase proliferation, increase the degree of proliferation, survival and/or efficiency of the injected cells, for example, cells expressing the recombinant receptor.

[0457] In some aspects, after the introduction of cells to an individual (eg, human), the biological activity of cell populations is measured by any known methods. Evaluation parameters include specific binding of cells to an antigen in vivo , eg, by imaging, or ex vivo , eg, by ELISA or flow cytometry. In some embodiments, the ability of cells to destroy target cells can be assessed by any suitable method known to those skilled in the art, such as the cytotoxicity assays described, for example, in Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 ( 2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In some embodiments of the invention, the biological activity of cells can also be assessed by analyzing the expression and/or secretion of certain cytokines, such as CD107a, IFNγ, IL-2 and TNF. In some aspects of the invention, biological activity is measured by assessing clinical outcomes such as reduction in tumor burden or tumor volume. In some aspects, toxic effects, cell persistence and/or proliferation, and/or the presence or absence of an immune response in the host are assessed.

B. Compositions and preparations

[0458] In some embodiments of the invention, the dose of cells containing cells constructed using a recombinant antigen receptor, for example, CAR or TCR, is obtained in the form of a composition or preparation, such as a pharmaceutical composition or preparation. Such compositions may be used in accordance with the methods disclosed herein and/or as described in articles relating to formulation or compositions, for example, in the prevention or treatment of diseases, conditions and disorders, or in methods of detection, diagnosis and evaluation of prognosis.

[0459] The term "pharmaceutical composition" means a preparation that is prepared in a form that imparts effective biological activity to the active ingredient contained in the composition, and that does not contain any additional components that are unacceptably toxic to the individual who is administered this composition.

[0460] The term "pharmaceutically acceptable carrier" means an ingredient in a pharmaceutical composition that is not an active ingredient and that is non-toxic to an individual. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0461] In some aspects, the choice of carrier is determined in part by the particular cell or agent and/or route of administration. Accordingly, there are various suitable compositions. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may be, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of from about 0.0001% to about 2% by weight of the entire composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (approximately less than 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

[0462] In some aspects, the compositions include buffering agents. Suitable buffering agents are, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of from about 0.001% to about 4% by weight of the entire composition. Methods for preparing injectable pharmaceutical compositions are known. Representative methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).

[0463] The drug or composition may also include more than one active ingredient suitable for the treatment of a particular disease, disorder or condition that is being prevented or treated using cells or agents, where the respective activities do not adversely affect each other. Such active ingredients are preferably present in combination in amounts effective to achieve the desired objectives. Thus, in some embodiments of the invention, the pharmaceutical composition also includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the agents or cells are administered in the form of a salt, such as a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts are salts of mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids and organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic , succinic and arylsulfonic acids, for example, p-toluenesulfonic acid.

[0464] In some embodiments of the invention, the pharmaceutical composition contains agents or cells in an amount effective for the treatment or prevention of a disease or condition, for example, a therapeutically effective or prophylactically effective amount. In some embodiments of the invention, individuals undergoing treatment are periodically examined to evaluate therapeutic or prophylactic efficacy. For repeated administration over several days or more, depending on the condition, treatment is continued until the desired suppression of the symptoms of the disease is achieved. However, other dosing regimens may be suitable and may be developed. The desired dose may be delivered by a single bolus of the composition, multiple bolus of the composition, or by continuous infusion of the composition.

[0465] The agents or cells may be administered by any suitable route, e.g., by bolus infusion, by injection, e.g., by intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, vitreous injection, trans-septal injection, subscleral injections, intrachoroidal space injections, intracameral injections, subconjunctival injections, subtenon capsule injections, retrobulbar injections, peribulbar injections, or injections into the posterior space adjacent to the sclera. In some embodiments of the invention, these injections are administered parenterally, intrapulmonary and intranasally, and, if necessary, locally by injection into the affected area. Parenteral infusions are intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. In some embodiments of the invention, this dose is administered by introducing a single shock dose of cells or agent. In some embodiments of the invention, the dose is administered by administering multiple bolus doses of cells or agent, for example over a period of time not greater than 3 days, or by continuous infusion of cells or agent.

[0466] For the prevention or treatment of a disease, the appropriate dose may depend on the type of disease being treated, the type of agent or agents, the type of cell or recombinant receptor, the severity and course of the disease, whether the agent or cells are being administered prophylactically or therapeutically. , previous treatment, the patient's medical history, and response to the agent or cells, and at the discretion of the attending physician. In some embodiments of the invention, the compositions are suitable for administration to an individual immediately or as a course of treatment.

[0467] Cells or agents can be introduced by standard methods of administration, using drugs and/or devices. Formulations and devices such as syringes and vessels are used to store and administer the compositions. The cells introduced may be autologous or heterologous. Thus, for example, immunoresponsive or progenitor cells may be taken from one individual and administered to the same individual or another compatible individual. Immunoresponsive cells or their progeny derived from peripheral blood (for example, obtained in vivo , ex vivo or in vitro ) can be administered by local injection, including introduction by catheter, systemic injection, localized injection, intravenous injection or parenteral administration. When a therapeutic composition is administered (for example, a pharmaceutical composition containing genetically modified immunoresponsive cells or an agent that helps treat or alleviate the symptoms of neurotoxicity), such a composition is usually prepared in the form of an injectable unit dosage form (solution, suspension, emulsion).

[0468] Compositions are compositions for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments of the invention, the agent or cell populations are administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments of the invention, the agent or cell populations are administered to the individual via peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0469] In some embodiments of the invention, the compositions are prepared as sterile liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which, in some aspects, can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous formulations, and solid formulations. In addition, liquid formulations are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range so as to provide a longer period of contact with certain tissues. Liquid or viscous compositions may contain carriers, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0470] Sterile injectable solutions can be prepared by incorporating the agent or cells in a solvent, for example, in admixture with a suitable carrier, diluent, or excipient such as sterile water, saline, glucose, dextrose, or the like.

[0471] Compositions used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filter membranes.

C. Combination therapy, e.g., agents to modulate cell proliferation and activity

[0472] In some embodiments, the cells are administered as part of a combination therapy, eg, simultaneously or sequentially in any order, with another agent, eg, a therapeutic agent such as a drug. In some embodiments, the other agent, such as a drug, may be a therapeutic agent, such as an antibody or engineered cell, or a receptor or agent, such as a cytotoxic or therapeutic agent. In some embodiments of the invention, another agent, for example, a drug, may be an agent that enhances, increases or increases the rate of reproduction, proliferation, survival and/or efficiency of cells. In some cases, the cells are administered together with another therapy, after a sufficiently short period of time so that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the administration of one or more additional therapeutic agents. In some embodiments of the invention, the cells are administered after the administration of one or more additional therapeutic agents.

[0473] In some embodiments of the invention, such methods are methods of combined administration, for example, simultaneous or sequential administration together with a drug or agent capable of increasing, enhancing or increasing the degree of reproduction, proliferation, survival and / or efficiency of administered cells, for example, cells expressing the recombinant receptor. In some embodiments of the invention, such agents are administered simultaneously or sequentially in any order along with another agent, such as a drug. In some embodiments of the invention, the agent is administered before, during, during the course of treatment, or after the introduction of cells, for example, cells expressing a recombinant receptor, such as CAR. In some embodiments of the invention, such agents are agents that specifically increase, enhance or increase the rate of reproduction, proliferation, survival and/or efficiency of engineered cells due to the specific modulation of a transgene, for example, a transgene encoding a recombinant receptor. In some embodiments of the invention, such agents include agents that modulate cell proliferation and/or the activity of administered cells, for example, immune cells such as T cells.

[0474] In some embodiments, the administered cells, e.g., cells engineered to express the recombinant receptor, are modified to increase, enhance, or increase the expansion, proliferation, survival, and/or efficiency of the administered cells. In some embodiments, the administered cells, e.g., cells engineered to express a recombinant receptor, are modified such that the multiplication, proliferation, survival, and/or efficiency of the administered cells can be regulated and/or controlled, for example, by administering an agent.

[0475] In some embodiments, the methods include in vivo steps to reduce, inhibit, and/or minimize the effects of inhibitory factors that inhibit the proliferation, expansion, and/or survival of engineered cells in vivo . In some embodiments, the methods include in vivo steps to stimulate, maintain and/or enhance proliferation, expansion and/or survival of engineered cells in vivo .

[0476] In some embodiments, the additional agent is a small molecule, peptide, polypeptide, antibody or antigen-binding fragment thereof, antibody mimetic, aptamer or nucleic acid molecule (e.g., siRNA), lipid, polysaccharide, or any combination thereof. In some embodiments of the invention, the additional agent is an inhibitor or activator of a particular factor, molecule, receptor, function and/or enzyme. In some embodiments of the invention, the additional agent is an agonist or antagonist of a particular factor, molecule, receptor, function and/or enzyme. In some embodiments of the invention, the additional agent is an analogue or derivative of one or more factors and/or metabolites. In some embodiments of the invention, the additional agent is a protein or polypeptide. In some embodiments of the invention, the additional agent is a cell, for example, an engineered cell.

1. Agents for transgene-specific propagation

[0477] In some embodiments of the invention, the methods include the introduction of agents, for example, agents for combination therapy in addition to cells, such as cells engineered to express recombinant receptors. In some embodiments of the invention, agents specifically increase, enhance or increase the level of reproduction, proliferation, survival and/or efficiency of engineered cells due to specific modulation of a transgene, for example, a transgene encoding a recombinant receptor. In some embodiments of the invention, the agent specifically targets the transgene, for example, a recombinant receptor. In some embodiments of the invention, the agent specifically binds to a recombinant receptor, activates and/or increases the activity of this recombinant receptor and/or other functions of all or part of the recombinant molecule encoded by the transgene. In some embodiments of the invention, the introduction of an agent in combination with recombinant cells can increase, increase or increase the level of proliferation, expansion and/or survival of the administered cells, for example, increase the level of expansion of cells in vivo .

[0478] In some embodiments, representative methods or agents for transgene-specific propagation include treatment with an endogenous antigen, vaccination, treatment with anti-idiotypic antibodies or antigen-binding fragments thereof, and/or a regulated recombinant receptor. Thus, for example, in some embodiments of the invention, methods of transgene-specific reproduction include methods of vaccination. In some embodiments, the agent is a peptide or cell-based vaccine, e.g., cells engineered to express a particular antigen recognized by a recombinant receptor (see e.g. WO 2016/069647, WO 2011/066048, US 2016/0304624 , US Patent No. 9476028 and Hailemichael & Overwijk, Int J Biochem Cell Biol (2014) 53:46-50). In some embodiments of the invention, methods of transgene-specific propagation include the introduction of anti-idiotypic antibodies. Anti-idiotypic antibodies, including their antigen-binding fragments, specifically recognize, specifically target an antibody idiotope and/or an antigen-binding fragment thereof, and/or specifically bind to that idiotope or an antigen-binding fragment thereof, e.g., the antigen-binding domain of a recombinant receptor, such as a chimeric antigen receptor (CAR ). An idiotope is any one antigenic determinant or epitope in the variable region of an antibody. In some embodiments of the invention, anti-idiotypic antibodies or antigen-binding fragments thereof are agonists and/or have specific activity that contributes to the stimulation of cells expressing a particular antibody, including conjugates or recombinant receptors containing this antibody or its antigen-binding fragment (see, for example, publications US Patent Applications 2016/0096902; 2016/0068601; 2014/0322183; 2015/0175711; 2015/283178; US Patent No. 9102760; Jena et al. PloS one (2013) 8(3):e57838; Long et al ., Nature Medicine (2015) 21(6):581-590; Lee et al., The Lancet (2015) 385(9967):517-528; Zhao et al., PloS One (2014) 9(5): e96697; Leung et al., MAbs (2015) 7(1):66-76).

2. Agents for modulating cell proliferation or activity

[0479] In some embodiments, the methods include modulating the reproduction, proliferation, survival and/or activity of immune cells or immune function in general, including engineered cells being administered. In some embodiments of the invention, the methods include steps that essentially promote immunostimulation or, essentially, contribute to the stimulation, enhancement, increase and / or increase in the reproduction, proliferation, survival and / or activity of immune cells, including administered cells in vivo , for example, in the body of individuals. In some embodiments of the invention, the agent may reduce, inhibit and/or minimize the effects of inhibitory factors that suppress the proliferation, expansion and/or survival of immune cells, for example, administered cells in vivo .

a. Inhibition of negative regulators

[0480] In some embodiments, the methods include modulating engineered cell proliferation, eg, by inhibiting a negative regulator of proliferation, proliferation, and/or activation of administered cells, eg, engineered immune cells. In a specific case, in the body of an individual, the introduced cells expressing the recombinant receptor may interact with an environment that suppresses or inhibits the growth, proliferation, reproduction and/or survival of cells, for example, in an immunosuppressive environment. For example, immunosuppressive media may contain immunosuppressive cytokines, regulatory modulators, and co-inhibitory receptors. In some embodiments of the invention, an additional agent can be used to modulate the expansion of the injected cells, for example, to suppress the suppressive environment.

[0481] In some embodiments, the additional agent includes an immunomodulatory agent, an immune checkpoint inhibitor, metabolic pathway modulators, adenosine pathway or adenosine receptor antagonist or agonist, and signaling pathway modulators, e.g., kinase inhibitors.

[0482] In some embodiments of the invention, the additional agent is an immunomodulatory agent, such as an immune checkpoint inhibitor. In some examples, the additional agent (i.e., an immune checkpoint inhibitor) increases, enhances, or enhances the expansion and/or proliferation of the administered cells, and thereby increases, enhances, or enhances the immune response by blocking the immune checkpoint protein. In some embodiments of the invention, the additional agent is an agent that increases the activity of the engineered cell, for example, a cell expressing a recombinant receptor, and is a molecule that inhibits an immune suppressive molecule or an immune checkpoint molecule. Examples of immune inhibitory molecules are PD-1, PD-L1, CTLA4, TEVI3, CEACAM ( eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFRβ. In some embodiments, the immune checkpoint inhibitor may be an antibody directed against an immune checkpoint protein, such as an antibody directed against cytotoxic T-lymphocyte antigen 4 (CTLA4 or CD152), programmed cell death protein 1 (PD-1) , or programmed cell death protein ligand 1 (PD-L1) (see, eg, Pardoll, Nat Rev Cancer. 2012 Mar. 22; 12(4):252-264).

[0483] In some embodiments, the methods include contacting cells expressing the recombinant receptor with an agent that inhibits inhibitory cell surface receptors, such as transforming growth factor receptor beta (TGFβR). In some embodiments of the invention, the introduced cells, for example, cells expressing the recombinant receptor, can be engineered to block the effects of immunosuppressive cytokines that can inhibit the effector functions of these cells (see, for example, Foster et al., J Immunother. (2008) 31:500-505; Bollard et al., Molecular Therapy (2012) 20:S22; Bendle et al., J. Immunol. (2013) 191(6):3232-3239). In some embodiments of the invention, the additional agent is an anti-TGFβ or anti-TGFβR antibody (see, for example, WO 2011/109789).

[0484] In some embodiments of the invention, the additional agent modulates the metabolism, signaling and/or transport of immunosuppressive factors, such as adenosine. In some embodiments of the invention, the additional agent is an inhibitor of extracellular adenosine or adenosine receptor, or an agent that causes a decrease or decrease in extracellular levels of adenosine, such as an agent that prevents the formation of extracellular adenosine, degrades it, makes it inactive and / or reduces its levels . In some embodiments of the invention, the additional agent is an adenosine receptor antagonist, such as the A2a, A2b and/or A3 receptor.

[0485] In some embodiments of the invention, the additional agent is a modulator of levels of adenosine and/or a component of the adenosine pathway. Adenosine can function as an immunomodulatory agent in the body. For example, adenosine and certain adenosine analogs that non-selectively activate adenosine receptor subtypes reduce neutrophil production of inflammatory oxidation products (Cronstein et al., Ann. N.Y. Acad. Sci. 451:291, 1985; Roberts et al., Biochem. J. 227:669, 1985; Schrier et al., J. Immunol. 137:3284, 1986; Cronstein et al., Clinical Immunol. Immunopath. 42:76, 1987). In some cases, the concentration of extracellular adenosine or adenosine analogs may be increased in certain environments, such as the tumor microenvironment (TME). In some cases, adenosine or an adenosine analog signal in a manner dependent on hypoxia or factors involved in the development or regulation of hypoxia, such as hypoxia-inducing factor (HIF). In some embodiments of the invention, an increase in the level of adenosine signal can increase the level of intracellular cAMP and cAMP-dependent protein kinase, which will lead to inhibition of the production of pro-inflammatory cytokines, and can lead to the synthesis of immunosuppressive molecules and the development of Treg (Sitkovsky et al., Cancer Immunol Res ( 2014) 2(7):598-605). In some embodiments of the invention, an additional agent can reduce or abolish the immunosuppressive effects of adenosine, adenosine analogs and/or adenosine signals. In some embodiments of the invention, an additional agent can reduce or abolish A2-adenosynergic immunosuppression of T cells induced by hypoxia. In some embodiments, the additional agent is selected from adenosine receptor antagonists, extracellular adenosine degrading agents, inhibitors of adenosine production by CD39/CD73 ectoenzymes, and inhibitors of the HIF-1α signal that causes hypoxia. In some embodiments of the invention, the additional agent is an adenosine receptor antagonist or agonist.

[0486] Inhibition or reduction of extracellular adenosine or an adenosine receptor by an inhibitor of extracellular adenosine (such as an agent that prevents the formation of extracellular adenosine, degrades it, makes it inactive and / or reduces its levels) and / or an adenosine receptor inhibitor (such as an adenosine receptor antagonist ) may enhance the immune response, such as that mediated by macrophages, neutrophils, granulocytes, dendritic cells, T and/or B cells. In addition, inhibitors of the cAMP-dependent intracellular pathway mediated by Gs protein and inhibitors of the intracellular pathways mediated by Gi protein and triggered by the adenosine receptor can also increase acute and chronic inflammation.

[0487] In some embodiments of the invention, the additional agent is an adenosine receptor antagonist or agonist, for example, an antagonist or agonist of one or more adenosine receptors A2a, A2b, A1 and A3. A1 and A3 inhibit, and A2a and A2b stimulate, respectively, adenylate cyclase activity. Some adenosine receptors, such as A2a, A2b, and A3, can suppress or reduce the immune response during inflammation. Thus, inhibition of immunosuppressive adenosine receptors can enhance, enhance, or enhance the immune response, eg, the immune response to administered cells, eg, CAR-expressing T cells. In some embodiments of the invention, the additional agent inhibits the production of extracellular adenosines and signal transmission, triggered by adenosine, through adenosine receptors. Thus, for example, enhancement of the immune response, local tissue inflammation, and targeted tissue destruction can be further enhanced by inhibiting or reducing local adenosine-producing tissue hypoxia; destruction of accumulated extracellular adenosine (or blocking its activity); preventing or reducing the expression of adenosine receptors on cells of the immune system; and/or inhibition/blocking of signal transmission by adenosine ligands via adenosine receptors.

[0488] An antagonist is any substance that tends to nullify the effect of another agent, such as an agent that binds to a cellular receptor but does not produce a biological response. In some embodiments, the antagonist is a chemical compound that is an adenosine receptor antagonist, such as an A2a, A2b, or A3 receptor. In some embodiments, the antagonist is a peptide or peptidomimetic that binds to the adenosine receptor but does not trigger an intracellular G1 protein-dependent pathway. Examples of adenosine receptor antagonists are described in US Pat. Nos. 5,565,566; 5,545,627; and Jacobson and Gao, Nat Rev Drug Discov. (2006) 5(3): 247-264.

b. Initiation of immunostimulation

[0489] In some embodiments of the invention, the methods include the introduction of additional agents that are immunostimulatory. In some embodiments of the invention, the additional agent may essentially stimulate the proliferation, reproduction, survival and/or efficiency of immune cells. In some embodiments of the invention, the additional agent can specifically stimulate the injected cells, for example, cells expressing the recombinant receptor. In some embodiments of the invention, the additional agent is a cytokine. In some embodiments of the invention, the additional agent is a ligand.

[0490] In some embodiments of the invention, the additional agent is an immunostimulatory ligand, for example, CD40L. In some embodiments, the additional agent is a cytokine, such as IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha, beta or gamma (IFN) and erythropoietin (EPO).

3. Lymphatic depletion therapy

[0491] In some aspects, the methods contemplated herein may also include conducting one or more lymph-depleting therapies, for example, before or simultaneously with the start of administration of cells, for example, cells expressing a recombinant receptor. In some embodiments, the lymph depletion therapy comprises the administration of a phosphamide, such as cyclophosphamide. In some embodiments, the lymph depletion therapy may include the administration of fludarabine. In some embodiments, no fludarabine is administered during lymph depletion therapy. In some embodiments of the invention, no lymphatic depletion therapy is performed.

[0492] Preliminary administration of an immuno-depleting (eg, lymph-depleting) therapy to an individual may improve the effects of adoptive cell therapy (ACT). Pre-administration of lymph-depleting agents, including combinations of cyclosporine and fludarabine, has proven effective in increasing the efficiency of tumor infiltrating lymphocyte (TIL) transfer in cell therapy, including improving the response and/or persistence of transferred cells. See, for example, Dudley et al.Science, 298, 850-54 (2002); Rosenberg et al.,Clin Cancer Res, 17(13):4550-4557 (2011). The same way, in case of CAR⁺Several studies have been conducted on T-cells using lymph-depleting agents, mainly cyclophosphamide, fludarabine, bendamustine, or a combination thereof, sometimes together with low-dose irradiation. See Han et al.Journal of Hematology & Oncology, 6:47 (2013); Kochenderfer et al.blood, 119: 2709-2720 (2012); Kalos et al.,Sci Transl Med, 3(95):95ra73 (2011); Clinical Trial Study Record No.: NCT02315612; NCT01822652.

[0493] Such pre-treatment may be carried out in order to reduce the risk of one or more different outcomes that may reduce the effectiveness of therapy. These are the phenomenon known as the "cytokine shell" where T cells, B cells, NK cells compete with TIL for homeostasis and activation of cytokines such as IL-2, IL-7 and/or IL-15; suppression of TIL by regulatory T cells, NK cells, or other cells of the immune system; and exposure to negative regulators in the tumor microenvironment. Muranski et al., Nat Clin Pract Oncol. December; 3(12): 668-681 (2006).

[0494] Thus, in some embodiments of the invention, the contemplated method also includes providing an individual with lymph depletion therapy. In some embodiments, the method includes administering lymph depletion therapy to the individual prior to administration of the cell dose. In some embodiments, the lymph depletion therapy comprises the administration of a chemotherapeutic agent such as fludarabine and/or cyclophosphamide. In some embodiments of the invention, the administration of cells and/or the administration of lymphatic depletion therapy is carried out on an outpatient basis.

[0495] In some embodiments, the methods include administering to the individual a preconditioning agent, such as a lymph-depleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, prior to administration of the cell dose. Thus, for example, the subject may be administered a preconditioning agent at least 2 days, such as at least 3, 4, 5, 6, or 7 days prior to the first or subsequent dose. In some embodiments of the invention, the individual is administered a preconditioning agent no more than 7 days, for example, no more than 6, 5, 4, 3 or 2 days before the dose of cells.

[0496] In some embodiments, the individual is pre-administered with cyclophosphamide at a dose of from or about 20 mg/kg to 100 mg/kg, for example, from or about 40 mg/kg to 80 mg/kg. In some aspects, the individual is pre-administered with 60 mg/kg or about 60 mg/kg of cyclophosphamide. In some embodiments, the cyclophosphamide may be administered in a single dose or in divided doses, such as daily, every other day, or every three days. In some embodiments of the invention, cyclophosphamide is administered once a day for one or two days. In some embodiments, if the lymph-depleting agent comprises cyclophosphamide, then the subject is administered cyclophosphamide at a dose of from or about 100 mg/m ² to 500 mg/m ² , for example, at a dose of from or about 200 mg/m ² to 400 mg /m ² , or from 250 mg/m ² to 350 mg/m ² inclusive. In some cases, approximately 300 mg/m ² cyclophosphamide is administered to the subject. In some embodiments, the cyclophosphamide may be administered in a single dose or in divided doses, such as daily, every other day, or every three days. In some embodiments of the invention, cyclophosphamide is administered daily, for example, for 1-5 days, for example, for 3-5 days. In some cases, the subject is administered approximately 300 mg/m ² cyclophosphamide, daily for 3 days prior to the start of cell therapy.

[0497] In some embodiments, fludarabine may be administered in a single dose or in divided doses, such as daily, every other day, or every three days. In some embodiments of the invention, fludarabine is administered daily, for example, for 1-5 days, for example, for 3-5 days. In some cases, the subject is administered approximately 30 mg/m ² fludarabine, daily for 3 days prior to cell therapy. In some embodiments of the invention, cyclophosphamide is administered daily for one or two days.

[0498] In some embodiments, if the lymph-depleting agent includes fludarabine, then the individual is administered fludarabine at a dose of from or about 1 mg/m ² to 100 mg/m ² , for example, from or about 10 mg/m ² to 75 mg/m ² , 15 mg/m ² to 50 mg/m ² , 20 mg/m ² to 30 mg/m ² , or 24 mg/m ² to 26 mg/m ² . In some cases, the individual is administered 25 mg/m ² fludarabine. In some embodiments, fludarabine may be administered in a single dose or in divided doses, such as daily, every other day, or every three days. In some embodiments of the invention, fludarabine is administered daily, for example, for 1-5 days, for example, for 3-5 days.

[0499] In some embodiments, the lymph depleting agent includes a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or schedule, such as the doses and schedules described above, and fludarabine at any dose or schedule, such as the doses and schedules described above. Thus, for example, in some aspects, the individual is administered 60 mg/kg (~2 g/m ² ) cyclophosphamide and 3 to 5 doses of 25 mg/m ² fludarabine prior to administration of the cell dose.

[0500] In one representative dosing regimen, prior to the first dose, the subject is given prior lympho-depleting chemotherapy with cyclophosphamide and fludarabine (cyc/flu) at least two days prior to the first dose of CAR-expressing cells and, in generally, no more than 7 days before the introduction of cells. After pre-treatment, the individual is dosed with CAR-expressing T cells as described above.

[0501] In some embodiments of the invention, the introduction of a preconditioning agent prior to infusion of a dose of cells improves the outcome of treatment. Thus, for example, in some aspects, pre-treatment increases the efficacy of a dose treatment or increases the persistence of recombinant receptor-expressing cells (eg, CAR-expressing cells, such as CAR-expressing T cells) in an individual. In some embodiments of the invention, pre-treatment increases disease-free life, e.g., increases the percentage of surviving individuals who do not experience minimal residual or molecularly detectable signs of disease for a certain period of time after a dose of cells. In some embodiments of the invention, median disease-free life has increased.

[0502] After the cells are administered to an individual (eg, a human), the biological activity of the engineered cell populations is, in some aspects, measured by any of a number of known methods. Evaluation parameters include specific binding of engineered or native T cells or other immune cells to the antigen in vivo , eg, by imaging, or ex vivo , eg, by ELISA or flow cytometry. In some embodiments of the invention, the ability of engineered cells to destroy target cells can be assessed by any suitable method known to those skilled in the art, such as cytotoxicity assays as described, for example, by Kochenderfer et al. , J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In some embodiments of the invention, the biological activity of cells can also be assessed by analyzing the expression and/or secretion of certain cytokines, such as CD107a, IFNγ, IL-2 and TNF. In some aspects of the invention, biological activity is measured by assessing clinical outcomes such as reduction in tumor burden or tumor volume. In some aspects, toxic effects, cell persistence and/or proliferation, and/or the presence or absence of an immune response in the host are assessed.

[0503] In some embodiments, administration of a preconditioning agent prior to cell dose infusion improves treatment outcome, e.g., increases the efficacy of dose treatment or increases the persistence of cells expressing the recombinant receptor (e.g., CAR-expressing cells, such as CAR-expressing T cells ) in an individual.

D. Cell modification

[0504] In some embodiments of the invention, the cells are modified in any way, so that it leads to an increase in their therapeutic or prophylactic effectiveness, and / or to modulation of reproduction, proliferation, survival and / or efficiency. In some embodiments of the invention, cells are modified to modulate reproduction, proliferation, survival and/or efficiency, for example, to increase, enhance and/or increase in vivo after administration of cells to an individual. In some embodiments of the invention, cells are modified so that modulation and/or regulation of the expression of transgenes and/or immunomodulatory factors can be carried out. In some embodiments of the invention, the cells are modified to modulate the expression and/or activity of specific components of the recombinant receptor. In some embodiments of the invention, cells are modified to increase or decrease the level of expression of an agent, for example, a nucleic acid, such as inhibitory nucleic acids. In some embodiments of the invention, cells are modified to express and/or secrete an agent.

[0505] In some embodiments, a recombinant engineered receptor, eg, CAR, expressed by engineered cells can be conjugated either directly or indirectly through a linker to a target molecule. A practical method for contacting compounds, for example a recombinant receptor, with target molecules is known to those skilled in the art. See, for example, Wadwa et al. , J. Drug Targeting 3: 111 (1995) and US Patent 5,087,616.

1. Inhibitory nucleic acids and gene modification

[0506] In some embodiments of the invention, the methods include modifying the administered cells by contacting the cells with an agent that reduces or has the ability to effectively reduce the level of expression of negative regulators by the administered cells, for example, an engineered T cell expressing the recombinant receptor. Negative cellular regulators include any of the regulators described herein, such as an immune checkpoint inhibitor, inhibitory receptors, and/or adenosine modulators. In some embodiments of the invention, an agent that reduces or has the ability to effectively reduce the level of expression of negative regulators includes agents that are or include an inhibitory nucleic acid molecule, such as a molecule that is complementary to a gene or nucleic acid encoding a negative regulator, and that targets, inhibits, and/or binds to that gene or nucleic acid. In some embodiments, the agent is or includes a complex containing a ribonucleoprotein (RNP) complex that includes Cas9, such as, in some cases, enzymatically inactive Cas9, and rRNA targeted to a gene encoding a negative regulator.

[0507] In some of any such embodiments of the invention, the inhibitory nucleic acid molecule comprises an RNA interference agent. In some of any such embodiments, the inhibitory nucleic acid is or contains or encodes a short interfering RNA (siRNA), a shRNA, an adapted miRNA, a short hairpin RNA (shRNA), a hairpin shRNA, a precursor microRNA (pre-miRNA), or a microRNA. (siRNA). Methods for constructing inhibitory nucleic acids and modifying cells to express inhibitory nucleic acids are known to those skilled in the art (see, for example, WO 2004/0455543 and WO 2004/048566).

[0508] In some embodiments, the engineered cell is subjected to gene modification or gene editing that targets a locus encoding a gene involved in immunomodulation, immune cell downregulation, and/or immunosuppression. In some embodiments of the invention, gene editing results in an insertion or deletion at the target locus, or in "knockout" of the target locus and elimination of expression of the encoded protein. In some embodiments of the invention, gene editing is achieved by joining non-homologous ends (NHEJ) under the action of the CRISPR/Cas9 system. In some embodiments, one or more guide RNA (rRNA) molecules can be used in conjunction with one or more Cas9 nucleases, Cas9 nickases, enzymatically inactive Cas9, or variants thereof, or with engineered zinc finger proteins or with TALE systems. Gene modification methods are known to those skilled in the art (see, for example, WO 2015/161276; US Pat. , WO 03/016496 and US Application Publication 2011/0301073).

2. Modification for the expression of additional agents

[0509] In some embodiments of the invention, cells, for example, cells expressing a recombinant receptor, are further modified to express and/or secrete an additional agent that stimulates, enhances, increases and/or increases the proliferation, reproduction, survival and/or efficacy of administered cells. For example, cells expressing the recombinant receptor, such as CAR expressing cells, can be further engineered to express and/or secrete additional agents that block immunosuppressive effects and/or increase proliferation and/or improve T cell and recombinant receptor function. . In some embodiments of the invention, the cells can be engineered to express cytokines that promote the expansion of the injected cells. In some embodiments, such additional agents may be operably linked to an inducible expression system, such as an inducible promoter.

[0510] In some embodiments, the administered cells may be modified to express and/or secrete an agent that inhibits immunosuppressive factors, such as any of the factors described herein, and/or stimulates an immunostimulatory factor. In some embodiments, an additional agent expressed by the introduced cell reduces or prevents immunosuppression of said cell in the tumor microenvironment (see, for example, US Patent Application Publication No. 2016/0045551). In some embodiments of the invention, the additional agent that is encoded and/or secreted by the administered cells may include any of the additional agents described here.

[0511] In some embodiments of the invention, the additional agent that is encoded by the introduced cell is soluble and secreted. In some embodiments of the invention, the additional agent is a soluble ScFv. In some embodiments of the invention, the additional agent is a cytokine.

3. Regulation of the expression and/or activity of the recombinant receptor

[0512] In some embodiments of the invention, the methods include modifying cells to regulate the expression and/or activity of a recombinant receptor, for example, CAR, and thereby regulate the signal through the recombinant receptor. In some embodiments of the invention, regulated expression and/or activity is achieved by constructing a recombinant receptor so that it contains specific regulatory elements and/or systems or is under the control of such elements and/or systems, such as any of the elements or systems described here. In some embodiments of the invention, the introduction of the engineered cell into the body of the individual and/or its treatment with a specific ligand can regulate the expression and/or activity of a recombinant receptor, for example, CAR. In some embodiments, regulation of the expression and/or activity of the recombinant receptor is achieved by administering an additional agent that can regulate the expression of the recombinant receptor, such as CAR. In some embodiments, regulated expression of a recombinant receptor, such as CAR, is achieved through a regulated transcription factor release system or by administering an additional agent that can induce conformational changes and/or multimerization of polypeptides, such as a recombinant receptor. In some embodiments of the invention, the additional agent is a chemical inducer.

IV. DEFINITIONS

[0513] Unless otherwise stated, all terms used in the literature, explanatory terms, and other technical and scientific terms or terminology used in this application have generally accepted meanings that are understood by a person skilled in the field to which the claimed subject matter belongs. In some cases, terms having conventional meanings are defined herein for clarity and/or as a reference, and such definitions included in this specification should not necessarily be construed as substantially different from definitions commonly used in the art.

[0514] As used herein, singular objects with the articles "a", "an" and "the" include referents in the plural, unless the context of the description implies otherwise. So, for example, "a" or "an" means "at least one" or "one or more".

[0515] Throughout the description of the invention, various aspects of the claimed subject matter are presented in a systematic format. It should be noted that the description in a systematic format is provided only for convenience and brevity of description and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of the intervals should be considered as a specific disclosure of all possible sub-intervals, as well as individual numerical values in this interval. Thus, for example, if a range of values is specified, then it should be understood that every intermediate value between the upper and lower limits of that range and any other specified or intermediate value within that specified range is covered by the claimed subject matter. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges and are also covered by the claimed subject matter, subject to any specifically excluded limit in the specified range. If a specified range includes one or both of the limits, then ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the latitude of the interval.

[0516] The term "approximately" as used herein refers to the typical error interval for the corresponding value, well known to a person skilled in the art. Reference to an "approximate" value or parameter in this specification includes (and describes) variations relating to that value or parameter per se .

[0517] As used herein, the term "individual" includes any living organism, such as humans and other mammals. Mammals include, but are not limited to, humans and non-human animals, including farm animals, competitive animals, rodents, and pets.

[0518] As used herein, the term "depletion", when referring to one or more specific cell type or cell population, means a decrease in the number or percentage of cells of a particular type or population, for example, compared to the total number of cells in a composition or within the scope of the composition, or compared to other cell types, for example, as a result of negative selection based on markers expressed by the population or cell, or positive selection based on markers not present in the depleted cell population or cell. This term does not imply the complete removal of cells, cells of a certain type or population from the composition.

[0519] As used herein, the term "enrichment", when referring to one or more specific cell type or cell population, means an increase in the number or percentage of cells of a particular cell type or population, for example, compared to the total number of cells in a composition or within the scope of the composition, or compared to other cell types, for example, as a result of positive selection based on markers expressed by the population or cell, or negative selection based on markers not present in the depleted cell population or cell. The term does not imply complete removal of other cells, cell types or populations from the composition, and does not imply 100% or even nearly 100% enrichment of cells in an enriched composition.

[0520] As used herein, a statement that a cell or population of cells is "positive" or "+" for a particular marker means the detectable presence on or within a cell of a particular marker, usually a cell surface marker. If we are talking about a surface marker, then this term means the presence of expression on the surface, detected, in some embodiments of the invention using flow cytometry, for example, by staining with an antibody that specifically binds to the marker, and detecting said antibody, where such staining is detected using flow cytometry at a level significantly higher than the staining detected by the same procedure but in the presence of an appropriate isotype control, all other things being equal, and/or at a level substantially similar to that of cells known to be positive for the marker, and/or at a level significantly higher than for cells known to be negative for the marker.

[0521] As used herein, the statement that a cell or population of cells is "negative" for a particular marker means the absence of any detectable presence on or within a cell of a particular marker, usually a cell surface marker. When it comes to a surface marker, this term means the absence of surface expression, detected, in some embodiments, by flow cytometry, for example, by staining with an antibody that specifically binds to the marker, and detecting said antibody, where such staining is not detected with using flow cytometry at a level significantly higher than the staining detected by the same procedure but in the presence of an appropriate isotype control, all other things being equal, and/or at a level that is essentially significantly lower for cells known to , are positive for the marker, and/or at a level substantially similar to that of cells known to be negative for the marker.

[0522] As used herein, the term "percent (%) amino acid sequence identity" and "percent identity" when referring to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., Vpx or Vpr protein) , which are identical to amino acid residues in the reference polypeptide sequence, after sequence alignment and gap insertion, if necessary to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways that are known to those skilled in the art, for example using publicly available computer programs such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). The person skilled in the art is free to determine the appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences.

[0523] An amino acid substitution may involve replacing one amino acid in a polypeptide with another amino acid. Amino acids can generally be classified according to the following general side chain properties:

[0524] (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0525] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0526] (3) acidic: Asp, Glu;

[0527] (4) main: His, Lys, Arg;

[0528] (5) residues affecting chain orientation: Gly, Pro;

[0529] (6) aromatic: Trp, Tyr, Phe.

[0530] Non-conservative amino acid substitutions include replacing a member of one of these classes with a member of another class.

[0531] As used herein, the term "at a position corresponding to" or the statement that nucleotides or amino acid positions "correspond" to nucleotides or amino acid positions in a disclosed sequence, such as the sequence specified in a sequence listing, refers to the nucleotide or amino acid positions identified after open-sequence alignments to maximize identity according to a standard alignment algorithm such as the GAP algorithm. In some embodiments, representative corresponding Vpx or Vpr protein residues can be identified by sequence alignment with a representative Vpx sequence set forth in SEQ ID NO: 1 or with another Vpx or Vpr sequence described herein. In some embodiments, representative corresponding SAMHD1 residues can be identified by sequence alignment with the representative SAMHD1 sequence shown in SEQ ID NO: 19. residues as a reference. Generally speaking, in order to determine the corresponding positions, the amino acid sequences are aligned so that the best match is achieved (see, for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48 : 1073).

[0532] As used herein, the term "vector" means a nucleic acid molecule capable of replicating another nucleic acid to which it is attached. This term includes a vector as a self-replicating nucleic acid structure, as well as a vector incorporated into the genome of the host cell into which it has been introduced. Some vectors are capable of directing the expression of the nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors". Vectors include viral vectors, such as retroviral vectors, eg lentiviral or gamma-retroviral vectors, having a genome carrying a different nucleic acid and capable of integrating into the host genome and replicating them.

[0533] As used herein, the term "composition" means any mixture of two or more products, substances, or compounds, including cells. The composition may be a solution, suspension, liquid, powder, paste, aqueous, anhydrous, or any combination thereof.

[0534] As used herein, the terms "treatment", "treat", and "therapy" refer to the total or partial alleviation or reduction in the severity of a disease or condition or disorder or symptom, adverse effect or outcome, or associated phenotype. In some embodiments of the invention, the effect is therapeutic, that is, it consists in the partial or complete cure of a disease or condition, or an adverse symptom associated with them.

[0535] As used herein, the term "therapeutically effective amount" of a compound or composition or combination means an amount that, at specified doses and for a specified period of time, is effective to achieve a desired therapeutic result, e.g., to treat a disease, condition, or disorder and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary depending on such factors as the pathological condition, age, sex and body weight of the individual and the population of cells administered.

[0536] All publications, including patent documents, scientific articles and databases referred to in this application, are incorporated by reference in their entirety for all purposes as if each individual publication were separately incorporated by reference. If the definitions presented here contradict or otherwise do not correspond to the definition presented in patents, applications, published applications and other publications, which are incorporated into this description by reference, then the definitions presented in this application should be preferred, but not the definitions included in the present description by reference.

[0537] The section headings provided herein are for organizational purposes only and should not be construed as limiting the subject matter described herein.

V. Representative Options

[0538] The options presented here are:

1. A method for transducing T cells, wherein said method comprises incubating a viral vector particle containing a recombinant nucleic acid and an initial composition containing a plurality of T cells, wherein said plurality of T cells was obtained from a sample containing cells taken from an individual, where:

incubation begins no later than 24 hours after taking the sample from the individual; and/or

prior to incubation, the T cells were not brought to a temperature greater than or greater than about 15°C, about 18°C, about 22°C, or about 25°C for more than 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, or 24 hours after sampling from an individual; and/or

prior to incubation, T cells were not brought to a temperature of greater than or greater than approximately 37°C ± 2.0°C for a period of time greater than 15 minutes, 30 minutes, 1 hour, or 2 hours after sampling from an individual.

2. The method according to option 1, where the incubation begins no later than or no later than about 1 hour, 3 hours, 6 hours, 12 hours or 18 hours after taking the sample from the individual.

3. The method according to option 1 or option 2, where before said incubation, the method does not include stimulation of T cells under conditions that stimulate cell activation.

4. The method according to any of the options 1-3, where prior to said incubation, the original composition was not subjected to ex vivo stimulation, including incubation at a temperature of more than or about more than 37°C ± 2.0°C and/or incubation in the presence of an agent or agents capable of activating T cells, CD4 ⁺ -T cells and/or CD8 ⁺ -T cells, incubation in the presence of an agent or agents capable of activating the signal through the TCR complex and/or incubation in the presence of an agent or agents capable of inducing the proliferation of T cells, CD4 ⁺ -T cells and/or CD8 ⁺ -T cells; CD3 binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7.

5. The method according to any one of options 1-4, wherein prior to said incubation, no more than 5%, 10%, 20%, 30%, or 40% of activated T cells express a surface marker selected from the group , consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB; contain an intracellularly expressed cytokine selected from the group consisting of IL-2, IFN-gamma, TNF-alpha; are present in the G1 phase or in a later phase of the cell cycle and/or are capable of proliferating.

6. A method for transducing T cells, wherein said method comprises incubating a viral vector particle containing a recombinant nucleic acid and an initial composition containing T cells, wherein said T cells were obtained from a sample taken from an individual, where prior to said incubation, The T cells or parent composition have not been subjected to ex vivo stimulation, including incubation at a temperature greater than or approximately greater than 37° C.±2.0° C. and/or incubation in the presence of an agent or agents capable of activating T cells , CD4 ⁺ -T cells and/or CD8 ⁺ -T cells; incubation in the presence of an agent or agents capable of inducing a signal through the TCR complex; and/or incubation in the presence of an agent or agents capable of inducing the proliferation of T cells, CD4 ⁺ -T cells and/or CD8 ⁺ -T cells; CD3 binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7.

7. The method according to option 4 or option 6, where one or more agents contain an anti-CD3 antibody and/or an anti-CD28 antibody.

8. A method for transducing T cells, wherein said method comprises incubating a viral vector particle containing a recombinant nucleic acid and an initial composition containing T cells, wherein said T cells were obtained from a sample taken from an individual and where prior to said incubation , not more than 5%, 10%, 20%, 30% or 40% of activated T cells express a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB; contain an intracellularly expressed cytokine selected from the group consisting of IL-2, IFN-gamma, TNF-alpha; and/or are present in the G1 phase or in a later phase of the cell cycle.

9. The method according to option 5 or option 8, where immediately before incubation, no more than 10% of the T cells in the initial composition contain a T cell activation marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB.

10. The method of any one of embodiments 1-9, wherein prior to said incubation, more than 5%, 10%, 20%, 30%, or 40% of the T cells express the low density lipid receptor (LDL-R).

11. The method according to any one of embodiments 1-9, wherein the subject is a human.

12. The method according to any one of options 1-8, wherein the T cells were not brought to a temperature of 2°C to 8°C and/or maintained at this temperature for more than 48 hours prior to incubation.

13. The method according to any one of embodiments 1-12, wherein the sample is a blood sample.

14. The method according to any one of options 1-12, where the sample is a sample taken after leukapheresis.

15. The method of embodiments 1-14 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.

16. The method according to any one of embodiments 1-15, wherein the T cells were selected or enriched from a sample taken from an individual.

17. The method according to any one of options 1-16, which further comprises, prior to incubation, taking a sample from an individual and optionally collecting or enriching T cells from the sample, where these cells represent, but not necessarily, an enriched composition and/or produce original composition.

18. The method according to any one of options 1-17, wherein the percentage of T cells in the original composition is greater than or approximately greater than 75%, 80%, 85%, 90%, 95% T cells.

19. The method according to any of embodiments 3-18, wherein the T cells contain CD4 ⁺ or CD8 ⁺ cells.

20. The method according to any one of embodiments 3-18, wherein the T cells comprise CD4 ⁺ and CD8 ⁺ cells.

21. The method of embodiment 20, wherein the ratio of CD4 ⁺ cells to CD8 ⁺ cells is, or is about, 1:1, 1:2, 2:1, 1:3, or 3:1.

22. The method according to any of options 1-21, where:

the sample contains serum or plasma at a concentration of at least or at least about 10% (v/v), at least or at least about 15% (v/v), at least or at least about 20% ( v/v), at least or at least about 25% (v/v), at least or at least about 30% (v/v), at least or at least about 33% (v/v). v), at least or at least about 35% (v/v), or at least or at least about 40% (v/v); and/or

prior to incubation, the sample is not contacted ex vivo with serum or plasma at a concentration of at least or at least about 10% (v/v), at least or at least about 15% (v/v), at least at least or at least about 20% (v/v), at least or at least about 25% (v/v), at least or at least about 30% (v/v), at least or at least about 33% (v/v), at least or at least about 35% (v/v), or at least or at least about 40% (v/v).

23. The method according to any of options 1-22, where:

the sample contains serum or plasma at a concentration of at least or at least about 30% (v/v); and/or

prior to incubation, the sample is not contacted ex vivo with serum or plasma at a concentration of at least or at least about 30% (v/v).

24. The method according to option 22 or option 23, where the serum or plasma is human.

25. The method according to any one of embodiments 22-24, wherein the serum or plasma is autologous to the individual.

26. The method according to any one of options 1-22, where the sample contains an anticoagulant.

27. The method according to option 26, where the anticoagulant contains a free citrate ion.

28. The method according to any one of embodiments 1-27, wherein, prior to incubation, the method comprises cryopreserving T cells, optionally in a sample or enriched composition in the presence of a cryoprotectant, and thereby producing a cryopreserved composition.

29. The method according to option 28, where before incubation, the cryopreserved composition is washed under conditions that reduce the amount or remove the cryoprotectant and/or produce the original composition.

30. The method according to any of options 1-29, where the original composition contains N-acetylcysteine (NAC); serum, optionally human serum; recombinant interleukin-2 (IL-2), recombinant interleukin-15 (IL-15) and/or recombinant interleukin-7 (IL-7).

31. The method according to any of options 1-30, where:

the original composition contains N-acetylcysteine at a concentration of from or about 0.4 mg/ml to 4 mg/ml, from 0.8 mg/ml to 3.6 mg/ml, or from 1.6 mg/ml to 2.4 mg/ml, inclusive; or

the original composition contains N-acetylcysteine at a concentration of at least or at least about or about 0.4 mg/ml, 0.8 mg/ml, 1.2 mg/ml, 1.6 mg/ml, 2.0 mg /ml, 2.4 mg/ml, 2.8 mg/ml, 3.2 mg/ml, 3.6 mg/ml or 4.0 mg/ml.

32. The method according to any of options 1-31, where:

the initial composition contains serum, optionally human serum, at a concentration of from or about 0.5% to 25% (v/v), from 1.0% to 10% (v/v), or from 2.5% to 5.0 % (v/v), inclusive; or

the starting composition contains serum, optionally human serum, at a concentration of at least or at least about or about 0.5%, 1.%, 2.5%, 5% (v/v), or 10%.

33. The method according to any of options 1-32, where:

the original composition contains recombinant IL-2, optionally recombinant human IL-2 at a concentration of from or about 10 IU/ml to 500 IU/ml, from 50 IU/ml to 250 IU/ml, or from 100 IU/ml to 200 IU/ml ml, inclusive; or at a concentration of at least or at least about 10 IU/ml, 50 IU/ml, 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, or 500 IU/ml; and/or

the original composition contains recombinant IL-15, optionally recombinant human IL-15, at a concentration of from or about 1 IU/ml to 100 IU/ml, from 2 IU/ml to 50 IU/ml, or from 5 IU/ml to 10 IU /ml, inclusive; or at a concentration of at least or at least about 1 IU/ml, 2 IU/ml, 5 IU/ml, 10 IU/ml, 25 IU/ml, or 50 IU/ml; and/or

the original composition contains recombinant IL-7, optionally recombinant human IL-7, at a concentration of from or about 50 IU/ml to 1500 IU/ml, from 100 IU/ml to 1000 IU/ml, from 200 IU/ml to 600 IE/ml, inclusive; or at a concentration of at least or at least about 50 IU/ml, 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml , 800 IU/ml, 900 IU/ml or 1000 IU/ml.

34. The method according to any one of options 1-33, where: the incubation includes the step of centrifugal inoculation of viral vector particles with the original composition.

35. The method according to option 34, where centrifugal inoculation includes rotation in the inner compartment of the centrifugal chamber of the particles of the viral vector and the original composition, where the rotation is carried out at a relative centrifugal force applied to the inner surface of the side wall of the compartment, and the component:

from or approximately 500 g to 2500 g, 500 g to 2000 g, 500 g to 1600 g, 500 g to 1000 g, 600 g to 1600 g, 600 g to 1000 g, 1000 g to 2000 g or from 1000 g to 1600 g inclusive; or

at least or at least about 600 g, 800 g, 1000 g, 1200 g, 1600 g or 2000 g.

36. The method according to option 34 or option 35, where centrifugal inoculation is carried out for a period of time that is:

more than or about 5 minutes, more than or about 10 minutes, more than or about 15 minutes, more than or about 20 minutes, more than or about 30 minutes, more than or about 45 minutes, more, than or about 60 minutes, more than or about 90 minutes, or more than or about 120 minutes; or

from or about 5 minutes to 60 minutes, 10 minutes to 60 minutes, 15 minutes to 60 minutes, 15 minutes to 45 minutes, 30 minutes to 60 minutes, or 45 minutes to 60 minutes, inclusive.

37. The method of any one of embodiments 1-36 further comprising contacting the parent composition and/or viral vector particles with a transduction adjuvant.

38. The method according to option 37, where the contact is carried out before, during or after the centrifugal inoculation of the viral vector particles with the original composition.

39. The method according to any one of options 1-38, where at least part of the incubation is carried out at or about 37°C ± 2°C.

40. The method according to any one of embodiments 34-39, wherein at least a portion of the incubation is carried out after centrifugal inoculation.

41. The method according to option 39 or option 40, where at least part of the incubation is carried out for a period of time 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.

42. The method of any one of embodiments 39-41 wherein at least a portion of the incubation is for or about 24 hours.

43. The method according to any one of embodiments 1-42, wherein the total incubation period is no more than 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours.

44. The method of any one of embodiments 1-43, wherein the viral vector particle is a lentiviral vector particle.

45. The method of embodiment 44 wherein the lentiviral vector particle is derived from HIV-1.

46. The method of any one of embodiments 1-45, wherein the viral vector particle is pseudotyped with a viral envelope glycoprotein.

47. The method of embodiment 46 wherein the viral envelope glycoprotein is VSV-G.

48. The method according to any one of embodiments 1-47, wherein the viral vector particle contains a lentiviral protein having SAMHD1 inhibitory activity, wherein said protein is packaged in a viral particle.

49. The method of embodiment 48 wherein the SAMHD1 inhibitory protein is a wild type Vpx protein, a wild type Vpr protein, or a wild type Vpx or Vpr protein variant or portion having SAMHD1 inhibitory activity.

50. The method according to variant 48 or variant 49, wherein the SAMHD1 inhibitory protein is heterologous to the retroviral vector particle.

51. The method of any one of embodiments 48-50, wherein the SAMHD1 inhibitory protein is a wild type Vpx protein or a wild type Vpx variant or portion having SAMHD1 inhibitory activity.

52. The method of any one of embodiments 1-51, wherein the viral vector particle is incubated at an MOI of less than or less than about 20.0 or less than or less than about 10.0.

53. The method according to any of options 1-52, where:

the viral vector particle is incubated at a multiplicity of infection in the range or approximately in the range from 1.0 IU/cell to 10 IU/cell, or from 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/cage or 3.6 IU/cage, 4.0 IU/cage, 5.0 IU/cage, 6.0 IU/cage, 7.0 IU/cage, 8.0 IU/cage, 9 .0 IU/cage or 10.0 IU/cage.

54. The method according to any of options 1-53, where the original composition contains:

at least or approximately at least or approximately 50 x 10 ⁶ cells, 100 x 10 ⁶ cells, or 200 x 10 ⁶ cells.

55. The method of any one of embodiments 1-54, wherein the recombinant nucleic acid encodes an antigen receptor.

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

57. The method according to option 55 or option 56, where the antigen receptor is a chimeric antigen receptor (CAR).

58. The method of embodiment 57, wherein the chimeric antigen receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to the target antigen and to an intracellular signal transduction domain containing ITAM.

59. The method according to option 58, where the intracellular signal-transmitting domain contains an intracellular domain of the CD3-zeta chain (CD3ζ).

60. The method according to option 58 or option 59, further comprising a transmembrane domain binding an extracellular domain and an intracellular signal-transmitting domain.

61. The method of embodiment 60 wherein the transmembrane domain comprises the transmembrane portion of CD28.

62. The method of any one of embodiments 58-61, wherein the intracellular signal transduction domain also comprises an intracellular signal transduction domain of a T cell costimulatory molecule.

63. The method of embodiment 62 wherein the T cell co-stimulatory molecule is selected from the group consisting of CD28 and 41BB.

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

65. The method of embodiment 64 wherein the disease or condition is cancer and an autoimmune disease or disorder or infectious disease.

66. The method according to any one of options 1-65, where the method includes obtaining a final composition containing T cells transduced with a recombinant nucleic acid.

67. The method according to option 66, where at least 30%, or at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of T cells in the final composition transduce recombinant nucleic acid.

68. The method according to option 66 or option 67, further comprising receiving or isolating from the final composition transduced T cells obtained by this method.

69. The method according to option 66 or option 68, further comprising activating or expanding the cells of the final composition or cells transduced by this method.

70. The method according to option 69, where the activation and/or propagation is carried out ex vivo .

71. The method according to option 69 or option 70, wherein, after incubation, the cells in the final composition are also incubated in the presence of one or more stimulatory agents capable of activating T cells, inducing signaling through the TCR complex, and/or inducing T cell proliferation.

72. The method according to option 71, where one or more stimulating agents are selected from the group consisting of CD3-binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7.

73. The method of option 71 or option 72, wherein the one or more stimulatory agents comprise an anti-CD3 antibody and/or an anti-CD28 antibody.

74. The method according to option 69, where the activation and/or propagation is carried out in vivo .

75. The method according to option 69 or option 74, where the activation and/or propagation is carried out in the presence of an antigen that specifically binds to the antigen receptor, and/or the activation and/or propagation are transgene-specific.

76. The method according to option 72 or option 75, where after incubation, the cells in the final composition are not further incubated ex vivo in the presence of one or more stimulatory agents, optionally consisting of CD3-binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7, and/or the cells in the final composition are not additionally incubated at a temperature of more than 30°C for more than 24 hours.

77. Genetically engineered T cell obtained by the method of any one of embodiments 1-76.

78. Composition containing a genetically engineered T cell according to option 77 and a pharmaceutically acceptable carrier.

79. A method of treatment comprising administering to an individual suffering from a disease or condition a composition according to option 78.

80. The method according to option 79, where the composition is administered to the individual no later than 5 days after taking the sample from the individual.

81. The method according to option 80, where the composition is administered to the individual no later than 1 day, 2 days, 3 days or 4 days after sampling from the individual.

82. The method of adoptive cell therapy, including:

(a) enriching or isolating T cells from a sample taken from an individual suffering from a disease or condition;

(b) transducing the initial composition containing T cells with a viral vector particle by the method according to any of options 1-71 to obtain a final composition containing transduced cells, where the viral vector particle contains a recombinant nucleic acid encoding an antigen receptor that specifically binds to the antigen associated with the disease or disorder;

(c) administering the final composition containing the transduced cells to an individual for the treatment of a disease or condition, wherein the final composition is administered to the individual no more than 9 days after the individual has been sampled.

83. An adoptive cell therapy method comprising administering a final composition comprising T cells transduced with a recombinant nucleic acid to an individual for the treatment of a disease or condition, wherein the final composition is obtained by the method of any one of embodiments 1-71.

84. The method according to option 82 or option 83, where the final composition is administered to the individual no later than 1 day, 2 days, 3 days, 4 days or 5 days after sampling from the individual.

85. The method according to any one of embodiments 82-84, wherein prior to administration of the composition, the transduced cells or cells of the final composition are prepared in a pharmaceutically acceptable buffer.

86. The method according to any one of embodiments 82-85, wherein prior to administration of the composition containing the transduced cells, the cells are cultured ex vivo for up to 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days , 8 days or 9 days after transduction, where the specified cultivation is carried out at a temperature of more than 30°C.

87. The method according to any one of embodiments 82-86 wherein, after transduction, the final composition or cells containing the transduced cells are cultured in the presence of one or more stimulatory agents capable of activating T cells, inducing signal transduction through the TCR complex, and/or inducing T proliferation -cells to obtain a composition containing transduced cells.

88. The method according to any of the options 82-85, where before the introduction of the transduced cells, the final composition or cells containing the transduced cells are not additionally incubated ex vivo in the presence of one or more stimulating agents, and / or are not additionally incubated at a temperature of more than 30 °C for more than 24 hours.

89. The method according to option 87 or option 88, where one or more stimulating agents are selected from the group consisting of CD3-binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7; a vaccine containing an antigen specifically recognized by an antigen receptor; and an anti-idiotypic antibody that specifically binds to an antigen receptor.

90. The method of embodiment 89, wherein the one or more stimulatory agents comprise an anti-CD3 antibody and/or an anti-CD28 antibody.

91. The method of any one of embodiments 82-90, wherein the final composition cells or transduced cells are administered at a suboptimal dose.

92. The method of any one of embodiments 82-91, further comprising administering to the individual one or more agents to induce or enhance stimulation and/or expand the transduced T cells in vivo .

93. The method according to option 92, where one or more agents are transgene-specific and/or stimulate or activate cells through an expressed transgene, which is or optionally contains an antigen receptor.

94. The method according to option 92 or option 93, where one or more agents are selected from a vaccine containing an antigen specifically recognized by the antigen receptor; an anti-idiotypic antibody that specifically binds to an antigen receptor, or an agent capable of chemically inducing antigen receptor dimerization.

95. The method according to option 92, where one or more agents are an immunomodulatory agent; immune checkpoint inhibitor; an inhibitor of extracellular adenosine or an adenosine receptor, optionally an A2aR receptor; a kynurenine pathway modulator; and signal transduction pathway modulators, such as kinase inhibitors.

96. A composition containing a population of primary human T cells genetically engineered to express a chimeric antigen receptor (CAR) or transgenic TCR that specifically binds to a target antigen, where:

such a population contains many resting T cells, and

the plurality of resting T cells contains at least 7.5% of the genetically engineered cells in the composition.

97. The composition according to option 96, where genetically engineered resting T cells contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% , at least 70%, at least 80%, or at least 90% of the genetically engineered cells in the composition.

98. A composition according to option 96 or option 97, wherein the resting T cells are negative for a T cell activation surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L (CD154) and 4-1BB ( CD137); do not contain an intracellularly expressed cytokine selected from the group consisting of IL-2, IFN-gamma and TNF-alpha; are present in the G0 or G ₀ G _1a phase of the cell cycle; and/or contain active SAMHD1.

99. Composition according to variant 98, wherein resting T cells are negative for surface CD25 and CD69 (CD25 ^- /CD69 ^- ).

100. A composition according to any one of embodiments 96-99, wherein the resting T cells contain CD4 ⁺ and/or CD8 ⁺ T cells.

101. A composition according to any one of embodiments 96-100, wherein the target antigen is associated with a disease or disorder.

102. The composition of embodiment 100, wherein the disease or disorder is an infectious disease or condition, an autoimmune disease, an inflammatory disease, or cancer.

103. A composition according to any one of embodiments 96-102, wherein the target antigen is selected from the group consisting of B cell maturation antigen (BCMA), carbonic acid anhydrase 9 (CAIX), tEGFR, HER2/Neu (erbB2 tyrosine kinase receptor), CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40) , EPHa2, erb-B2, erb-B3, erb-B4, ErbB dimers, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R- alpha, IL-13R-alpha2, kinase insert domain receptor (kdr), kappa light chain, Lewis Y antigen, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE -A6, predominantly expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE Al, HLA-A2, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, fetal AchR, NKG2D ligands, CD44v6, dual antigen, testicular cancer antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, G protein-coupled receptor 5D (GPCR5D), ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, pathogen specific antigen and universal label associated antigen.

104. A composition according to any one of embodiments 96-103, wherein primary human T cells are genetically engineered to express a CAR containing an extracellular antigen recognition domain that specifically binds to a target antigen and to an intracellular signal transduction domain containing ITAM.

105. The composition of embodiment 104, wherein the intracellular signal-transmitting domain comprises an intracellular domain of the CD3-zeta (CD3ζ) chain.

106. The composition according to variant 104 or variant 105, where CAR additionally contains a transmembrane domain that binds an extracellular domain and an intracellular signal-transmitting domain.

107. A composition according to variant 106, wherein the transmembrane domain contains the transmembrane portion of CD28.

108. A composition according to any one of embodiments 104-107, wherein the intracellular signal transduction domain of CAR further comprises an intracellular signal transduction domain of a T cell costimulatory molecule.

109. The composition of embodiment 108 wherein the T cell co-stimulatory molecule is selected from the group consisting of CD28 and 41BB.

110. A composition according to any one of options 96-108 containing a pharmaceutically acceptable carrier.

VI. EXAMPLES

[0539] The following examples are included for illustrative purposes only and should not be construed as limiting the scope of the invention.

Example 1 Evaluation of Transduction of Primary Human T Cells by Lentivirus without Cell Preactivation

[0540] Transduction of primary CD4 ⁺ - and CD8 ⁺ -T cells enriched from a sample taken from a human after leukapheresis, lentivirus, was carried out without a preliminary stage of cell activation by ex vivo T cells before transduction.

[0541] A leukapheresis human sample enriched in mononuclear cells was obtained from an individual's whole blood sample using a leukapheresis sample collection system. The blood was subjected to leukapheresis, resulting in a sample containing mononuclear cells, autologous plasma and the anticoagulant, ACD-A (dextrose A citrate anticoagulant).

[0542] Leukapheresis sample cells were washed and resuspended in buffer for use in affinity selection, where the buffer contained phosphate buffered saline (PBS), EDTA, and human serum albumin. For selection of T cells based on immunoaffinity, washed cells in selection buffer were incubated for 30 minutes at room temperature with magnetic beads coupled to selection monoclonal antibodies and then subjected to selection on a magnetic separation column. The enriched T cells were resuspended in cryopreservation medium and the cells were cryopreserved and stored in liquid nitrogen until further use. Cryopreserved CD4 ⁺ - and CD8 ⁺ -T cells were thawed, washed and resuspended in transduction medium.

[0543] The T cells are then contacted with the lentiviral vector particles without incubating the cells with a T cell activating agent, for example, without incubating the cells with anti-CD3 and anti-CD28 reagents. For transduction, T cells were added to individual wells of a 24-well plate in a volume of 200 μl. Lentiviral vector particles containing a nucleic acid encoding a transgene (in this case encoding a representative chimeric antigen receptor (CAR) and a truncated EGFR, EGFRt, a sequence for use as a transduction marker separated from the CAR sequence by a T2A self-cleaving sequence) that has been premixed with polycationic adjuvant for transduction, was added to the wells in 2-fold serial dilutions. The final volume per well was adjusted to 1.1 ml. The compositions were subjected to centrifugation for approximately 1 hour, and then incubated for 24 hours at 37°C.

[0544] As a control, transduction was also performed as described above using CD4 ⁺ - and CD8 ⁺ -T cells thawed from the cryopreserved composition, except that prior to transduction, T cells were activated by culturing at 37 °C for approximately 24 hours with one of two different activation reagents, each containing magnetic spheres coated with anti-CD3 and anti-CD28 antibody fragments (referred to here as reagent 1 or reagent 2).

[0545] After transduction under each of the above conditions, the cells of each composition were cultured at 37°C in the presence of medium and IL-2 for 48 hours.

[0546] As an indicator of transduction efficiency, flow cytometric analysis was performed to determine the percentage of cells positive for surface CAR expression (in this case, expression detected using an anti-EGFR antibody that recognizes the EGFRt surrogate marker). As shown in FIG. 1A-1B, in this study, transduction efficiency (as measured by surface EGFRt expression) without activation was comparable to transduction efficiency in cells that were activated with anti-CD3/anti-CD28 reagents prior to transduction. The results showed that comparable T cell transduction can be achieved using lentiviral vectors without prior incubation with an ex vivo T cell activating agent.

Example 2: Large Scale Method for Transducing Primary Human T Cells with Lentivirus Without Prior Cell Activation

[0547] This example describes the transduction of CD4 ⁺ - and CD8 ⁺ -T cells taken after apheresis from a healthy donor, using a large-scale procedure, where the obtained cells were subjected to affinity selection, transduced (with or without pre-activation) and cultured or incubated under various conditions. Washing, selection based on affinity and transduction was carried out mainly in a vertical centrifuge for processing, for example, as described in the publication of International application WO2016/073602.

1. Sample collection and leukapheresis

[0548] Autologous peripheral blood mononuclear cells (PBMCs) were collected from a patient using a cell collection system after leukapheresis. The blood was processed to obtain a sample containing mononuclear cells and approximately 1/3 volume of autologous plasma and the anticoagulant, ACD-A (anticoagulant dextrose A citrate). The leukapheresis sample was sealed at 2-8° C. for approximately 24 hours.

2. Washing after leukapheresis

[0549] The leukapheresis sample was sterile transferred into a transfer bag. The leukapheresis sample cells were washed and resuspended in selection buffer for affinity selection, where said buffer contained PBS, EDTA and human serum albumin. Washing was performed using a sterile, disposable kit purchased from Biosafe SA, used in regenerative medicine, where said kit included a centrifuge processing chamber (A-200F). The transfer bag containing the cells and the bag containing the buffer were sterilely connected to the kit, which was placed in the ^Sepax® 2 processor. Two (2) washes were performed, each with RCF applied to the inner wall of the compartment at approximately 200 g for 180 seconds, followed by a final cell resuspension in 20 ml. At the end of the protocol, the cells were kept in the processing compartment of the centrifuge chamber for subsequent incubation with affinity-based selection reagents.

3. Selection based on affinity

[0550] CD4 and CD8 T cells were enriched by immunoaffinity selection using magnetic spheres coupled to monoclonal antibodies, which were added to the washed sample taken after leukapheresis in a centrifuge chamber. The spheres were mixed in the selection buffer described above and then connected to the device. The program was carried out in a Sepax® 2 device, whereby the mixture of spheres and selection buffer entered the chamber with washed cells, and the contents of the chamber were mixed for 30 minutes.

[0551] At the end of the program, in the Sepax® 2 device, cells were precipitated and excess buffer/spheres were ejected, the precipitated cells were washed, and resuspended in selection buffer. The program started collecting the washed cells into a transfer bag.

[0552] The cells were then removed from the transfer bag using a closed sterile tubing system and separation column in the presence of a magnetic field using standard cell separation methods that were coupled with CD4 and/or CD8 specific reagents. These magnetically labeled cells were then collected into a transfer bag for further processing.

4. Transduction

[0553] Transduction with the lentiviral vector was performed in a centrifuge chamber built into the kit placed in the Sepax® processing device. Selected cells were either transduced (transduced at t=0) or activated using anti-CD3/CD28 reagent(s) (activated at t=0 and transduced at t=24 h).

[0554] For cells that were first activated, selected cells were washed and resuspended in complete medium using the Sepax® 2 system, and this medium was then combined with anti-CD3/28 reagent(s) in the chamber compartment at room temperature. After incubation, the incubated material was transferred via a Sepax® 2 device to a cell culture bag and then incubated at 37° C. for 24 hours.

[0555] The following steps were performed to initiate transduction.

[0556] A transduction reagent solution was prepared containing complete medium (X-VIVO-15, comprising 5% (v/v) human serum, 1.6 mg/ml N-acetylcysteine (NAC) and 100 IU/ml IL- 2) and sufficient polycation to achieve a final concentration during transduction initiation of 10 μg/ml), viral vector particles (approximately 3.2 IU/cell), and, if necessary, air. The transduction reagent solution was aseptically transferred to a centrifuge bag.

[0557] A product bag containing a composition comprising approximately 200×10 ⁶ selected cells or 200×10 ⁶ activated cells was sterile connected to a disposable kit that was placed in a ^Sepax® 2 processing device. An automated cycle was run on Sepax® 2, which facilitates the entry of the cell-containing composition into the chamber compartment, centrifugation of the composition on the Sepax® 2 to pellet the cells, and removal of the appropriate volume of liquid necessary to achieve the desired volume, eg 10 ml. Then, the entire contents of the transduction reagent solution entered the compartment of the cell chamber. Thereafter, a final volume of 200 ml (containing cells and virus) was transferred to a centrifuge bag.

[0558] To initiate transduction, the centrifuge bag containing the virus and cells was removed and sterile attached to the initial position of the kit, and then transferred to the chamber compartment. Virus and cells were centrifuged in the chamber compartment with RCF applied to the inner wall of the compartment and approximately equal to 1600 g. Centrifugation was carried out at the indicated speed for 1 hour. The transduced material was then transferred to the output package. The complete medium was transferred into the chamber, mixed for 60 seconds, and then transferred from the processing compartment to the output pouch until a total volume of 200 ml was reached in the output pouch containing the transduced material.

[0559] Then, cells transduced after selection, without prior activation step, (1) were immediately activated with anti-CD3/anti-CD28 reagent in culture medium supplemented with FBS, 100 IU/ml IL-2 and 10 IU/ml IL- 15 and collected on the 9th day; (2) incubated at 37°C, 5% CO ₂ for 24 hours and then activated with anti-CD3/anti-CD28 reagent in culture medium, and harvested on day 9; (3) maintained without activation by cultivation at 37°C, 5% CO ₂ for 3 days; (4) incubated at 37°C, 5% CO ₂ for 24 hours, cryopreserved, thawed and activated with anti-CD3/anti-CD28 reagent in culture medium and harvested on day 14. In the case of control cells that were activated prior to transduction and then transduced 24 hours later, these cells were expanded in the presence of culture medium (no activation) and harvested on day 9. For each condition, cells were cultured at 37°C, 5% CO ₂ .

[0560] Table 1 summarizes study groups and incubation conditions after transduction.

Table 1: Transduction groups

Terms Description Activation at t=0, and transduction at t=24 h
(Act0_Tx24; control) Activation after selection, transduction 24 h later and harvest on day 9 Transduction at t=0 and activation at t=0
(Tx_Act_0) Transduction after selection, then incubation with activating reagent immediately after transduction and harvest on day 9 Transduction at t=0 and activation at t=24h (Tx0_Act24) Transduction after selection, incubation with activating reagent after 24 hours and harvest on day 9 Transduction at t=0, cryopreservation, activation at t=48 h (Tx0_Cryopreserved) Transduction after selection, cryopreservation after 24 hours, thawing, incubation with activating reagent on day 2 and harvest on day 14 Transduction at t=0, not activated (Tx_No_Act) Transduction after selection (no activation) and cultivation for 3 days.

[0561] The frequency of transduction on different days after cell collection after leukapheresis was measured as described in Example 1. As shown in FIG. 2, comparable transduction frequencies (as measured by surface marker expression) were observed when T cells were first transduced and then immediately activated compared to T cells that were first activated for 24 hours and then transduced (63% compared to 68%, respectively, in the cryopreserved product collected 9 days after collection). This result demonstrated that a relatively high transduction efficiency could be achieved without T cell activation prior to transduction.

[0562] Given that transduction (gene integration) does not require prior activation of T cells, the results obtained in this study suggest that incubation of cells with an activating agent is essential for stable surface expression of the recombinant protein. In cells that were not activated, surface expression of EGFRt (a surface surrogate marker for CAR expression) was transient; and the maximum detectable frequency of surface expression of EGFRt (indicative of transduction) was reached 24 hours after transduction at the level of 29% and decreased to <1% on the 3rd day after transduction.

[0563] In addition, in FIG. 2 shows that even if incubation with the activating reagent was carried out after 24 hours (compared to cells activated before transduction), this gave a fairly high frequency of expression of the marker (an indicator of the frequency of transduction), which is 43%. Immediate incubation with an activating reagent (with magnetic spheres containing an anti-CD3/CD28 antibody) after transduction resulted in a higher frequency of cells with surface expression of the transduction marker than when activated 24 hours later (63% versus 43% corresponding frequency measured in a cryopreserved product harvested 9 days after collection). Cryopreservation of transduced cells (24 hours after transduction) prior to thawing and incubation with activating reagent resulted in a decrease in the frequency of transduction marker expression compared to cells transduced and incubated with activating reagent without cryopreservation (16% versus 43% corresponding frequency). transduction measured in a cryopreserved product).

[0564] The present invention should not be construed as limiting the scope of the specifically disclosed embodiments of the invention, which are presented, for example, to illustrate various aspects of the invention. Various modifications introduced into the compositions and methods described here will be apparent from the description and summary of the invention presented here. Such variations may be made without departing from the true spirit and scope of the invention and are within the scope of the disclosure of the present invention.

Sequences

SEQID NO: Subsequence Description one MSDPRERIPPGNSGEETIGEAFEWLNRTVEEINREAVNHLPRELIFQVWQRSWEYWHDEQGMSQSYVKYRYLCLMQKALFMHCKKGCRCLGEGHGAGGWRPGPPPPPPPGLA SIVmac Vpx 2 MTDPRERIPPGNSGEETIGEAFEWLHNTVEALNQTAVQHLPRELIFQVWRRCWEYWVDEQGYSPSYAKYRYVQLMQKAMFQHFRKGCTCRGEGHSQGGWRTGPPPPPPPGLA SIVsm
Vpx 3 MSDPRERIPPGNSGEETIGEAFDWLDRTVEEINRAAVNHLPRELIFQVWRRSWEYWHDEMGMSVSYTKYRYLCLIQKAMFMHCKKGCRCLGGEHGAGGWRPGPPPPPPPGLA SIVrcm Vpx four MADPRERVPPGNSGEETIGEAFEWLDRTIEALNREAVNHLPRELIFQVWQRSWAYWHDEQGMSTSYTKYRYLCIMQKAVYIHFKKGCTCLGRGHGPGGWRPGPPPPPPPGLV HIV-2
Vpx 5 MERYPPSHPPHFTSRTVPMTRLALQQAMQDLNEEALKHFTREELWGVWNHCVDLPAQPDWTGEQAWAASVIDYIKIVQRMLWLHLREACFHREREATRRYPNIRPLTGRNREVRDGE SIVdeb Vpr 6 MERVPPSHRPPWHSRVVPTTMQQAQQAMWDLNEEAEKHFSREELRGIWNDVTELPADPNWTVDQAAIACAIDYIRRTQTLLFRHYREGCYHRYSNTIRRYPNIRPLRGTQAPPSNSMPNADPTPPLRPSRYRMDE SIVmus Vpr 7 MASGRDPREERPGELEIWDLSREPWDEWLRDMLTELNQEAQRHFGRELLFQVWNYCQEEGERQNVPMQERAYKYYKLVQRALFVHFRCGCRRRQPFEPYEERRNGQGGGRAGRVPPGLD SIVagm Vpr eight MASGRDPREERPGEVEIWDLSREPWDEWLRDMLAELNREAQRHFGRELLFQVWNYCQEEGERQNIPMQERAYKYYRLVQKALFVHIRCGCRRRQPFEPYEERRNGQGGGRAERVPPGLD SIVagm Vpr 9 MERYPPSHPPHFTSRTVPMTRLALQQAMQDLNEEALKHFTREELWGVWNHCVDLPAQPDWTGEQAWAASVIDYIKIVQRMLWLHLREACFHREREATRRYPNIRPLTGRNREVRDGE SIVdeb Vpr ten MERLPPSHPPALVSRMAETTQRQLQEAVWDITEEARKHFSKEEITGIWDHCWSLPALPHWTEGQVMAAAVIDFIRIMQKEIWKHYRVGCFHREAERVRHYPNIRPLRPRREEP SIVden Vpr eleven MERLPPSHPLPLTSRLIPTPRPQLERLMREVRDEMLIHFTRDEVIGVWNHCVELPADPDWTGEQAWAASVIDFARKGNQMLLLHFQQGCYHEQQRRTPHYPNIRPLSGRGERTMQ SIVtal Vpr 12 MERIPPSHPMPWLSRRVPTTMAIAQNALWEINEEAEKHFSRDELRGIWHDVTELPADPDWTVDQAAIACAIDYIRVQTLLFRHFKDGCFHRVNLSGRYPAIRPSRGTAPPDSNSVSHADPEQPRRPSRYRMDE SIVgsn Vpr 13 MERTPPSHPLPWISRRVPTTMAVAQSAMWEINEEAEKHFSREELRGIWHDVTELPADPDWTVDQAAIACAIDYVRRVQTLLFRHFRDGCFHRYNRIVRRYPVIRPLRGTAPPDSNSVPHADPEQPRRPSRYRMDE SIVgsn Vpr fourteen MEQPPQSHPLHWTSRMVPIERQALQAAIWELNEEALKHFSREELRGIWEQVTELPADPAWNADQAWAACAIDYTRWVQTILYRHYREGCYHRYAEQIRRYPVLRPMRGTAPGPTSSVPQADPDNPRRPSRYRMDE SIVmon Vpr fifteen MAEAFFNPSQHVQGTPWFFIPRNVELTPNVINVTVKAELVVTEASKHFTPQEIYGVWNQSLNEEAGTDSPTMAWERTMLDMVRALNLMLFFEHFAAGCPQRTRYARHRGYPHPS SIVsyk Vpr 16 MAERAPEAPEGAGEVGLEQWLETSLERINREARLHFHPEFLFRRLWNTCVEHWHDRHQRSLDYAKYRYLLLMHKAMYTHMQQGCPCRNGRPRGPPPPGMA SIVmnd2 Vpx 17 MAERQSVERAPAEPMGAGEVELEEWLQRSLLRINQEARLHFHPEFLFRLWNTCMEHYHDALQLSFTYSKYRYLLLLQKAMFMHFQQGCSCLQGRHPPPLRPAGDRLPPPPPPP SIVdrl Vpx eighteen MSDPRERIPPGNSGEETIGEAFEWLNRTVEEINREAVNHLPRELIFQVWQRSWEYWHDEQGMSPSYVKYRYLCLIQKALFMHCKKGCRCLGEGHGAGGWRPGPPPPPPPGLA SIVmne Vpx 19 MQRADSEQPSKRPRCDDSPRTPSNTPSAEADWSPGLELHPDYKTWGPEQVCSFLRRGGFEEPVLLKNIRENEITGALLPCLDESRFENLGVSSLGERKKLLSYIQRLVQIHVDTMKVINDPIHGHIELHPLLVRIIDTPQFQRLRYIKQLGGGYYVFPGASHNRFEHSLGVGYLAGCLVHALGEKQPELQISERDVLCVQIAGLCHDLGHGPFSHMFDGRFIPLARPEVKWTHEQGSVMMFEHLINSNGIKPVMEQYGLIPEEDICFIKEQIVGPLESPVEDSLWPYKGRPENKSFLYEIVSNKRNGIDVDKWDYFARDCHHLGIQNNFDYKRFIKFARVCEVDNELRICARDKEVGNLYDMFHTRNSLHRRAYQHKVGNIIDTMITDAFLKADDYIEITGAGGKKYRISTAIDDMEAYTKLTDNIFLEILYSTDPKLKDAREILKQIEYRNLFKYVGETQPTGQIKIKREDYESLPKEVASAKPKVLLDVKLKAEDFIVDVINMDYGMQEKNPIDHVSFYCKTAPNRAIRITKNQVSQLLPEKFAEQLIRVYCKKVDRKSLYAARQYFVQWCADRNFTKPQDGDVIAPLITPQKKEWNDSTSVQNPTRLREASKSRVQLFKDDPM Human SAMHD1 twenty LQSRPEPTAPPEESFRFGVETTTPPQKQEPIDKELYPLTSLRSFGNDPSSQ HIV-1 p6 21 PMAQVHQGLMPTAPPEDPAVDLLKNYMQLGKQQREKQRESREKPYKEVTEDLLHLNSLFGGDQ P6 Gag 22 DXAXXLL Packing Motif P6 Gag 23 DPAVDLL Packing motif P6 24 DPAVDLLKNY Packing motif P6 25 LQSRPEPTAPPEESDPAVDLLTTPPQKQEPIDKELYPLTSLRSFGNDPSSQ Hybrid or chimeric p6 domain 26 WXXXXXXXXXXXXXXXX Motive Wx4Φx2Φx3AΦxH 27 ESKYGPPCPPCP Spacer (IgG4 hinge region) (a.k.) 28 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT Spacer (IgG4 hinge region) (nuk.) 29 ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Spacer Hinge-CH3 thirty ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Spacer Hinge-CH2-CH3 31 RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH IgD-hinge-Fc 32 LEGGGEGRGSLTCGDVEENPGPR T2A 33 MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR 34 FWVLVVVGGVLACYSLLVTVAFIFWV CD28 (amino acids 153-179, accession no. P10747) 35 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 114-179 accession No. P10747) 36 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (amino acids 180-220, P10747) 37 RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL - GG) 38 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB (amino acids 214-255, Q07011.1) 39 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta 40 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta 41 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta 42 RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR 43 EGRGSLLTCGDVEENPGP T2A 44 GSGATNFSLLKQAGDVEENPGP P2A 45 ATNFSLLKQAGDVEENPGP P2A 46 QCTNYALLKLAGDVESNPGP E2A 47 VKQTLNFDLLKLAGDVESNPGP F2A 48 -PGGG-(SGGGG)5-P- where P is proline, G is glycine and S is serine Linker 49 GSADDAKKDAAKKDGKS Linker fifty atgcttctcctggtgacaagccttctgctctgtgagtttaccacacccagcattcctcctgatccca GMCSFR alpha chain signal sequence 51 MLLLVTSLLLCELPHPAFLLIP GMCSFR alpha chain signal sequence 52 MALPVTALLLPLALLLHA Signal peptide CD8 alpha

[0565] The present invention should not be construed as limiting the scope of the specifically disclosed embodiments of the invention, which are presented, for example, to illustrate various aspects of the invention. Various modifications introduced into the compositions and methods described here will be apparent from the description and summary of the invention presented here. Such variations may be made without departing from the true spirit and scope of the invention and are within the scope of the disclosure of the present invention.

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SEQUENCE LIST

<110> Juno Therapeutics, Inc.

Tareen, Semih U

Beaushesne, Pascal

<120> PRODUCTION OF DESIGNED CELLS FOR

ADOPTIONAL CELL THERAPY

<130> 735042005040

<150> US 62/430,349

<151> 2016-12-05

<160> 52

<170>PatentIn version 3.5

<210> 1

<211> 112

<212> protein

<213> Simian immunodeficiency virus

<400> 1

Met Ser Asp Pro Arg Glu Arg Ile Pro Pro Gly Asn Ser Gly Glu Glu

1 5 10 15

Thr Ile Gly Glu Ala Phe Glu Trp Leu Asn Arg Thr Val Glu Glu Ile

20 25 30

Asn Arg Glu Ala Val Asn His Leu Pro Arg Glu Leu Ile Phe Gln Val

35 40 45

Trp Gln Arg Ser Trp Glu Tyr Trp His Asp Glu Gln Gly Met Ser Gln

50 55 60

Ser Tyr Val Lys Tyr Arg Tyr Leu Cys Leu Met Gln Lys Ala Leu Phe

65 70 75 80

Met His Cys Lys Lys Gly Cys Arg Cys Leu Gly Glu Gly His Gly Ala

85 90 95

Gly Gly Trp Arg Pro Gly Pro Pro Pro Pro Pro Pro Pro Gly Leu Ala

100 105 110

<210> 2

<211> 112

<212> protein

<213> Simian immunodeficiency virus

<400> 2

Met Thr Asp Pro Arg Glu Arg Ile Pro Pro Gly Asn Ser Gly Glu Glu

1 5 10 15

Thr Ile Gly Glu Ala Phe Glu Trp Leu His Asn Thr Val Glu Ala Leu

20 25 30

Asn Gln Thr Ala Val Gln His Leu Pro Arg Glu Leu Ile Phe Gln Val

35 40 45

Trp Arg Arg Cys Trp Glu Tyr Trp Val Asp Glu Gln Gly Tyr Ser Pro

50 55 60

Ser Tyr Ala Lys Tyr Arg Tyr Val Gln Leu Met Gln Lys Ala Met Phe

65 70 75 80

Gln His Phe Arg Lys Gly Cys Thr Cys Arg Gly Glu Gly His Ser Gln

85 90 95

Gly Gly Trp Arg Thr Gly Pro Pro Pro Pro Pro Pro Pro Gly Leu Ala

100 105 110

<210> 3

<211> 112

<212> protein

<213> Simian immunodeficiency virus

<400> 3

Met Ser Asp Pro Arg Glu Arg Ile Pro Pro Gly Asn Ser Gly Glu Glu

1 5 10 15

Thr Ile Gly Glu Ala Phe Asp Trp Leu Asp Arg Thr Val Glu Glu Ile

20 25 30

Asn Arg Ala Ala Val Asn His Leu Pro Arg Glu Leu Ile Phe Gln Val

35 40 45

Trp Arg Arg Ser Trp Glu Tyr Trp His Asp Glu Met Gly Met Ser Val

50 55 60

Ser Tyr Thr Lys Tyr Arg Tyr Leu Cys Leu Ile Gln Lys Ala Met Phe

65 70 75 80

Met His Cys Lys Lys Gly Cys Arg Cys Leu Gly Gly Glu His Gly Ala

85 90 95

Gly Gly Trp Arg Pro Gly Pro Pro Pro Pro Pro Pro Pro Gly Leu Ala

100 105 110

<210> 4

<211> 112

<212> protein

<213> Simian immunodeficiency virus

<400> 4

Met Ala Asp Pro Arg Glu Arg Val Pro Pro Gly Asn Ser Gly Glu Glu

1 5 10 15

Thr Ile Gly Glu Ala Phe Glu Trp Leu Asp Arg Thr Ile Glu Ala Leu

20 25 30

Asn Arg Glu Ala Val Asn His Leu Pro Arg Glu Leu Ile Phe Gln Val

35 40 45

Trp Gln Arg Ser Trp Ala Tyr Trp His Asp Glu Gln Gly Met Ser Thr

50 55 60

Ser Tyr Thr Lys Tyr Arg Tyr Leu Cys Ile Met Gln Lys Ala Val Tyr

65 70 75 80

Ile His Phe Lys Lys Gly Cys Thr Cys Leu Gly Arg Gly His Gly Pro

85 90 95

Gly Gly Trp Arg Pro Gly Pro Pro Pro Pro Pro Pro Pro Gly Leu Val

100 105 110

<210> 5

<211> 117

<212> protein

<213> Simian immunodeficiency virus

<400> 5

Met Glu Arg Tyr Pro Pro Ser His Pro Pro His Phe Thr Ser Arg Thr

1 5 10 15

Val Pro Met Thr Arg Leu Ala Leu Gln Gln Ala Met Gln Asp Leu Asn

20 25 30

Glu Glu Ala Leu Lys His Phe Thr Arg Glu Glu Leu Trp Gly Val Trp

35 40 45

Asn His Cys Val Asp Leu Pro Ala Gln Pro Asp Trp Thr Gly Glu Gln

50 55 60

Ala Trp Ala Ala Ser Val Ile Asp Tyr Ile Lys Ile Val Gln Arg Met

65 70 75 80

Leu Trp Leu His Leu Arg Glu Ala Cys Phe His Arg Glu Arg Glu Ala

85 90 95

Thr Arg Arg Tyr Pro Asn Ile Arg Pro Leu Thr Gly Arg Asn Arg Glu

100 105 110

Val Arg Asp Gly Glu

115

<210> 6

<211> 135

<212> protein

<213> Simian immunodeficiency virus

<400> 6

Met Glu Arg Val Pro Ser His Arg Pro Pro Trp His Ser Arg Val

1 5 10 15

Val Pro Thr Thr Met Gln Gln Ala Gln Gln Ala Met Trp Asp Leu Asn

20 25 30

Glu Glu Ala Glu Lys His Phe Ser Arg Glu Glu Leu Arg Gly Ile Trp

35 40 45

Asn Asp Val Thr Glu Leu Pro Ala Asp Pro Asn Trp Thr Val Asp Gln

50 55 60

Ala Ala Ile Ala Cys Ala Ile Asp Tyr Ile Arg Arg Thr Gln Thr Leu

65 70 75 80

Leu Phe Arg His Tyr Arg Glu Gly Cys Tyr His Arg Tyr Ser Asn Thr

85 90 95

Ile Arg Arg Tyr Pro Asn Ile Arg Pro Leu Arg Gly Thr Gln Ala Pro

100 105 110

Pro Ser Asn Ser Met Pro Asn Ala Asp Pro Thr Pro Pro Leu Arg Pro

115 120 125

Ser Arg Tyr Arg Met Asp Glu

130 135

<210> 7

<211> 119

<212> protein

<213> Simian immunodeficiency virus

<400> 7

Met Ala Ser Gly Arg Asp Pro Arg Glu Glu Arg Pro Gly Glu Leu Glu

1 5 10 15

Ile Trp Asp Leu Ser Arg Glu Pro Trp Asp Glu Trp Leu Arg Asp Met

20 25 30

Leu Thr Glu Leu Asn Gln Glu Ala Gln Arg His Phe Gly Arg Glu Leu

35 40 45

Leu Phe Gln Val Trp Asn Tyr Cys Gln Glu Glu Gly Glu Arg Gln Asn

50 55 60

Val Pro Met Gln Glu Arg Ala Tyr Lys Tyr Tyr Lys Leu Val Gln Arg

65 70 75 80

Ala Leu Phe Val His Phe Arg Cys Gly Cys Arg Arg Arg Gln Pro Phe

85 90 95

Glu Pro Tyr Glu Glu Arg Arg Asn Gly Gln Gly Gly Gly Arg Ala Gly

100 105 110

Arg Val Pro Pro Gly Leu Asp

115

<210> 8

<211> 119

<212> protein

<213> Simian immunodeficiency virus

<400> 8

Met Ala Ser Gly Arg Asp Pro Arg Glu Glu Arg Pro Gly Glu Val Glu

1 5 10 15

Ile Trp Asp Leu Ser Arg Glu Pro Trp Asp Glu Trp Leu Arg Asp Met

20 25 30

Leu Ala Glu Leu Asn Arg Glu Ala Gln Arg His Phe Gly Arg Glu Leu

35 40 45

Leu Phe Gln Val Trp Asn Tyr Cys Gln Glu Glu Gly Glu Arg Gln Asn

50 55 60

Ile Pro Met Gln Glu Arg Ala Tyr Lys Tyr Tyr Arg Leu Val Gln Lys

65 70 75 80

Ala Leu Phe Val His Ile Arg Cys Gly Cys Arg Arg Arg Gln Pro Phe

85 90 95

Glu Pro Tyr Glu Glu Arg Arg Asn Gly Gln Gly Gly Gly Arg Ala Glu

100 105 110

Arg Val Pro Pro Gly Leu Asp

115

<210> 9

<211> 117

<212> protein

<213> Simian immunodeficiency virus

<400> 9

Met Glu Arg Tyr Pro Pro Ser His Pro Pro His Phe Thr Ser Arg Thr

1 5 10 15

Val Pro Met Thr Arg Leu Ala Leu Gln Gln Ala Met Gln Asp Leu Asn

20 25 30

Glu Glu Ala Leu Lys His Phe Thr Arg Glu Glu Leu Trp Gly Val Trp

35 40 45

Asn His Cys Val Asp Leu Pro Ala Gln Pro Asp Trp Thr Gly Glu Gln

50 55 60

Ala Trp Ala Ala Ser Val Ile Asp Tyr Ile Lys Ile Val Gln Arg Met

65 70 75 80

Leu Trp Leu His Leu Arg Glu Ala Cys Phe His Arg Glu Arg Glu Ala

85 90 95

Thr Arg Arg Tyr Pro Asn Ile Arg Pro Leu Thr Gly Arg Asn Arg Glu

100 105 110

Val Arg Asp Gly Glu

115

<210> 10

<211> 113

<212> protein

<213> Simian immunodeficiency virus

<400> 10

Met Glu Arg Leu Pro Pro Ser His Pro Pro Ala Leu Val Ser Arg Met

1 5 10 15

Ala Glu Thr Thr Gln Arg Gln Leu Gln Glu Ala Val Trp Asp Ile Thr

20 25 30

Glu Glu Ala Arg Lys His Phe Ser Lys Glu Glu Ile Thr Gly Ile Trp

35 40 45

Asp His Cys Trp Ser Leu Pro Ala Leu Pro His Trp Thr Glu Gly Gln

50 55 60

Val Met Ala Ala Ala Val Ile Asp Phe Ile Arg Ile Met Gln Lys Glu

65 70 75 80

Ile Trp Lys His Tyr Arg Val Gly Cys Phe His Arg Glu Ala Glu Arg

85 90 95

Val Arg His Tyr Pro Asn Ile Arg Pro Leu Arg Pro Arg Arg Glu Glu

100 105 110

Pro

<210> 11

<211> 115

<212> protein

<213> Simian immunodeficiency virus

<400> 11

Met Glu Arg Leu Pro Pro Ser His Pro Leu Pro Leu Thr Ser Arg Leu

1 5 10 15

Ile Pro Thr Pro Arg Pro Gln Leu Glu Arg Leu Met Arg Glu Val Arg

20 25 30

Asp Glu Met Leu Ile His Phe Thr Arg Asp Glu Val Ile Gly Val Trp

35 40 45

Asn His Cys Val Glu Leu Pro Ala Asp Pro Asp Trp Thr Gly Glu Gln

50 55 60

Ala Trp Ala Ala Ser Val Ile Asp Phe Ala Arg Lys Gly Asn Gln Met

65 70 75 80

Leu Leu Leu His Phe Gln Gln Gly Cys Tyr His Glu Gln Gln Arg Arg

85 90 95

Thr Pro His Tyr Pro Asn Ile Arg Pro Leu Ser Gly Arg Gly Glu Arg

100 105 110

Thr Met Gln

115

<210> 12

<211> 133

<212> protein

<213> Simian immunodeficiency virus

<400> 12

Met Glu Arg Ile Pro Ser His Pro Met Pro Trp Leu Ser Arg Arg

1 5 10 15

Val Pro Thr Thr Met Ala Ile Ala Gln Asn Ala Leu Trp Glu Ile Asn

20 25 30

Glu Glu Ala Glu Lys His Phe Ser Arg Asp Glu Leu Arg Gly Ile Trp

35 40 45

His Asp Val Thr Glu Leu Pro Ala Asp Pro Asp Trp Thr Val Asp Gln

50 55 60

Ala Ala Ile Ala Cys Ala Ile Asp Tyr Ile Arg Val Gln Thr Leu Leu

65 70 75 80

Phe Arg His Phe Lys Asp Gly Cys Phe His Arg Val Asn Leu Ser Gly

85 90 95

Arg Tyr Pro Ala Ile Arg Pro Ser Arg Gly Thr Ala Pro Pro Asp Ser

100 105 110

Asn Ser Val Ser His Ala Asp Pro Glu Gln Pro Arg Arg Pro Ser Arg

115 120 125

Tyr Arg Met Asp Glu

130

<210> 13

<211> 135

<212> protein

<213> Simian immunodeficiency virus

<400> 13

Met Glu Arg Thr Pro Ser His Pro Leu Pro Trp Ile Ser Arg Arg

1 5 10 15

Val Pro Thr Thr Met Ala Val Ala Gln Ser Ala Met Trp Glu Ile Asn

20 25 30

Glu Glu Ala Glu Lys His Phe Ser Arg Glu Glu Leu Arg Gly Ile Trp

35 40 45

His Asp Val Thr Glu Leu Pro Ala Asp Pro Asp Trp Thr Val Asp Gln

50 55 60

Ala Ala Ile Ala Cys Ala Ile Asp Tyr Val Arg Arg Val Gln Thr Leu

65 70 75 80

Leu Phe Arg His Phe Arg Asp Gly Cys Phe His Arg Tyr Asn Arg Ile

85 90 95

Val Arg Arg Tyr Pro Val Ile Arg Pro Leu Arg Gly Thr Ala Pro Pro

100 105 110

Asp Ser Asn Ser Val Pro His Ala Asp Pro Glu Gln Pro Arg Arg Pro

115 120 125

Ser Arg Tyr Arg Met Asp Glu

130 135

<210> 14

<211> 135

<212> protein

<213> Simian immunodeficiency virus

<400> 14

Met Glu Gln Pro Pro Gln Ser His Pro Leu His Trp Thr Ser Arg Met

1 5 10 15

Val Pro Ile Glu Arg Gln Ala Leu Gln Ala Ala Ile Trp Glu Leu Asn

20 25 30

Glu Glu Ala Leu Lys His Phe Ser Arg Glu Glu Glu Leu Arg Gly Ile Trp

35 40 45

Glu Gln Val Thr Glu Leu Pro Ala Asp Pro Ala Trp Asn Ala Asp Gln

50 55 60

Ala Trp Ala Ala Cys Ala Ile Asp Tyr Thr Arg Trp Val Gln Thr Ile

65 70 75 80

Leu Tyr Arg His Tyr Arg Glu Gly Cys Tyr His Arg Tyr Ala Glu Gln

85 90 95

Ile Arg Arg Tyr Pro Val Leu Arg Pro Met Arg Gly Thr Ala Pro Gly

100 105 110

Pro Thr Ser Ser Val Pro Gln Ala Asp Pro Asp Asn Pro Arg Arg Pro

115 120 125

Ser Arg Tyr Arg Met Asp Glu

130 135

<210> 15

<211> 113

<212> protein

<213> Simian immunodeficiency virus

<400> 15

Met Ala Glu Ala Phe Phe Asn Pro Ser Gln His Val Gln Gly Thr Pro

1 5 10 15

Trp Phe Phe Ile Pro Arg Asn Val Glu Leu Thr Pro Asn Val Ile Asn

20 25 30

Val Thr Val Lys Ala Glu Leu Val Val Thr Glu Ala Ser Lys His Phe

35 40 45

Thr Pro Gln Glu Ile Tyr Gly Val Trp Asn Gln Ser Leu Asn Glu Glu

50 55 60

Ala Gly Thr Asp Ser Pro Thr Met Ala Trp Glu Arg Thr Met Leu Asp

65 70 75 80

Met Val Arg Ala Leu Asn Leu Met Leu Phe Glu His Phe Ala Ala Gly

85 90 95

Cys Pro Gln Arg Thr Arg Tyr Ala Arg His Arg Gly Tyr Pro His Pro

100 105 110

Ser

<210> 16

<211> 99

<212> protein

<213> Simian immunodeficiency virus

<400> 16

Met Ala Glu Arg Ala Pro Glu Ala Pro Glu Gly Ala Gly Glu Val Gly

1 5 10 15

Leu Glu Gln Trp Leu Glu Thr Ser Leu Glu Arg Ile Asn Arg Glu Ala

20 25 30

Arg Leu His Phe His Pro Glu Phe Leu Phe Arg Leu Trp Asn Thr Cys

35 40 45

Val Glu His Trp His Asp Arg His Gln Arg Ser Leu Asp Tyr Ala Lys

50 55 60

Tyr Arg Tyr Leu Leu Leu Met His Lys Ala Met Tyr Thr His Met Gln

65 70 75 80

Gln Gly Cys Pro Cys Arg Asn Gly Arg Pro Arg Gly Pro Pro Pro Pro

85 90 95

Gly Met Ala

<210> 17

<211> 113

<212> protein

<213> Simian immunodeficiency virus

<400> 17

Met Ala Glu Arg Gln Ser Val Glu Arg Ala Pro Ala Glu Pro Met Gly

1 5 10 15

Ala Gly Glu Val Glu Leu Glu Glu Trp Leu Gln Arg Ser Leu Leu Arg

20 25 30

Ile Asn Gln Glu Ala Arg Leu His Phe His Pro Glu Phe Leu Phe Arg

35 40 45

Leu Trp Asn Thr Cys Met Glu His Tyr His Asp Ala Leu Gln Leu Ser

50 55 60

Phe Thr Tyr Ser Lys Tyr Arg Tyr Leu Leu Leu Leu Gln Lys Ala Met

65 70 75 80

Phe Met His Phe Gln Gln Gly Cys Ser Cys Leu Gln Gly Arg His Pro

85 90 95

Pro Pro Leu Arg Pro Ala Gly Asp Arg Leu Pro Pro Pro Pro Pro Pro

100 105 110

Pro

<210> 18

<211> 112

<212> protein

<213> Simian immunodeficiency virus

<400> 18

Met Ser Asp Pro Arg Glu Arg Ile Pro Pro Gly Asn Ser Gly Glu Glu

1 5 10 15

Thr Ile Gly Glu Ala Phe Glu Trp Leu Asn Arg Thr Val Glu Glu Ile

20 25 30

Asn Arg Glu Ala Val Asn His Leu Pro Arg Glu Leu Ile Phe Gln Val

35 40 45

Trp Gln Arg Ser Trp Glu Tyr Trp His Asp Glu Gln Gly Met Ser Pro

50 55 60

Ser Tyr Val Lys Tyr Arg Tyr Leu Cys Leu Ile Gln Lys Ala Leu Phe

65 70 75 80

Met His Cys Lys Lys Gly Cys Arg Cys Leu Gly Glu Gly His Gly Ala

85 90 95

Gly Gly Trp Arg Pro Gly Pro Pro Pro Pro Pro Pro Pro Gly Leu Ala

100 105 110

<210> 19

<211> 626

<212> protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<223> Human SAMHD1

<300>

<308> UniProt:Q9Y3Z3

<309> 1999-11-01

<400> 19

Met Gln Arg Ala Asp Ser Glu Gln Pro Ser Lys Arg Pro Arg Cys Asp

1 5 10 15

Asp Ser Pro Arg Thr Pro Ser Asn Thr Pro Ser Ala Glu Ala Asp Trp

20 25 30

Ser Pro Gly Leu Glu Leu His Pro Asp Tyr Lys Thr Trp Gly Pro Glu

35 40 45

Gln Val Cys Ser Phe Leu Arg Arg Gly Gly Phe Glu Glu Pro Val Leu

50 55 60

Leu Lys Asn Ile Arg Glu Asn Glu Ile Thr Gly Ala Leu Leu Pro Cys

65 70 75 80

Leu Asp Glu Ser Arg Phe Glu Asn Leu Gly Val Ser Ser Leu Gly Glu

85 90 95

Arg Lys Lys Leu Leu Ser Tyr Ile Gln Arg Leu Val Gln Ile His Val

100 105 110

Asp Thr Met Lys Val Ile Asn Asp Pro Ile His Gly His Ile Glu Leu

115 120 125

His Pro Leu Leu Val Arg Ile Ile Asp Thr Pro Gln Phe Gln Arg Leu

130 135 140

Arg Tyr Ile Lys Gln Leu Gly Gly Gly Tyr Tyr Val Phe Pro Gly Ala

145 150 155 160

Ser His Asn Arg Phe Glu His Ser Leu Gly Val Gly Tyr Leu Ala Gly

165 170 175

Cys Leu Val His Ala Leu Gly Glu Lys Gln Pro Glu Leu Gln Ile Ser

180 185 190

Glu Arg Asp Val Leu Cys Val Gln Ile Ala Gly Leu Cys His Asp Leu

195 200 205

Gly His Gly Pro Phe Ser His Met Phe Asp Gly Arg Phe Ile Pro Leu

210 215 220

Ala Arg Pro Glu Val Lys Trp Thr His Glu Gln Gly Ser Val Met Met

225 230 235 240

Phe Glu His Leu Ile Asn Ser Asn Gly Ile Lys Pro Val Met Glu Gln

245 250 255

Tyr Gly Leu Ile Pro Glu Glu Asp Ile Cys Phe Ile Lys Glu Gln Ile

260 265 270

Val Gly Pro Leu Glu Ser Pro Val Glu Asp Ser Leu Trp Pro Tyr Lys

275 280 285

Gly Arg Pro Glu Asn Lys Ser Phe Leu Tyr Glu Ile Val Ser Asn Lys

290 295 300

Arg Asn Gly Ile Asp Val Asp Lys Trp Asp Tyr Phe Ala Arg Asp Cys

305 310 315 320

His His Leu Gly Ile Gln Asn Asn Phe Asp Tyr Lys Arg Phe Ile Lys

325 330 335

Phe Ala Arg Val Cys Glu Val Asp Asn Glu Leu Arg Ile Cys Ala Arg

340 345 350

Asp Lys Glu Val Gly Asn Leu Tyr Asp Met Phe His Thr Arg Asn Ser

355 360 365

Leu His Arg Arg Ala Tyr Gln His Lys Val Gly Asn Ile Ile Asp Thr

370 375 380

Met Ile Thr Asp Ala Phe Leu Lys Ala Asp Asp Tyr Ile Glu Ile Thr

385 390 395 400

Gly Ala Gly Gly Lys Lys Tyr Arg Ile Ser Thr Ala Ile Asp Asp Met

405 410 415

Glu Ala Tyr Thr Lys Leu Thr Asp Asn Ile Phe Leu Glu Ile Leu Tyr

420 425 430

Ser Thr Asp Pro Lys Leu Lys Asp Ala Arg Glu Ile Leu Lys Gln Ile

435 440 445

Glu Tyr Arg Asn Leu Phe Lys Tyr Val Gly Glu Thr Gln Pro Thr Gly

450 455 460

Gln Ile Lys Ile Lys Arg Glu Asp Tyr Glu Ser Leu Pro Lys Glu Val

465 470 475 480

Ala Ser Ala Lys Pro Lys Val Leu Leu Asp Val Lys Leu Lys Ala Glu

485 490 495

Asp Phe Ile Val Asp Val Ile Asn Met Asp Tyr Gly Met Gln Glu Lys

500 505 510

Asn Pro Ile Asp His Val Ser Phe Tyr Cys Lys Thr Ala Pro Asn Arg

515 520 525

Ala Ile Arg Ile Thr Lys Asn Gln Val Ser Gln Leu Leu Pro Glu Lys

530 535 540

Phe Ala Glu Gln Leu Ile Arg Val Tyr Cys Lys Lys Val Asp Arg Lys

545 550 555 560

Ser Leu Tyr Ala Ala Arg Gln Tyr Phe Val Gln Trp Cys Ala Asp Arg

565 570 575

Asn Phe Thr Lys Pro Gln Asp Gly Asp Val Ile Ala Pro Leu Ile Thr

580 585 590

Pro Gln Lys Lys Glu Trp Asn Asp Ser Thr Ser Val Gln Asn Pro Thr

595 600 605

Arg Leu Arg Glu Ala Ser Lys Ser Arg Val Gln Leu Phe Lys Asp Asp

610 615 620

ProMet

625

<210> 20

<211> 52

<212> protein

<213> Human immunodeficiency virus

<220>

<221> MISC_FEATURE

<223> HIV-1 P6

<400> 20

Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Glu Glu Ser Phe Arg

1 5 10 15

Phe Gly Val Glu Thr Thr Thr Pro Pro Gln Lys Gln Glu Pro Ile Asp

20 25 30

Lys Glu Leu Tyr Pro Leu Thr Ser Leu Arg Ser Leu Phe Gly Asn Asp

35 40 45

Pro Ser Ser Gln

fifty

<210> 21

<211> 63

<212> protein

<213> Simian immunodeficiency virus

<220>

<221> MISC_FEATURE

<223> SIVmac P6

<400> 21

Pro Met Ala Gln Val His Gln Gly Leu Met Pro Thr Ala Pro Pro Glu

1 5 10 15

Asp Pro Ala Val Asp Leu Leu Lys Asn Tyr Met Gln Leu Gly Lys Gln

20 25 30

Gln Arg Glu Lys Gln Arg Glu Ser Arg Glu Lys Pro Tyr Lys Glu Val

35 40 45

Thr Glu Asp Leu Leu His Leu Asn Ser Leu Phe Gly Gly Asp Gln

50 55 60

<210> 22

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> synthetic construction

<220>

<221> MISC_FEATURE

<223> SIVmac P6

<220>

<221> OPTION

<222> 2, 4, 5

<223> Xaa can be any amino acid

<220>

<221> misc_feature

<222> (4)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 22

Asp Xaa Ala Xaa Xaa Leu Leu

fifteen

<210> 23

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> synthetic construction

<220>

<221> MISC_FEATURE

<223> packing motif

<400> 23

Asp Pro Ala Val Asp Leu Leu

fifteen

<210> 24

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> synthetic construction

<220>

<221> MISC_FEATURE

<223> packing motif

<400> 24

Asp Pro Ala Val Asp Leu Leu Lys Asn Tyr

1 5 10

<210> 25

<211> 52

<212> protein

<213> Artificial sequence

<220>

<223> synthetic construction

<220>

<221> MISC_FEATURE

<223> P6 hybrid domain

<400> 25

Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Glu Glu Ser Asp Pro

1 5 10 15

Ala Val Asp Leu Leu Thr Thr Pro Pro Gln Lys Gln Glu Pro Ile Asp

20 25 30

Lys Glu Leu Tyr Pro Leu Thr Ser Leu Arg Ser Leu Phe Gly Asn Asp

35 40 45

Pro Ser Ser Gln

fifty

<210> 26

<211> 16

<212> protein

<213> Artificial sequence

<220>

<223> synthetic construction

<220>

<221> MISC_FEATURE

<223> Binding motif

<220>

<221> OPTION

<222> 6, 9, 14

<223> Xaa can be Val, Ile, Leu, Phe, Trp, Tyr, or Met

<220>

<221> OPTION

<222> 2, 3, 4, 5, 7, 8, 10, 11, 12, 15

<223> Xaa can be any amino acid

<400> 26

Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa His

1 5 10 15

<210> 27

<211> 12

<212> protein

<213> Artificial sequence

<220>

<223> spacer (IgG4 hinge region) (a.k.)

<400> 27

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 28

<211> 36

<212> protein

<213> Artificial sequence

<220>

<223> spacer (IgG4 hinge region) (nuc.)

<400> 28

Gly Ala Ala Thr Cys Thr Ala Ala Gly Thr Ala Cys Gly Gly Ala Cys

1 5 10 15

Cys Gly Cys Cys Cys Thr Gly Cys Cys Cys Cys Cys Cys Thr Thr Gly

20 25 30

Cys Cys Cys Thr

35

<210> 29

<211> 119

<212> protein

<213> Artificial sequence

<220>

<223> Spacer "hinge-CH3"

<400> 29

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg

1 5 10 15

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

20 25 30

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

35 40 45

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

50 55 60

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

65 70 75 80

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

85 90 95

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

100 105 110

Leu Ser Leu Ser Leu Gly Lys

115

<210> 30

<211> 229

<212> protein

<213> Artificial sequence

<220>

<223> Spacer "hinge-CH2-CH3"

<400> 30

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 31

<211> 282

<212> protein

<213> Artificial sequence

<220>

<223> IgD-hinge region-Fc

<400> 31

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210> 32

<211> 24

<212> protein

<213> Artificial sequence

<220>

<223> T2A

<400> 32

Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

1 5 10 15

Val Glu Glu Asn Pro Gly Pro Arg

twenty

<210> 33

<211> 357

<212> protein

<213> Artificial sequence

<220>

<223>tEGFR

<400> 33

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala

325 330 335

Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly

340 345 350

Ile Gly Leu Phe Met

355

<210> 34

<211> 27

<212> protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<223> CD28 (amino acids 153-179, registration No. P10747)

<400> 34

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 35

<211> 66

<212> protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<223> CD28 (amino acids 114-179, registration No. P10747)

<400> 35

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly

35 40 45

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

50 55 60

Trp Val

65

<210> 36

<211> 41

<212> protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<223> CD28 (amino acids 180-220, P10747)

<400> 36

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 37

<211> 41

<212> protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<223> CD28 (LL-GG)

<400> 37

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 38

<211> 42

<212> protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<223> 4-1BB (amino acids 214-255, Q07011.1)

<400> 38

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 39

<211> 112

<212> protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<223> CD3 zeta

<400> 39

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 40

<211> 112

<212> protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<223> CD3 zeta

<400> 40

Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 41

<211> 112

<212> protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<223> CD3 zeta

<400> 41

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 42

<211> 335

<212> protein

<213> Artificial sequence

<220>

<223>tEGFR

<400> 42

Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

145 150 155 160

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His

275 280 285

Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro

290 295 300

Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala

305 310 315 320

Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

325 330 335

<210> 43

<211> 18

<212> protein

<213> Artificial sequence

<220>

<223> T2A

<400> 43

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

GlyPro

<210> 44

<211> 22

<212> protein

<213> Artificial sequence

<220>

<223> P2A

<400> 44

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

twenty

<210> 45

<211> 19

<212> protein

<213> Artificial sequence

<220>

<223> P2A

<400> 45

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 46

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223>E2A

<400> 46

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

twenty

<210> 47

<211> 22

<212> protein

<213> Artificial sequence

<220>

<223> F2A

<400> 47

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

twenty

<210> 48

<211> 30

<212> protein

<213> Artificial sequence

<220>

<223> Linker

<400> 48

Pro Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro

20 25 30

<210> 49

<211> 17

<212> protein

<213> Artificial sequence

<220>

<223> Linker

<400> 49

Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys

1 5 10 15

Ser

<210> 50

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> GMCSFR alpha chain signal sequence

<400> 50

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcca 66

<210> 51

<211> 22

<212> protein

<213> Artificial sequence

<220>

<223> GMCSFR alpha chain signal sequence

<400> 51

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

twenty

<210> 52

<211> 18

<212> protein

<213> Artificial sequence

<220>

<223> CD8-alpha signal peptide

<400> 52

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala

<---

Claims (135)

1. A method for transducing T cells, comprising: (a) incubating a viral vector particle containing a recombinant nucleic acid encoding a heterologous molecule and an initial composition containing T cells, where said T cells were obtained from a sample taken from an individual, thereby obtaining the final composition, and incubation in step (a) begins no later than 24 hours after sampling from the individual, and prior to incubation in (a) the original composition has not been subjected to ex vivo stimulation, including incubation in the presence of one or more agents capable of activating T cells; and (b) ex vivo incubation of the final composition in the presence of one or more stimulatory agents capable of activating T cells, signal induction by the TCR complex, and/or induction of T cell proliferation, wherein the incubation occurs for a period of time up to 10 days after incubation in (a). 2. The method of claim 1, wherein the one or more agents capable of activating T cells prior to incubation in (a) comprise an anti-CD3 antibody and/or an anti-CD28 antibody. 3. The method according to claim 1 or 2, where before incubation in (a) not more than 5%, 10%, 20%, 30% or 40% of T cells: (i) are activated cells; (ii) expressing a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB; (iii) contain an expressed intracellular cytokine selected from the group consisting of IL-2, IFN-gamma and TNF-alpha; and/or (iv) are present in the G1 phase or in a later phase of the cell cycle. 4. A method for transducing T cells, comprising: (a) incubating a viral vector particle containing a recombinant nucleic acid encoding a heterologous molecule and an initial composition containing a plurality of T cells, where said plurality of T cells was obtained from a sample taken from an individual, thereby obtaining the final composition , and (b) ex vivo incubation of the final composition in the presence of one or more stimulatory agents capable of activating T cells, signal induction by the TCR complex, and/or induction of T cell proliferation, wherein the incubation occurs for a period of time up to 10 days after incubation in (a), and moreover: (i) incubation in (a) begins no later than 24 hours after the sample is taken from the individual; and/or (ii) before incubation in (a) the T cells were not brought to a temperature of more than or more than about 15°C for more than 24 hours after sampling from the individual; and/or (iii) prior to incubation in (a), the T cells were not brought to a temperature of approximately 37° C. ± 2.0° C. or more for more than 15 minutes after sampling from the individual. 5. The method according to any one of paragraphs. 1-4, where the incubation begins no later than or no later than about 1 hour, 3 hours, 6 hours, 12 hours or 18 hours after the sample is taken from the individual. 6. The method according to claim 4 or 5, wherein prior to incubation in (a), the method does not include stimulation of T cells under conditions that stimulate cell activation. 7. The method according to any one of paragraphs. 4-6, where prior to incubation in (a) the original composition was not subjected to ex vivo stimulation, including: (i) incubation at a temperature greater than or greater than about 37° C. ± 2.0° C.; (ii) incubation in the presence of one or more agents capable of activating T cells; (iii) incubation in the presence of one or more agents capable of inducing a signal through the TCR complex; (iv) incubation in the presence of one or more agents capable of inducing T cell proliferation; and/or (v) incubation in the presence of CD3 binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15 and/or recombinant IL-7. 8. The method according to any one of paragraphs. 4-7, where before incubation in (a) no more than 5%, 10%, 20%, 30% or 40% of T cells: (a) are activated cells; (b) expressing a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB; (c) contain an expressed intracellular cytokine selected from the group consisting of IL-2, IFN-gamma and TNF-alpha; (d) are present in the G1 phase or in a later phase of the cell cycle; and/or (e) capable of proliferating. 9. The method according to claim 3 or 8, wherein immediately prior to incubation in (a) no more than 10% of the T cells in the initial composition contain a T cell activation marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB. 10. The method according to any one of paragraphs. 1-9, wherein prior to incubation in (a), more than 5%, 10%, 20%, 30%, or 40% of the T cells express the low density lipid receptor (LDL-R). 11. The method according to any one of paragraphs. 1-10, further comprising activating or expanding the cells of the final composition. 12. The method according to any one of paragraphs. 1-11, where one or more stimulatory agents in (b) are selected from the group consisting of CD3-binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7. 13. The method according to any one of paragraphs. 1-12, wherein one or more of the stimulatory agents in (b) comprise an anti-CD3 antibody and/or an anti-CD28 antibody. 14. The method according to any one of paragraphs. 1-13, where the individual is a human. 15. The method according to any one of paragraphs. 1-14, where T cells were not brought to a temperature of 2°C to 8°C and/or maintained at this temperature for more than 48 hours prior to incubation in (a). 16. The method according to any one of paragraphs. 1-15, where the sample is a blood sample. 17. The method according to any one of paragraphs. 1-15, where the sample is a sample taken after leukapheresis. 18. The method according to paragraphs. 1-17, where 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. 19. The method according to any one of paragraphs. 1-18, where T cells were selected or enriched from a sample taken from an individual. 20. The method according to any one of paragraphs. 1-19, which further comprises, prior to incubation in (a), selecting or enriching T cells from the sample, thereby obtaining an enriched composition and/or initial composition. 21. The method according to any one of paragraphs. 1-20, wherein the percentage of T cells in the original composition is greater than or approximately greater than 75%, 80%, 85%, 90%, 95% T cells. 22. The method according to any one of paragraphs. 1-21, where T cells contain CD4 ⁺ or CD8 ⁺ cells. 23. The method according to any one of paragraphs. 1-22, where T cells contain CD4 ⁺ - and CD8 ⁺ cells. 24. The method of claim 23, wherein the ratio of CD4 ⁺ cells to CD8 ⁺ cells is or approximately 1:1, 1:2, 2:1, 1:3, or 3:1. 25. The method according to any one of paragraphs. 1-24 where: the sample contains serum or plasma at a concentration of at least or at least about 10% (v/v), at least or at least about 15% (v/v), at least or at least about 20% (v/v), at least or at least about 25% (v/v), at least or at least about 30% (v/v), at least or at least about 33% (v/v), at least or at least about 35% (v/v), or at least or at least about 40% (v/v) ; and/or prior to incubation in (a), the sample has been contacted ex vivo with serum or plasma at a concentration of at least or at least about 10% (v/v), at least or at least about 15% (v/v). vol.), at least or at least about 20% (v/v), at least or at least about 25% (v/v), at least or at least about 30% (v/v), at least or at least about 33% (v/v), at least or at least about 35% (v/v), or at least or at least about 40% (v/v). 26. The method according to any one of paragraphs. 1-25 where: the sample contains serum or plasma at a concentration of at least or at least about 30% (v/v); and/or prior to incubation in (a), the sample has been contacted ex vivo with serum or plasma at a concentration of at least or at least about 30% (v/v). 27. The method of claim 25 or 26 wherein the serum or plasma is human. 28. The method according to any one of paragraphs. 25-27, wherein the serum or plasma is autologous to the individual. 29. The method according to any one of paragraphs. 1-25, where the sample contains an anticoagulant. 30. The method of claim 29, wherein the anticoagulant contains a free citrate ion. 31. The method according to any one of paragraphs. 1-30, wherein, prior to incubation in (a), the method comprises cryopreserving T cells, optionally T cells, in a sample or enriched composition in the presence of a cryoprotectant, and thereby producing a cryopreserved composition. 32. The method of claim 31 wherein, prior to incubation in (a), the method comprises washing the cryopreserved composition under conditions that reduce or remove the cryoprotectant and/or produce the original composition. 33. The method according to any one of paragraphs. 1-32, where the original composition contains N-acetylcysteine (NAC); serum, optionally human serum; recombinant interleukin-2 (IL-2), recombinant interleukin-15 (IL-15) and/or recombinant interleukin-7 (IL-7). 34. The method according to any one of paragraphs. 1-33 where: the original composition contains N-acetylcysteine at a concentration of from or about 0.4 mg/ml to 4 mg/ml, from 0.8 mg/ml to 3.6 mg/ml, or from 1.6 mg/ml to 2.4 mg/ml inclusive; or the original composition contains N-acetylcysteine at a concentration of at least, or at least about, or about 0.4 mg/ml, 0.8 mg/ml, 1.2 mg/ml, 1.6 mg/ml, 2, 0 mg/ml, 2.4 mg/ml, 2.8 mg/ml, 3.2 mg/ml, 3.6 mg/ml or 4.0 mg/ml. 35. The method according to any one of paragraphs. 1-34 where: the initial composition contains serum, optionally human serum, at a concentration of from or about 0.5% to 25% (v/v), from 1.0% to 10% (v/v), or from 2.5% up to 5.0% (v/v) inclusive; or the starting composition contains serum, optionally human serum, at a concentration of at least, or at least about, or about 0.5%, 1.%, 2.5%, 5% (v/v), or 10%. 36. The method according to any one of paragraphs. 1-35 where: the original composition contains recombinant IL-2, optionally recombinant human IL-2, at a concentration of from or about 10 IU/ml to 500 IU/ml, from 50 IU/ml to 250 IU/ml, or from 100 IU/ml to 200 IU /ml inclusive; or at a concentration of at least or at least about 10 IU/ml, 50 IU/ml, 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, or 500 IU/ml; and/or the original composition contains recombinant IL-15, optionally recombinant human IL-15, at a concentration of from or about 1 IU/ml to 100 IU/ml, from 2 IU/ml to 50 IU/ml, or from 5 IU/ml to 10 IU /ml inclusive; or at a concentration of at least or at least about 1 IU/ml, 2 IU/ml, 5 IU/ml, 10 IU/ml, 25 IU/ml, or 50 IU/ml; and/or the original composition contains recombinant IL-7, optionally recombinant human IL-7, at a concentration of from or about 50 IU/ml to 1500 IU/ml, from 100 IU/ml to 1000 IU/ml, from 200 IU/ml to 600 IU /ml inclusive; or at a concentration of at least or at least about 50 IU/ml, 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml , 800 IU/ml, 900 IU/ml or 1000 IU/ml. 37. The method according to any one of paragraphs. 1-36 wherein the incubation in (a) includes the step of centrifugally inoculating the viral vector particles with the parent composition. 38. The method according to p. 37, where the centrifugal inoculation includes the rotation of the particles of the viral vector and the original composition in the inner compartment of the centrifuge chamber, where the rotation is carried out with a relative centrifugal force applied to the inner surface of the side wall of the compartment and component: from or approximately 500 g to 2500 g, 500 g to 2000 g, 500 g to 1600 g, 500 g to 1000 g, 600 g to 1600 g, 600 g to 1000 g, 1000 g to 2000 g or from 1000 g to 1600 g inclusive; or at least or at least about 600 g, 800 g, 1000 g, 1200 g, 1600 g or 2000 g. 39. The method according to claim 37 or 38, where centrifugal inoculation is carried out for a period of time that is: more than or about 5 minutes, more than or about 10 minutes, more than or about 15 minutes, more than or about 20 minutes, more than or about 30 minutes, more than or about 45 minutes, more than or about 60 minutes, more than or about 90 minutes or more than or about 120 minutes; or from or about 5 minutes to 60 minutes, 10 minutes to 60 minutes, 15 minutes to 60 minutes, 15 minutes to 45 minutes, 30 minutes to 60 minutes, or 45 minutes to 60 minutes, inclusive. 40. The method according to any one of paragraphs. 1-39, further comprising contacting the parent composition and/or viral vector particles with a transduction adjuvant. 41. The method of claim 40, wherein the contact is made before, during, or after centrifugal inoculation of the viral vector particles with the parent composition. 42. The method according to any one of paragraphs. 1-41, where at least part of the incubation in (a) is carried out at or approximately at 37°C ± 2°C. 43. The method according to any one of paragraphs. 37-42, where at least part of the incubation in (a) is carried out after centrifugal inoculation. 44. The method according to p. 42 or 43, where at least part of the incubation in (a) is carried out for a period of time 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. 45. The method according to any one of paragraphs. 42-44, wherein at least a portion of the incubation in (a) is for or about 24 hours. 46. The method according to any one of paragraphs. 1-45, where the entire incubation period in (a) is no more than 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours. 47. The method according to any one of paragraphs. 1-46, where the incubation in step (b) occurs for up to 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, or 9 days after the incubation in (a) . 48. The method according to any one of paragraphs. 1-47, where the viral vector particle is a lentiviral vector particle. 49. The method of claim 48, wherein the lentiviral vector particle is derived from HIV-1. 50. The method according to any one of paragraphs. 1-49, where the viral vector particle is pseudotyped with a viral envelope glycoprotein. 51. The method of claim 48, wherein the viral envelope glycoprotein is VSV-G. 52. The method according to any one of paragraphs. 1-51, where the viral vector particle contains a lentiviral protein with SAMHD1 inhibitory activity, where the specified protein is packaged in a viral particle. 53. The method of claim 52, wherein the SAMHD1 inhibitory protein is a wild type Vpx protein, a wild type Vpr protein, or a wild type Vpx or Vpr protein variant or portion having SAMHD1 inhibitory activity. 54. The method of claim 52 or 53, wherein the SAMHD1 inhibitory protein is heterologous to the retroviral vector particle. 55. The method according to any one of paragraphs. 52-54, wherein the SAMHD1 inhibitory protein is a wild type Vpx protein or a variant or portion of a wild type Vpx protein having SAMHD1 inhibitory activity. 56. The method according to any one of paragraphs. 1-55, wherein the viral vector particle is incubated at an MOI of less than or less than about 20.0, or less than or less than about 10.0. 57. The method according to any one of paragraphs. 1-56 where: the viral vector particle is incubated at a multiplicity of infection ranging from or about 1.0 IU/cell to 10 IU/cell, or from 2.0 IU/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/cage or 3.6 IU/cage, 4.0 IU/cage, 5.0 IU/cage, 6.0 IU/cage, 7.0 IU/cage, 8.0 IU/cage, 9 .0 IU/cage or 10.0 IU/cage. 58. The method according to any one of paragraphs. 1-57, where the original composition contains at least, or at least about, or about 50 x 10 ⁶ cells, 100 x 10 ⁶ cells or 200 x 10 ⁶ cells. 59. The method according to any one of paragraphs. 1-58, where the recombinant nucleic acid encodes an antigen receptor. 60. The method of claim 59 wherein the antigen receptor is a transgenic T cell receptor (TCR). 61. The method of claim 59 or 60, wherein the antigen receptor is a chimeric antigen receptor (CAR). 62. The method of claim 61, wherein the chimeric antigen receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to the target antigen and to an intracellular signal transduction domain containing ITAM. 63. The method of claim 62, wherein the intracellular signal-transmitting domain comprises an intracellular domain of the CD3-zeta chain (CD3ζ). 64. The method according to claim 62 or 63, further comprising a transmembrane domain that binds an extracellular domain and an intracellular signal-transmitting domain. 65. The method of claim 64, wherein the transmembrane domain contains the transmembrane portion of CD28. 66. The method according to any one of paragraphs. 62-65, wherein the intracellular signal-transmitting domain further comprises an intracellular signal-transmitting domain of a T-cell costimulatory molecule. 67. The method of claim 66, wherein the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB. 68. The method according to any one of paragraphs. 1-67, where the antigen receptor specifically binds to an antigen associated with a disease or condition, or specifically binds to a universal label. 69. The method of claim 68 wherein the disease or condition is cancer and an autoimmune disease or disorder or infectious disease. 70. The method according to any one of paragraphs. 1-69, wherein the method results in a transduced composition comprising T cells transduced with the recombinant nucleic acid. 71. The method of claim 70, wherein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the T cells in the transduced composition transduce recombinant nucleic acid. 72. The method of claim 70 or 71, further comprising obtaining or isolating from the transduced composition transduced T cells obtained by said method. 73. The method according to any one of paragraphs. 11-72, where activation and/or propagation is carried out ex vivo . 74. The method according to any one of paragraphs. 1-73, where the final composition is activated and/or propagated in vivo . 75. The method according to any one of paragraphs. 11-74, where the activation and/or propagation is carried out in the presence of an antigen that specifically binds to the antigen receptor, and/or the activation and/or propagation is transgene-specific. 76. The method according to any one of paragraphs. 1-75, where after incubation in (a) the final composition is not further incubated at a temperature of more than 30°C for more than 24 hours. 77. The method of adoptive cell therapy, including: (a) enriching or isolating T cells from a sample taken from an individual suffering from a disease or condition; (b) transduction of the original composition containing T-cells, a viral vector particle by the method according to any one of paragraphs. 1-76 to obtain a final composition containing transduced cells, where (i) the viral vector particle contains a recombinant nucleic acid encoding an antigenic receptor that specifically binds to an antigen associated with a disease or disorder, and (ii) the disease or condition is cancer , an autoimmune disease or disorder or infectious disease; (c) administering the final composition containing the transduced cells to an individual for the treatment of a disease or condition, wherein the final composition is administered to the individual no later than 9 days after the individual has been sampled. 78. An adoptive cell therapy method comprising administering a final composition comprising T cells transduced with a recombinant nucleic acid encoding a heterologous molecule to an individual for the treatment of a disease or condition wherein (i) the final composition is obtained by carrying out the method of transduction of T cells according to any one of paragraphs. 1-76; (ii) the disease or condition is cancer, an autoimmune disease or disorder, or an infectious disease; and (iii) the antigen receptor specifically binds to an antigen associated with the disease or condition. 79. The method according to p. 77 or 78, where the final composition is administered to the individual no later than 1 day, 2 days, 3 days, 4 days or 5 days after taking the sample from the individual. 80. The method according to any one of paragraphs. 77-79, wherein prior to administration of the composition, the transduced cells or cells of the final composition are prepared in a pharmaceutically acceptable buffer. 81. The method according to any one of paragraphs. 77-80, where prior to the administration of the composition containing the transduced cells, these cells are cultured ex vivo for a period of time up to 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or 9 days after transduction, where the specified cultivation is carried out at a temperature of more than 30°C. 82. The method according to any one of paragraphs. 77-81, where after transduction, the final composition or cells containing the transduced cells are cultured in the presence of one or more stimulatory agents capable of activating T cells, inducing signal transmission through the TCR complex, and/or inducing T cell proliferation to obtain a composition containing transduced cells. 83. The method according to any one of paragraphs. 77-80, wherein prior to the administration of the transduced cells, the final composition or cells containing the transduced cells are not further incubated ex vivo in the presence of one or more stimulating agents and/or are not further incubated at more than 30°C for more than 24 hours. 84. The method according to p. 82 or 83, where one or more stimulating agents are selected from the group consisting of CD3-binding molecules; CD28 binding molecules; recombinant IL-2; recombinant IL-15 and recombinant IL-7; a vaccine containing an antigen specifically recognized by an antigen receptor; and an anti-idiotypic antibody that specifically binds to an antigen receptor. 85. The method of claim 84 wherein the one or more stimulatory agents comprise an anti-CD3 antibody and/or an anti-CD28 antibody. 86. The method according to any one of paragraphs. 77-85, where the cells of the final composition or transduced cells are administered at a suboptimal dose. 87. The method according to any one of paragraphs. 77-86, further comprising administering to the individual one or more agents to induce or enhance stimulation and/or expand the transduced T cells in vivo . 88. The method according to p. 87, where one or more agents are transgene-specific and/or stimulate or activate cells through an expressed transgene, which optionally is or contains an antigen receptor. 89. The method according to p. 87 or 88, where one or more agents are selected from a vaccine containing an antigen specifically recognized by the antigen receptor; an anti-idiotypic antibody that specifically binds to an antigen receptor, or an agent capable of chemically inducing antigen receptor dimerization. 90. The method according to p. 89, where one or more agents are an immunomodulatory agent; immune checkpoint inhibitor; an inhibitor of extracellular adenosine or an adenosine receptor, optionally an A2aR receptor; a kynurenine pathway modulator; and signaling pathway modulators, such as kinase inhibitors.

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