Phenotypic markers for cell therapy and related procedures

Description

Field

[0001] The present disclosure relates to cell therapy involving the administration of one or more doses of a therapeutic T cell composition, and methods, compositions and articles of manufacture for use in the same. The cells of the T cell composition express recombinant receptors such as chimeric receptors, e.g. chimeric antigen receptors (CARs) or other transgenic receptors such as T cell receptors (TCRs). Features of the embodiments of the present disclosure, including the dose of cells or units of cells administered and / or the phenotype of administered cells, provide various advantages, such as consistent dosing, lower risk of toxicity and / or increased response in subjects administered the T cell compositions.Background

[0002] Various immunotherapy and / or cell therapy methods are available for treating diseases and conditions. For example, adoptive cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and / or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies) can be beneficial in the treatment of cancer or other diseases or disorders. Improved approaches are needed. Disclosed are methods and articles of manufacture that meet such needs. WO2018157171 describes compositions, articles of manufacture and methods related to dosing in cell therapy. WO2019032927 describes methods for producing genetically engineered cell compositions and related compositions. REBECCA A. GARDNER ET AL (BLOOD, vol. 129, no. 25, 13 April 2017) describes intent to treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults. Y. XU ET AL (BLOOD, vol. 123, no. 24, 12 June 2014) describes that closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15. JOSEPH FRAIETTA ET AL (Blood Journal", 1 January 2016) describes biomarkers of response to anti-CD19 chimeric antigen receptor (CAR) T-Cell Therapy in Patients with Chronic Lymphocytic Leukemia. FRAIETTA J A ET AL (EMBASE, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, 1 December 2017) describes identification of functional determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T-cell therapy of chronic lymphocytic leukemia. WO2019032929 describes methods and compositions for preparing genetically engineered cells. WO2020033927 describes processes for generating engineered cells and compositions thereof.Summary

[0003] The present invention is defined in the appended set of claims. In particular the present invention provides a method of determining a unit dose of engineered T cells for treating a subject, the method comprising: (a) assessing, in a therapeutic composition comprising a plurality of CD8 +< and / or CD4 +< T cells engineered to express a recombinant receptor, the number, percentage or ratio of T cells that express the recombinant receptor and CCR7 and CD27 (receptor +< / CCR7 +< / CD27 +< ); (b) based on the number, percentage or ratio of receptor +< / CCR7 +< / CD27 +< cells, determining one or more unit doses of cells for administration to a subject having a disease or condition, wherein the unit dose comprises a defined number of recombinant receptor-expressing CD8 +< T cells that express CCR7 and CD27 (receptor +< / CD8 +< / CCR7 +< / CD27 +< cells) and / or a defined number of recombinant receptor-expressing CD4 +< T cells that express CCR7 and CD27 (receptor +< / CD4 +< / CCR7 +< / CD27 +< cells) and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or receptor +< / CD4 +< / CCR7 +< / CD27 +< cells to another subset of cells in the composition; wherein the recombinant receptor is a chimeric antigen receptor (CAR). The present invention also provides a method of producing a composition comprising a unit dose of a T cell composition, the method comprising: (a) assessing, in a therapeutic composition comprising a plurality of CD8 +< and / or CD4 +< T cells engineered to express a recombinant receptor, the number, percentage or ratio of T cells that express the recombinant receptor and CCR7 and CD27 (receptor +< / CCR7 +< / CD27 +< ); and (b) filling a container with all or a portion of the therapeutic composition and optionally another solution to achieve a unit dose of the T cell composition, wherein the unit dose comprises a defined number of recombinant receptor-expressing CD8 +< T cells that express CCR7 and CD27 (receptor +< / CD8 +< / CCR7 +< / CD27 +< cells) and / or a defined number of recombinant receptor-expressing CD4 +< T cells that express CCR7 and CD27 (receptor +< / CD4 +< / CCR7 +< / CD27 +< cells) and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or receptor +< / CD4 +< / CCR7 +< / CD27 +< cells to another subset of cells in the composition; wherein the recombinant receptor is a chimeric antigen receptor (CAR). The present invention also provides a therapeutic composition comprising a plurality of CD8 +< and CD4 +< T cells engineered to express a recombinant receptor for use in a method of treating a subject having a disease or condition, wherein the method comprises administering one or more unit doses of the therapeutic composition to the subject, and wherein at least 50%, 60%, 70%, 80%, or 90% of the total receptor +< / CD8 +< cells in the composition are receptor +< / CD8 +< / CCR7 +< / CD27 +< and at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total receptor +< / CD4 +< cells in the composition are receptor +< / CD4 +< / CCR7 +< / CD27 +< ; wherein the recombinant receptor is a chimeric antigen receptor (CAR). The present invention also provides a therapeutic composition comprising T cells expressing a recombinant receptor, wherein at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of T cells in the composition, or the total number of T cells in the composition expressing the recombinant receptor, are surface positive for CCR7 and CD27, wherein: i) the T cells in the composition and / or expressing the recombinant receptor comprise or consist of CD8 +< and CD4 +< T cells; and ii) at least 50%, 60%, 70%, 80%, or 90% of the total receptor +< / CD8 +< cells in the composition are receptor +< / CD8 +< / CCR7 +< / CD27 +< and at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total receptor +< / CD4 +< cells in the composition are receptor +< / CD4 +< / CCR7 +< / CD27 +< ; wherein the recombinant receptor is a chimeric antigen receptor (CAR). The present invention also provides a method of predicting likelihood of response to a therapeutic T cell composition, the method comprising: (a) assaying an engineered cell composition comprising T cells expressing a recombinant receptor for a factor indicative of the function or phenotype of engineered cells in the composition, the factor being the percentage of T cells expressing the recombinant receptor that are surface positive for CCR7 and CD27; and (b) determining the likelihood of response following administration of a cell therapy comprising a dose of the engineered cells, wherein if the factor is at or above a threshold value identifying the subject as likely to achieve a durable response or progression free survival, optionally for at least 3 months, following administration the therapy; or if the factor is below a threshold value identifying the subject as not likely to exhibit a durable response or progression free survival to the therapy, wherein the threshold value of the factor is 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of T cells in the composition or of the total number of T cells in the composition expressing the recombinant receptor, are surface positive for CCR7 and CD27; and wherein the recombinant receptor is a chimeric antigen receptor (CAR).

[0004] Provided herein are methodsinvolving or comprising a unit dose of cells based on a defined number, such as a subtype of CD8 +< T cells or a subtype of CD4 +< T cells or cells thereof expressing a recombinant receptor, or a ratio of such subtype of CD8 +< and CD4 +< T cells, as defined in the claims. In some embodiments, the total number of T cells in the composition, or the total number of T cells in the composition expressing the recombinant receptor, are surface positive for CCR7. In some embodiments, the total number of T cells in the composition, or the total number of T cells in the composition expressing the recombinant receptor, are surface positive for CD27. In some embodiments, the total number of T cells in the composition, or the total number of T cells in the composition expressing the recombinant receptor, are surface positive for CCR7 and CD27. In some embodiments, the composition comprises one or more unit doses of cells.

[0005] In some embodiments, the provided methods as defined in the claims are for use in connection with cell therapy, such as engineered T cell therapy for the treatment of diseases and conditions, including various tumors.

[0006] Also provided herein are therapeutic compositions, as defined in the claims. In the invention, the therapeutic composition, as defined in the claims, comprises T cells expressing a recombinant receptor, wherein at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of T cells in the composition (or of the total number of T cells in the composition expressing the recombinant receptor), are surface positive for CCR7 and / or CD27..

[0007] In some embodiments, the unit dose of cells comprises any of the therapeutic compositions described herein.

[0008] In some embodiments, the unit dose of cells comprises a defined number of CD8 +< / CCR7 +< cells; and / or wherein the unit dose of cells comprises a defined number of CD4 +< / CCR7 +< cells. In some embodiments, the unit dose of cells comprises a defined number of CD8 +< / CD27 +< cells; and / or wherein the unit dose of cells comprises a defined number of CD4 +< / CD27 +< cells. In some embodiments, the unit dose of cells comprises a defined number of CD8 +< / CCR7 +< / CD27 +< cells; and / or wherein the unit dose of cells comprises a defined number of CD4 +< / CCR7 +< / CD27 +< cells.

[0009] In some embodiments, the unit dose or composition comprises between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD8 +< cells that express the recombinant receptor (receptor +< / CD8 +< cells) or total CD4 +< cells that express the recombinant receptor (receptor +< / CD4 +< cells), total receptor +< / CD8 +< / CCR7 +< cells, total receptor +< / CD4 +< / CCR7 +< cells, total receptor +< / CD8 +< / CD27 +< cells, or total receptor +< / CD4 +< / CD27 +< cells, each inclusive, and / or the unit dose or composition comprises no more than about 1 x 10 8< , no more than about 5 x 10 7< , no more than about 1 x 10 7< , no more than about 5 x 10 6< , no more than about 1 x 10 6< , or no more than about 5 x 10 5< total receptor +< / CD8 +< cells or total receptor +< / CD4 +< cells, total receptor +< / CD8 +< / CCR7 +< cells, total receptor +< / CD4 +< / CCR7 +< cells, total receptor +< / CD8 +< / CD27 +< cells, or total receptor +< / CD4 +< / CD27 +< cells.

[0010] In some embodiments, the unit dose comprises at least about 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< cells or total receptor +< / CD8 +< / CD27 +< cells and / or at least about 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< cells or total receptor +< / CD4 +< / CD27 +< . In some embodiments, the unit dose comprises between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< cells or total receptor +< / CD8 +< / CD27 +< cells and / or between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< cells or total receptor +< / CD4 +< / CD27 +< , each inclusive.

[0011] In some embodiments, the unit dose of cells or composition comprises a defined ratio of receptor +< / CD8 +< / CCR7 +< cells to receptor +< / CD4 +< / CCR7 +< cells, which ratio optionally is or is approximately 1:1 or is between approximately 1:3 and approximately 3:1.

[0012] In some embodiments, the unit dose of cells or composition comprises a defined ratio of receptor +< / CD8 +< / CD27 +< cells to receptor +< / CD4 +< / CD27 +< cells, which ratio optionally is or is approximately 1:1 or is between approximately 1:3 and approximately 3:1. In some embodiments, the defined number or ratio is further based on expression or absence of expression of CD45RA on the cells. In some embodiments, the defined number of cells further express or do not express CD45RA, optionally wherein the defined number of cells further are CD45RA -< cells. In some embodiments, the unit dose of cells or composition comprises a defined number of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or receptor +< / CD8 +< / CCR7 +< / CD45RA -< cells. In some embodiments, the unit dose of cells or composition comprises a defined number of receptor +< / CD4 +< / CCR7 +< / CD27 +< cells and / or receptor +< / CD4 +< / CCR7 +< / CD45RA -< cells.

[0013] In some embodiments, the unit dose comprises at least about 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or at least about 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells. In some embodiments, the unit dose comprises between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, each inclusive.

[0014] In some embodiments, the unit dose of cells comprises a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, which ratio optionally is or is approximately 1:1 or is between approximately 1:3 and approximately 3:1.

[0015] In some embodiments, the defined number or ratio is further based on expression or absence of expression of CD45RA on the cells. In some embodiments, the defined number of cells further express or do not express CD45RA, optionally wherein the defined number of cells further are CD45RA -< cells. In some embodiments, the defined number further comprises cells that express or do not express CD45RA, optionally wherein the defined number further comprises CD45RA -< cells.

[0016] In some embodiments, among a plurality of unit doses or compositions, optionally produced according to the same method, the number or ratio of cells that express CCR7 (CCR7 +< cells) in the unit dose varies by no more than 40%, by no more than 30%, by no more than 20%, by no more than 10% or by no more than 5%.

[0017] In some embodiments, the unit dose or number of cells expressing the recombinant receptor comprises between at or about 1 x 10 5< and at or about 5 x 10 8< , between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD3 +< cells that express the recombinant receptor (receptor +< / CD3 +< cells) or total CD3 +< cells, each inclusive. In some embodiments, the unit dose or number of cells expressing the recombinant receptor comprises no more than about 5 x 10 8< , no more than about 1 x 10 8< , no more than about 5 x 10 7< , no more than about 1 x 10 7< , no more than about 5 x 10 6< , no more than about 1 x 10 6< , or no more than about 5 x 10 5< total receptor +< / CD3 +< cells or total CD3 +< cells.

[0018] In some embodiments, the total number of CD3 +< cells, total number of receptor +< / CD3 +< cells, total number of receptor +< / CD8 +< cells, total number of receptor +< / CD4 +< cells, total number of receptor +< / CD8 +< / CCR7 +< cells, total number of receptor +< / CD4 +< / CCR7 +< cells, total number of receptor +< / CD8 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD45RA -< cells and / or receptor +< / CD4 +< / CCR7 +< / CD45RA -< cells is the total number of such cells that are live or viable. In some embodiments, the total number of CD3 +< cells, total number of receptor +< / CD3 +< cells, total number of receptor +< / CD8 +< cells, total number of receptor +< / CD4 +< cells, total number of receptor +< / CD8 +< / CCR7 +< cells, total number of receptor +< / CD4 +< / CCR7 +< cells, total number of receptor +< / CD8 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD45RA -< cells and / or receptor +< / CD4 +< / CCR7 +< / CD45RA -< cells is the total number of such cells that do not express an apoptotic marker and / or is the total number of such cells that are apoptotic marker negative ( -< ), wherein the apoptotic marker is Annexin V or activated Caspase 3.

[0019] In the invention, the recombinant receptor is a chimeric receptor. In some embodiments, the recombinant receptor is capable of binding to a target antigen that is associated with, specific to, and / or expressed on a cell or tissue of a disease, disorder or condition. In some embodiments, the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or a cancer. In some embodiments, the target antigen is a tumor antigen.

[0020] In some embodiments, the target antigen is selected from among αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD138, CD171, epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), G Protein Coupled Receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erbB2), 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-AI), 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 (L1CAM), CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific antigen, or an antigen associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0021] In the invention, the recombinant receptor is a chimeric antigen receptor (CAR).

[0022] In some embodiments, the recombinant receptor comprises an extracellular domain comprising an antigen-binding domain. In some embodiments, the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, which optionally is a single chain fragment. In some embodiments, the fragment comprises antibody variable regions joined by a flexible linker. In some embodiments, the fragment comprises an scFv.

[0023] In some embodiments, the recombinant receptor comprises an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

[0024] In some embodiments, the recombinant receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some embodiments, the intracellular signaling region further comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling domain is between the transmembrane domain and the intracellular signaling domain.

[0025] In some embodiments, the T cells are primary T cells obtained from a subject. In some embodiments, the T cells are autologous to the subject. In some embodiments, the T cells are allogeneic to the subject.

[0026] In some embodiments, also provided are compositions for use in methods of treatment, wherein the methods involve administering to a subject having a disease or condition one or more unit doses of any compositions, as defined in the claims.

[0027] In some embodiments, the unit dose of cells comprises a defined number of recombinant receptor-expressing CD8 +< T cells that express CCR7 and CD27 (receptor +< / CD8 +< / CCR7 +< / CD27 +< cells) and / or a defined number of recombinant receptor-expressing CD4 +< T cells that express CCR7 and CD27 cells) and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or receptor +< / CD4 +< / CCR7 +< / CD27 +< cells to another subset of cells in the composition; and among a group of subjects treated according to the method, the number or ratio of cells that express CCR7 (CCR7 +< cells) in the unit dose varies by no more than 40%, by no more than 30%, by no more than 20%, by no more than 10% or by no more than 5%.

[0028] In some embodiments, the unit dose of cells comprises a defined number of CD8 +< / CCR7 +< / CD27 +< cells. In some embodiments, the unit dose of cells comprises a defined number of CD4 +< / CCR7 +< / CD27 +< cells. In some embodiments, the unit dose comprises between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD8 +< cells that express the recombinant receptor (receptor +< / CD8 +< cells) or total CD4 +< cell that express the recombinant receptor (receptor +< / CD4 +< cells), total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, or total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, each inclusive. In some embodiments, the unit dose comprises no more than about 1 x 10 8< , no more than about 5 x 10 7< , no more than about 1 x 10 7< , no more than about 5 x 10 6< , no more than about 1 x 10 6< , or no more than about 5 x 10 5< total receptor +< / CD8 +< cells or total receptor +< / CD4 +< cells, total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, or total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells.

[0029] In some embodiments, the unit dose comprises at least about 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells; and / or at least about 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells. In some embodiments, the unit dose comprises between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells; and / or between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, each inclusive.

[0030] In some embodiments, the unit dose of cells comprises a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, which ratio optionally is or is approximately 1:1 or is between approximately 1:3 and approximately 3:1.

[0031] In some embodiments, the defined number or ratio is further based on expression or absence of expression of CD45RA on the cells. In some embodiments, the defined number or ratio is further based on the number of CD45RA -< cells. In some embodiments, the defined number of cells further express or do not express CD45RA, optionally wherein the defined number of cells further are CD45RA -< cells.

[0032] In some embodiments, the unit dose comprises between at or about 1 x 10 5< and at or about 5 x 10 8< , between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD3 +< cells that express the recombinant receptor (receptor +< / CD3 +< cells) or total CD3 +< cells, each inclusive. In some embodiments, the unit dose comprises no more than about 5 x 10 8< , no more than about 1 x 10 8< , no more than about 5 x 10 7< , no more than about 1 x 10 7< , no more than about 5 x 10 6< , no more than about 1 x 10 6< , or no more than about 5 x 10 5< total receptor +< / CD3 +< cells or total CD3 +< cells.

[0033] In some embodiments, the total number of CD3 +< cells, total number of receptor +< / CD3 +< cells, total number of receptor +< / CD8 +< cells, total number of receptor +< / CD4 +< cells, total number of receptor +< / CD8 +< / CCR7 +< cells, total number of receptor +< / CD4 +< / CCR7 +< cells, total number of receptor +< / CD8 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD45RA cells and / or receptor +< / CD4 +< / CCR7 +< / CD45RA -< cells is the total number of such cells that are live or viable. In some embodiments, the total number of CD3 +< cells, total number of receptor +< / CD3 +< cells, total number of receptor +< / CD8 +< cells, total number of receptor +< / CD4 +< cells, total number of receptor +< / CD8 +< / CCR7 +< cells, total number of receptor +< / CD4 +< / CCR7 +< cells, total number of receptor +< / CD8 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD45RA -< cells and / or receptor +< / CD4 +< / CCR7 +< / CD45RA -< cells is the total number of such cells that do not express an apoptotic marker and / or is the total number of such cells that are apoptotic marker negative ( -< ), wherein the apoptotic marker is Annexin V or activated Caspase 3.

[0034] In some embodiments, the method involves administering a plurality of unit doses contained in a plurality of separate compositions.

[0035] In some embodiments, the plurality of separate compositions comprise a first composition comprising one of the CD8 +< T cells and the CD4 +< T cells and a second composition comprising the other of the CD8 +< T cells and the CD4 +< T cells. In some embodiments, the first composition comprises the CD8 +< T cells. In some embodiments, the first composition comprises the CD4 +< T cells.

[0036] In some embodiments, the method involves administering the composition containing CD8 +< T cells or one or more unit doses thereof and / or a volume corresponding to such one or more of unit doses and the composition containing CD4 +< T cells or one or more unit doses thereof and / or a volume corresponding to such one or more of unit doses, no more than 2 hours, no more than 1 hour, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes or no more than 5 minutes apart or simultaneously.

[0037] In some embodiments, the method involves administering the composition containing CD8 +< T cells or one or more unit doses thereof and / or a volume corresponding to such one or more of unit doses, prior to administering the composition containing CD4 +< cells or one or more unit doses thereof and / or a volume corresponding to such one or more of unit doses.

[0038] In some embodiments, the method involves administering the composition containing CD4 +< T cells or one or more unit doses thereof and / or a volume corresponding to such one or more of unit doses, prior to administering the composition containing CD8 +< cells or one or more unit doses thereof and / or a volume corresponding to such one or more of unit doses.

[0039] In the invention, the recombinant receptor is a chimeric receptor. In some embodiments, the recombinant receptor is capable of binding to a target antigen that is associated with, specific to, and / or expressed on a cell or tissue of a disease, disorder or condition. In some embodiments, the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or a cancer. In some embodiments, the target antigen is a tumor antigen.

[0040] In some embodiments, the target antigen is selected from among αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), , type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase 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), CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific antigen, or an antigen associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0041] In the invention, the recombinant receptor is a chimeric antigen receptor (CAR).

[0042] In some embodiments, the recombinant receptor comprises an extracellular domain comprising an antigen-binding domain. In some embodiments, the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, which optionally is a single chain fragment. In some embodiments, the fragment comprises antibody variable regions joined by a flexible linker. In some embodiments, wherein the fragment comprises an scFv.

[0043] In some embodiments, the recombinant receptor comprises an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

[0044] In some embodiments, the recombinant receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some embodiments, the intracellular signaling region further comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof.

[0045] In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling domain is between the transmembrane domain and the intracellular signaling domain.

[0046] In some embodiments, the T cells are primary T cells obtained from a subject. In some embodiments, the T cells are autologous to the subject. In some embodiments, the T cells are allogeneic to the subject.

[0047] Also provided herein are methods of determining a unit dose of engineered T cells for treating a subject, as defined in the claims. In the invention, the method, as defined in the claims, comprises assessing, in a therapeutic composition comprising a plurality of CD8 +< and / or CD4 +< T cells engineered to express a recombinant receptor, the number, percentage or ratio of T cells that express the recombinant receptor and CCR7 and CD27 (receptor +< / CCR7 +< / CD27 +< ). In the invention, the method, as defined in the claims, comprises, based on the number, percentage or ratio of receptor +< / CCR7 +< / CD27 +< cells, determining one or more unit doses of cells for administration to a subject having a disease or conditions, wherein the unit dose comprises a defined number of recombinant receptor-expressing CD8 +< T cells that express CCR7 and CD27 (receptor +< / CD8 +< / CCR7 +< / CD27 +< cells) and / or a defined number of recombinant receptor-expressing CD4 +< T cells that express CCR7 and CD27 cells) and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or receptor +< / CD4 +< / CCR7 +< / CD27 +< cells to another subset of cells in the composition.

[0048] Also provided herein are methods of producing a composition comprising a unit dose of a T cell composition, as defined in the claims. In the invention, the method, as defined in the claims, comprises assessing, in a therapeutic composition comprising a plurality of CD8 +< and / or CD4 +< T cells engineered to express a recombinant receptor, the number, percentage or ratio of T cells that express the recombinant receptor and CCR7 and CD27 (receptor +< / CCR7 +< / CD27 +< ). In the invention, the method, as defined in the claims, comprises filling a container with all or a portion of the composition and optionally another solution to achieve a unit dose of the T cell composition, wherein the unit dose comprises a defined number of recombinant receptor-expressing CD8 +< T cells that express CCR7 and CD27 (receptor +< / CD8 +< / CCR7 +< / CD27 +< cells) and / or a defined number of recombinant receptor-expressing CD4 +< T cells that express CCR7 and CD27 cells) and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or receptor +< / CD4 +< / CCR7 +< / CD27 +< cells to another subset of cells in the composition.

[0049] In some embodiments, the unit dose of cells comprises a defined number of CD8 +< / CCR7 +< / CD27 +< cells. In some embodiments, the unit dose of cells comprises a defined number of CD4 +< / CCR7 +< / CD27 +< cells.

[0050] In some embodiments, the unit dose comprises between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD8 +< cells that express the recombinant receptor (receptor +< / CD8 +< cells) or total CD4 +< cell that express the recombinant receptor (receptor +< / CD4 +< cells), total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, or total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, each inclusive. In some embodiments, the unit dose comprises no more than about 1 x 10 8< , no more than about 5 x 10 7< , no more than about 1 x 10 7< , no more than about 5 x 10 6< , no more than about 1 x 10 6< , or no more than about 5 x 10 5< total receptor +< / CD8 +< cells or total receptor +< / CD4 +< cells, total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, or total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells.

[0051] In some embodiments, the unit dose comprises at least about 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or at least about 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells. In some embodiments, the unit dose comprises between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, each inclusive.

[0052] In some embodiments, the unit dose of cells comprises a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, which ratio optionally is or is approximately 1:1 or is between approximately 1:3 and approximately 3:1.

[0053] In some embodiments, the unit dose further comprises a defined number of cells comprising cells that express or do not express CD45, optionally wherein the unit dose further comprises a defined number of cells comprising CD45RA- cells. In some embodiments, the method further comprises assessing in the therapeutic composition the number, percentage or ratio of T cells that express or do not express CD45RA, optionally the number, percentage or ratio of T cells that are CD45RA-.

[0054] In some embodiments, the therapeutic composition is any of the compositions defined in the claims.

[0055] In some embodiments, the unit dose comprises between at or about 1 x 10 5< and at or about 5 x 10 8< , between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD3 +< cells that express the recombinant receptor (receptor +< / CD3 +< cells) or total CD3 +< cells, each inclusive. In some embodiments, the unit dose comprises no more than about 5 x 10 8< , no more than about 1 x 10 8< , no more than about 5 x 10 7< , no more than about 1 x 10 7< , no more than about 5 x 10 6< , no more than about 1 x 10 6< , or no more than about 5 x 10 5< total receptor +< / CD3 +< cells or total CD3 +< cells.

[0056] In some embodiments, the total number of CD3 +< cells, total number of receptor +< / CD3 +< cells, total number of receptor +< / CD8 +< cells, total number of receptor +< / CD4 +< cells, total number of receptor +< / CD8 +< / CCR7 +< cells, total number of receptor +< / CD4 +< / CCR7 +< cells, total number of receptor +< / CD8 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD45RA -< cells and / or receptor +< / CD4 +< / CCR7 +< / CD45RA -< cells is the total number of such cells that are live or viable. In some embodiments, the total number of CD3 +< cells, total number of receptor +< / CD3 +< cells, total number of receptor +< / CD8 +< cells, total number of receptor +< / CD4 +< cells, total number of receptor +< / CD8 +< / CCR7 +< cells, total number of receptor +< / CD4 +< / CCR7 +< cells, total number of receptor +< / CD8 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD45RA -< cells and / or receptor +< / CD4 +< / CCR7 +< / CD45RA -< cells is the total number of such cells that do not express an apoptotic marker and / or is the total number of such cells that are apoptotic marker negative ( -< ), wherein the apoptotic marker is Annexin V or activated Caspase 3.

[0057] In the invention, the recombinant receptor is a chimeric receptor. In some embodiments, the recombinant receptor is capable of binding to a target antigen that is associated with, specific to, and / or expressed on a cell or tissue of a disease, disorder or condition. In some embodiments, the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or a cancer. In some embodiments, the target antigen is a tumor antigen.

[0058] In some embodiments, the target antigen is selected from among αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase 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), CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific antigen, or an antigen associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0059] In the invention, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor comprises an extracellular domain comprising an antigen-binding domain. In some embodiments, the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, which optionally is a single chain fragment. In some embodiments, the fragment comprises antibody variable regions joined by a flexible linker. In some embodiments, the fragment comprises an scFv.

[0060] In some embodiments, the recombinant receptor comprises an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

[0061] In some embodiments, the recombinant receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

[0062] In some embodiments, the intracellular signaling region further comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling domain is between the transmembrane domain and the intracellular signaling domain.

[0063] In some embodiments, the T cells are primary T cells obtained from a subject. In some embodiments, the T cells are autologous to the subject. In some embodiments, the T cells are allogeneic to the subject.

[0064] In some aspects of any of the embodiments provided herein, the cells in a provided therapeutic composition or a unit dose are associated with certain functional attributes, including certain recombinant receptor (e.g. CAR)- dependent activities that depend on an activity or presence of the recombinant receptor, e.g. an activity induced or mediated in the presence of antigen recognized by the recombinant receptor. In some embodiments, a functional attribute includes production of higher levels of a cytokine, e.g., IL-2. In some aspects of any of the embodiments provided herein, the functional attribute includes production of lower levels of a cytokine, e.g., IFNγ or IL-13. In some embodiments, the higher or lower levels of such cytokines are with reference to a reference composition in which is contained a non-defined number of cells of a particular phenotype and / or in which cells of a particular phenotype are not at a particular defined ratio according to embodiments provided herein. In some embodiments, a reference composition is one in which is contained a greater frequency or number of T cells having an effector or differentiated phenotype.

[0065] In some aspects, upon administration of a therapeutic composition, as defined in the claims, or a unit dose to one of a group of subjects according to any of the embodiments provided herein, a plurality of subjects among the group of subjects exhibits one or more features associated with increased efficacy, persistence and / or reduced toxicity following the administration. In some embodiments, a plurality of subjects among the group of subjects is likely to exhibit and / or is associated with, pharmacokinetic parameters, reduced likelihood of development of a toxicity or an adverse event, and / or a particular response, efficacy or survival outcome. In some embodiments, the subject is a subject suspected of having a disease or condition. In some embodiments, the one or more features are improved compared to a similar group of subjects treated with the reference composition in which is contained a non-defined number of cells of a particular phenotype and / or in which cells of a particular phenotype are not at a particular defined ratio according to embodiments provided herein. In some embodiments, a reference composition is one in which is contained a greater frequency or number of T cells having an effector or differentiated phenotype.

[0066] In some aspects, upon administration of a therapeutic composition or a unit dose to one of a group of subjects according to any of the embodiments provided herein, a plurality of subjects among the group of subjects is likely to exhibit and / or is associated with particular pharmacokinetic parameters, such as maximum (peak) plasma concentration (C max ), the peak time (i.e. when maximum plasma concentration (C max ) occurs; T max ), the minimum plasma concentration (i.e. the minimum plasma concentration between doses of a therapeutic agent, e.g., CAR +< T cells; C min ), the elimination half-life (T 1 / 2 ) and area under the curve (i.e. the area under the curve generated by plotting time versus plasma concentration of the therapeutic agent CAR +< T cells; AUC). In some aspects, upon administration of a therapeutic composition or a unit dose, a plurality of subjects among the group is likely to exhibit or is associated with increased maximum (peak) plasma concentration (C max ) of the administered cells. In some embodiments, long term persistence of cells of the therapeutic composition or unit dose are observed in a plurality of subjects among the group.

[0067] In some aspects, upon administration of a therapeutic composition or a unit dose to one of a group of subjects according to any of the embodiments provided herein, a plurality of subjects among the group of subjects is likely to exhibit and / or is associated with a reduced likelihood of development of a toxicity or an adverse event, such as cytokine release syndrome (CRS) or severe CRS (sCRS) or neurotoxicity (NT) or severe NT (sNT).

[0068] In some aspects, upon administration of a therapeutic composition or a unit dose to one of a group of subjects according to any of the embodiments provided herein, a plurality of subjects among the group of subjects is likely to exhibit and / or is associated with a particular response, efficacy or survival outcome, such as partial response (PR) or partial remission, complete response (CR) or complete remission, progression-free survival (PFS), objective response (OR), overall survival (OS), event-free survival (EFS), increased duration of response (DOR) or increased survival rate. In some aspects, upon administration of a therapeutic composition or a unit dose, a plurality of subjects among the group is likely to exhibit or is associated with increased progression-free survival (PFS).

[0069] In some embodiments of any of the provided embodiments, the methods, as defined in the claims, and / or compositions for use, according to the claims , achieve certain outcomes and / or are associated with certain reduced risks of toxicity, e.g., in the population of subjects treated according to the methods. In some aspects, at least 35%, at least 40% at least 50%, or at least 60%, of subjects treated according to the method achieve a complete response (CR) or complete remission, progression-free survival (PFS), objective response (OR), overall survival (OS), event-free survival (EFS), or a response that is durable for greater than 3 months, greater than 6 months or greater than 12 months. In certain embodiments of any of the provided methods, greater than or greater than about 50% of the subjects treated according to the method do not exhibit a grade 3 or greater cytokine release syndrome (CRS) or a grade 3 or greater neurotoxicity. In some embodiments, such subjects do not exhibit early onset CRS and / or neurotoxicity, such as do not exhibit onset of CRS earlier than 3 days following initiation of the administration and / or do not exhibit onset of neurotoxicity earlier than 5 days following initiation of the administration. In some embodiments of any of the provided methods, greater than or greater than about 30%, 35%, 40%, or 50% of the subjects treated according to the method do not exhibit any grade of cytokine release syndrome (CRS) or neurotoxicity.

[0070] Provided herein are methods, as defined in the claims, of predicting likelihood of response to a therapeutic T cell composition, the method including: (a) assaying an engineered cell composition containing T cells expressing a recombinant receptor for a factor indicative of the function or phenotype of engineered cells in the composition, the factor selected from (i) the percentage of T cells expressing the recombinant receptor that are surface positive for CCR7 and CD27, and optionally surface negative for CD45RA; and (b) determining the likelihood of response following administration of a cell therapy containing a dose of the engineered cells, wherein if the factor is at or above a threshold value identifying the subject as likely to achieve a durable response or progression free survival, optionally for at least 3 months, following administration the therapy; or if the factor is below a threshold value identifying the subject as not likely to exhibit a durable response or progression free survival to the therapy, wherein the threshold value of the factor is: (i) at or about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of T cells in the composition or of the total number of T cells in the composition expressing the recombinant receptor, are surface positive for CCR7 and CD27; and wherein the recombinant receptor is a CAR.

[0071] In some embodiments, cells of the cell composition are autologous to the subject. In some embodiments, if the subject is identified as likely to achieve a durable response or progression free survival to the therapy, administering to the subject one or more unit doses of cells of the engineered T cell composition. In some embodiments, the one or more unit doses contain between at or about 1 x 10 5< and at or about 5 x 10 8< , between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total T cells that express the recombinant receptor (receptor +< / T cells) or total T cells, each inclusive. In some embodiments, the one or more unit doses contains no more than about 5 x 10 8< , no more than about 1 x 10 8< , no more than about 5 x 10 7< , no more than about 1 x 10 7< , no more than about 5 x 10 6< , no more than about 1 x 10 6< , or no more than about 5 x 10 5< total receptor +< / T cells or total T cells. In some embodiments, the threshold value of the factor is at or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of CD4+ T cells in the composition expressing the recombinant receptor are able to produce a cytokine selected from interleukin 2 (IL-2) and / or TNF-alpha. In some embodiments, the threshold value of the factor is at or about, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or more of the total number of CD4+ T cells in the composition expressing the recombinant receptor are polyfunctional for producing two or more cytokines selected from among interferon-gamma (IFN-gamma), interleukin 2 (IL-2) and TNF-alpha. In some embodiments, the threshold value of the factor is at or about, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or more of the total number of CD4+ T cells in the composition expressing the recombinant receptor are polyfunctional for producing the cytokines interferon-gamma (IFN-gamma), interleukin 2 (IL-2) and TNF-alpha.

[0072] In some embodiments, the subject is a subject that, at the time of the assaying and / or prior to the administering, is identified or known to have a high tumor burden. In some embodiments, the subject has high tumor burden if a sum of product dimensions (SPD) of a tumor in the subject is above at or about 30 cm 2< , 40 cm 2< , 50 cm 2< , 60 cm 2< or 70 cm 2< ; and / or the subject has high tumor burden if C reactive protein (CRP) in a biological sample, optionally a serum sample, from the subject is above at or about 5 miligrams per liter, 10 miligrams per liter, 15 miligrams per liter, 20 miligrams per liter, 25 miligrams per liter, 30 miligrams per liter, 40 miligrams per liter or 50 miligrams per liter. In some embodiments, the subject has high tumor burden if the SPD of a tumor in the subject is above at or about 50 cm 2< . In some embodiments, the subject has high tumor burden if the CRP is above at or about 20 miligrams per liter in the biological sample.

[0073] In the invention, the recombinant receptor is a chimeric receptor. In some embodiments, the recombinant receptor is capable of binding to a target antigen that is associated with, specific to, and / or expressed on a cell or tissue of a disease, disorder or condition. In some embodiments, the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or a cancer. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the target antigen is selected from among αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase 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-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0074] In the invention, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor contains an extracellular domain containing an antigen-binding domain and an intracellular signaling region containing an intracellular signaling domain. In some embodiments, the antigen-binding domain is or contains an antibody or an antibody fragment thereof, which optionally is a single chain fragment. In some embodiments, the fragment contains antibody variable regions joined by a flexible linker. In some embodiments, the fragment comprises an scFv. In some embodiments, the intracellular signaling domain is or contains a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or contains an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof. In some embodiments, the recombinant receptor further contains a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some embodiment, the intracellular signaling region further contains a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain contains an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling domain contains an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling domain is between the transmembrane domain and the intracellular signaling domain.

[0075] In some embodiments, the T cells are primary T cells obtained from a subject. In some embodiments, the T cells are autologous to the subject.Brief Description of the Drawings

[0076] FIG. 1A shows the number of CD3 +< CAR +< T cells present in CAR T cell compositions for administration at DL1 and DL2. FIG. 1B shows the number of CD4 +< CAR +< and CD8 +< CAR +< cells, CD4 +< CAR +< TNF-α +< cells and CD8 +< CAR +< TNF-α +< , CD4 +< CAR +< CCR7 +< cells and CD8 +< CAR +< CCR7 +< cells present in CAR T cell compositions for administration at DL1 and DL2. FIG. 1C shows the number of CD4 +< CAR +< and CD8 +< CAR +< cells, CD4 +< CAR +< CCR7 +< cells and CD8 +< CAR +< CCR7 +< cells present in CAR T cell compositions for administration at DL1 and DL2. FIG. 2 depicts the median (±quartiles) number of CAR-expressing CD3 +< cells / µL blood, assessed by flow cytometry using an antibody specific for a truncated receptor (CD3, circle; N=87); or median (±quartiles) number of copies integrated CAR transgene / µg genomic DNA, assessed by quantitative polymerase chain reaction (qPCR) using primers specific for a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) present in the vector encoding the CAR (qPCR, square; N=85) in blood samples from 87 subjects that have been administered anti-CD19 CAR-expressing cells. The cutoff for CAR +< cell detection in flow cytometry was set at ≥ 25 events in the CAR +< gate, and limit of detection for qPCR was ≥ 12.5 copies of CAR transgene per µg of genomic DNA. FIG. 3A shows a plot of percentage CCR7 +< cells (among CD4 +< CAR +< and CD8 +< CAR +< cell populations) against pharmacokinetic parameters of CD4 +< CAR +< cells and CD8 +< CAR +< cells, respectively (as indicated by AUC 0-28 of CD4 +< CAR +< or AUC 0-28 of CD8 +< CAR +< cells in blood for subjects administered the cell therapy). Each dot represents an individual patient, with shading of individual dots indicating whether the patient received dose level 1 (DL1) or dose level 2 (DL2). FIG. 3B shows similar plots, indicated separately for subjects observed to exhibit high (in this assessment, indicated by a sum of the products of diameters (SPD) value of greater than or equal to 50 cm 2< ) or low (SPD value of less than 50 cm 2< ) pre-treatment tumor burden (assessed at the time of pre-treatment lymphodepleting chemotherapy). FIG. 3C depicts a plot of the number of CD4 +< CAR +< CCR7 +< cells and CD8 +< CAR +< CCR7 +< cells against AUC 0-28 of CD4 +< CAR +< or AUC 0-28 of CD8 +< CAR +< cells in blood for subjects administered the cell therapy), with the correlation coefficients and associated p-values. FIGS. 3D-3J, depict plots of the percentage CCR7 +< or CCR7 +< CD27 +< CD4 +< or CD8 +< CAR +< T cells against pharmacokinetic parameters (AUC 0-28 and C max ) of CD4 +< CAR +< T cells (FIGS. 3A, 3D-3F) and CD8 +< CAR +< T cells (FIGS. 3G-3J), with the correlation coefficients and associated p-values. FIGS. 4A-4B depict a plot of the percentage of CCR7 +< CD27 +< cells among CD4 +< CAR +< cells against IFNγ (FIG. 4A) and IL-2 (FIG. 4B) secretion by the CD4 +< CAR +< composition upon stimulation with the antigen recognized by the CAR (CD19). FIGS. 5A-5B depict a plot of the percentage of CCR7 +< CD27 +< cells among CD8 +< CAR +< cells against IL-5 (FIG. 5A) and IL-13 (FIG. 5B) secretion by the CD4 +< CAR +< composition upon stimulation with the antigen recognized by the CAR (CD19). FIGS. 6A-6D show the Kaplan-Meier survival curves for subjects who were administered CAR +< T cell compositions, divided into groups that were administered compositions containing a percentage of CCR7 +< CD27 +< CAR +< T cells among CD4 +< CAR +< T cells (FIG. 6A for progression free survival, FIG. 6C for duration of response) and among CD8 +< CAR +< T cells (FIG. 6B for progression free survival, FIG. 6D for duration of response) that is above or below a certain threshold level. FIGS. 7A-7B show boxplots depicting the percentage of CCR7 +< CD27 +< cells among CD4 +< CAR +< cells (FIG. 7A) or IFNγ secretion by CD4 +< CAR +< cells (FIG. 7B) in cell compositions administered to for subjects as a function of whether the subject went on to develop cytokine release syndrome (CRS; grade 0 vs. grade 1 or higher). FIGS. 7C-7D show boxplots depicting the percentage of CCR7 +< CD27 +< cells among CD4 +< CAR +< cells (FIG. 7C) or IFNγ secretion by CD4 +< CAR +< cells (FIG. 7D) in cell compositions administered to for subjects as a function of whether the subject went on to develop neurotoxicity (NT; grade 0-2 vs. grade 3 or higher). FIG. 8A shows an exemplary multiparameter phenotype analysis based on a t-distributed stochastic neighbor embedding (tSNE). FIG. 8B shows an exemplary flow cytometry plot for a cell subtype identified from the analysis (within the boundary of the shape drawn on FIG. 8A) for CCR7 and CD27 expression. FIGS. 9A-9C show exemplary partial least squares-discriminant analysis (PLS-DA) assessment of phenotypic and functional attributes (FIG. 9A) and transcriptomic (FIG. 9B) and epigenomic analyses (FIG. 9C) to identify variables that discriminate between subjects that exhibited complete response (CR) and subjects that exhibited progressive disease (PD) or partial response (PR), at 6 months after administration of the CAR +< T cell composition. FIG. 10A shows a plot of the frequency of CD4 +< CCR7 +< CD27 +< cells in pre-engineering compositions against the frequency of CD4 +< CAR +< CCR7 +< CD27 +< cells in the engineered cell compositions (Spearman's rank-order correlation coefficient: 0.591). FIG. 10B shows a plot of the frequency of CD8 +< CCR7 +< CD27 +< cells in pre-engineering compositions and the frequency of CD8 +< CAR +< CCR7 +< CD27 +< cells in the engineered cell compositions (Spearman's rank-order correlation coefficient: 0.357). FIGS. 11A-11D depict results from CAR+ T cell compositions generated from exemplary expanded and non-expanded processes involving a bead-based stimulatory reagent (Bead) and incubation in basal media (Bead-Basal Media), or an oligomeric stimulatory reagent (Oligomer). FIG. 11A depicts the percentage of CD4+ and CD8+ T cells positive for both CCR7 and CD27. FIG. 11B depicts the percentage of CCR7+CD27+cells for CD4+CAR+T cells. FIG. 11C depicts the percentage of CCR7+CD27+cells for CD8+CAR+T cells. FIG. 11D displays the percentage of CCR7+ CD27+ cells generated from a representative donor from an exemplary expanded process at various days during the process of manufacture, including activation at day 1(activation d1), transduction at day 2 (transduction d2), and at various timed after initiation of cultivation (d4 INOC+2, d6 INOC+4, d7 INOC+5). FIG. 12 shows the T cell clonality of the isolated CD4+ and CD8+ T cell compositions before engineering and of the CD4+ and CD8+ therapeutic CAR+T cell compositions after engineering (Shannon index applied). FIGS. 13A and 13B show progression free survival curves (Cox Proportional Hazard (top) and optimal split (bottom)) for patients with a "high" or "low" percentage of cells producing IL-2 or TNFα, respectively, in CD4 -< / CAR +< T cells of the therapeutic composition as determined using maximally selected rank statistics. FIG. 14 shows the relationship of clinical outcome to baseline sum of the products of diameters (SPD) and baseline C-reactive protein (CRP) levels in subjects exhibiting a complete response (CR) (triangles) or loss of response / no response (circles)) at 6 months. FIG. 15 shows the percentage of IL-2 / CD4+ / CAR+ cells in therapeutic T cell compositions generated from high baseline SPD / CRP subjects exhibiting a complete response (CR) (triangles; 1) or loss of response / no response (circles; 0)) at 6 months. FIG. 16 shows progression free survival curves for exemplary tertile cut-off values for cells positive for the apoptotic marker activated caspase 3 in the input compositions. FIG. 17 shows the percent of cells positive for apoptotic marker activated caspase 3 caspase 3 in enriched input T cells compositions as a function of age of the starting apheresis sample, i.e. samples varying in the length of time (e.g. in hours) between apheresis and its further processing before enrichment of T cells. The left plot shows the percentage of CD4+ / activated caspase 3+ cells and the right plot shows the percentage of CD8+ / activated caspase 3+ cells. Detailed Description

[0077] References to methods of treatment are intended to refer to products for use in said methods. Disclosed herein are methods, compositions, and articles of manufacture for use in connection with cell therapy, such as engineered T cell therapy for the treatment of diseases and conditions, including various tumors. The disclosurerelates to therapeutic T cell compositions containing engineered T cells such as those engineered to express recombinant proteins such as expressing recombinant receptors designed to recognize and / or specifically bind to molecules associated with the disease or condition and result in a response, such as an immune response against such molecules upon binding to such molecules. The receptors may include chimeric receptors, e.g., chimeric antigen receptors (CARs), and other transgenic antigen receptors including transgenic T cell receptors (TCRs).

[0078] The disclosurein some aspects relate to aspects in which cell phenotypes, e.g., the presence and / or expression of a surface marker, and / or the absence or lack of expression of a surface marker, is related to function or activity of the therapeutic T cell composition, pharmacokinetic parameters, e.g., exposure or maximum cell concentration, risk of the likelihood of developing a toxicity, such as cytokine release syndrome (CRS) or neurotoxicity (NT) and / or an outcome of the cell therapy, e.g., response to the cell therapy, in a subject administered the T cell composition and / or may provide information about the potency of the therapeutic T cell composition. In some aspects, the disclosed instances are based on the observation that certain cell phenotypes, e.g., expression of one or more surface markers, production of one or more cytokines are associated with pharmacokinetic parameters, likelihood of response and / or likelihood of developing a toxicity. In some instances, the expression and / or absence of expression of cell surface markers such as C-C chemokine receptor type 7 (CCR7), CD27 and CD45RA or combinations thereof in the T cell composition for administration, e.g., drug composition, are positively or negatively correlated with pharmacokinetic parameters and / or response or toxicity outcomes. In some aspects, it is observed herein that phenotype and functional attributes associated with a less differentiated T cell product or of a product enriched in naive-like or central memory T cell subsets correlate with or exhibit a relationship with improved pharmacokinetic properties or responses, such as durability of response and / or progression free survival, following administration to a subject.

[0079] Aspects of the disclosure also relate to administering or providing a unit dose containing a number, ratio, percentage and / or proportion of cells, that is a function of the number of cells of a certain phenotype, such as a phenotype indicative of a cell population, including for use in connection with dosing of the therapeutic T cell composition. In some aspects, the disclosure allow for consistent dosing and / or administration. In some aspects, the disclosure may be used to assess the likelihood of a certain outcome and / or value of a certain pharmacokinetic parameter, risk of toxicity and / or response, potency or efficacy of the cell therapy. In some aspects, the disclosed methods can control, ameliorate or reduce the risk of toxicity in a subject administered a dose of the therapeutic T cell composition, while ensuring potency of the T cell composition. Also disclosed are articles of manufacture containing the cells and designed for administration according to such dosing regimens.

[0080] In some instances, the disclosed methods, doses, unit doses, compositions, and articles of manufacture are based on observations that it can be advantageous to take into account certain phenotypes, e.g., expression of surface markers, and combinations thereof when determining appropriate dose of cell therapy and / or releasing or generating cell compositions for therapy. In certain available methods, doses are based on numbers of particular cell types, such as those engineered to exhibit a particular activity, such as those positive for an engineered receptor. For example, in certain available methods and doses, dose is based upon an observed or suspected relationship between the number (or number per patient weight) of viable engineered T cells, or of a subset thereof, such as of viable, cytotoxic (e.g., CD8 +< ) engineered T cells. In various contexts, such numbers can have a relationship with efficacy and / or safety outcomes, such as response and / or risk of toxicities, such as neurotoxicity, cerebral edema and CRS. Disclosed herein are instances based on the observation that nonetheless, such metrics in some contexts do not consistently adequately associate with pharmacological parameters in the subject, risk of toxicities, CRS or NT, particularly without taking into account other variables. Accordingly, approaches for defining dose and evaluating product for release that rely on such metrics alone may not be entirely satisfactory. For example, such approaches may in some contexts fail to reach a certain therapeutic range or window, for safe and effective cell therapy. Disclosed herein are methods (including treatment, dosing, dose-determination and assay methods), therapeutic compositions, and articles of manufacture that address such shortcomings.

[0081] In some aspects, the disclosure permit the administration of a controlled and consistent dose of cells, thereby minimizing variation in efficacy and / or safety outcomes in the subjects. In some aspects, controlling the dose of cells based on a defined number, ratio, percentage and / or proportions of particular subset of cells, e.g., based on cell phenotypes, permit the understanding of the impact of a subset of cells having particular phenotypes on the health, potency and / or efficacy of the cells contained in the therapeutic compositions. Such approaches can be used to determine and / or calculate consistent and precise effective doses of the cells in the cell therapy and / or control the pharmacokinetic parameters of the cell therapy. Disclosed are methods of determining such doses, including unit doses for administration, based on the number, ratio, percentage and / or proportions of cells expressing particular surface markers and / or having particular phenotypes, articles of manufacture and kits containing doses determined using such methods and instructions for administration, and related methods of treatment. In some aspects, the disclosure allow the identification of attributes of cells in the engineered cell composition and potential association with pharmacokinetics (PK) and clinical outcomes such as response and toxicity, based on the consistency and tight control of the number of CD4 +< and CD8 +< CAR +< T cells, and optionally subsets thereof that are CCR7 +< , CD27 +< and / or CD45RA-, in the therapeutic composition for administration in subjects. Such consistency and tight control of the total number of CAR +< cells in the cell composition for administration can provide the basis for strategies to assess and control other aspects of the cells in the engineered cell composition for administration.

[0082] In some aspects, the disclosure permits the administration of CAR +< T cell compositions that exhibit low variability, are pure and contain a precise dose, allowing identification of relationships between various cell attributes and clinical outcomes, such as response and toxicity. In some aspects, the disclosure are based on the observation that cell populations expressing markers associated with certain phenotypes, such as a less differentiated phenotype, exhibit certain functional profiles (e.g., cytokine production). In some aspects, CCR7 +< CD27 +< CD4 +< CAR T cells produce reduced IFNγ and increased IL-2, and, in some aspects, CCR7 +< CD27 +< CD8 +< CAR T cells produce reduced IL-5 and IL-13, such as upon antigen-specific stimulation of the CAR. In some instances, administered cell compositions containing CAR +< T cells in which a certain percentage of cells in the composition exhibit a less differentiated state and / or are CCR7 +< CD27 +< are associated with desirable or improved pharmacokinetic parameters, safety events and increased durable response, such as compared to compositions containing a lower percentage of such cell populations.

[0083] If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein disclosed, the definition set forth herein prevails .

[0084] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. DOSES AND DOSE DETERMINATION IN THERAPEUTIC T CELL COMPOSITIONS

[0085] Disclosed herein are methods, compositions, and articles of manufacture for use in connection with cell therapy, such as engineered T cell therapy for the treatment of diseases and conditions, including various tumors. The disclosure relate to methods of determining a dose for administration to a subject, related methods of treatment, compositions containing doses of cells and related article of manufacture and kits. The disclosure in some aspects relate to aspects in which cell phenotypes, e.g., the presence and / or expression of a surface marker, the absence or lack of expression of a surface marker and / or the ability to produce one or more cytokines (e.g. IL-2), of the cells in the composition for administration, is related to, correlated with or associated with values or measurements of pharmacokinetic parameters, e.g., exposure or maximum cell concentration. In some aspects, the phenotypes and / or parameters are also related to correlated with or associated with activity and / or function of the cells and / or the likelihood of developing a toxicity, such as cytokine release syndrome (CRS) or neurotoxicity (NT) and / or an outcome of the cell therapy, e.g., response to the cell therapy, in a subject administered the T cell composition, such as durability of response and progression free survival.

[0086] In some aspects, the disclosure, involve determining and / or administering a dose of the cell composition, e.g., one or more unit doses of a cell composition and / or a volume corresponding to such unit doses. In some aspects, the dose of cells is enriched for, e.g. has a high or relatively high percentage of, biologically active engineered T cells having a particular phenotype. In some aspects, the dose is based upon the number of biologically active engineered T cells having a particular phenotype. In some aspects, biologically active refers to a property of cells not programmed to undergo cell death, e.g., non-apoptotic cells or cells not showing indications of entry into an apoptotic pathway. In some aspects, dose is based upon number of biologically active engineered CD8 +< T cells having a particular phenotype, and / or, where dose is based upon total numbers of biologically active engineered T cells having a particular phenotype.

[0087] In some instances, the methods ensure that a unit dose encompasses a relatively consistent numbers, proportion, ratio and / or percentage of engineered cells having a particular phenotype in one or more particular compositions. In some aspects, the consistency is associated with or related to a relatively consistent activity, function, pharmacokinetic parameters, toxicity outcome and / or response outcome. In some instances, the percentage of engineered cells capable of consistent activity, function, pharmacokinetic parameters, toxicity outcome, and / or response outcome (e.g., durable response, durable progression free survival) is or includes at least at or about, or at or about, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of engineered cells (e.g., T cells) in the composition or of the total number of engineered cells (e.g., T cells) in the composition expressing the recombinant receptor, surface positive for CCR7 and / or CD27, optionally further negative for surface expression of CD45RA. In some instances, the engineered cells (e.g., T cells) are further negative for an apoptotic marker, such as activated caspase 3 or annexin V. In some instances, the percentage of engineered cells capable of consistent activity, function, pharmacokinetic parameters, toxicity outcome, and / or response outcome (e.g., durable response, durable progression free survival) is or includes at least at or about, or at or about, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the total number of engineered cells (e.g., T cells) in the composition or of the total number of engineered cells (e.g., T cells) in the composition expressing the recombinant receptor, negative for markers of apoptosis (e.g., caspase-3, annexin V). In some instances, the percentage of engineered cells capable of consistent activity, function, pharmacokinetic parameters, toxicity outcome, and / or response outcome (e.g., durable response, durable progression free survival) is or includes at least at or about, or at or about, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of engineered CD4+ T cells in the composition or the total number of engineered CD4+ T cells expressing the recombinant receptor capable of producing a cytokine selected from interleukin 2 (IL-2) and / or TNF-alpha. In some instances, the percentage of engineered cells capable of consistent activity, function, pharmacokinetic parameters, toxicity outcome, and / or response outcome (e.g., durable response, durable progression free survival) is or includes at least at or about, or at or about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or more of the total number of engineered CD4+ T cells in the composition or the total number of engineered CD4+ T cells expressing the recombinant receptor are polyfunctional for producing two or more cytokines selected from among interferon-gamma (IFN-gamma), interleukin 2 (IL-2) and TNF-alpha. In some instances, the engineered cells of the composition capable of consistent activity, function, pharmacokinetic parameters, toxicity outcome, and / or response outcome (e.g., durable response, durable progression free survival) are or include one or more phenotypes described herein. Thus, in some instances, the percentage of engineered cells capable of consistent activity, function, pharmacokinetic parameters, toxicity outcome, and / or response outcome (e.g., durable response, durable progression free survival) is or includes a combination of percentages of phenotypes. In some instances, the composition to be used as a therapeutic composition is selected based on the percentage of cells including one or more phenotypes in the composition (e.g., output composition).

[0088] In some aspects, in a plurality of subjects, compositions and / or doses, the numbers, proportion, ratio and / or percentage, are relatively consistent, e.g., the number or ratio of cells that have a particular phenotype, e.g., express CCR7 (CCR7 +< ) and / or have the ability to produce one or more cytokines, in the composition or unit dose, varies by no more than 40%, by no more than 30%, by no more than 20%, by no more than 10% or by no more than 5%. In some aspects the number or ratio of cells that have a particular phenotype, e.g., express CCR7 (CCR7 +< ) and / or the ability to produce one or more cytokines, in the composition or unit dose, varies by no more than 20% or no more than 10% or no more than 5% from an average of said number or ratio in a plurality of T cell compositions produced by the process and / or varies from such average by no more than one standard deviation or varies by no more than 20% or no more than 10% or no more than 5% among a plurality of T cell compositions or doses determined.

[0089] In some instances, the methods involve determining and / or administering one or more unit doses of cells, e.g., engineered T cells expressing a recombinant receptor such as a CAR. In some aspects, the methods involve administering one or more unit doses of cells in which the one or more unit doses of cells are enriched in, and / or contain a high or relatively high percentage of cells having a particular phenotype, such as particular surface marker phenotype and / or indicating functional activity, such as the ability to produce one or more cytokines. In some aspects, the unit dose is determined based on the number, percentage, ratio, frequency and / or proportion of a particular subset of engineered T cells, e.g., cells having a particular phenotype, such as particular surface marker phenotype and / or indicating functional activity, such as the ability to produce one or more cytokines. In some instances, an engineered or therapeutic cell composition can be produced by a method that results in an output composition containing or enriched for cells having such phenotypes or functional attributes, such as compared to the starting material or input composition used to generate the engineered output composition. Exemplary phenotype and functional attributes of cells of an engineered or therapeutic cell composition, such as an output composition produced in accord with methods for generating engineered cells, including in one or more unit doses of any such composition, are described herein.

[0090] In some aspects, the cell marker includes markers indicative of cell health, viability and / or apoptotic state of the cells. In some aspects, exemplary markers include CD3, CD4, CD8, CCR7, CD27, CD45RA, annexin V, or activated caspase 3. In some aspects, exemplary markers include CCR7, CD27 and / or CD45RA. In some aspects, exemplary markers include CCR7 and / or CD27. In some aspects, an exemplary marker is CCR7. In some aspects, an exemplary marker is CD27.

[0091] In some aspects, the disclosed methods also involve assessing the therapeutic composition for cell phenotypes, e.g., expression of the markers, e.g. CD3, CD4, CD8, CCR7, CD27, CD45RA, annexin V, or activated caspase 3. In some instances, the phenotype includes antigen-specific functions. In some instances, the phenotype includes production or secretion of a cytokine, e.g., upon stimulation with an antigen that the recombinant receptor, e.g., CAR, specifically binds and / or recognizes. In some instances, the phenotype includes production of cytokines, e.g., cytokines associated with particular cell types, such as cytokines associated with Th1, Th2, Th17 and / or Treg subtypes.

[0092] In some aspects, the disclosed methods involve determining a suitable dose, e.g., one or more unit doses of a therapeutic composition containing engineered T cells for administration to a subject having a disease or condition. In some aspects, the methods also involve assessing the number, percentage, ratio, frequency and / or proportion of a particular subset of engineered T cells, e.g., cells having a particular phenotype, such as particular surface marker phenotype, in a therapeutic composition containing engineered T cells. In some instances, the disclosed methods involve assessing, in a therapeutic composition comprising a plurality of CD8 +< and / or CD4 +< T cells engineered to express a recombinant receptor, the number, percentage or ratio of T cells that express the recombinant receptor and CCR7 (receptor +< / CCR7 +< ).

[0093] In some instances, the disclosed methods involve assessing, in a therapeutic composition comprising a plurality of CD8 +< and / or CD4 +< T cells engineered to express a recombinant receptor, the number, percentage or ratio of T cells that express or do not express CD27 and / or CD45RA. Other exemplary phenotypic markers for assessment and / or methods for assessment are described below, e.g., in Section I.A. In some aspects, the unit dose can be determined based on any of the assessments. In some aspects, the methods also involve assessing the number, percentage, ratio, frequency and / or proportion of the cells expressing the recombinant receptor (e.g., CAR) or a surrogate marker of the recombinant receptor. The doses, e.g., one or more unit doses, can be determined based on the assessment for the number, percentage, ratio, frequency and / or proportion any of the phenotypic markers described herein.A. Determining Cell Phenotypes

[0094] In some instances, cell phenotypes, or the level or percentage of such phenotypes in a composition of cells, correlate to or are associated with activity and / or function of the cells and / or the likelihood of developing a toxicity, such as cytokine release syndrome (CRS) or neurotoxicity (NT) and / or an outcome of the cell therapy, e.g., response to the cell therapy, in a subject administered the T cell composition, such as durability of response and progression free survival. In some instances, compositions including cells with specific phenotypes, and, more particularly, percentages of cells with such specific phenotypes, correlate with clinical outcomes, such as durable response and / or progression free survival. In some instances, the phenotype is determined by assessing the presence or absence of one or more specific molecules, including surface molecules and / or molecules that may accumulate or be produced by the cells or a subpopulation of cells within a T cell composition. In some instances, the phenotype, directly or inversely, indicates or is indicative of a biological activity of the cells or of a population of cells within the T cell composition. In some instances, phenotype may include cell activity, such as production of a factor (e.g., cytokine) in response to a stimulus. In certain instances, assessment of a cell composition is performed to identify, detect, or quantify a phenotype of the cell composition. In particular instances, a measurement of a cell composition is performed to identify, detect, or quantify the presence, absence, degree of expression or level of a specific molecule.

[0095] It is contemplated that the phenotype of the cell composition (e.g., engineered T cell composition) can, in some cases, depend upon many factors, including, but not limited to, the phenotype of the starting cellular material (e.g., apheresis product or leukapheresis product) used to generate the cell composition and the phenotype of the cellular material undergoing processing to generate the cell composition. Thus, in some instances, phenotype is assessed in cells of the starting material and / or the material undergoing processing to generate the cell composition, as well as the final cell composition.

[0096] In some instances, the phenotype is indicative of viability of a cell. In some instances, the phenotype is indicative of absence of apoptosis, absence of early stages of apoptosis or absence of late stages of apoptosis. In some instances, the phenotype is the absence of a factor indicative of absence of apoptosis, early apoptosis or late stages of apoptosis. In some instances, the phenotype is a phenotype of a sub-population or subset of T cells, such as recombinant receptor-expressing T cells (e.g. CAR +< T cells), CD8 +< T cells, or CD4 +< T cells. In some instances, the phenotype is a phenotype of cells that are not activated and / or that lack or are reduced for or low for expression of one or more activation marker. In some instances, the phenotype is a phenotype of cells that are not exhausted and / or that lack or are reduced for or low for expression of one or more exhaustion markers.

[0097] In some instances, the phenotype is indicated by the presence, absence, or level of expression in a cell of one or more specific molecules, such as certain surface markers indicative of the phenotype, e.g., surface proteins, intracellular markers indicative of the phenotype, or nucleic acids indicative of the phenotype or other molecules or factors indicative of the phenotype. In some instances, the phenotype is or comprises a positive or negative expression of the one or more of specific molecules. In some instances, the specific molecules include, but are not limited to, a surface marker, e.g., a membrane glycoprotein or a receptor; a marker associated with apoptosis or viability; or a specific molecule that indicates the status of an immune cells, e.g., a marker associated with activation, exhaustion, or a mature or naive phenotype. In some instances, any known method for assessing or measuring, counting, and / or quantifying cells based on specific molecules can be used to determine the number of cells of the phenotype.

[0098] In some instances, a phenotype is or includes a positive or negative expression of one or more specific molecules in a cell. In some instances, the positive expression is indicated by a detectable amount of the specific molecule in the cell. In certain instances, the detectable amount is any detected amount of the specific molecule in the cell. In particular instances, the detectable amount is an amount greater than a background, e.g., background staining, signal, etc., in the cell. In certain instances, the positive expression is an amount of the specific molecule that is greater than a threshold, e.g., a predetermined threshold. Likewise, in particular instances, a cell with negative expression of a specific molecule may be any cell not determined to have positive expression, or is a cell that lacks a detectable amount of the specific molecule or a detectable amount of the specific molecule above background. In some instances, the cell has negative expression of a specific molecule if the amount of the specific molecule is below a threshold. One of skill in the art will understand how to define a threshold to define positive and / or negative expression for a specific molecule as a matter of routine skill, and that the thresholds may be defined according to specific parameters of, for example, but not limited to, the assay or method of detection, the identity of the specific molecule, reagents used for detection, and instrumentation.

[0099] Examples of methods that can be used to detect a specific molecule and / or analyze a phenotype of the cells include, but are not limited to, biochemical analysis; immunochemical analysis; image analysis; cytomorphological analysis; molecule analysis such as PCR, sequencing, high-throughput sequencing, determination of DNA methylation; proteomics analysis such as determination of protein glycosylation and / or phosphorylation pattern; genomics analysis; epigenomics analysis (e.g., ChIP-seq or ATAC-seq); transcriptomics analysis (e.g., RNA-seq); and any combination thereof. In some instances, the methods can include assessment of immune receptor repertoire, e.g., repertoire of T cell receptors (TCRs). In some aspects, determination of any of the phenotypes can be assessed in high-throughput, automated and / or by single-cell-based methods. In some aspects, large-scale or genome-wide methods, can be used to identify one or more molecular signatures. In some aspects, large-scale or genome-wide methods, can be used to identify molecular signatures that are associated with outcomes of therapy, e.g., efficacy and safety, or pharmacokinetic parameters. In some aspects, one or more molecular signatures, e.g., expression of specific RNA or proteins in the cell, can be determined. In some instances, molecular features of the phenotype analyzed by image analysis, PCR (including the standard and all variants of PCR), microarray (including, but not limited to DNA microarray, MMchips for microRNA, protein microarray, cellular microarray, antibody microarray, and carbohydrate array), sequencing, biomarker detection, or methods for determining DNA methylation or protein glycosylation pattern. In particular instances, the specific molecule is a polypeptide, i.e. a protein. In some instances, the specific molecule is a polynucleotide.

[0100] In some instances, positive or negative expression of a specific molecule is determined 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 the positively or negatively selected cells, respectively. In particular instances, the positive or negative expression is determined by flow cytometry, immunohistochemistry, or any other suitable method for detecting specific markers.

[0101] In particular instances, expression of a specific molecule is assessed with flow cytometry. Flow cytometry is a laser- or impedance-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second.

[0102] The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates." Specific gating protocols exist for diagnostic and clinical purposes especially in relation to immunology. Plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap, signals at the detectors have to be compensated electronically as well as computationally. Data accumulated using the flow cytometer can be analyzed using software, e.g., JMP (statistical software), WinMDI, Flowing Software, and web-based Cytobank), Cellcion, FCS Express, FlowJo, FACSDiva, CytoPaint (aka Paint-A-Gate), VenturiOne, CellQuest Pro, Infinicyt or Cytospec.

[0103] Flow Cytometry is a standard technique in the art and one of skill would readily understand how to design or tailor protocols to detect one or more specific molecules and analyze the data to determine the expression of one or more specific molecules in a population of cells. Standard protocols and techniques for flow cytometry are found in Loyd "Flow Cytometry in Microbiology; Practical Flow Cytometry by Howard M. Shapiro; Flow Cytometry for Biotechnology by Larry A. Sklar, Handbook of Flow Cytometry Methods by J. Paul Robinson, et al., Current Protocols in Cytometry, Wiley-Liss Pub, Flow Cytometry in Clinical Diagnosis, v4, (Carey, McCoy, and Keren, eds), ASCP Press, 2007, Ormerod, M.G. (ed.) (2000) Flow Cytometry -A practical approach. 3rd edition. Oxford University Press, Oxford, UK, Ormerod, M.G. (1999) Flow Cytometry. 2nd edition. BIOS Scientific Publishers, Oxford., and Flow Cytometry -A basic introduction. Michael G. Ormerod, 2008.

[0104] In some instances, cells are sorted by phenotype for further analysis. In some instances, cells of different phenotypes within the same cell composition are sorted by Fluorescence-activated cell sorting (FACS). FACS is a specialized type of flow cytometry that allows for sorting a heterogeneous mixture of cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.

[0105] In some instances, the phenotype includes the number of total T cells or the number of total CD3 +< T cells. In particular instances, a T cell composition, e.g., a therapeutic T cell composition that contains cells that express a recombinant receptor or a CAR, may include one or more different subtypes of T cells. In some instances, the phenotype is or includes the identity of a T cell subtype. Different populations or subtypes of T cells include, but are not limited to effector T cells, helper T cells, memory T cell, Regulatory T cells, naive T cells, CD4 +< cells, and CD8 +< T cells. In certain instances, a T cell sub-type may be identified by detecting the presence or absence of a specific molecule. In certain instances, the specific molecule is a surface marker that can be used to identify a T cell subtype.

[0106] In some instances, the phenotype is positive or high level expression of one or more specific molecule that are surface markers, e.g., CD3, CD4, CD8, CD28, CD62L, CCR7, CD27, CD127, CD4, CD8, CD45RA, and / or CD45RO. In certain instances, the phenotype is a surface marker of T cells or of a subpopulation or subset of T cells, such as based on positive surface marker expression of one or more surface markers, e.g., CD3 +< , CD4 +< , CD8 +< , CD28 +< , CD62L +< , CCR7 +< , CD27 +< , CD127 +< , CD4 +< , CD8 +< , CD45RA +< , and / or CD45RO +< . In some instances, the phenotype is positive or high level expression of one or more specific molecule that are surface markers, e.g., C-C chemokine receptor type 7 (CCR7), Cluster of Differentiation 27 (CD27), Cluster of Differentiation 28 (CD28), and Cluster of Differentiation 45 RA (CD45RA). In certain instances, the phenotype markers include CCR7, CD27, CD28, CD44, CD45RA, CD62L, and L-selectin. In some instances, the phenotype is negative or the absence of expression of one or more specific molecule that are surface markers, e.g., CD3, CD4, CD8, CD28, CD62L, CCR7, CD27, CD127, CD4, CD8, CD45RA, and / or CD45RO. In certain instances, the phenotype is a surface marker of T cells or of a subpopulation or subset of T cells, such as based on the absence of surface marker expression of one or more surface markers, e.g., CD3 -< , CD4 -< , CD8-, CD28 -< , CD62L -< , CCR7 -< , CD27 -< , CD127 -< , CD4 -< , CD8 -< , CD45RA -< , and / or CD45RO -< . In some instances, the phenotype is negative or the absence of expression of one or more specific molecule that are surface markers, e.g., C-C chemokine receptor type 7 (CCR7), Cluster of Differentiation 27 (CD27), Cluster of Differentiation 28 (CD28), and Cluster of Differentiation 45 RA (CD45RA). In certain instances, the phenotype markers include CCR7, CD27, CD28, CD44, CD45RA, CD62L, and L-selectin.

[0107] In certain instances, the phenotype is or includes positive or negative expression of CD27, CCR7 and / or CD45RA. In some instances, the phenotype is CCR7 +< . In some instances, the phenotype is CD27 +< . In some instances, the phenotype is CCR7 +< / CD27 +< . In some instances, the phenotype is CD45RA -< . In some instances, the phenotype is CCR7 +< / CD45RA -< . In some instances, the phenotype is CD27 +< / CD45RA -< . In some instances, the phenotype is CCR7 +< / CD27 +< / CD45RA -< .

[0108] In certain instances, the surface marker indicates expression of a recombinant receptor, e.g., a CAR. In particular instances, the surface marker is expression of the recombinant receptor, e.g. CAR, which, in some aspects, can be determined using an antibody, such as an anti-idiotype antibody. In some instances, the surface marker that indicates expression of the recombinant receptor is a surrogate marker. In particular instances, such a surrogate marker is a surface protein that has been modified to have little or no activity. In certain instances, the surrogate marker is encoded on the same polynucleotide that encodes the recombinant receptor. In some instances, the nucleic acid sequence encoding the recombinant receptor is operably linked to a nucleic acid sequence encoding a marker, optionally separated by an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence, such as a T2A (e.g., SEQ ID NOS: 6 and 17), a P2A (e.g., SEQ ID NOS: 18 and 19), a E2A (e.g., SEQ ID NO: 20) or a F2A (e.g., SEQ ID NO: 21). Extrinsic marker genes may in some cases be utilized in connection with engineered cells to permit detection or selection of cells and, in some cases, also to promote cell suicide.

[0109] Exemplary surrogate markers can include truncated cell surface polypeptides, such as a truncated human epidermal growth factor receptor 2 (tHER2), a truncated epidermal growth factor receptor (EGFRt, exemplary EGFRt sequence set forth in SEQ ID NO:7 or 16) or a prostate-specific membrane antigen (PSMA) or modified form thereof. EGFRt may contain an epitope recognized by the antibody cetuximab (Erbitux ®< ) or other therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been engineered with the EGFRt construct and a recombinant receptor, such as a chimeric antigen receptor (CAR), and / or to eliminate or separate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). In some aspects, the marker, e.g. surrogate marker, includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some instances, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in PCT Pub. No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 7 or 16 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16.

[0110] In some instances, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP, red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of the fluorescent proteins. In some instances, the marker is or comprises an enzyme, such as a luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT). Exemplary light-emitting reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS) or variants thereof.

[0111] In certain instances, the phenotype comprises expression, e.g. surface expression, of one or more of the surface markers CD3, CD4, CD8, and / or a recombinant receptor (e.g. CAR) or its surrogate marker indicating or correlating to expression of a recombinant receptor (e.g. CAR).

[0112] In particular instances, the phenotype is identified by the expression of one or more specific molecules that are surface markers. In certain instances, the phenotype is or includes positive or negative expression of CD3, CD4, CD8, and / or a recombinant receptor, e.g. a CAR. In certain instances, the recombinant receptor is a CAR. In particular instances the phenotype comprises CD3 +< / CAR +< , CD4 +< / CAR +< , and / or CD8 +< / CAR +< .

[0113] In certain instances, the phenotype is or includes positive or negative expression of CD27, CCR7 and / or CD45RA, and / or a recombinant receptor, e.g. a CAR. In some instances, the phenotype is CCR7 +< / CAR +< . In some instances, the phenotype is CD27 +< / CAR +< . In some instances, the phenotype is CCR7 +< / CD27 +< / CAR +< . In some instances, the phenotype is CD45RA -< / CAR +< . In some instances, the phenotype is CCR7 +< / CD45RA -< / CAR +< . In some instances, the phenotype is CD27 +< / CD45RA -< / CAR +< . In some instances, the phenotype is CCR7 +< / CD27 +< / CD45RA -< / CAR +< .

[0114] In some instances, the phenotype is viability. In certain instances, the phenotype is the positive expression of a marker that indicates that the cell undergoes normal functional cellular processes and / or has not undergone or is not under the process of undergoing necrosis or programmed cell death. In some instances, viability can be assessed by the redox potential of the cell, the integrity of the cell membrane, or the activity or function of mitochondria. In some instances, viability is the absence of a specific molecule associated with cell death, or the absence of the indication of cell death in an assay.

[0115] In some instances, the phenotype is or comprises cell viability. In certain instances, the viability of cells can be detected, measured, and / or assessed by a number of means that are routine in the art. Non-limiting examples of such viability assays include, but are not limited to, dye uptake assays (e.g., calcein AM assays), XTT cell viability assays, and dye exclusion assays (e.g., trypan blue, Eosin, or propidium dye exclusion assays). Viability assays are useful for determining the number or percentage (e.g., frequency) of viable cells in a cell dose, a cell composition, and / or a cell sample. In particular instances, the phenotype comprises cell viability along with other features, e.g., recombinant receptor expression.

[0116] In certain instances, the phenotype is or includes cell viability, viable CD3 +< , viable CD4 +< , viable CD8 +< , viable CD3 +< / CAR +< , viable CD4 +< / CAR +< , viable CD8 +< / CAR +< , viable CD4 +< / CCR7 +< / CAR +< , viable CD8 +< / CD27 +< / CAR +< , viable CD4 +< / CD27 +< / CAR +< , viable CD8 +< / CCR7 +< / CD27 +< / CAR +< , viable CD4 +< / CCR7 +< / CD27 +< / CAR +< , viable CD8 +< / CCR7 +< / CD45RA -< / CAR +< or viable CD4 +< / CCR7 +< / CD45RA -< or a combination thereof.

[0117] In particular instances, the phenotype is or includes an absence of apoptosis and / or an indication the cell is undergoing the apoptotic process. Apoptosis is a process of programmed cell death that includes a series of stereotyped morphological and biochemical events that lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay. Apoptosis is a well characterized process, and specific molecules associated with various stages are well known in the art.

[0118] In some instances, the phenotype is the absence of an early stage of apoptosis, and / or an absence of an indicator and / or a specific molecule associated with an early stage of apoptosis. In the early stages of apoptosis, changes in the cellular and mitochondrial membrane become apparent. Biochemical changes are also apparent in the cytoplasm and nucleus of the cell. For example, the early stages of apoptosis can be indicated by activation of certain caspases, e.g., 2, 8, 9, and 10. In particular instances, the phenotype is the absence of a late stage of apoptosis, and / or an absence of an indicator and / or a specific molecule associated with a late stage of apoptosis. The middle to late stages of apoptosis are characterized by further loss of membrane integrity, chromatin condensation and DNA fragmentation, and include biochemical events such as activation of caspases 3, 6, and 7.

[0119] In certain instances, the phenotype is the negative expression of one or more factors associated with apoptosis, including pro-apoptotic factors known to initiate apoptosis, e.g., members of the death receptor pathway, activated members of the mitochondrial (intrinsic) pathway, such as Bcl-2 family members, e.g., Bax, Bad, and Bid, and caspases. In some instances, the phenotype is a negative or low amount of a marker of apoptosis. In certain instances, the phenotype is the negative expression of a marker of apoptosis. In certain instances, the phenotype is the absence of an indicator, e.g., an Annexin V molecule, which will preferentially bind to cells undergoing apoptosis when incubated with or contacted to a cell composition. In some instances, the phenotype is or includes the expression of one or more markers that are indicative of an apoptotic state in the cell.

[0120] In some instances, the phenotype is the negative (or low) expression of a specific molecule that is a marker for apoptosis. Various apoptosis markers are known to those of ordinary skill in the art and include, but are not limited to, an increase in activity of one or more caspases i.e. an activated caspase (e.g., an active caspase), an increase in PARP cleavage, activation and / or translocation of Bcl-2 family proteins, members of the cell death pathway, e.g., Fas and FADD, presence of nuclear shrinkage (e.g., monitored by microscope) and presence of chromosome DNA fragmentation (e.g., presence of chromosome DNA ladder) or with apoptosis assays that include TUNEL staining, and Annexin V staining.

[0121] Caspases are enzymes that cleave proteins after an aspartic acid residue, the term is derived from "cysteine-aspartic acid proteases." Caspases are involved in apoptosis, thus activation of caspases, such as caspase-3 is indicative of an increase or revival of apoptosis. In certain instances, caspase activation can be detected by methods known to the person of ordinary skill. In some instances, an antibody that binds specifically to an activated caspase (i.e., binds specifically to the cleaved polypeptide) can be used to detect caspase activation. In another example, a fluorochrome inhibitor of caspase activity (FLICA) assay can be utilized to detect caspase-3 activation by detecting hydrolysis of acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin (Ac-DEVD-AMC) by caspase-3 (i.e., detecting release of the fluorescent 7-amino-4-methylcoumarin (AMC)). FLICA assays can be used to determine caspase activation by a detecting the product of a substrate processed by multiple caspases (e.g., FAM-VAD-FMK FLICA). Other techniques include The CASPASE-GLO ®< caspase assays (PROMEGA) that use luminogenic caspase-8 tetrapeptide substrate (Z-LETD-aminoluciferin), the caspase-9 tetrapeptide substrate (Z-LEHD-aminoluciferin), the caspase-3 / 7 substrate (Z-DEVD-aminoluciferin), the caspase-6 substrate (Z-VEID-aminoluciferin), or the caspase-2 substrate (Z-VDVAD-aminoluciferin).

[0122] In certain instances, the phenotype is or includes negative expression of activated caspase-1, activated caspase-2, activated caspase-3, activated caspase-7, activated caspase-8, activated caspase-9, activated caspase-10 and / or activated caspase-13 in a cell. In particular instances, the phenotype is or includes activated caspase 3 -< . In some instances, the proform (zymogen cleaved) form of a caspase, such as any above, also is a marker indicating the presence of apoptosis. In some instances, the phenotype is or includes the absence of or negative expression of a proform of a caspase, such as the proform of caspase-3.

[0123] In some instances, the marker of apoptosis is cleaved the Poly ADP-ribose polymerase 1 (PARP). PARP is cleaved by caspase during early stages of apoptosis. Thus, detection of a cleaved PARP peptide is a marker for apoptosis. In particular instances, the phenotype is or includes positive or negative expression of cleaved PARP.

[0124] In some instances, the marker of apoptosis is a reagent that detects a feature in a cell that is associated with apoptosis. In certain instances, the reagent is an annexin V molecule. During the early stages of apoptosis the lipid phosphatidylserine (PS) translocates from the inner to the outer leaflet of the plasma membrane. PS is normally restricted to the internal membrane in healthy and / or non-apoptotic cells. Annexin V is a protein that preferentially binds phosphatidylserine (PS) with high affinity. When conjugated to a fluorescent tag or other reporter, Annexin V can be used to rapidly detect this early cell surface indicator of apoptosis. In some instances, the presence of PS on the outer membrane will persist into the late stages of apoptosis. Thus in some instances, annexin V staining is an indication of both early and late stages of apoptosis. In certain instances, an Annexin, e.g. Annexin V, is tagged with a detectable label and incubated with, exposed to, and / or contacted with cells of a cell composition to detect cells that are undergoing apoptosis, for example by flow cytometry. In some instances, fluorescence tagged annexins, e.g., annexin V, are used to stain cells for flow cytometry analysis, for example with the annexin -< V / 7 -< AAD assay. Alternative protocols suitable for apoptosis detection with annexin include techniques and assays that utilize radiolabeled annexin V. In certain instances, the phenotype is or includes negative staining by annexin, e.g. annexin V -< . In particular instances, the phenotype is or includes the absence of PS on the outer plasma membrane. In certain instances, the phenotype is or includes cells that are not bound by annexin e.g. annexin V. In certain instances, the cell that lacks detectable PS on the outer membrane is annexin V -< . In particular instances, the cell that is not bound by annexin V -< in an assay, e.g., flow cytometry after incubation with labeled annexin V, is annexin V -< .

[0125] In particular instances, the phenotype is annexin V -< , annexin V -< CD3 +< , annexin V -< CD4 +< , annexin V -< CD8 +< , annexin V -< CD3 +< / CAR +< , annexin V -< CD4 +< / CAR +< , annexin V -< CD8 +< / CAR +< , activated caspase 3 -< , activated caspase 3 -< / CD3 +< , activated caspase 3 -< / CD4 +< , activated caspase 3 -< / CD8 +< , activated caspase 3 -< / CD3 +< / CAR +< , activated caspase 3 -< / CD4 +< / CAR +< , activated caspase 3 -< / CD8 +< / CAR +< , annexin V -< / CD4 +< / CCR7 +< / CAR +< , annexin V -< / CD8 +< / CD27 +< / CAR +< , annexin V- / CD4 +< / CD27 +< / CAR +< , annexin V -< / CD8 +< / CCR7 +< / CD27 +< / CAR +< , annexin V- / CD4 +< / CCR7 +< / CD27 +< / CAR +< , annexin V -< / CD8 +< / CCR7 +< / CD45RA -< / CAR +< or annexin V- / CD4 +< / CCR7 +< / CD45RA -< ; activated caspase 3 -< / CD4 +< / CCR7 +< / CAR +< , activated caspase 3- / CD8 +< / CD27 +< / CAR +< , activated caspase 3 -< / CD4 +< / CD27 +< / CAR +< , activated caspase 3 -< / CD8 +< / CCR7 +< / CD27 +< / CAR +< , activated caspase 3 -< / CD4 +< / CCR7 +< / CD27 +< / CAR +< , activated caspase 3 -< / CD8 +< / CCR7 +< / CD45RA -< / CAR +< or activated caspase 3 -< / CD4 +< / CCR7 +< / CD45RA -< or a combination thereof.

[0126] Particular instances contemplate that cells positive for expression of a marker for apoptosis are undergoing programmed cell death, show reduced or no immune function, and have diminished capabilities if any to undergo activation, expansion, and / or bind to an antigen to initiate, perform, or contribute to an immune response or activity. In particular instances, the phenotype is defined by negative expression for an activated caspase and / or negative staining with annexin V.

[0127] In certain instances, the phenotype is or includes activated caspase 3 -< (caspase 3 -< ) and / or annexin V -< .

[0128] Among the phenotypes are the expression or surface expression of one or more markers generally associated with one or more sub-types or subpopulations of T cells, or phenotypes thereof. T cell subtypes and subpopulations may include CD4 +< and / or of CD8 +< T cells and subtypes thereof that may include naïve T (T N ) cells, effector T cells (T EFF ), memory T cells and sub-types thereof, such as stem cell memory T (T SCM ), central memory T (T CM ), effector memory T (T EM ), T EMRA cells or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0129] In some aspects, among the phenotypes include expression or markers or functions, e.g. antigen-specific functions such as cytokine secretion, that are associated with a less differentiated cell subset or a more differentiated subset. In some instances, the phenotypes are those associated with a less differentiated subset, such as one or more of CCR7 +< , CD27 +< and interleukin-2 (IL-2) production. In some aspects, less differentiated subsets can also be related to therapeutic efficacy, self-renewal, survival functions or graft-versus-host disease. In some instances, the phenotypes are those associated with a more differentiated subset, such as one or more of interferon-gamma (IFN-γ) or IL-13 production. In some aspects, more differentiated subsets can also be related to senescence and effector function.

[0130] In some instances, the phenotype is or includes a phenotype of a memory T cell or memory T cell subset exposed to their cognate antigen. In some instances the phenotype is or includes a phenotype of a memory T cell (or one or more markers associated therewith), such as a T CM cell, a T EM cell, or a T EMRA cell, a T SCM cell, or a combination thereof. In particular instances, the phenotype is or includes the expression of one or more specific molecules that is a marker for memory and / or memory T cells or subtypes thereof. In some aspects, exemplary phenotypes associated with T CM cells can include one or more of CD45RA -< , CD62L +< , CCR7 +< and CD95 +< . In some aspects, exemplary phenotypes associated with T EM cells can include one or more of CD45RA -< , CD62L -< , CCR7 -< and CD95 +< .

[0131] In particular instances, the phenotype is or includes the expression of one or more specific molecules that is a marker for naive T cells.

[0132] In some instances, the phenotype is or includes a memory T cell or a naive T cell. In certain instances, the phenotype is the positive or negative expression of one or more specific molecules that are markers for memory. In some instances, the memory marker is a specific molecule that may be used to define a memory T cell population.

[0133] In some instances, the phenotype is or includes a phenotype of or one or more marker associated with a non-memory T cell or sub-type thereof; in some aspects, it is or includes a phenotype or marker(s) associated with a naive cell. In some instances, the phenotype is CCR7 +< / CD27 +< / CD28 +< / CD45RA +< . In certain instances, the phenotype is or includes CCR7 +< / CD45RA +< . In some instances, the phenotype is or includes a phenotype of a central memory T cell. In particular instances, the phenotype is or includes CCR7 +< / CD27 +< / CD28 +< / CD45RA -< . In some instances, the phenotype is or includes CCR7 -< / CD27 +< / CD28 +< / CD45RA -< . In certain instances, the phenotype is or includes that of a T EMRA cell or a T SCM cell. In certain instances, the phenotype is or includes CD45RA +< . In particular instances, the phenotype is or includes CCR7 -< / CD27 -< / CD28 -< / CD45RA +< . In some instances, the phenotype is or includes one of CD27 +< / CD28 +< , CD27 -< / CD28 +< , CD27 +< / CD28 -< , or CD27 -< / CD28 -< . In some instances, the phenotype is CCR7 +< / CD27 +< / CD45RA +< . In certain instances, the phenotype is or includes CCR7 +< / CD45RA +< . In particular instances, the phenotype is or includes CCR7 +< / CD27 +< / CD45RA -< . In some instances, the phenotype is or includes CCR7 -< / CD27 +< / CD45RA -< . In certain instances, the phenotype is or includes CD45RA +< . In particular instances, the phenotype is or includes CCR7 -< / CD27 -< / CD45RA +< .

[0134] In some instances the phenotype is or includes any of the foregoing phenotypic properties and further includes the expression of a recombinant receptor, such as phenotype associated with a memory T cell or memory subtype and that expresses a CAR, or a phenotype associated with a naive cell that expresses a CAR. In certain instances, the phenotype is or includes that of a central memory T cell or stem central memory T cell that expresses a CAR. In particular instances, the phenotype is or includes that of an effector memory cell that expresses a CAR. In some instances, the phenotype is or includes that of a T EMRA cell that expresses a CAR. In particular instances, the phenotype is or includes CAR +< / CCR7 +< / CD27 +< / CD28 +< / CD45RA -< ; CAR +< / CCR7 -< / CD27 +< / CD28 +< / CD45RA -< ; CAR -< / CCR7 -< / CD27 -< / CD28 -< / CD45RA +< ; CAR +< / CD27 +< / CD28 +< ; CAR +< / CD27 -< / CD28 +< ; CAR +< / CD27 +< / CD28 -< ; or CAR +< / CD27 -< / CD28 -< . In particular instances, the phenotype is or includes CAR +< / CCR7 +< / CD27 +< / CD45RA -< ; CAR +< / CCR7 -< / CD27 +< / CD45RA -< ; CAR +< / CCR7 -< / CD27 -< / CD28 -< / CD45RA +< ; CAR +< / CD27 +< ; CAR +< / CD27 -< ; CAR +< / CD27 +< / CD28 -< ; or CAR +< / CD27 -< / CD28 -< .

[0135] In certain instances, the phenotype is or includes a phenotype of a T cell that is negative for a marker of apoptosis. In certain instances, the phenotype is or includes a naive cell that is negative for a marker of apoptosis. In some instances, the marker of apoptosis is activated caspase 3. In some instances, the marker of apoptosis is positive staining by annexin V.

[0136] In particular instances, the phenotype is or includes that of a memory T cell or subtype thereof that is negative for a marker of apoptosis that expresses a CAR. In particular instances, the phenotype is or includes that of a memory T cell or particular subtype that is negative for a marker of apoptosis that expresses a CAR. In certain instances, the phenotype is or includes a naive cell that is negative for a marker of apoptosis that expresses a CAR. In certain instances, the phenotype is or includes that of a central memory T cell or T SCM cell or naive cell that is negative for a marker of apoptosis that expresses a CAR. In particular instances, the phenotype is or includes that of an effector memory cell that is negative for a marker of apoptosis that expresses a CAR. In certain instances, the phenotype is or includes annexin V -< / CAR +< / CCR7 +< / CD27 +< / CD28 +< / CD45RA -< ; annexin V -< / CAR +< / CCR7 -< / CD27 +< / CD28 +< / CD45RA -< ; annexin V -< / CAR +< / CCR7 -< / CD27 -< / CD28 -< / CD45RA +< ; annexin V -< / CAR +< / CD27 +< / CD28 +< ; annexin V -< / CAR +< / CD27 -< / CD28 +< ; annexin V -< / CAR +< / CD27 +< / CD28 -< ; or annexin V -< / CAR +< / CD27 -< / CD28 -< . In certain instances, the phenotype is or includes activated caspase 3 -< / CAR +< / CCR7 +< / CD27 +< / CD28 +< / CD45RA -< ; activated caspase 3 -< / CAR +< / CCR7 -< / CD27 +< / CD28 +< / CD45RA -< ; activated caspase 3 -< / CAR +< / CCR7 -< / CD27 -< / CD28 -< / CD45RA +< ; activated caspase 3 -< / CAR +< / CD27 +< / CD28 +< ; activated caspase 3 -< / CAR +< / CD27 -< / CD28 +< ; activated caspase 3 -< / CAR +< / CD27 +< / CD28 -< ; or activated caspase 3 -< / CAR +< / CD27 -< / CD28 -< . In certain instances, the phenotype is or includes annexin V -< / CAR +< / CCR7 +< / CD27 +< / CD45RA -< ; annexin V -< / CAR +< / CCR7 -< / CD27 +< / CD45RA -< ; annexin V -< / CAR +< / CCR7 -< / CD27 -< / CD45RA +< ; annexin V -< / CAR +< / CD27 +< / CD28 +< ; annexin V -< / CAR +< / CD27 -< / CD28 +< ; annexin V -< / CAR +< / CD27 +< ; or annexin V -< / CAR +< / CD27 -< . In certain instances, the phenotype is or includes activated caspase 3 -< / CAR +< / CCR7 +< / CD27 +< / CD45RA -< ; activated caspase 3 -< / CAR +< / CCR7 -< / CD27 +< / CD45RA -< ; activated caspase 3 -< / CAR +< / CCR7 -< / CD27 -< / CD45RA +< ; activated caspase 3 -< / CAR +< / CD27 +< / CD28 +< ; activated caspase 3 -< / CAR +< / CD27 -< / CD28 +< ; activated caspase 3 -< / CAR +< / CD27 +< ; or activated caspase 3 -< / CAR +< / CD27 -< .

[0137] In particular instances, the phenotype is or includes CD27 +< / CD28 +< , CD27 -< / CD28 +< , CD27 +< / CD28 -< , CD27 -< / CD28 -< , or a combination thereof. In some instances, the phenotype is or includes CAR +< / CD27 +< / CD28 +< , CAR +< / CD27 -< / CD28 +< , CAR +< / CD27 +< / CD28 -< , CAR +< / CD27 -< / CD28 -< , or a combination thereof. In certain instances, the phenotype is or includes activated caspase 3 -< / CAR +< / CD27 +< / CD28 +< , activated caspase 3 -< / CAR +< / CD27 -< / CD28 +< , activated caspase 3 -< / CAR +< / CD27 +< / CD28 -< , activated caspase 3 -< / CAR +< / CD27 -< / CD28 -< , or a combination thereof. In particular instances, the phenotype is or includes annexin V -< / CAR +< / CD27 +< / CD28 +< , annexin V -< / CAR +< / CD27 -< / CD28 +< , annexin V -< / CAR +< / CD27 +< / CD28 -< , annexin V -< / CAR +< / CD27 -< / CD28 -< , or a combination thereof. In particular instances, the phenotype is or includes CD27 +< , CD27 -< , CD27 +< , CD27 -< , or a combination thereof. In some instances, the phenotype is or includes CAR +< / CD27 +< , CAR +< / CD27 -< , CAR +< / CD27 +< , CAR +< / CD27 -< , or a combination thereof. In certain instances, the phenotype is or includes activated caspase 3 -< / CAR +< / CD27 +< , activated caspase 3 -< / CAR +< / CD27 -< , activated caspase 3 -< / CAR +< / CD27 +< , activated caspase 3 -< / CAR +< / CD27 -< , or a combination thereof. In particular instances, the phenotype is or includes annexin V -< / CAR +< / CD27 +< , annexin V -< / CAR +< / CD27 -< , annexin V -< / CAR +< / CD27 +< , annexin V -< / CAR +< / CD27 -< , or a combination thereof.

[0138] In particular instances, the phenotype is or includes CCR7 +< / CD28 +< , CCR7 -< / CD28 +< , CCR7 +< / CD28 -< , CCR7 -< / CD28 -< , or a combination thereof. In some instances, the phenotype is or includes CAR +< / CCR7 +< / CD28 +< , CAR +< / CCR7 -< / CD28 +< , CAR +< / CCR7 +< / CD28 -< , CAR +< / CCR7 -< / CD28 -< , or a combination thereof. In certain instances, the phenotype is or includes activated caspase 3 -< / CAR +< / CCR7 +< / CD28 +< , activated caspase 3 -< / CAR +< / CCR7 -< / CD28 +< , activated caspase 3 -< / CAR +< / CCR7 +< / CD28 -< , activated caspase 3 -< / CAR +< / CCR7 -< / CD28 -< , or a combination thereof. In particular instances, the phenotype is or includes annexin V -< / CAR +< / CCR7 +< / CD28 +< , annexin V -< / CAR +< / CCR7 -< / CD28 +< , annexin V -< / CAR +< / CCR7 +< / CD28 -< , annexin V -< / CAR +< / CCR7 -< / CD28 -< , or a combination thereof. In particular instances, the phenotype is or includes CCR7 +< , CCR7 -< , CCR7 +< , CCR7 -< , or a combination thereof. In some instances, the phenotype is or includes CAR +< / CCR7 +< , CAR +< / CCR7 -< , CAR +< / CCR7 +< , CAR +< / CCR7 -< , or a combination thereof. In certain instances, the phenotype is or includes activated caspase 3 -< / CAR +< / CCR7 +< , activated caspase 3 -< / CAR +< / CCR7 -< , activated caspase 3 -< / CAR +< / CCR7 +< , activated caspase 3 -< / CAR +< / CCR7 -< , or a combination thereof. In particular instances, the phenotype is or includes annexin V -< / CAR +< / CCR7 +< , annexin V -< / CAR +< / CCR7 -< , annexin V -< / CAR +< / CCR7 +< , annexin V -< / CAR +< / CCR7 -< , or a combination thereof.

[0139] In some instances, the phenotype is or includes positive or negative expression of a marker of exhaustion. In certain instances, the phenotype is or includes positive or negative expression of a specific molecule that is associated with exhaustion. In certain instances, the specific molecule is any molecule that is associated with exhaustion or a quality associated with exhaustion, e.g., poor effector function or inhibitory receptor expression. In particular instances, the phenotype is positive or negative expression of an immune checkpoint inhibitor. In particular instances, marker of exhaustion is CTLA-4, FOXP3, PD-1, TIGIT, LAB-3, 2B4, BTLA, TIM3, VISTA, or CD96, or a combination thereof. In certain instances, the phenotype is the positive or negative expression of CTLA-4, FOXP3, PD-1, TIGIT, LAB-3, 2B4, BTLA, TIM3, VISTA, or CD96, or a combination thereof. In particular instances, the phenotype is positive or negative expression of PD1 and / or FOXP3.

[0140] In some instances, the phenotype is or includes positive or negative expression of an exhaustion marker in a CD3 +< cell that expresses a recombinant receptor or a CAR. In particular instances, the phenotype is or includes positive or negative expression of an exhaustion marker in a CD4 +< cell that expresses a recombinant receptor or a CAR. In some instances, the phenotype is or includes positive or negative expression of an exhaustion marker and CD3 +< and positive expression of a recombinant receptor or a CAR. In particular instances, the phenotype is or includes positive or negative expression of an exhaustion marker in a CD4 +< cell that expresses a recombinant receptor or a CAR. In some instances, the phenotype is or includes positive or negative expression of an exhaustion marker in a CD8 +< cell that expresses a recombinant receptor or a CAR. In particular instances, the exhaustion marker is one or more of CTLA-4, FOXP3, PD-1, TIGIT, LAB-3, 2B4, BTLA, TIM3, VISTA, or CD96. In particular instances, the exhaustion marker is PD1 and / or FOXP3.

[0141] In particular instances, the phenotype is or includes PD1 -< / CD3 +< , PD1 -< / CD4 +< , PD1 -< / CD8 +< , PD1 -< / CD3 +< / CAR +< , PD1 -< / CD4 +< / CAR +< , PD1 -< / CD8 +< / CAR +< , PD1 -< / annexin V -< , PD1 -< / annexin V -< / CD3 +< , PD1 -< / annexin V -< / CD4 +< , PD1 -< / annexin V -< / CD8 +< , PD1 -< / annexin V / -< CD3 +< / CAR +< , PD1 -< / annexin V -< / CD4 +< / CAR +< , PD1 -< / annexin V -< / CD8 +< / CAR +< , PD1 -< / activated caspase 3 -< , PD1 -< / activated caspase 3 -< / CD3 +< , PD1 -< / activated caspase 3 -< / CD4 +< , PD1 -< / activated caspase 3 -< / CD8 +< , PD1 -< / activated caspase 3 -< / CD3 +< / CAR +< , PD1 -< / activated caspase 3 -< / CD4 +< / CAR +< , PD1 -< / activated caspase 3 -< / CD8 +< / CAR +< , or a combination thereof.

[0142] In certain instances, the phenotype is or includes FOXP3 -< / CD3 +< , FOXP3 -< CD4 +< , FOXP3 -< / CD8 +< , FOXP3 -< / CD3 +< / CAR +< , FOXP3 -< / CD4 +< / CAR +< , FOXP3 -< / CD8 +< / CAR +< , FOXP3 -< / annexin V -< , FOXP3 -< / annexin V -< / CD3 +< , FOXP3 -< / annexin V -< / CD4 +< , FOXP3 -< / annexin V -< / CD8 +< , FOXP3 -< / annexin V / -< CD3 +< / CAR +< , FOXP3 -< / annexin V -< / CD4 +< / CAR +< , FOXP3 -< / annexin V -< / CD8 +< / CAR +< , FOXP3 -< / activated caspase 3 -< , FOXP3 -< / activated caspase 3 -< / CD3 +< , FOXP3 / activated caspase 3 -< / CD4 +< , FOXP3 -< / activated caspase 3 -< / CD8 +< , FOXP3 -< / activated caspase 3 -< / CD3 +< / CAR +< , FOXP3 -< / activated caspase 3 -< / CD4 +< / CAR +< , FOXP3 -< / activated caspase 3 -< / CD8 +< / CAR +< , or a combination thereof.

[0143] In certain instances, the phenotype is the negative expression of a specific molecule that is associated with T cell activation. In some instances, the phenotype is or includes the negative expression of one or more of a specific molecule that is an activation marker. In general, T cell activation requires two simultaneous signals. The first is binding of the T cell receptor complex (TCR) to a major histocompatibility complex (MHC) molecule carrying a peptide antigen. The second is provided by the binding of the co-stimulatory receptor CD28 to proteins in the surface of the APC, such as B7-2 or B7-1. In certain instances, the specific molecule is associated with TCR activation, e.g., is activated, altered, or expressed as a result of T cell activation. In some instances, the specific molecule is associated with activation of a CD28 receptor, e.g., a molecule that is activated, altered, or expressed as a result of T cell activation.

[0144] In particular instances, the phenotype is or includes the negative expression of one or more of a specific molecule that is an activation marker. In certain instances, the activation marker is one or more of CD25, CD26, CD27, CD28, CD30, CD71, CD154, CD40L, CD127, LAG3, Ki67, or a combination thereof. In certain instances, the phenotype is the negative or positive expression of one or more of CD25, CD26, CD27, CD28, CD30, CD71, CD154, CD40L, CD127, LAG3, or Ki67. In certain instances, the phenotype is or includes the expression of CD25, CD127, LAG3, Ki67, or a combination thereof.

[0145] In some instances, the phenotype is or includes positive or negative expression of an activation marker in a CD3 +< cell that expresses a recombinant receptor or a CAR. In particular instances, the phenotype is or includes positive or negative expression of activation marker in a CD4 +< cell that expresses a recombinant receptor or a CAR. In some instances, the phenotype is or includes positive or negative expression of activation marker in a CD8 +< cell that expresses a recombinant receptor or a CAR. In particular instances, the activation marker is one or more of CD25, CD26, CD27, CD28, CD30, CD71, CD154, CD40L, CD127, LAG3, or Ki67. In particular instances, the activation marker is CD25, CD127, LAG3, Ki67, or a combination thereof.

[0146] In particular instances, the phenotype is or includes positive or negative expression of an activation marker and CD3 +< , CD4 +< , CD8 +< , CD3 +< / CAR +< , CD4 +< / CAR +< , CD8 +< / CAR +< , annexin V -< , annexin V -< / CD3 +< , annexin V -< / CD4 +< , annexin V -< / CD8 +< , annexin V / -< CD3 +< / CAR +< , annexin V -< / CD4 +< / CAR +< , annexin V -< / CD8 +< / CAR +< , activated caspase 3 -< , activated caspase 3 -< / CD3 +< , activated caspase 3 -< / CD4 +< , activated caspase 3 -< / CD8 +< , activated caspase 3 -< / CD3 +< / CAR +< , activated caspase 3 -< / CD4 +< / CAR +< , activated caspase 3 -< / CD8 +< / CAR +< , or a combination thereof.

[0147] In some instances, the phenotype is assessed by a response to a stimulus, for example a stimulus that triggers, induces, stimulates, or prolongs an immune cell function. In certain instances, the cells are incubated in the presence of stimulating conditions or a stimulatory agent, the phenotype is or includes the response to the stimulation. In particular instances, the phenotype is or includes the production or secretion of a soluble factor in response to one or more stimulations. In some instances, the phenotype is or includes a lack or production or secretion of a soluble factor in response to one or more stimulations. In certain instances, the soluble factor is a cytokine. In some instances, the cytokine is IL-2. In some instances, the cytokine is TNFa.

[0148] The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., 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 the cells. In some instances, the cells are stimulated and the phenotype is determined by whether or not a soluble factor, e.g., a cytokine or a chemokine, is produced or secreted. In some instances, the stimulation is nonspecific, i.e., is not an antigen-specific stimulation. In some instances, the stimulation comprises PMA and ionomycin. In some instances, cells are incubated in the presence of stimulating conditions or a stimulatory agent for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 24 hours, about 48 hours, or for a duration of time between 1 hour and 4 hours, between 1 hour and 12 hours, between 12 hours and 24 hours, or for more than 24 hours.

[0149] In some instances, the cells are stimulated with an agent that is an antigen or an epitope thereof that is specific to the recombinant receptor, or is an antibody or fragment thereof that binds to and / or recognizes the recombinant receptor, or a combination thereof. In some instances, the recombinant receptor is a CAR, and the agent is an antigen or an epitope thereof that is specific to the CAR, or is an antibody or fragment thereof that binds to and / or recognizes the CAR, or a combination thereof. In particular instances, the cells are stimulated by incubating the cells in the presence of target cells with surface expression of the antigen that is recognized by the CAR. In certain instances, the recombinant receptor is a CAR, and the agent is an antibody or an active fragment, variant, or portion thereof that binds to the CAR. In certain instances, the antibody or the active fragment, variant, or portion thereof that binds to the CAR is an anti-idiotypic (anti-ID) antibody.

[0150] In some instances, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of stimulating or activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to a solid support such as a bead, and / or one or more cytokines. In some instances, the one or more agents are PMA and ionomycin.

[0151] In particular instances, the phenotype is or includes the production or secretion of a cytokine in response to one or more of stimulations. The production and / or the secretion of cytokines contributes to immune responses, and is involved in different processes including the induction of anti-viral proteins and the induction of T cell proliferation. Cytokines are not preformed factors but are rapidly produced and secreted in response to cellular activation. The production or secretion of cytokines may be measured, detected, and / or quantified by any suitable technique known in the art.

[0152] In certain instances, the phenotype is the production of one or more cytokines. In some instances, the production of two or more cytokines from the same cell can be indicative of polyfunctional features of such cells. In particular instances, the production of one or more cytokines is measured, detected, and / or quantified by intracellular cytokine staining. Intracellular cytokine staining (ICS) by flow cytometry is a technique well-suited for studying cytokine production at the single-cell level. It detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation, allowing for the identification of cell populations that are positive or negative for production of a particular cytokine or for the separation of high producing and low producing cells based on a threshold. In some instances, as described above, the stimulation can be performed using nonspecific stimulation, e.g., is not an antigen-specific stimulation. For example, PMA / ionomycin can be used for nonspecific cell stimulation. In some instances, the stimulation can be performed by an agent that is an antigen or an epitope thereof that is specific to the recombinant receptor (e.g., CAR), or is an antibody or fragment thereof that binds to and / or recognizes the recombinant receptor, or a combination thereof. ICS can also be used in combination with other flow cytometry protocols for immunephenotyping using cell surface markers or with MHC multimers to access cytokine production in a particular subgroup of cells, making it an extremely flexible and versatile method. Other single-cell techniques for measuring or detecting cytokine production include, but are not limited to ELISPOT, limiting dilution, and T cell cloning.

[0153] In some instances, the phenotype is the production of a cytokine, such as following stimulation of the recombinant receptor with an antigen specific to and / or recognized by the recombinant receptor. In particular instances, the phenotype is the lack of the production of the cytokine, such as following stimulation of the recombinant receptor with an antigen specific to and / or recognized by the recombinant receptor. In particular instances, the phenotype is positive for or is a high level of production of a cytokine. In certain instances, the phenotype is negative for or is a low level of production of a cytokine. Cytokines may include, but are not limited to, interleukin-1 (IL-1), IL-1β, IL-2, sIL-2Ra, IL-3, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL 27, IL-33, IL-35, TNF, tumor necrosis factor alpha (TNF-α), CXCL2, CCL2, CCL3, CCL5, CCL17, CCL24, PGD2, LTB4, interferon gamma (IFN-γ), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1α, MIP-1β, Flt-3L, fracktalkine, and / or IL-5. In some instances, the phenotype includes production of cytokines, e.g., cytokines associated with particular cell types, such as cytokines associated with Th1, Th2, Th17 and / or Treg subtypes. In some instances, exemplary Th1-related cytokines include IL-2, IFN-γ, and transforming growth factor beta (TGF-β), and in some cases are involved in cellular immune responses. In some instances, exemplary Th2-related cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and in some cases are associated with humoral immunity and antiinflammatory properties. In some instances, exemplary Th17-related cytokines include IL-17A and IL-17F, and in some cases are involved in recruiting neutrophils and macrophages, e.g., during an inflammatory reaction.

[0154] In some instances, the phenotype is or includes the production of a cytokine. In certain instances, the phenotype is or includes the production of more than one cytokine(e.g., polyfunctional). In certain instances, the phenotype is or includes a lack of a production of one or more cytokines. In certain instances, the phenotype is or includes the production, or lack thereof, of one or more of IL-2, IL-5, IL-13, IFN-gamma, or TNF-alpha. In certain instances, the phenotype is or includes the production, or lack thereof, of one or more of IL-2, IL-13, IFN-gamma, or TNF-alpha. In some instances, the phenotype is the presence of a production, and / or the presence of a high level of production of the cytokine. In some instances, the phenotype is a low, reduced, or absent production of a cytokine.

[0155] In some instances, the phenotype is or includes the internal (intracellular) production of a cytokine, for example, as assessed in the presence of a stimulatory agent or under stimulatory conditions when secretion is prevented or inhibited. In some instances, the stimulatory agent is nonspecific stimulatory agent, e.g., a stimulatory agent that does not bind to an antigen binding domain, for example on a recombinant receptor (e.g., CAR). In some embodimetns, the stimulatory agent is PMA / ionomycin, which can act as a nonspecific stimulatory agent. In some instances, the stimulatory agent is a specific stimulatory agent, e.g., is a stimulatory agent that is an antigen or an epitope thereof that is specific to the recombinant receptor (e.g., CAR), or is an antibody or fragment thereof that binds to and / or recognizes the recombinant receptor, or a combination thereof. In particular instances, the phenotype is or includes the lack or absence of an internal production of a cytokine. In certain instances, the phenotype is or includes the internal amount of one or more cytokines when the production of more than one cytokines as assessed with an ICS assay. In certain instances, the phenotype is or includes the internal amount of one or more of IL-2,IL-5, IL-13, IFN-gamma, or TNF-alpha as assessed with an ICS assay. In some instances, the phenotype is or includes a low internal amount or a lack of a detectable amount of one or more cytokines as assessed with an ICS assay. In certain instances, phenotype is or includes a low internal amount or a lack of a detectable amount of IL-2, IL-5, IL-13, IFN-gamma, or TNF-alpha as assessed with an ICS assay. In some instances, the phenotype includes assessment of multiple cytokines, e.g., by multiplexed assays or assays to assess polyfunctionality (see, e.g., Xue et al., (2017) Journal for ImmunoTherapy of Cancer 5:85). In some instances, the lack of cytokine expression is inversely correlated with or associated with activity and / or function of the cells and / or durability of response and progression free survival. In some instances, cells with reduced, minimal or no cytokine production, assessed according to any known method or method described herein, are reduced in the cell composition (e.g., output composition, therapeutic cell composition).

[0156] Particular instances contemplate that the phenotype may include the production of a cytokine or a lack of or a low amount of production for a cytokine. This may depend on several factors that include, but are not limited to, the identity of the cytokine, the assay performed to detect the cytokine, and the stimulatory agent or condition used with the assay. For example, in some instances it is contemplated that the phenotype is or includes a lack of, or a low level of IL-13 production as indicated by ICS while in some instances, the phenotype is or includes production of IFN-gamma as indicated by ICS.

[0157] In some instances, the phenotype is or includes production of one or more cytokines and either CD3 +< , CD4 +< , CD8 +< , CD3 +< / CAR +< , CD4 +< / CAR +< , CD8 +< / CAR +< , annexin V -< , annexin V -< CD3 +< , annexin V -< CD4 +< , annexin V -< CD8 +< , annexin V -< CD3 +< / CAR +< , annexin V -< CD4 +< / CAR +< , annexin V -< CD8 +< / CAR +< , activated caspase 3 -< , activated caspase 3 -< / CD3 +< , activated caspase 3 -< / CD4 +< , activated caspase 3 -< / CD8 +< , activated caspase 3 -< / CD3 +< / CAR +< , activated caspase 3 -< / CD4 +< / CAR +< , or activated caspase 3 -< / CD8 +< / CAR +< , or a combination thereof. In particular instances, the phenotype is or includes production of one or more cytokines in CD4 +< / CAR +< and / or CD8 +< / CAR +< . In some instances, the one or more cytokines are IL-2, IFN-gamma, and / or TNF-alpha. In some instances, the phenotype is or includes production of IL-2 in CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of TNF-alpha in CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of IL-2 and TNF-alpha in CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of IL-2 and IFN-gamma in CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of TNF-alpha in CD8 +< / CAR +< cells. In some instances, the phenotype is or includes production of IFN-gamma and TNF-alpha in CD8 +< / CAR +< cells. In some instances, the phenotype is or includes production of IL-2 in activated caspase 3 -< / CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of TNF-alpha in activated caspase 3 -< / CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of IL-2 and TNF-alpha in activated caspase 3 -< / CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of IL-2 and IFN-gamma in activated caspase 3 -< / CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of TNF-alpha in activated caspase 3 -< / CD8 +< / CAR +< cells. In some instances, the phenotype is or includes production of IFN-gamma and TNF-alpha in activated caspase 3 -< / CD8 +< / CAR +< cells. In some instances, the phenotype is or includes production of TNF-alpha in annexin V -< / CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of IL-2 and TNF-alpha in annexin V -< / CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of IL-2 and IFN-gamma in annexin V -< / CD4 +< / CAR +< cells. In some instances, the phenotype is or includes production of TNF-alpha in annexin V -< / CD8 +< / CAR +< cells. In some instances, the phenotype is or includes production of IFN-gamma and TNF-alpha in annexin V -< / CD8 +< / CAR +< cells. In some instances, the phenotypes described in this paragraph are positively correlated with durable response and progression free survival. Thus, in some instances, cells including these phenotypes are maximized or increased in the cell composition (e.g., output composition, therapeutic cell composition). In some instances, the cell composition includes at least at or about, or at or about, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of CD4+ T cells, such as engineered CD4 T cells expressing a recombinant receptor (e.g., CAR), in the composition that are capable of producing a cytokine selected from interleukin 2 (IL-2) and / or TNF-alpha. In some instances, the cell composition includes at least at or about, or at or about, 10%, 15%, 20%, 25%, 30%, 40% or more, of the total number of CD4+ T cells, such as engineered CD4 T cells expressing a recombinant receptor (e.g., CAR), in the composition polyfunctional for producing two or more cytokines selected from among interferon-gamma (IFN-gamma), interleukin 2 (IL-2) and TNF-alpha.

[0158] In some instances, the phenotype is or includes a lack of production of one or more cytokines. In certain instances, the phenotype is or includes a lack of a production of one or more cytokines and either CD3 +< , CD4 +< , CD8 +< , CD3 +< / CAR +< , CD4 +< / CAR +< , CD8 +< / CAR +< , annexin V -< , annexin V -< CD3 +< , annexin V -< CD4 +< , annexin V -< CD8 +< , annexin V -< CD3 +< / CAR +< , annexin V -< CD4 +< / CAR +< , annexin V -< CD8 +< / CAR +< , activated caspase 3 -< , activated caspase 3 -< / CD3 +< , activated caspase 3 -< / CD4 +< , activated caspase 3 -< / CD8 +< , activated caspase 3 -< / CD3 +< / CAR +< , activated caspase 3 -< / CD4 +< / CAR +< , or activated caspase 3 -< / CD8 +< / CAR +< , or a combination thereof. In some instances, the one or more cytokines are IL-2, IFN-gamma, and / or TNF-alpha. In some instances, the phenotype is or includes the lack of production of IL-2 in activated caspase 3 -< / CD4 +< / CAR +< cells. In some instances, the phenotype is or includes the lack of production of TNF-alpha in activated caspase 3 -< / CD4 +< / CAR -< cells. In some instances, the phenotype is or includes the lack of production of IL-2 and TNF-alpha in activated caspase 3 -< / CD4 +< / CAR +< cells. In some instances, the phenotype is or includes the lack of production of IL-2 and IFN-gamma in activated caspase 3 -< / CD4 +< / CAR +< cells. In some instances, the phenotype is or includes the lack of production of TNF-alpha in activated caspase 3 -< / CD8 +< / CAR +< cells. In some instances, the phenotype is or includes the lack of production of INF-gamma and TNF-alpha in activated caspase 3 -< / CD8 +< / CAR +< cells. In some instances, the phenotypes described in this paragraph are negatively correlated with durable response and progression free survival. Thus, in some instances, cells including these phenotypes are minimized or reduced in the cell composition (e.g., output composition, therapeutic cell composition). For example, in some instances, cells including the phenotype of this paragraph compose less than 2%, 5%, 10%, 15,%, 20,%, or 25% of the total cells in the cell composition (e.g., output composition, therapeutic cell composition).

[0159] In particular instances, the phenotype is or includes the presence or absence of an internal amount of one or more of IL-2, IL-13, IFN-gamma, or TNF-alpha as assessed with an ICS assay and one or more specific markers for a subset of cells or cells of a particular cell type. In some instances, the phenotype is or includes production, or lack thereof, of one or more of IL-2, IL-13, IFN-gamma, or TNF-alpha and CD4 +< / CAR +< and / or CD8 +< / CAR +< . In certain instances, the phenotype is or includes production of IL-2 and CD4 +< / CAR +< and / or CD8 +< / CAR +< . In some instances, the phenotype is or includes a lack of or low production of IL-2 and CD4 +< / CAR +< and / or CD8 +< / CAR +< . In some instances, the phenotype is or includes production of IL-13 and CD4 +< / CAR +< and / or CD8 +< / CAR +< . In some instances, the phenotype is or includes production of IL-13 and CD4 +< / CAR +< and / or CD8 +< / CAR +< . In certain instances, the phenotype is or includes the lack of or low production of IL -< 13 and CD4 +< / CAR +< and / or CD8 +< / CAR +< . In some instances, the phenotype is or includes production of IFN-gamma and CD4 +< / CAR +< and / or CD8 +< / CAR +< . In certain instances, the phenotype is or includes production of TNF-alpha and CD4 +< / CAR +< and / or CD8 +< / CAR +< . In certain instances, the phenotype is or includes a lack of or low production of TNF-alpha and CD4 +< / CAR +< and / or CD8 +< / CAR +< .

[0160] Any one or more the phenotypes, alone or in combination, can be assessed or determined in accord with the disclosed methods. In some instances, the phenotype is CD3 +< , CD3 +< / CAR +< , CD4 +< / CAR +< , CD8 +< / CAR +< , or a combination thereof.

[0161] In certain instances, the phenotype is or includes CD3 +< . In certain instances, the phenotype is or includes CD3 +< / CAR +< . In some instances, the phenotype is or includes CD8 +< / CAR +< . In certain instances, the phenotype is or includes CD4+ / CAR+.

[0162] In particular instances, the phenotype is or includes Annexin -< / CD3 +< / CAR +< . In some instances, the phenotype is or includes Annexin -< / CD4 +< / CAR +< In particular instances, the phenotype is Annexin -< / CD8 +< / CAR.

[0163] In particular instances, the phenotype is or includes a lack of or a low amount of intracellular IL-2 and CD4 +< / CAR +< . In particular instances, the phenotype is a lack of or a low amount of intracellular IL-13 and CD4 +< / CAR +< . In some instances, the phenotype is a lack of or a low amount of intracellular expression of IL-13 and CD8 +< / CAR +< cells. In particular instances, the phenotype is a lack of or a low amount of intracellular TNF-alpha CD4 +< / CAR +< .

[0164] In certain instances, the phenotype is or includes CD8 +< / CAR +< . In certain instances, the phenotype is or includes annexin -< / CD8 +< / CAR +< .

[0165] In some instances, the phenotype comprises an indicator of production of one or a combination of cytokines, optionally non-specific to the antigen or the recombinant receptor and / or that is polyclonally produced, wherein the one or more cytokines is IL-2, IL-13, IL-17, IFN-gamma or TNF-alpha. In some instances, the indicator of production is measured in an assay, optionally an intracellular cytokine staining assay, comprising incubating a sample of the T cell composition with a polyclonal agent, an antigen-specific agent or an agent that binds the recombinant receptor, optionally CAR. In some instances, the agent is or comprises PMA and ionomycin or is or comprises a T cell receptor or T cell receptor complex agonist. In some instances, the phenotype comprises negative expression of an activation marker, wherein the activation marker is selected from among CD25, CD127, LAG3, Ki67 and combinations thereof. In some instances, the phenotype comprises negative expression of an exhaustion marker, wherein the exhaustion maker is a PD1 or FOXP3 gene product or a combination thereof. In some instances, the phenotype comprises a naive phenotype or a memory phenotype, optionally wherein the memory phenotype comprises a T effector memory phenotype, a T central memory phenotype, or a T effector memory phenotype expressing CD45RA (Temra).

[0166] In some instances, the recombinant receptor-dependent (e.g., CAR) activity is a measure of the production or accumulation of a proinflammatory cytokine, optionally, one of or a combination of TNF-alpha, IFN-gamma, and IL-2. In some instances, a reference measure is the average of the measure among a plurality, optionally at least 10, at least 15, at least 20, of reference therapeutic T cell compositions comprising the recombinant receptor (e.g., CAR) in which: (i) each of the reference therapeutic T cell compositions has been observed or determined to result in an acceptable safety profile following administration to a subject, optionally wherein the subject has a disease or condition expressing or associated with the antigen; and / or (ii) each of the reference therapeutic T cell compositions has been observed or determined to result in a desired efficacy following administration to a subject, optionally wherein the subject has a disease or condition expressing or associated with the antigen.

[0167] In certain instances, a number, multiple, or fraction of cells of a particular phenotype of a cell composition is determined, measured, obtained, detected, observed, and / or identified. In some instances, the cell composition is a T cell composition. In certain instances, the cell composition contains cells that express a recombinant receptor, e.g., a CAR. In particular instances, the cell composition is a therapeutic T composition containing cells that express a recombinant receptor that may be administered to a subject to treat a disease or condition. In certain instances, the number of cells of the phenotype is the total amount of cells of the phenotype of the cell composition. In certain instances, the number of cells of the phenotype is the total number of cells of the phenotype present in a dose of the cell composition. In particular instances, the number of cells of the phenotype is the number of cells of the phenotype present in a sample of the cell composition. In some instances, the number of the cells of the phenotype may be expressed as a frequency, ratio, and / or a percentage of cells of the phenotype present in the cell composition, or a dose or a sample thereof.

[0168] In particular instances, the number, multiple, or fraction of the cells of a phenotype is transformed, for example to compress the range of relevant values of the number, multiple, or fraction. In some instances, the transformation is any application of a deterministic mathematical function to each point in a data set, such as, each data point x is replaced with the transformed value y = f(x), where f is a function. In general, transforms may be applied so that the data appear to more closely meet the assumptions of a statistical inference procedure that is to be applied, or to improve the interpretability or appearance of graphs. In most cases the function that is used to transform the data is invertible, and generally is continuous. The transformation is usually applied to a collection of comparable measurements. Examples of suitable transformations include, but are not limited to, logarithm and square root transformation, reciprocal transformations, and power transformations. In certain instances, the number, multiple, or fraction of the cells of a phenotype is transformed by a logarithmic transformation. In certain instances, the logarithmic transformation is a common log (log 10 (x)), a natural log (ln(x)) or a binary log (log 2 (x)).B. Determining Dosing and Administration

[0169] The therapeutic composition or a dose thereof, in some instances contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. In some instances, the composition includes the cells in an amount effective to reduce burden of the disease or condition. In some instances, the composition includes cells in an amount that provides more consistent outcome, e.g., response and / or safety outcomes, among a group of subjects administered the composition, and / or more consistent pharmacokinetic parameters. In some instances, the composition includes the cells in an amount effective to promote durable response and / or progression free survival. In some aspects, the disclosed methods involve assessing a therapeutic composition containing T cells for cell phenotypes, and determining doses based on such outcomes.

[0170] In some instances, the dose is determined to encompass a relatively consistent number, proportion, ratio and / or percentage of engineered cells having a particular phenotype in one or more particular compositions. In some aspects, the consistency is associated with or related to a relatively consistent activity, function, pharmacokinetic parameters, toxicity outcome and / or response outcome. In some aspects, in a plurality of subjects, compositions and / or doses the numbers, proportion, ratio and / or percentage, are relatively consistent, e.g., the number or ratio of cells that have a particular phenotoype, e.g., express CCR7 (CCR7 +< ) or, that produce a cytokine, for example, produce IL-2, TNF-alpha, or IFN-gamma, in the composition or unit dose, varies by no more than 40%, by no more than 30%, by no more than 20%, by no more than 10% or by no more than 5%. In some aspects the number or ratio of cells that have a particular phenotoype, e.g., express CCR7 (CCR7 +< ), in the composition or unit dose, varies by no more than 20% or no more than 10% or no more than 5% from an average of said number or ratio in a plurality of T cell compositions produced by the process and / or varies from such average by no more than one standard deviation or varies by no more than 20% or no more than 10% or no more than 5% among a plurality of T cell compositions or doses determined. In some instances, the plurality of subjects includes at least 10 subjects, such as at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more subjects.

[0171] In some aspects, the dose, e.g., one or more unit dose(s) is determined based on the number, percentage, ratio, frequency and / or proportion of a particular subset of engineered T cells, e.g., cells having a particular phenotype, such as particular surface marker phenotype. In some aspects, the cell phenotype is determined based on expression and / or absence of expression of particular cell markers, e.g., surface markers. In some aspects, the cell marker includes markers indicative of viability and / or apoptotic state of the cells. In some aspects, exemplary markers include CD3, CD4, CD8, CCR7, CD27, CD45RA, annexin V, or activated caspase 3. In some aspects, an exemplary marker is CCR7. In some aspects, an exemplary marker is CD27. In some aspects, exemplary markers include CCR7 and / or CD27. In some aspects, exemplary markers include CCR7, CD27 and / or CD45RA.

[0172] In some instances, disclosed are methods involving administering to a subject one or more unit doses of a therapeutic T cell composition, such as any described herein and / or any unit dose determined by the methods disclosed herein.

[0173] In some instances, disclosed are methods involving administering to a subject having a disease or condition a unit dose of a T cell composition comprising cells comprising a recombinant receptor, such as a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with the disease or condition, wherein either a defined number of total recombinant receptor-expressing cells (receptor +< ) of the therapeutic composition, total CD8 +< recombinant receptor-expressing cells (receptor +< / CD8 +< ) are administered and / or a unit dose of such cells is administered in which the unit dose contains a defined number, percentage, ratio, frequency and / or proportion of cells with a certain phenotype, e.g., CCR7 +< / CD4 +< , CCR7 +< / CD8 +< , CD27 +< / CD4 +< , CD27 +< / CD8 +< , CD45RA +< / CD4 +< , CD45RA +< / CD8 +< , CCR7 -< / CD4 +< , CCR7 / CD8 +< , CD27 -< / CD4 +< , CD27 / CD8 +< , CD45RA -< / CD4 +< , CD45RA / CD8 +< , CCR7 +< / CD27 +< / CD4 +< , CCR7 +< / CD27 +< / CD8 +< , CCR7 +< / CD45RA -< / CD4 +< , CCR7 +< / CD45RA -< / CD8 +< , CCR7 -< / CD45RA - -< / CD4 +< , CCR7 -< / CD45RA -< / CD8 +< , CCR7 -< / CD27 -< / CD4 +< , CCR7 -< / CD27 -< / CD8 +< .

[0174] In some instances, the unit dose of cells comprises a defined number of recombinant receptor-expressing CD8 +< T cells that express C-C chemokine receptor type 7 (CCR7) (receptor +< / CD8 +< / CCR7 +< cells) and / or a defined number of recombinant receptor-expressing CD4 +< T cells that express CCR7 (receptor +< / CD4 +< / CCR7 +< cells) and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< cells to receptor +< / CD4 -< / CCR7 +< cells and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< cells and / or receptor +< / CD4 +< / CCR7 +< cells to another subset of cells in the composition. In some instances, the unit dose of cells comprises a defined number of CD8 +< / CCR7 +< cells. In some instances, the unit dose of cells comprises a defined number of CD4 +< / CCR7 +< cells. In some instances, the defined number or ratio is further based on expression or absence of expression of CD27 and / or CD45RA on the cells.

[0175] In some instances, the unit dose of cells comprises a defined number of recombinant receptor-expressing CD8 +< T cells that express cluster of differentiation 27 (CD27) (receptor +< / CD8 +< / CD27 +< cells) and / or a defined number of recombinant receptor-expressing CD4 +< T cells that express CD27 (receptor +< / CD4 +< / CD27 +< cells) and / or a defined ratio of receptor +< / CD8 +< / CD27 +< cells to receptor +< / CD4 +< / CD27 +< cells and / or a defined ratio of receptor +< / CD8 +< / CD27 +< cells and / or receptor +< / CD4 +< / CD27 +< cells to another subset of cells in the composition. In some instances, the unit dose of cells comprises a defined number of CD8 +< / CD27 +< cells. In some instances, the unit dose of cells comprises a defined number of CD4 +< / CD27 +< cells. In some instances, the defined number or ratio is further based on expression or absence of expression of CCR7 and / or CD45RA on the cells.

[0176] In some instances, the unit dose of cells comprises a defined number of recombinant receptor-expressing CD8 +< T cells that express CCR7 and CD27 (receptor +< / CD8 +< / CCR7 +< / CD27 +< cells) and / or a defined number of recombinant receptor-expressing CD4 +< T cells that express CCR7 and CD27 (receptor +< / CD4 +< / CCR7 +< / CD27 +< cells) and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells and / or a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or receptor +< / CD4 +< / CCR7 +< / CD27 +< cells to another subset of cells in the composition. In some instances, the unit dose of cells comprises a defined number of CD8 +< / CCR7 +< / CD27 +< cells. In some instances, the unit dose of cells comprises a defined number of CD4 +< / CCR7 +< / CD27 +< cells. In some instances, the defined number or ratio is further based on expression or absence of expression of CD45RA on the cells.

[0177] In some instances, the number of cells in the unit dose is the number of cells or number of recombinant receptor-expressing or CAR-expressing cells, or number, percentage, ratio, frequency and / or proportion of such cells of a certain phenotype, e.g. cells that express or do not express one or more markers selected from CD3 CD4, CD8, CCR7, CD27, CD45RA, annexin V, or activated caspase 3, that it is desired to administer to a particular subject in a dose, such as a subject from which the cells have been derived. In some instances, the number of cells in the unit dose is the number of cells or number of recombinant receptor-expressing or CAR-expressing cells, or number, percentage, ratio, frequency and / or proportion of such cells of a certain phenotype, e.g., CCR7 +< , CD27 +< , CD45RA +< , CD45RA -< , CD4 +< , CD8 +< , CD3 +< , apoptosis marker negative (e.g. Annexin V -< or Caspase 3 -< ) cells, or cells that are positive or negative for one or more of any of the foregoing.

[0178] In some instances, the number of cells in the unit dose is the number of cells or number of recombinant receptor-expressing or CAR-expressing cells, or number, percentage, ratio and / or proportion of such cells of a certain phenotype, e.g., CCR7 +< / CD4 +< , CCR7 +< / CD8 +< , CD27 +< / CD4 +< , CD27 +< / CD8 +< , CD45RA +< / CD4 +< , CD45RA +< / CD8 +< , CCR7 -< / CD4 +< , CCR7 -< / CD8 +< , CD27 -< / CD4 +< , CD27 -< / CD8 +< , CD45RA -< / CD4 +< , CD45RA -< / CD8 +< , CCR7 +< / CD27 +< / CD4 +< , CCR7 +< / CD27 +< / CD8 +< , CCR7 +< / CD45RA -< / CD4 +< , CCR7 +< / CD45RA / CD8 +< , CCR7 -< / CD45RA - -< / CD4 +< , CCR7 -< / CD45RA -< / CD8 +< , CCR7 -< / CD27 -< / CD4 +< , CCR7 -< / CD27 -< / CD8 +< ; and apoptosis marker negative (e.g. Annexin V -< or Caspase 3 -< ) cells, that it is desired to administer to a particular subject in a dose, such as a subject from which the cells have been derived. In some instances, the unit dose contains a defined number of cells or number of recombinant receptor-expressing or CAR-expressing cells, or number, percentage, ratio and / or proportion of such cells of a certain phenotype e.g., CCR7 +< / CD4 +< , CCR7 +< / CD8 +< , CD27 +< / CD4 +< , CD27 +< / CD8 +< , CD45RA +< / CD4 +< , CD45RA +< / CD8 +< , CCR7 -< / CD4 +< , CCR7 -< / CD8 +< , CD27 -< / CD4 +< , CD27 -< / CD8 +< , CD45RA -< / CD4 +< , CD45RA -< / CD8 +< , CCR7 +< / CD27 +< / CD4 +< , CCR7 +< / CD27 +< / CD8 +< , CCR7 +< / CD45RA -< / CD4 +< , CCR7 +< / CD45RA / CD8 +< , CCR7 -< / CD45RA -< / CD4 +< , CCR7 -< / CD45RA -< / CD8 +< , CCR7 -< / CD27 -< / CD4 +< , CCR7 -< / CD27 -< / CD8 +< ; and apoptosis marker negative (e.g. Annexin V -< or Caspase 3 -< ) cells, and / or any subset thereof.

[0179] In some instances, the unit dose is determined based on the number of cells or cell type(s) and / or a frequency, ratio, and / or percentage of cells or cell types, e.g., individual populations, phenotypes, or subtypes, in the cell composition, such as those with the phenotypes of annexin V -< / CCR7 +< / CAR +< ; annexin V -< / CCR7 +< / CAR +< / CD4 +< ; annexin V -< / CCR7 +< / CAR +< / CD8 +< ; annexin V -< / CD27 +< / CAR +< ; annexin V -< / CD27 +< / CAR +< / CD4 +< ; annexin V -< / CD27 +< / CAR +< / CD8 +< ; annexin V -< / CCR7 +< / CD27 +< / CAR +< ; annexin V -< / CCR7 +< / CD27 +< / CAR +< / CD4 +< ; annexin V -< / CCR7 +< / CD27 +< / CAR +< / CD8 +< ; annexin V -< / CCR7 +< / CD45RA -< / CAR +< ; annexin V -< / CCR7 +< / CD45RA -< / CAR +< / CD4 +< ; annexin V -< / CCR7 +< / CD45RA -< / CAR +< / CD8 +< ; annexin V -< / CCR7 -< / CD45RA -< / CAR +< ; annexin V -< / CCR7 -< / CD45RA -< / CAR +< / CD4 +< ; annexin V -< / CCR7 -< / CD45RA -< / CAR +< / CD8 +< ; annexin V -< / CCR7 -< / CD27 -< / CAR +< , annexin V -< / CCR7 -< / CD27 -< / CAR +< / CD4 +< ; annexin V -< / CCR7 -< / CD27 / CAR +< / CD8 +< ; activated caspase 3 -< / CCR7 +< / CAR +< ; activated caspase 3 -< / CCR7 +< / CAR +< / CD4 +< ; activated caspase 3 -< / CCR7 +< / CAR +< / CD8 +< ; activated caspase 3 -< / CD27 +< / CAR +< ; activated caspase 3 -< / CD27 +< / CAR +< / CD4 +< ; activated caspase 3 -< / CD27 +< / CAR +< / CD8 +< ; activated caspase 3 -< / CCR7 +< / CD27 +< / CAR +< ; activated caspase 3 -< / CCR7 +< / CD27 +< / CAR +< / CD4 +< ; activated caspase 3 -< / CCR7 +< / CD27 +< / CAR +< / CD8 +< ; activated caspase 3 -< / CCR7 +< / CD45RA -< / CAR +< ; activated caspase 3 -< / CCR7 +< / CD45RA -< / CAR +< / CD4 +< ; activated caspase 3 -< / CCR7 +< / CD45RA -< / CAR +< / CD8 +< ; activated caspase 3 / CCR7 -< / CD45RA -< / CAR +< ; activated caspase 3 -< / CCR7 -< / CD45RA -< / CAR +< / CD4 +< ; activated caspase 3 -< / CCR7 -< / CD45RA -< / CAR +< / CD8 +< ; activated caspase 3 -< / CCR7 -< / CD27 -< / CAR +< ; activated caspase 3 -< / CCR7 -< / CD27 -< / CAR +< / CD4 +< ; and / or activated caspase 3 -< / CCR7 -< / CD27 / CAR +< / CD8 +< ; or a combination thereof.

[0180] In some instances, the unit dose comprises between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD8 +< cells that express the recombinant receptor (receptor +< / CD8 +< cells) or total CD4 +< cell that express the recombinant receptor (receptor +< / CD4 +< cells), total receptor +< / CD8 +< / CCR7 +< cells, total receptor +< / CD4 +< / CCR7 +< cells, total receptor +< / CD8 +< / CD27 +< cells, or total receptor +< / CD4 +< / CD27 +< cells, each inclusive. In some instances, the unit dose comprises no more than about 1 x 10 8< , no more than about 5 x 10 7< , no more than about 1 x 10 7< , no more than about 5 x 10 6< , no more than about 1 x 10 6< , or no more than about 5 x 10 5< total receptor +< / CD8 +< cells or total receptor +< / CD4 +< cells, total receptor +< / CD8 +< / CCR7 +< cells, total receptor +< / CD4 +< / CCR7 +< cells, total receptor +< / CD8 +< / CD27 +< cells, or total receptor +< / CD4 +< / CD27 +< cells.

[0181] In some instances, the unit dose comprises between at or about 5 x 10 5< and at or about 5 x 10 7< , between at or about 1 x 10 6< and at or about 1 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< cells or receptor +< / CD4 +< / CCR7 +< cells, each inclusive. In some instances, the unit dose comprises at least or at least about 5 x 10 7< , 1 x 10 7< , 5 x 10 6< , 1 x 10 6< , or at least about 5 x 10 5< total receptor +< / CD8 +< / CCR7 +< cells or receptor +< / CD4 +< / CCR7 +< cells.

[0182] In some instances, the unit dose comprises between at or about 5 x 10 5< and at or about 5 x 10 7< , between at or about 1 x 10 6< and at or about 1 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD8 +< / CD27 +< cells or receptor +< / CD4 +< / CD27 +< cells, each inclusive. In some instances, the unit dose comprises at least or at least about 5 x 10 7< , 1 x 10 7< , 5 x 10 6< , 1 x 10 6< , or at least about 5 x 10 5< total receptor +< / CD8 +< / CD27 +< cells or receptor +< / CD4 +< / CD27 +< cells.

[0183] In some instances, the unit dose comprises at least at or about 1 x 10 6< , 2 x 10 6< , 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< cells and / or at least at or about 1 x 10 6< , 2 x 10 6< , 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< cells, each inclusive. In some instances, the unit dose comprises between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< cells and / or between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< cells, each inclusive.

[0184] In some instances, the unit dose comprises at least at or about 1 x 10 6< , 2 x 10 6< , 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD8 +< / CD27 +< cells and / or at least at or about 1 x 10 6< , 2 x 10 6< , 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD4 +< / CD27 +< cells, each inclusive. In some instances, the unit dose comprises unit dose comprises between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD8 +< / CD27 +< cells and / or between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD4 +< / CD27 +< cells, each inclusive.

[0185] In some instances, the unit dose comprises between at or about 5 x 10 5< and at or about 5 x 10 7< , between at or about 1 x 10 6< and at or about 1 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells or receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, each inclusive. In some instances, the unit dose comprises at least or at least at or about 5 x 10 7< , 1 x 10 7< , 5 x 10 6< , 1 x 10 6< , or at least at or about 5 x 10 5< total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells or receptor +< / CD4 +< / CCR7 +< / CD27 +< cells.

[0186] In some instances, the unit dose comprises at least at or about 1 x 10 6< , 2 x 10 6< , 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or at least at or about 1 x 10 6< , 2 x 10 6< , 3 x 10 6< , 4 x 10 6< , 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , or 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, each inclusive. In some instances, the unit dose comprises between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells and / or between at or about 3 x 10 6< and at or about 2.5 x 10 7< , between at or about 4 x 10 6< and at or about 2 x 10 7< , or between at or about 5 x 10 6< and at or about 1 x 10 7< total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, each inclusive.

[0187] In some instances, the unit dose of cells comprises a defined ratio of receptor +< / CD8 +< / CCR7 +< cells to receptor +< / CD4 -< / CCR7 +< cells, which ratio optionally is or is approximately 1:1 or is between approximately 1:3 and approximately 3:1.

[0188] In some instances, the unit dose of cells comprises a defined ratio of receptor +< / CD8 +< / CD27 +< cells to receptor +< / CD4 +< / CD27 +< cells, which ratio optionally is or is approximately 1: 1 or is between approximately 1:3 and approximately 3: 1.

[0189] In some instances, the unit dose comprises between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD8 +< cells that express the recombinant receptor (receptor +< / CD8 +< cells) or total CD4 +< cell that express the recombinant receptor (receptor +< / CD4 +< cells), total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, or total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, each inclusive. In some instances, the unit dose comprises no more than at or about 1 x 10 8< , no more than at or about 5 x 10 7< , no more than at or about 1 x 10 7< , no more than at or about 5 x 10 6< , no more than at or about 1 x 10 6< , or no more than at or about 5 x 10 5< total receptor +< / CD8 +< cells or total receptor +< / CD4 +< cells, total receptor +< / CD8 -< / CCR7 +< / CD27 +< cells, or total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells.

[0190] In some instances, the unit dose of cells comprises a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, which ratio optionally is or is approximately 1: 1 or is between approximately 1:3 and approximately 3: 1.

[0191] In some instances, the unit dose comprises between at or about 1 x 10 5< and at or about 5 x 10 8< , between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD3 +< cells that express the recombinant receptor (receptor +< / CD3 +< cells) or total CD3 +< cells, each inclusive. In some instances, the unit dose comprises no more than at or about 5 x 10 8< , no more than at or about 1 x 10 8< , no more than at or about 5 x 10 7< , no more than at or about 1 x 10 7< , no more than at or about 5 x 10 6< , no more than at or about 1 x 10 6< , or no more than at or about 5 x 10 5< total receptor +< / CD3 +< cells or total CD3 +< cells.

[0192] In some instances, the total number of CD3 +< cells, total number of receptor +< / CD3 +< cells, total number of receptor +< / CD8 +< cells, total number of receptor +< / CD4 +< cells, total number of receptor +< / CD8 +< / CCR7 +< cells, total number of receptor +< / CD4 +< / CCR7 +< cells, total number of receptor +< / CD8 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD45RA -< cells and / or receptor +< / CD4 +< / CCR7 +< / CD45RA -< cells is the total number of such cells that are live or viable. In some instances, the total number of CD3 +< cells, total number of receptor +< / CD3 +< cells, total number of receptor +< / CD8 +< cells, total number of receptor +< / CD4 +< cells, total number of receptor +< / CD8 +< / CCR7 +< cells, total number of receptor +< / CD4 +< / CCR7 +< cells, total number of receptor +< / CD8 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, total number of receptor +< / CD8 +< / CCR7 +< / CD45RA -< cells and / or receptor +< / CD4 +< / CCR7 +< / CD45RA -< cells is the total number of such cells that do not express an apoptotic marker and / or is the total number of such cells that are apoptotic marker negative ( -< ), wherein the apoptotic marker is Annexin V or activated Caspase 3.

[0193] In some instances, in any of the composition comprising T cells expressing a recombinant receptor disclosed herein, at least at or about, or at or about, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of T cells in the composition (or of the total number of T cells in the composition expressing the recombinant receptor), are able to produce a cytokine selected from interleukin 2 (IL-2) and / or TNF-alpha.In some instances, the T cell able to produce IL-2 and / or TNF-alpha is a CD4+ T cell.

[0194] In some instances, the unit dose comprises between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD8 +< cells that express the recombinant receptor (receptor +< / CD8 +< cells) or total CD4 +< cell that express the recombinant receptor (receptor +< / CD4 +< cells), total receptor +< / CD8 +< / CCR7 +< cells, total receptor +< / CD4 +< / CCR7 +< cells, total receptor +< / CD8 +< / CD27 +< cells, or total receptor +< / CD4 +< / CD27 +< cells, each inclusive. In some instances, the unit dose comprises no more than at or about 1 x 10 8< , no more than at or about 5 x 10 7< , no more than at or about 1 x 10 7< , no more than at or about 5 x 10 6< , no more than at or about 1 x 10 6< , or no more than at or about 5 x 10 5< total receptor +< / CD8 +< cells or total receptor +< / CD4 +< cells, total receptor +< / CD8 +< / CCR7 +< cells, total receptor +< / CD4 +< / CCR7 +< cells, total receptor +< / CD8 +< / CD27 +< cells, or total receptor +< / CD4 +< / CD27 +< cells.

[0195] In some instances, the unit dose of cells comprises a defined ratio of receptor +< / CD8 +< / CCR7 +< cells to receptor +< / CD4 -< / CCR7 +< cells, which ratio optionally is or is approximately 1: 1 or is between approximately 1:3 and approximately 3: 1.

[0196] In some instances, the unit dose comprises between at or about 1 x 10 5< and at or about 1 x 10 8< , between at or about 5 x 10 5< and at or about 1 x 10 7< , or between at or about 1 x 10 6< and at or about 1 x 10 7< total CD8 +< cells that express the recombinant receptor (receptor +< / CD8 +< cells) or total CD4 +< cell that express the recombinant receptor (receptor +< / CD4 +< cells), total receptor +< / CD8 +< / CCR7 +< / CD27 +< cells, or total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, each inclusive. In some instances, the unit dose comprises no more than at or about 1 x 10 8< , no more than at or about 5 x 10 7< , no more than at or about 1 x 10 7< , no more than at or about 5 x 10 6< , no more than at or about 1 x 10 6< , or no more than at or about 5 x 10 5< total receptor +< / CD8 +< cells or total receptor +< / CD4 +< cells, total receptor +< / CD8 -< / CCR7 +< / CD27 +< cells, or total receptor +< / CD4 +< / CCR7 +< / CD27 +< cells.

[0197] In some instances, the unit dose of cells comprises a defined ratio of receptor +< / CD8 +< / CCR7 +< / CD27 +< cells to receptor +< / CD4 +< / CCR7 +< / CD27 +< cells, which ratio optionally is or is approximately 1: 1 or is between approximately 1:3 and approximately 3: 1.

[0198] In some instances, the disclosed methods involve administering a dose containing a defined number of cells. In some instances, the dose, such as the defined number of cells, such as a defined number of CAR +< cells that are CCR7 +< / CD4 +< , CCR7 +< / CD8 +< , CD27 +< / CD4 +< , CD27 +< / CD8 +< , CD45RA +< / CD4 +< , CD45RA +< / CD8 +< , CCR7 -< / CD4 +< , CCR7 -< / CD8 +< , CD27 -< / CD4 +< , CD27 -< / CD8 +< , CD45RA -< / CD4 +< , CD45RA -< / CD8 +< , CCR7 +< / CD27 +< / CD4 +< , CCR7 +< / CD27 +< / CD8 +< , CCR7 +< / CD45RA -< / CD4 +< , CCR7 +< / CD45RA / CD8 +< , CCR7 -< / CD45RA -< / CD4 +< , CCR7 -< / CD45RA -< / CD8 +< , CCR7 -< / CD27 -< / CD4 +< , or CCR7 -< / CD27 -< / CD8 +< , is between or between about 5.0 x 10 6< and 2.25 x 10 7< , 5.0 x 10 6< and 2.0 x 10 7< , 5.0 x 10 6< and 1.5 x 10 7< , 5.0 x 10 6< and 1.0 x 10 7< , 5.0 x 10 6< and 7.5 x 10 6< , 7.5 x 10 6< and 2.25 x 10 7< , 7.5 x 10 6< and 2.0 x 10 7< , 7.5 x 10 6< and 1.5 x 10 7< , 7.5 x 10 6< and 1.0 x 10 7< , 1.0 x 10 7< and 2.25 x 10 7< , 1.0 x 10 7< and 2.0 x 10 7< , 1.0 x 10 7< and 1.5 x 10 7< , 1.5 x 10 7< and 2.25 x 10 7< , 1.5 x 10 7< and 2.0 x 10 7< , 2.0 x 10 7< and 2.25 x 10 7< . In some instances, such dose, such as such defined number of cells refers to the total recombinant-receptor expressing cells in the administered composition. In some aspects, the defined number of recombinant receptor-expressing cells that are administered are cells that are apoptotic marker negative(-) and optionally wherein the apoptotic marker is Annexin V or activated Caspase 3.

[0199] In some instances, the dose of cells of the unit dose contains a number of cells, such as a defined number of cells, between at least or at least about 5 x 10 6< , 6 x 10 6< , 7 x 10 6< , 8 x 10 6< , 9 x 10 6< , 10 x 10 6< and about 15 x10 6< recombinant-receptor expressing cells, such as recombinant-receptor expressing cells that are CCR7 +< / CD4 +< , CCR7 +< / CD8 +< , CD27 +< / CD4 +< , CD27 +< / CD8 +< , CD45RA +< / CD4 +< , CD45RA +< / CD8 +< , CCR7 -< / CD4 +< , CCR7 -< / CD8 +< , CD27 -< / CD4 +< , CD27 -< / CD8 +< , CD45RA -< / CD4 +< , CD45RA -< / CD8 +< , CCR7 +< / CD27 +< / CD4 +< , CCR7 +< / CD27 +< / CD8 +< , CCR7 +< / CD45RA -< / CD4 +< , CCR7 +< / CD45RA -< / CD8 +< , CCR7 -< / CD45RA -< / CD4 +< , CCR7 -< / CD45RA -< / CD8 +< , CCR7 -< / CD27 -< / CD4 +< , or CCR7 -< / CD27 -< / CD8 +< , and / or that are apoptotic marker negative(-) and CD8 +< , optionally wherein the apoptotic marker is Annexin V or activated Caspase 3.

[0200] In some instances, a dose of cells is administered to subjects in accord with the disclosed methods, and / or with the disclosed articles of manufacture or compositions. In some instances, the size or timing of the doses is determined as a function of the particular disease or condition in the subject. In some cases, the size or timing of the doses for a particular disease in view of the provided description may be empirically determined.

[0201] In some instances, the dose of cells comprises between at or about 2 x 10 5< of the cells / kg and at or about 2 x 10 6< of the cells / kg, such as between at or about 4 x 10 5< of the cells / kg and at or about 1 x 10 6< of the cells / kg or between at or about 6 x 10 5< of the cells / kg and at or about 8 x 10 5< of the cells / kg. In some instances, the dose of cells comprises no more than 2 x 10 5< of the cells (e.g. antigen-expressing, such as CAR-expressing cells) per kilogram body weight of the subject (cells / kg), such as no more than at or about 3 x 10 5< cells / kg, no more than at or about 4 x 10 5< cells / kg, no more than at or about 5 x 10 5< cells / kg, no more than at or about 6 x 10 5< cells / kg, no more than at or about 7 x 10 5< cells / kg, no more than at or about 8 x 10 5< cells / kg, no more than at or about 9 x 10 5< cells / kg, no more than at or about 1 x 10 6< cells / kg, or no more than at or about 2 x 10 6< cells / kg. In some instances, the dose of cells comprises at least or at least about or at or about 2 x 10 5< of the cells (e.g. antigen-expressing, such as CAR-expressing cells) per kilogram body weight of the subject (cells / kg), such as at least or at least about or at or about 3 x 10 5< cells / kg, at least or at least about or at or about 4 x 10 5< cells / kg, at least or at least about or at or about 5 x 10 5< cells / kg, at least or at least about or at or about 6 x 10 5< cells / kg, at least or at least about or at or about 7 x 10 5< cells / kg, at least or at least about or at or about 8 x 10 5< cells / kg, at least or at least about or at or about 9 x 10 5< cells / kg, at least or at least about or at or about 1 x 10 6< cells / kg, or at least or at least about or at or about 2 x 10 6< cells / kg.

[0202] In certain instances, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of at or about 0.1 million to at or about 100 billion cells and / or that amount of cells per kilogram of body weight of the subject, such as, e.g., at or about 0.1 million to at or about 50 billion cells (e.g., at or about 5 million cells, at or about 25 million cells, at or about 500 million cells, at or about 1 billion cells, at or about 5 billion cells, at or about 20 billion cells, at or about 30 billion cells, at or about 40 billion cells, or a range defined by any two of the foregoing values), at or about 1 million to at or about 50 billion cells (e.g., at or about 5 million cells, at or about 25 million cells, at or about 500 million cells, at or about 1 billion cells, at or about 5 billion cells, at or about 20 billion cells, at or about 30 billion cells, at or about 40 billion cells, or a range defined by any two of the foregoing values), such as at or about 10 million to at or about 100 billion cells (e.g., at or about 20 million cells, at or about 30 million cells, at or about 40 million cells, at or about 60 million cells, at or about 70 million cells, at or about 80 million cells, at or about 90 million cells, at or about 10 billion cells, at or about 25 billion cells, at or about 50 billion cells, at or about 75 billion cells, at or about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases at or about 100 million cells to at or about 50 billion cells (e.g., at or about 120 million cells, at or about 250 million cells, at or about 350 million cells, at or about 450 million cells, at or about 650 million cells, at or about 800 million cells, at or about 900 million cells, at or about 3 billion cells, at or about 30 billion cells, at or about 45 billion cells) or any value in between these ranges and / or per kilogram of body weight of the subject. Dosages may vary depending on attributes particular to the disease or disorder and / or patient and / or other treatments. In some instances, the dose of cells is a flat dose of cells or fixed dose of cells such that the dose of cells is not tied to or based on the body surface area or weight of a subject.

[0203] In some instances, for example, where the subject is a human, the dose includes fewer than about 5 x 10 8< total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of at or about 1 x 10 6< to at or about 5 x 10 8< such cells, such as at or about 2 x 10 6< , 5 x 10 6< , 1 x 10 7< , 5 x 10 7< , 1 x 10 8< , 1.5 x 10 8< , or 5 x 10 8< total such cells, or the range between any two of the foregoing values. In some instances, for example, where the subject is a human, the dose includes more than at or about 1 x 10 6< total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) and fewer than at or about 2 x 10 9< total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of at or about 2.5 x 10 7< to at or about 1.2 x 10 9< such cells, such as at or about 2.5 x 10 7< , 5 x 10 7< , 1 x 10 8< , 1.5 x 10 8< total such cells, or the range between any two of the foregoing values.

[0204] In some instances, the dose of genetically engineered cells comprises from at or about 1 x 10 5< to at or about 5 x 10 8< total CAR-expressing (CAR-expressing) T cells, from at or about 1 x 10 5< to at or about 2.5 x 10 8< total CAR-expressing T cells, from at or about 1 x 10 5< to at or about 1 x 10 8< total CAR-expressing T cells, from at or about 1 x 10 5< to at or about 5 x 10 7< total CAR-expressing T cells, from at or about 1 x 10 5< to at or about 2.5 x 10 7< total CAR-expressing T cells, from at or about 1 x 10 5< to at or about 1 x 10 7< total CAR-expressing T cells, from at or about 1 x 10 5< to at or about 5 x 10 6< total CAR-expressing T cells, from at or about 1 x 10 5< to at or about 2.5 x 10 6< total CAR-expressing T cells, from at or about 1 x 10 5< to at or about 1 x 10 6< total CAR-expressing T cells, from at or about 1 x 10 6< to at or about 5 x 10 8< total CAR-expressing T cells, from at or about 1 x 10 6< to at or about 2.5 x 10 8< total CAR-expressing T cells, from at or about 1 x 10 6< to at or about 1 x 10 8< total CAR-expressing T cells, from at or about 1 x 10 6< to at or about 5 x 10 7< total CAR-expressing T cells, from at or about 1 x 10 6< to at or about 2.5 x 10 7< total CAR-expressing T cells, from at or about 1 x 10 6< to at or about 1 x 10 7< total CAR-expressing T cells, from at or about 1 x 10 6< to at or about 5 x 10 6< total CAR-expressing T cells, from at or about 1 x 10 6< to at or about 2.5 x 10 6< total CAR-expressing T cells, from at or about 2.5 x 10 6< to at or about 5 x 10 8< total CAR-expressing T cells, from at or about 2.5 x 10 6< to at or about 2.5 x 10 8< total CAR-expressing T cells, from at or about 2.5 x 10 6< to at or about 1 x 10 8< total CAR-expressing T cells, from at or about 2.5 x 10 6< to at or about 5 x 10 7< total CAR-expressing T cells, from at or about 2.5 x 10 6< to at or about 2.5 x 10 7< total CAR-expressing T cells, from at or about 2.5 x 10 6< to at or about 1 x 10 7< total CAR-expressing T cells, from at or about 2.5 x 10 6< to at or about 5 x 10 6< total CAR-expressing T cells, from at or about 5 x 10 6< to at or about 5 x 10 8< total CAR-expressing T cells, from at or about 5 x 10 6< to at or about 2.5 x 10 8< total CAR-expressing T cells, from at or about 5 x 10 6< to at or about 1 x 10 8< total CAR-expressing T cells, from at or about 5 x 10 6< to at or about 5 x 10 7< total CAR-expressing T cells, from at or about 5 x 10 6< to at or about 2.5 x 10 7< total CAR-expressing T cells, from at or about 5 x 10 6< to at or about 1 x 10 7< total CAR-expressing T cells, from at or about 1 x 10 7< to at or about 5 x 10 8< total CAR-expressing T cells, from at or about 1 x 10 7< to at or about 2.5 x 10 8< total CAR-expressing T cells, from at or about 1 x 10 7< to at or about 1 x 10 8< total CAR-expressing T cells, from at or about 1 x 10 7< to at or about 5 x 10 7< total CAR-expressing T cells, from at or about 1 x 10 7< to at or about 2.5 x 10 7< total CAR-expressing T cells, from at or about 2.5 x 10 7< to at or about 5 x 10 8< total CAR-expressing T cells, from at or about 2.5 x 10 7< to at or about 2.5 x 10 8< total CAR-expressing T cells, from at or about 2.5 x 10 7< to at or about 1 x 10 8< total CAR-expressing T cells, from at or about 2.5 x 10 7< to at or about 5 x 10 7< total CAR-expressing T cells, from at or about 5 x 10 7< to at or about 5 x 10 8< total CAR-expressing T cells, from at or about 5 x 10 7< to at or about 2.5 x 10 8< total CAR-expressing T cells, from at or about 5 x 10 7< to at or about 1 x 10 8< total CAR-expressing T cells, from at or about 1 x 10 8< to at or about 5 x 10 8< total CAR-expressing T cells, from at or about 1 x 10 8< to at or about 2.5 x 10 8< total CAR-expressing T cells, from at or about or 2.5 x 10 8< to at or about 5 x 10 8< total CAR-expressing T cells. In some instances, the dose of genetically engineered cells comprises from or from about 2.5 x 10 7< to at or about 1.5 x 10 8< total CAR-expressing T cells, such as from or from about 5 x 10 7< to or to about 1 x 10 8< total CAR-expressing T cells.

[0205] In some instances, the dose of genetically engineered cells comprises at least at or about 1 x 10 5< CAR-expressing cells, at least at or about 2.5 x 10 5< CAR-expressing cells, at least at or about 5 x 10 5< CAR-expressing cells, at least at or about 1 x 10 6< CAR-expressing cells, at least at or about 2.5 x 10 6< CAR-expressing cells, at least at or about 5 x 10 6< CAR-expressing cells, at least at or about 1 x 10 7< CAR-expressing cells, at least at or about 2.5 x 10 7< CAR-expressing cells, at least at or about 5 x 10 7< CAR-expressing cells, at least at or about 1 x 10 8< CAR-expressing cells, at least at or about 1.5 x 10 8< CAR-expressing cells, at least at or about 2.5 x 10 8< CAR-expressing cells, or at least at or about 5 x 10 8< CAR-expressing cells.

[0206] In some instances, the cell therapy comprises administration of a dose comprising a number of cell from or from about 1 x 10 5< to or to about 5 x 10 8< total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), from or from about 5 x 10 5< to or to about 1 x 10 7< total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs) or from or from about 1 x 10 6< to or to about 1 x 10 7< total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), each inclusive. In some instances, the cell therapy comprises administration of a dose of cells comprising a number of cells at least or at least about 1 x 10 5< total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), such at least or at least 1 x 10 6< , at least or at least about 1 x 10 7< , at least or at least about 1 x 10 8< of such cells. In some instances, the number is with reference to the total number of CD3 +< or CD8 +< , in some cases also recombinant receptor-expressing (e.g. CAR +< ) cells. In some instances, the cell therapy comprises administration of a dose comprising a number of cell from or from about 1 x 10 5< to or to about 5 x 10 8< CD3 +< or CD8 +< total T cells or CD3 +< or CD8 +< recombinant receptor-expressing cells, from or from about 5 x 10 5< to or to about 1 x 10 7< CD3 +< or CD8 +< total T cells or CD3 +< or CD8 +< recombinant receptor-expressing cells, or from or from about 1 x 10 6< to or to about 1 x 10 7< CD3 +< or CD8 +< total T cells or CD3 +< or CD8 +< recombinant receptor-expressing cells, each inclusive. In some instances, the cell therapy comprises administration of a dose comprising a number of cell from or from about 1 x 10 5< to or to about 5 x 10 8< total CD3 +< / CAR +< or CD8 +< / CAR +< cells, from or from about 5 x 10 5< to or to about 1 x 10 7< total CD3 +< / CAR +< or CD8 +< / CAR +< cells, or from or from about 1 x 10 6< to or to about 1 x 10 7< total CD3 +< / CAR +< or CD8 +< / CAR +< cells, each inclusive.

[0207] In some instances, the T cells of the dose include CD4+ T cells, CD8+ T cells or CD4+ and CD8+ T cells.

[0208] In some instances, for example, where the subject is human, the CD8+ T cells of the dose, including in a dose including CD4+ and CD8+ T cells, includes between at or about 1 x 10 6< and at or about 5 x 10 8< total recombinant receptor (e.g., CAR)-expressing CD8+cells, e.g., in the range of from at or about 5 x 10 6< to at or about 1 x 10 8< such cells, such as 1 x 10 7< , 2.5 x 10 7< , 5 x 10 7< , 7.5 x 10 7< , 1 x 10 8< , 1.5 x 10 8< , or 5 x 10 8< total such cells, or the range between any two of the foregoing values. In some instances, the patient is administered multiple doses, and each of the doses or the total dose can be within any of the foregoing values. In some instances, the dose of cells comprises the administration of from or from about 1 x 10 7< to or to about0.75 x 10 8< total recombinant receptor-expressing CD8+ T cells, from or from about 1 x 10 7< to or to about 5 x 10 7< total recombinant receptor-expressing CD8+ T cells, from or from about 1 x 10 7< to or to about 0.25 x 10 8< total recombinant receptor-expressing CD8+ T cells, each inclusive. In some instances, the dose of cells comprises the administration of at or about 1 x 10 7< , 2.5 x 10 7< , 5 x 10 7< , 7.5 x 10 7< , 1 x 10 8< , 1.5 x 10 8< , 2.5 x 10 8< , or 5 x 10 8< total recombinant receptor-expressing CD8+ T cells.

[0209] In some instances, the dose of cells, e.g., recombinant receptor-expressing T cells, is administered to the subject as a single dose or is administered only one time within a period of two weeks, one month, three months, six months, 1 year or more.

[0210] In the context of adoptive cell therapy, administration of a given "dose" encompasses administration of the given amount or number of cells as a single composition and / or single uninterrupted administration, e.g., as a single injection or continuous infusion, and also encompasses administration of the given amount or number of cells as a split dose or as a plurality of compositions, provided in multiple individual compositions or infusions, over a specified period of time, such as over no more than 3 days. Thus, in some contexts, the dose is a single or continuous administration of the specified number of cells, given or initiated at a single point in time. In some contexts, however, the dose is administered in multiple injections or infusions over a period of no more than three days, such as once a day for three days or for two days or by multiple infusions over a single day period.

[0211] Thus, in some aspects, the cells of the dose are administered in a single pharmaceutical composition. In some instances, the cells of the dose are administered in a plurality of compositions, collectively containing the cells of the dose.

[0212] In some instances, the term "split dose" refers to a dose that is split so that it is administered over more than one day. This type of dosing is encompassed by the present methods and is considered to be a single dose.

[0213] Thus, the dose of cells may be administered as a split dose, e.g., a split dose administered over time. For example, in some instances, the dose may be administered to the subject over 2 days or over 3 days. Exemplary methods for split dosing include administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other instances, 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 instances, the split dose is not spread over more than 3 days.

[0214] In some instances, cells of the dose may be administered by administration of a plurality of compositions or solutions, such as a first and a second, optionally more, each containing some cells of the dose. In some aspects, the plurality of compositions, each containing a different population and / or sub-types of cells, are administered separately or independently, optionally within a certain period of time. For example, the populations or sub-types of cells can include CD8 +< and CD4 +< T cells, respectively, and / or CD8 +< - and CD4 +< -enriched populations, respectively, e.g., CD4 +< and / or CD8 +< T cells each individually including cells genetically engineered to express the recombinant receptor. In some instances, the administration of the dose comprises administration of a first composition comprising a dose of CD8 +< T cells or a dose of CD4 +< T cells and administration of a second composition comprising the other of the dose of CD4 +< T cells and the CD8 +< T cells.

[0215] In some instances, the administration of the composition or dose, e.g., administration of the plurality of cell compositions, involves administration of the cell compositions separately. In some aspects, the separate administrations are carried out simultaneously, or sequentially, in any order. In some instances, the dose comprises a first composition and a second composition, and the first composition and second composition are administered 0 to 12 hours apart, 0 to 6 hours apart or 0 to 2 hours apart. In some instances, the initiation of administration of the first composition and the initiation of administration of the second composition are carried out no more than 2 hours, no more than 1 hour, or no more than 30 minutes apart, no more than 15 minutes, no more than 10 minutes or no more than 5 minutes apart. In some instances, the initiation and / or completion of administration of the first composition and the completion and / or initiation of administration of the second composition are carried out no more than 2 hours, no more than 1 hour, or no more than 30 minutes apart, no more than 15 minutes, no more than 10 minutes or no more than 5 minutes apart.

[0216] In some instances , the first composition, e.g., first composition of the dose, comprises CD4 +< T cells. In some instances, the first composition, e.g., first composition of the dose, comprises CD8 +< T cells. In some instances, the first composition is administered prior to the second composition. In some instances, the second composition, e.g., second composition of the dose, comprises CD4+ T cells. In some instances, the second composition, e.g., second composition of the dose, comprises CD8+ T cells.

[0217] In some instances, the dose or composition of cells includes a defined or target ratio of CD4 +< cells expressing a recombinant receptor to CD8 +< cells expressing a recombinant receptor and / or of CD4 +< cells to CD8 +< cells, which ratio optionally is approximately 1:1 or is between approximately 1:3 and approximately 3:1, such as approximately 1:1. In some aspects, the administration of a composition or dose with the target or desired ratio of different cell populations (such as CD4 +< :CD8 +< ratio or CAR +< CD4 +< :CAR+CD8 +< ratio, e.g., 1:1) involves the administration of a cell composition containing one of the populations and then administration of a separate cell composition comprising the other of the populations, where the administration is at or approximately at the target or desired ratio. In some aspects, administration of a dose or composition of cells at a defined ratio leads to improved expansion, persistence and / or antitumor activity of the T cell therapy.

[0218] In some instances, the subject receives multiple doses, e.g., two or more doses or multiple consecutive doses, of the cells. In some instances, two doses are administered to a subject. In some instances, the subject receives the consecutive dose, e.g., 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 instances, multiple consecutive doses are administered following the first dose, such that an additional dose or doses are administered following administration of the consecutive dose. In some aspects, the number of cells administered to the subject in the additional dose is the same as or similar to the first dose and / or consecutive dose. In some instances, the additional dose or doses are larger than prior doses.

[0219] In some aspects, the size of the first and / or consecutive dose is determined based on one or more criteria such as response of the subject to prior treatment, e.g. chemotherapy, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and / or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or a host immune response against the cells and / or recombinant receptors being administered.

[0220] In some aspects, the time between the administration of the first dose and the administration of the consecutive dose is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some instances, the administration of the consecutive dose is at a time point more than about 14 days after and less than about 28 days after the administration of the first dose. In some aspects, the time between the first and consecutive dose is about 21 days. In some instances, an additional dose or doses, e.g. consecutive doses, are administered following administration of the consecutive dose. In some aspects, the additional consecutive dose or doses are administered at least about 14 and less than about 28 days following administration of a prior dose. In some instances, the additional dose is administered less than about 14 days following the prior dose, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the prior dose. In some instances, no dose is administered less than about 14 days following the prior dose and / or no dose is administered more than about 28 days after the prior dose.

[0221] In some instances, the dose of cells, e.g., recombinant receptor-expressing cells, comprises two doses (e.g., a double dose), comprising a first dose of the T cells and a consecutive dose of the T cells, wherein one or both of the first dose and the second dose comprises administration of the split dose of T cells.

[0222] In some instances, the dose of cells is generally large enough to be effective in reducing disease burden.

[0223] In some instances, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell types. Thus, the dosage of cells in some instances is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4 +< to CD8 +< ratio. In some instances, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some instances, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

[0224] In some instances, the populations or sub-types of cells, such as CD8 +< and CD4 +< T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio (such as CD4 +< to CD8 +< ratio), e.g., within a certain tolerated difference or error of such a ratio.

[0225] In some instances, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4 +< cells and / or a desired dose of CD8 +< cells. In some aspects, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells of the population or sub-type, or minimum number of cells of the population or sub-type per unit of body weight.

[0226] Thus, in some instances, the dosage is based on a desired fixed dose of total cells and a desired ratio, and / or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub-populations. Thus, in some instances, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4 +< to CD8 +< cells, and / or is based on a desired fixed or minimum dose of CD4 +< and / or CD8 +< cells.

[0227] In some instances, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4 +< and CD8 +< cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios. For example, in some instances, the desired ratio (e.g., ratio of CD4 +< to CD8 +< cells) is between at or about 1:5 and at or about 5:1 (or greater than about 1:5 and less than about 5:1), or between at or about 1:3 and at or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between at or about 2:1 and at or about 1:5 (or greater than about 1:5 and less than about 2:1), such as at or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In some aspects, the tolerated 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 between these ranges.

[0228] In particular instances, the numbers and / or concentrations of cells refer to the number of recombinant receptor (e.g., CAR)-expressing cells. In other instances, the numbers and / or concentrations of cells refer to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

[0229] In some aspects, the size of the dose is determined based on one or more criteria such as response of the subject to prior treatment, e.g. chemotherapy, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and / or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or a host immune response against the cells and / or recombinant receptors being administered.

[0230] In some instances, the methods also include administering one or more additional doses of cells expressing a chimeric antigen receptor (CAR) and / or lymphodepleting therapy, and / or one or more steps of the methods are repeated. In some instances, the one or more additional dose is the same as the initial dose. In some instances, the one or more additional dose is different from the initial dose, e.g., higher, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold or more higher than the initial dose, or lower, such as e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold or more lower than the initial dose. In some instances, administration of one or more additional doses is determined based on response of the subject to the initial treatment or any prior treatment, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and / or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or a host immune response against the cells and / or recombinant receptors being administered.C. Methods of Treatment

[0231] Disclosed herein are methods of treatment, e.g., including administering any of the engineered cells or compositions containing engineered cells described herein, including one or more doses, e.g., a unit doses or any compositions containing one or more unit doses described herein. In some aspects, also disclosed are methods of administering any of the engineered cells or compositions containing engineered cells or unit doses described herein to a subject, such as a subject that has a disease or disorder. In some aspects, also disclosed are uses of any of the engineered cells or compositions containing engineered cells described herein for treatment of a disease or disorder. In some aspects, also disclosed are uses of any of the engineered cells or compositions containing engineered cells or unit doses described herein for the manufacture of a medicament for the treatment of a disease or disorder. In some aspects, also disclosed are any of the engineered cells or compositions containing engineered cells or unit doses described herein, for use in treatment of a disease or disorder, or for administration to a subject having a disease or disorder.

[0232] The engineered cells expressing a recombinant receptor, such as a chimeric antigen receptor (CAR), or compositions comprising the same, are useful in a variety of therapeutic, diagnostic and prophylactic indications. For example, the engineered cells or compositions comprising the engineered cells are useful in treating a variety of diseases and disorders in a subject. Such methods and uses include therapeutic methods and uses, for example, involving administration of the engineered cells, or compositions containing the same, to a subject having a disease, condition, or disorder, such as a tumor or cancer. In some instances, the engineered cells or compositions comprising the same are administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the engineered cells or compositions in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some instances, the methods are carried out by administering the engineered cells, or compositions comprising the same, to the subject having or suspected of having the disease or condition. In some instances, the methods thereby treat the disease or condition or disorder in the subject.

[0233] Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the disclosed methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., 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.

[0234] The disease or condition that is treated can be any in which expression of an antigen is associated with and / or involved in the etiology of a disease condition or disorder, e.g. causes, exacerbates or otherwise is involved in such disease, condition, or disorder. Exemplary diseases and conditions can include diseases or conditions associated with malignancy or transformation of cells (e.g. cancer), autoimmune or inflammatory disease, or an infectious disease, e.g. caused by a bacterial, viral or other pathogen. Exemplary antigens, which include antigens associated with various diseases and conditions that can be treated, are described above. In particular instances, the chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with the disease or condition.

[0235] Among the diseases, conditions, and disorders are tumors, including solid tumors, hematologic malignancies, and melanomas, and including localized and metastatic tumors, infectious diseases, such as infection with a virus or other pathogen, e.g., HIV, HCV, HBV, CMV, HPV, and parasitic disease, and autoimmune and inflammatory diseases. In some instances, the disease, disorder or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease or disorder. In some instances, the disease or condition is a B cell malignancy. In some instances, the B cell malignancy is a leukemia or a lymphoma. Such diseases include but are not limited to leukemia, lymphoma, e.g., acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), Anaplastic large cell lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM). In some instances, disease or condition is a B cell malignancy selected from among acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), and Diffuse Large B-Cell Lymphoma (DLBCL). In some instances, the disease or condition is NHL and the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), primary mediastinal large B cell lymphoma (PMBCL), T cell / histocyte-rich large B cell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL), and / or follicular lymphoma (FL), optionally, follicular lymphoma Grade 3B (FL3B).

[0236] In some aspects, the disease or condition is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), or Diffuse Large B-Cell Lymphoma (DLBCL). In some cases, the disease or condition is an NHL, such as or including an NHL that is an aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), primary mediastinal large B cell lymphoma (PMBCL), T cell / histocyte-rich large B cell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL), and / or follicular lymphoma (FL), optionally, follicular lymphoma Grade 3B (FL3B). In some aspects, the recombinant receptor, such as a CAR, specifically binds to an antigen associated with the disease or condition or expressed in cells of the environment of a lesion associated with the B cell malignancy. Antigens targeted by the receptors in some instances include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some instances, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30, or combinations thereof.

[0237] In some instances, the disease or condition is a myeloma, such as a multiple myeloma. In some aspects, the recombinant receptor, such as a CAR, specifically binds to an antigen associated with the disease or condition or expressed in cells of the environment of a lesion associated with the multiple myeloma. Antigens targeted by the receptors in some instances include antigens associated with multiple myeloma. In some aspects, the antigen, e.g., the second or additional antigen, such as the disease-specific antigen and / or related antigen, is expressed on multiple myeloma, such as B cell maturation antigen (BCMA), G protein-coupled receptor class C group 5 member D (GPRC5D), CD38 (cyclic ADP ribose hydrolase), CD138 (syndecan-1, syndecan, SYN-1), CS-1 (CS1, CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), BAFF-R, TACI and / or FcRH5. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, CD44, CD20, CD40, CD74, CD200, EGFR, β2-Microglobulin, HM1.24, IGF-1R, IL-6R, TRAIL-R1, and the activin receptor type IIA (ActRIIA). See Benson and Byrd, J. Clin. Oncol. (2012) 30(16): 2013-15; Tao and Anderson, Bone Marrow Research (2011):924058; Chu et al., Leukemia (2013) 28(4):917-27; Garfall et al., Discov Med. (2014) 17(91):37-46. In some instances, the antigens include those present on lymphoid tumors, myeloma, AIDS-associated lymphoma, and / or post-transplant lymphoproliferations, such as CD38. Antibodies or antigen-binding fragments directed against such antigens are known and include, for example, those described in U.S. Patent No. 8,153,765; 8,603477, 8,008,450; U.S. Pub. No. US20120189622 or US20100260748; and / or International PCT Publication Nos. WO2006099875, WO2009080829 or WO2012092612 or WO2014210064. In some instances, such antibodies or antigen-binding fragments thereof (e.g. scFv) are contained in multispecific antibodies, multispecific chimeric receptors, such as multispecific CARs, and / or multispecific cells.

[0238] In some instances, the disease or disorder is associated with expression of G protein-coupled receptor class C group 5 member D (GPRC5D) and / or expression of B cell maturation antigen (BCMA).

[0239] In some instances, the disease or disorder is a B cell-related disorder. In some of any of the disclosure of the methods, the disease or disorder associated with BCMA is an autoimmune disease or disorder. In some of any of the disclosure of the methods, the autoimmune disease or disorder is systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease, rheumatoid arthritis, ANCA associated vasculitis, idiopathic thrombocytopenia purpura (ITP), thrombotic thrombocytopenia purpura (TTP), autoimmune thrombocytopenia, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, vasculitis, diabetes mellitus, Reynaud's syndrome, anti-phospholipid syndrome, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, myasthenia gravis, or progressive glomerulonephritis.

[0240] In some instances, the disease or disorder is a cancer. In some instances, the cancer is a GPRC5D-expressing cancer. In some instances, the cancer is a plasma cell malignancy and the plasma cell malignancy is multiple myeloma (MM) or plasmacytoma. In some instances, the cancer is multiple myeloma (MM). In some instances, the cancer is a relapsed / refractory multiple myeloma.

[0241] In some instances, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In some instances, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, asthma, and / or a disease or condition associated with transplant.

[0242] In some instances, the antigen associated with the disease or disorder or includes αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase 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-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by the receptors in some instances include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some instances, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.

[0243] In some instances, the antigen is or includes a pathogen-specific or pathogen-expressed antigen. In some instances, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and / or parasitic antigens.

[0244] In some instances, the cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0245] In some instances, the cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such instances, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some instances, the first and second subjects are genetically identical. In some instances, the first and second subjects are genetically similar. In some instances, the second subject expresses the same HLA class or supertype as the first subject.

[0246] The cells can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some instances, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some instances, a given dose is administered by a single bolus administration of the cells. In some instances, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells. In some instances, administration of the cell dose or any additional therapies, e.g., the lymphodepleting therapy, intervention therapy and / or combination therapy, is carried out via outpatient delivery.

[0247] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some instances suitably administered to the subject at one time or over a series of treatments.

[0248] In some instances, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some instances are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some instances, the cells are administered prior to the one or more additional therapeutic agents. In some instances, the cells are administered after the one or more additional therapeutic agents. In some instances, the one or more additional agents include a cytokine, such as IL-2, for example, to enhance persistence. In some instances, the methods comprise administration of a chemotherapeutic agent.

[0249] In some instances, the methods comprise administration of a chemotherapeutic agent, e.g., a conditioning chemotherapeutic agent, for example, to reduce tumor burden prior to the administration.

[0250] Preconditioning subjects with immunodepleting (e.g., lymphodepleting) therapies in some aspects can improve the effects of adoptive cell therapy (ACT).

[0251] Thus, in some instances, the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a subject prior to the initiation of the cell therapy. For example, the subject may be administered a preconditioning agent at least 2 days prior, such as at least 3, 4, 5, 6, or 7 days prior, to the initiation of the cell therapy. In some instances, the subject is administered a preconditioning agent no more than 7 days prior, such as no more than 6, 5, 4, 3, or 2 days prior, to the initiation of the cell therapy.

[0252] In some instances, the subject is preconditioned with cyclophosphamide at a dose between or between about 20 mg / kg and 100 mg / kg, such as between or between about 40 mg / kg and 80 mg / kg. In some aspects, the subject is preconditioned with or with about 60 mg / kg of cyclophosphamide. In some instances, the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some instances, the cyclophosphamide is administered once daily for one or two days. In some instances, where the lymphodepleting agent comprises cyclophosphamide, the subject is administered cyclophosphamide at a dose between or between about 100 mg / m 2< and 500 mg / m 2< , such as between or between about 200 mg / m 2< and 400 mg / m 2< , or 250 mg / m 2< and 350 mg / m 2< , inclusive. In some instances, the subject is administered about 300 mg / m 2< of cyclophosphamide. In some instances, the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some instances, cyclophosphamide is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some instances, the subject is administered about 300 mg / m 2< of cyclophosphamide, daily for 3 days, prior to initiation of the cell therapy.

[0253] In some instances, where the lymphodepleting agent comprises fludarabine, the subject is administered fludarabine at a dose between or between about 1 mg / m 2< and 100 mg / m 2< , such as between or between about 10 mg / m 2< and 75 mg / m 2< , 15 mg / m 2< and 50 mg / m 2< , 20 mg / m 2< and 40 mg / m 2< , or 24 mg / m 2< and 35 mg / m 2< , inclusive. In some instances, the subject is administered about 30 mg / m 2< of fludarabine. In some instances, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some instances, fludarabine is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some instances, the subject is administered about 30 mg / m 2< of fludarabine, daily for 3 days, prior to initiation of the cell therapy.

[0254] In some instances, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some aspects, the subject is administered 60 mg / kg (~2 g / m 2< ) of cyclophosphamide and 3 to 5 doses of 25 mg / m 2< fludarabine prior to the first or subsequent dose.

[0255] Following administration of the cells, the biological activity of the engineered cell populations in some instances is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain instances, the ability of the engineered cells to destroy target cells can be measured using any suitable known methods, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain instances, the biological activity of the cells is measured by assaying expression and / or secretion of one or more cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

[0256] In certain instances, the engineered cells are further modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population can be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known. See, for instance, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Patent 5,087,616.

[0257] In some instances, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some instances are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some instances, the cells are administered prior to the one or more additional therapeutic agents. In some instances, the cells are administered after the one or more additional therapeutic agents. In some instances, the one or more additional agent includes a cytokine, such as IL-2, for example, to enhance persistence.D. Monitoring or Assessing Therapy with Therapeutic T Cell Compositions1. Assessing Pharmacokinetics (PK) of Engineered Cells, e.g. Peak Cell Levels and Patient Factors

[0258] In some instances, the method includes assessment of the exposure, number, concentration, persistence and proliferation of the T cells, e.g., T cells administered for the T cell based therapy. In some instances, the method includes assessment of the exposure, number or level of engineered T cells, e.g., T cells administered for the T cell based therapy, or subset thereof, such as CD3 +< cells, CD4 +< cells, CD8 +< cells, CD3 +< CAR +< cells, CD4 +< CAR +< cells or CD8 +< CAR +< cells. In some instances, the exposure, or prolonged expansion and / or persistence of the cells, and / or changes in cell phenotypes or functional activity of the cells, e.g., cells administered for immunotherapy, e.g. T cell therapy, in the methods disclosed herein, can be measured by assessing the characteristics of the T cells in vitro or ex vivo. In some instances, such assays can be used to determine or confirm the function of the T cells used for the immunotherapy, e.g. T cell therapy, before or after administering the cell therapy disclosed herein.

[0259] In some aspects, the exposure, number, concentration, persistence and proliferation relate to pharmacokinetic parameters. In some cases, pharmacokinetics can be assessed by measuring such parameters as the maximum (peak) plasma concentration (C max ), the peak time (i.e. when maximum plasma concentration (C max ) occurs; T max ), the minimum plasma concentration (i.e. the minimum plasma concentration between doses of a therapeutic agent, e.g., CAR +< T cells; C min ), the elimination half-life (T 1 / 2 ) and area under the curve (i.e. the area under the curve generated by plotting time versus plasma concentration of the therapeutic agent CAR +< T cells; AUC), following administration. The concentration of a particular therapeutic agent, e.g., CAR +< T cells, in the plasma following administration can be measured using any method known in the art suitable for assessing concentrations of the therapeutic agents, e.g., CAR +< T cells, in samples of blood, or any methods described herein. For example, nucleic acid-based methods, such as quantitative PCR (qPCR) or flow cytometry-based methods, or other assays, such as an immunoassay, ELISA, or chromatography / mass spectrometry-based assays can be used.

[0260] In some instances, the pharmacokinetics (PK) of administered cells, e.g., CAR +< T cell composition, are determined to assess the availability, e.g., bioavailability, of the administered cells. In some instances, the determined pharmacokinetic parameters of the administered cells include maximum (peak) plasma concentrations (C max ), such as C max of CD3 +< CAR +< cells, CD4 +< CAR +< cells and or CD8 +< CAR +< T cells; the time point at which C max is achieved (T max ), such as the T max of CD3 +< CAR +< cells, CD4 +< CAR +< cells and or CD8 +< CAR +< T cells, and or area under the curve (AUC), such as the AUC 0-28 , of CD3 +< CAR +< cells, CD4 +< CAR +< cells and or CD8 +< CAR +< T cells. In some instances, the pharmacokinetic parameter is peak CD3 +< CAR +< T cell concentration (C max CD3 +< CAR +< T cells), or CD8 +< CAR +< T cell concentration (C max CD8 +< CAR +< T cells). In some instances, the pharmacokinetic parameter is AUC 0-28 , of CD3 +< CAR +< T cells, (AUC 0-28 CD3 +< CAR +< T cells), or AUC 0-28 , of CD8 +< CAR +< T cells, (AUC0-28 CD8 +< CAR +< T cells),

[0261] In some instances, "exposure" can refer to the body exposure of a therapeutic agent, e.g., CAR +< T cells in the plasma (blood or serum) after administration of the therapeutic agent over a certain period of time. In some instances exposure can be set forth as the area under the therapeutic agent concentration-time curve (AUC) as determined by pharmacokinetic analysis after administration of a dose of the therapeutic agent, e.g., CAR +< T cells. In some cases, the AUC is expressed in cells*days / µL, for cells administered in cell therapy, or in corresponding units thereof. In some instances, the AUC is measured as an average AUC in a patient population, such as a sample patient population, e.g., the average AUC from one or more patient(s). In some instances, systemic exposure refers to the area under the curve (AUC) within a certain period of time, e.g., from day 0 to day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days or more, or week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, or month 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 48 or more. In some instances, the AUC is measured as an AUC from day 0 to day 28 (AUC 0-28 ) after administration of the therapeutic agent, e.g., CAR +< T cells, including all measured data and data extrapolated from measured pharmacokinetic (PK) parameters, such as an average AUC from a patient population, such as a sample patient population. In some instances, to determine exposure over time, e.g., AUC for a certain period of time, such as AUC 0-28 , a therapeutic agent concentration-time curve is generated, using multiple measurements or assessment of parameters, e.g., cell concentrations, over time, e.g., measurements taken every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21 or 28 days or more.

[0262] In some instances, the presence and / or amount of cells expressing the recombinant receptor (e.g., CAR-expressing cells administered for T cell based therapy) in the subject following the administration of the T cells and before, during and / or after the administration of the therapy is detected. In some aspects, nucleic acid-based methods, such as quantitative PCR (qPCR), are used to assess the quantity of cells expressing the recombinant receptor (e.g., CAR-expressing cells administered for T cell based therapy) in the blood or serum or organ or tissue sample (e.g., disease site, e.g., tumor sample) of the subject. In some aspects, persistence is quantified as copies of DNA or plasmid encoding the receptor, e.g., CAR, per microgram of DNA, or as the number of receptor-expressing, e.g., CAR-expressing, cells per microliter of the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of the sample. In some instances, the primers or probe used for qPCR or other nucleic acid-based methods are specific for binding, recognizing and / or amplifying nucleic acids encoding the recombinant receptor, and / or other components or elements of the plasmid and / or vector, including regulatory elements, e.g., promoters, transcriptional and / or post-transcriptional regulatory elements or response elements, or markers, e.g., surrogate markers. In some instances, the primers can be specific for regulatory elements, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some examples, the presence and / or amount of cells expressing the recombinant receptor is expressed as copies of the nucleic acid sequence (e.g., transgene sequence) encoding the CAR or a nucleic acid sequence operably connected to the CAR-encoding sequences, per mass of DNA (e.g., copies / µg of DNA); AUC of the curve of copies / µg of DNA over time, maximum or peak copies / µg of DNA following treatment, or copies / µg of DNA at day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, or week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more post-treatment or initiation thereof.

[0263] In some instances, the cells are detected in the subject at or at least at 4, 14, 15, 27, or 28 days following the administration of the T cells, e.g., CAR-expressing T cells. In some aspects, the cells are detected at or at least at 2, 4, or 6 weeks following, or 3, 6, or 12, 18, or 24, or 30 or 36 months, or 1, 2, 3, 4, 5, or more years, following the administration of the T cells, e.g., CAR-expressing T cells.

[0264] In some instances, the peak levels and / or AUC are assessed and / or the sample is obtained from the subject at a time that is at least 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days after initiation of administration of the genetically engineered cells. In some instances the peak levels and / or AUC are assessed and / or the sample is obtained from the subject at a time that is between or between about 11 to 22 days, 12 to 18 days or 14 to 16 days, each inclusive, after initiation of administration of the genetically engineered cells.

[0265] The exposure, e.g., number or concentration of cells, e.g. T cells administered for T cell therapy, indicative of expansion and / or persistence, may be stated in terms of maximum numbers or concentration of the cells to which the subject is exposed, duration of detectable cells or cells above a certain number or percentage, area under the curve (AUC) for number or concentration of cells over time, and / or combinations thereof and indicators thereof. Such outcomes may be assessed using known methods, such as qPCR to detect copy number of nucleic acid encoding the recombinant receptor compared to total amount of nucleic acid or DNA in the particular sample, e.g., blood, serum, plasma or tissue, such as a tumor sample, and / or flow cytometric assays detecting cells expressing the receptor generally using antibodies specific for the receptors. Cell-based assays may also be used to detect the number or percentage or concentration of functional cells, such as cells capable of binding to and / or neutralizing and / or inducing responses, e.g., cytotoxic responses, against cells of the disease or condition or expressing the antigen recognized by the receptor.

[0266] In some aspects, increased exposure of the subject to the cells includes increased expansion of the cells. In some instances, the receptor expressing cells, e.g. CAR-expressing cells, expand in the subject following administration of the T cells, e.g., CAR-expressing T cells.

[0267] In some instances, cells expressing the receptor are detectable in the serum, plasma, blood or tissue, e.g., tumor sample, of the subject, e.g., by a specified method, such as qPCR or flow cytometry-based detection method, at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 or more days following administration of the T cells, e.g., CAR-expressing T cells, for at least at or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or more weeks following the administration of the T cells, e.g., CAR-expressing T cells.

[0268] In some aspects, at least at or about 1 x 10 2< , at least at or about 1 x 10 3< , at least at or about 1 x 10 4< , at least at or about 1 x 10 5< , or at least at or about 1 x 10 6< or at least at or about 5 x 10 6< or at least at or about 1 x 10 7< or at least at or about 5 x 10 7< or at least at or about 1 x 10 8< recombinant receptor-expressing, e.g., CAR-expressing cells, and / or at least 10, 25, 50, 100, 200, 300, 400, or 500, or 1000 receptor-expressing cells per microliter, e.g., at least 10 per microliter, are detectable or are present in the subject or fluid, plasma, serum, tissue, or compartment thereof, such as in the blood, e.g., peripheral blood, or disease site, e.g., tumor, thereof. In some instances, such a number or concentration of cells is detectable in the subject for at least at or about 20 days, at least at or about 40 days, or at least at or about 60 days, or at least at or about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 2 or 3 years, following administration of the T cells, e.g., CAR-expressing T cells. Such cell numbers may be as detected by flow cytometry-based or quantitative PCR-based methods and extrapolation to total cell numbers using known methods. See, e.g., Brentjens et al., Sci Transl Med. 2013 5(177), Park et al, Molecular Therapy 15(4):825-833 (2007), Savoldo et al., JCI 121(5):1822-1826 (2011), Davila et al., (2013) PLoS ONE 8(4):e61338, Davila et al., Oncoimmunology 1(9):1577-1583 (2012), Lamers, Blood 2011 117:72-82, Jensen et al., Biol Blood Marrow Transplant 2010 September; 16(9): 1245-1256, Brentjens et al., Blood 2011 118(18):4817-4828.

[0269] In some aspects, the copy number of nucleic acid encoding the recombinant receptor, e.g., vector copy number, per 100 cells, for example in the peripheral blood or bone marrow or other compartment, as measured by immunohistochemistry, PCR, and / or flow cytometry, is at least 0.01, at least 0.1, at least 1, or at least 10, at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at least about 6 weeks, or at least about 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, or 12 months or at least 2 or 3 years following administration of the cells, e.g., CAR-expressing T cells. In some instances, the copy number of the vector expressing the receptor, e.g. CAR, per microgram of genomic DNA is at least 100, at least 1000, at least 5000, or at least 10,000, or at least 15,000 or at least 20,000 at a time about 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeks following administration of the T cells, e.g., CAR-expressing T cells or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or at least 2 or 3 years following such administration.

[0270] In some aspects, the receptor, e.g. CAR, expressed by the cells, is detectable by quantitative PCR (qPCR) or by flow cytometry in the subject, plasma, serum, blood, tissue and / or disease site thereof, e.g., tumor site, at a time that is at least at or about 3 months, at least at or about 6 months, at least at or about 12 months, at least at or about 1 year, at least at or about 2 years, at least at or about 3 years, or more than 3 years, following the administration of the cells, e.g., following the initiation of the administration of the T cells. In some instances, the area under the curve (AUC) for concentration of receptor- (e.g., CAR-) expressing cells in a fluid, plasma, serum, blood, tissue, organ and / or disease site, e.g. tumor site, of the subject over time following the administration of the T cells, e.g., CAR-expressing T cells, is measured.

[0271] Also disclosed are methods of assessing likelihood of a response or a durable response. In some instances, the methods involve detecting, in a biological sample from a subject, peak levels of one or more inflammatory marker and / or peak levels of genetically engineered cells comprising T cells expressing a chimeric antigen receptor (CAR), wherein the subject has been previously administered a dose of the genetically engineered cells for treating a disease or condition. In some instances, the methods involve comparing, individually, the peak levels to a threshold value, thereby determining a likelihood that a subject will achieve a durable response to the administration of the genetically engineered cells.

[0272] In some instances, the subject is likely to achieve a response or a durable response if the peak levels of the one or more inflammatory marker is below a threshold value and the subject is not likely to achieve a durable response if the peak levels of the one or more inflammatory marker is above a threshold value. In some instances, the subject is likely to achieve a durable response if the peak level of the genetically engineered cells is within a therapeutic range between a lower threshold value and an upper threshold value and the subject is not likely to achieve a durable response if the peak level of the genetically engineered cells is below the lower threshold value or is above the upper threshold value.

[0273] In some cases, the disclosure involve assessing parameters, such as attributes, factors, characteristic of the patient and / or the disease or condition, and / or expression of biomarkers. In some instances, the assessed parameters are associated with and / or correlated with pharmacokinetic parameters, response, durable response and / or development of toxicity. In some instances, the parameters include patient factors or patient attributes. In some instances, the assessed parameters are correlated with the phenotype of the cell compositions disclosed herein. In some instances, the assessed paramters indicate whether the output composition will have the necessary cellular composition (e.g., percentage of cell phenotypes) correlated with progression free survival and / or durable response. In some instances, the assessed parameters can be used to predict the likelihood of producing an output composition useful for therapeutic administration. In some embodiemts, patients with high disease burden, for example those with high SPD (e.g., a value of greater than or equal to 50 cm 2< ) or CRP values (e.g., a value greater than or equal to 20 mg / L), are less likely to produce output compositions useful for therapeutic administration. In some instances, patients with low disease burden, for example those with low SPD (e.g., a value of less than 50 cm 2< ) or CRP values (e.g., a value less than 20 mg / L), are more likely to produce output compositions useful for therapeutic administration. In some instances, the parameters include attributes, factors, characteristic of the disease or condition. In some instances, the parameters are assessed prior to treatment, e.g., prior to administration of the cell therapy. In some instances, the parameters are assessed after treatment, e.g., after administration of one or more doses of the cell therapy.

[0274] In some instances, the parameter is or includes pharmacokinetic parameters, e.g., maximum (peak) plasma concentration (C max ), the peak time (i.e., when maximum plasma concentration (C max ) occurs; T max ), the minimum plasma concentration (i.e., the minimum plasma concentration between doses of a therapeutic agent, e.g., CAR +< T cells; C min ), the elimination half-life (T 1 / 2 ) and area under the curve (i.e., the area under the curve generated by plotting time versus plasma concentration of the therapeutic agent CAR +< T cells; AUC; such as AUC 0-28 ).

[0275] In some instances, the parameter is or includes one or more factors indicative of the state of the patient and / or the disease or condition of the patient. In some instances, the parameter is indicative of tumor burden. In some instances, the factor indicative of tumor burden is a volumetric measure of tumor(s). In some instances, the volumetric measure is a measure of the lesion(s), such as the tumor size, tumor diameter, tumor volume, tumor mass, tumor load or bulk, tumor-related edema, tumor-related necrosis, and / or number or extent of metastases. In some instances, the volumetric measure of tumor is a bidimensional measure. For example, in some instances, the area of lesion(s) are calculated as the product of the longest diameter and the longest perpendicular diameter of all measurable tumors. In some cases, the volumetric measure of tumor is a unidimensional measure. In some cases, the size of measurable lesions is assessed as the longest diameter. In some instances, the sum of the products of diameters (SPD), longest tumor diameters (LD), sum of longest tumor diameters (SLD), necrosis, tumor volume, necrosis volume, necrosis-tumor ratio (NTR), peritumoral edema (PTE), and edema-tumor ratio (ETR) is measured.

[0276] Exemplary methods for measuring and assessing tumor burden include those described in, e.g., Carceller et al., Pediatr Blood Cancer. (2016) 63(8):1400-1406 and Eisenhauer et al., Eur J Cancer. (2009) 45(2):228-247. In some instances, the volumetric is a sum of the products of diameters (SPD) measured by determining the sum of the products of the largest perpendicular diameters of all measurable tumors. In some aspects, the tumor or lesion is measured in one dimension with the longest diameter (LD) and / or by determining the sum of longest tumor diameters (SLD) of all measurable lesions. In some instances, the volumetric measure of tumor is a volumetric quantification of tumor necrosis, such as necrosis volume and / or necrosis-tumor ratio (NTR), see Monsky et al., Anticancer Res. (2012) 32(11): 4951-4961. In some aspects, the volumetric measure of tumor is a volumetric quantification of tumor-related edema, such as peritumoral edema (PTE) and / or edema-tumor ratio (ETR). In some instances, measuring can be performed using imaging techniques such as computed tomography (CT), positron emission tomography (PET), and / or magnetic resonance imaging (MRI) of the subject.

[0277] In some instances, the volumetric measure is SPD and in some cases, development of toxicity, e.g., CRS or NT, is correlated with the SPD value that is above a threshold value. In some instances, the volumetric measure is SPD, and the threshold value is or is about 30 cm 2< , is or is about 40 cm 2< , is or is about 50 cm 2< , is or is about 60 cm 2< , or is or is about 70 cm 2< . In some instances, the volumetric measure is SPD and the threshold value is or is about 30 cm 2< , is or is about 40 cm 2< , is or is about 50 cm 2< , is or is about 60 cm 2< , or is or is about 70 cm 2< .

[0278] In some instances, the volumetric measure of tumor is determined at a screening session, such as a routine assessment or blood draw to confirm and / or identify the condition or disease in the subject.

[0279] In some aspects, the parameter, e.g., measurements of tumor burden, correlates to and / or is associated with pharmacokinetic parameters. In some instances, the parameter, including pharmacokinetic parameters, is associated with response and / or durable response, and / or a risk for developing toxicity, e.g., CRS or neurotoxicity (NT).

[0280] In some instances, the parameter is or includes at least one or a panel of biomarkers. In some instances, expression and / or presence of the biomarker is associated with and / or correlated with pharmacokinetic parameters, response, durable response and / or development of toxicity. In some instances, the parameter is compared to a particular reference value, e.g., those associated with response and / or durable response, and / or a risk for developing toxicity, e.g., CRS or neurotoxicity (NT). In some instances, the methods also involve administering an agent capable of modulating CAR +< T cell expansion, proliferation, and / or activity, to the subject, based on the assessment of patient factors and / or biomarkers.

[0281] In some aspects, the instances involve obtaining a biological sample for detecting the parameter and / or assessing the presence of and / or or detecting the parameter. In some instances, the biological sample is obtained generally within 4 hours to 12 months of administration of the cell therapy, or a first administration or dose thereof, or after the initiation of any of the foregoing, such as generally within or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60 or 90 or more days, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 48 or more months, after administration of the cell therapy, or a first administration or dose thereof, or after the initiation of any of the foregoing. In some instances, the parameter is assessed or measured in a subject prior to administration of the cell therapy or soon after administration of the cell therapy, or a first administration or dose thereof, or after the initiation of any of the foregoing , such as generally within 4 hours to 3 days of administration of the cell therapy, or a first administration or dose thereof, or after the initiation of any of the foregoing, such as generally within or about 1 day, 2 days or 3 days after administration of the cell therapy, or a first administration or dose thereof, or after the initiation of any of the foregoing. In some instances, the parameter is assessed or measured. In some instances, the parameter is assessed generally within 4 hours to 12 months of administration of the cell therapy, or a first administration or dose thereof, or after the initiation of any of the foregoing, such as generally within or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60 or 90 or more days, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 48 or more months, after administration of the cell therapy, or a first administration or dose thereof, or after the initiation of any of the foregoing.

[0282] In some aspects, the parameter, e.g., patient factor and / or biomarker, correlates to and / or is associated with pharmacokinetic parameters. In some instances, the parameter, including pharmacokinetic parameters, is associated with response and / or durable response, and / or a risk for developing toxicity, e.g., CRS or neurotoxicity (NT).

[0283] In some instances, the parameter is a biomarker. In some instances, the parameter is or includes the expression of the biomarker and / or the number, concentration, and / or percentage of cells that express a particular biomarker. In some instances, the parameter includes biomarkers or each biomarker in a panel that comprises a plurality of biomarkers. In some instances, the biomarker is or comprises a cytokine and / or other serum or blood factor, such as any as described herein. In some instances, the biomarker or each biomarker in a panel is a cytokine, which, in some cases, can be a chemokine. In some instances, the biomarkers or each biomarker in a panel comprises a soluble receptor. In some instances, the biomarkers or each biomarker in a panel comprises a soluble serum protein. Exemplary biomarkers or panel of biomarkers is described herein.

[0284] In some instances, the parameter is or includes levels and / or concentrations of a blood analyte. In some instances, the parameter is or includes levels and / or concentrations of an inflammatory marker. In some instances, the blood analyte and / or inflammatory marker is or includes levels and / or concentrations of interleukin-7 (IL-7), IL-15, macrophage inflammatory protein (MIP-1α). In some instances, the blood analyte and / or inflammatory marker is or includes levels and / or concentrations of IL-6, IL-10, IL-16, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), MIP-1α, MIP-1β, Monocyte chemoattractant protein-1 (MCP-1), and C-X-C motif chemokine 10 (CXCL10). In some instances, the blood analyte and / or inflammatory marker is or includes levels and / or concentrations of ferritin, C-reactive protein (CRP), D-dimer (fibrin degradation product), IL-6, IL-10, IL-15, IL-16, TNF-α, MIP-1α, and MIP-1β. In some instances, the blood analyte and / or inflammatory marker is or includes levels and / or concentrations of LDH, Ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 and / or MIP-1β. In some instances, the blood analyte and / or inflammatory marker is or includes levels and / or concentrations of CRP, Serum Amyloid A1 (SAA-1), IL-2, IL-6, IL-10, IL-15, TNF-α, MIP-1α, MIP-1β, MCP-1, CXCL10 and C-C Motif Chemokine Ligand 13 (CCL13). In some instances, the blood analyte and / or inflammatory marker is or includes levels and / or concentrations of LDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ and / or MIP-1α.

[0285] In some instances, an inflammatory marker is or includes the level or presence of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin, ferritin, β2 microglobulin (β2-M), or lactate dehydrogenase (LDH) is detected and assessed. In some instances, the inflammatory marker is assessed using an immune assay. For example, an enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), surface plasmon resonance (SPR), Western Blot, Lateral flow assay, immunohistochemistry, protein array or immuno-PCR (iPCR) can be used to detect the inflammatory marker. In some instances, using the articles of manufacture include detecting an inflammatory marker indicative of tumor burden. In some cases, the assaying or assessing of an inflammatory marker is using flow cytometry. In some cases, the reagent is a soluble protein that binds the inflammatory marker. In some example, the reagent is a protein that binds C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin, ferritin, β2 microglobulin (β2-M), or lactate dehydrogenase (LDH).

[0286] In some instances, the biomarker, e.g., inflammatory marker is or includes C-reactive protein (CRP). In some instances, CRP is assessed using an in vitro enzyme-linked immunosorbent assay to obtain a quantitative measurement of human CRP from a sample such as serum, plasma, or blood. In some examples, CRP is detected using a human Enzyme- Linked Immunosorbent Assay (ELISA). In some instances, the biomarker, e.g. inflammatory marker is or includes erythrocyte sedimentation rate (ESR). In some instances, ESR is assessed by measuring the distance (in millimeters per hour) that red cells have fallen after separating from the plasma in a vertical pipette or tube. In some embodimments the biomarker is or includes albumin. In some aspects, albumin is assessed using a colorimetric test or an in vitro enzyme-linked immunosorbent assay. In some examples, albumin is detected using a human Enzyme-Linked Immunosorbent Assay (ELISA). In some instances, the biomarker, e.g., inflammatory marker, is or includes ferritin or β2 microglobulin. In some instances, ferritin or β2 microglobulin is assessed using an immunoassay or detected using an ELISA. In some aspects, the biomarker, e.g., inflammatory marker, is or includes lactate dehydrogenase (LDH), and LDH is assessed using a colorimetric test or an in vitro enzyme-linked immunosorbent assay.

[0287] In some instances, the parameter is LDH and in some cases, development of toxicity, e.g., CRS or NT, is correlated with the LDH value that is above a threshold value. In some instances, the inflammatory marker is LDH and the threshold value is or is about 300 units per liter, is or is about 400 units per liter, is or is about 500 units per liter or is or is about 600 units per liter.

[0288] In some instances, the parameter or biomarker is LDH. In some instances, the biomarker is LDH and the threshold value is 500 U / L or higher. In some instances, the parameter or biomarker is SPD. In some instances, the parameter is SPD, and the threshold value is or is about 50 cm 2< or higher. In some instances, biomarker or parameters are SPD and LDH, and the threshold values are SPD of 50 cm 2< or higher and LDH of 500 U / L or higher. In some instances, the biomarkers or parameters are associated with increased risk of developing CRS or NT. In some instances, a measurement of the parameter or marker that is above the threshold value, e.g., SPD of 50 cm 2< or higher and LDH of 500 U / L or higher, are associated with an approximately 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold or more increased risk of developing CRS or NT, such as any grade CRS or NT. In some instances, a measurement of the parameter or marker that is below the threshold value, e.g., SPD of lower than 500 cm 2< and LDH of lower than 500 U / L, are associated with an approximately 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold or more decreased risk of developing CRS or NT, such as any grade CRS or NT.2. Assessing Response, Efficacy and Survival

[0289] In some aspects, the methods involve determining the association or relationship between the dose of cells having a particular cell phenotype, e.g., CCR7 +< cells, and response, efficacy and / or survival in subjects administered such doses of cells.

[0290] In some aspects, response rates in subjects, such as subjects with NHL, are based on the Lugano criteria. (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin Clin Oncol 4(1):5). In some aspects, response assessment utilizes any of clinical, hematologic, and / or molecular methods. In some aspects, response assessed using the Lugano criteria involves the use of positron emission tomography (PET)-computed tomography (CT) and / or CT as appropriate. PET-CT evaluations may further comprise the use of fluorodeoxyglucose (FDG) for FDG-avid lymphomas. In some aspects, where PET-CT will be used to assess response in FDG-avid histologies, a 5-point scale may be used. In some respects, the 5-point scale comprises the following criteria: 1, no uptake above background; 2, uptake ≤ mediastinum; 3, uptake > mediastinum but ≤ liver; 4, uptake moderately > liver; 5, uptake markedly higher than liver and / or new lesions; X, new areas of uptake unlikely to be related to lymphoma.

[0291] In some aspects, a complete response (CR) as described using the Lugano criteria involves a complete metabolic response and a complete radiologic response at various measureable sites. In some aspects, these sites include lymph nodes and extralymphatic sites, wherein a CR is described as a score of 1, 2, or 3 with or without a residual mass on the 5-point scale, when PET-CT is used. In some aspects, in Waldeyer's ring or extranodal sites with high physiologic uptake or with activation within spleen or marrow (e.g., with chemotherapy or myeloid colony-stimulating factors), uptake may be greater than normal mediastinum and / or liver. In this circumstance, complete metabolic response may be inferred if uptake at sites of initial involvement is no greater than surrounding normal tissue even if the tissue has high physiologic uptake. In some aspects, response is assessed in the lymph nodes using CT, wherein a CR is described as no extralymphatic sites of disease and target nodes / nodal masses must regress to ≤ 1.5 cm in longest transverse diameter of a lesion (LDi). Further sites of assessment include the bone marrow wherein PET-CT-based assessment should indicate a lack of evidence of FDG-avid disease in marrow and a CT-based assessment should indicate a normal morphology, which if indeterminate should be IHC negative. Further sites may include assessment of organ enlargement, which should regress to normal. In some aspects, nonmeasured lesions and new lesions are assessed, which in the case of CR should be absent (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin Clin Oncol 4(1):5).

[0292] In some aspects, a partial response (PR) as described using the Lugano criteria involves a partial metabolic and / or radiological response at various measureable sites. In some aspects, these sites include lymph nodes and extralymphatic sites, wherein a PR is described as a score of 4 or 5 with reduced uptake compared with baseline and residual mass(es) of any size, when PET-CT is used. At interim, such findings can indicate responding disease. At the end of treatment, such findings can indicate residual disease. In some aspects, response is assessed in the lymph nodes using CT, wherein a PR is described as ≥50% decrease in SPD of up to 6 target measureable nodes and extranodal sites. If a lesion is too small to measure on CT, 5 mm × 5 mm is assigned as the default value; if the lesion is no longer visible, the value is 0 mm × 0 mm; for a node >5 mm × 5 mm, but smaller than normal, actual measurements are used for calculation. Further sites of assessment include the bone marrow wherein PET-CT-based assessment should indicate residual uptake higher than uptake in normal marrow but reduced compared with baseline (diffuse uptake compatible with reactive changes from chemotherapy allowed). In some aspects, if there are persistent focal changes in the marrow in the context of a nodal response, consideration should be given to further evaluation with MRI or biopsy, or an interval scan. In some aspects, further sites may include assessment of organ enlargement, where the spleen must have regressed by >50% in length beyond normal. In some aspects, nonmeasured lesions and new lesions are assessed, which in the case of PR should be absent / normal, regressed, but no increase. No response / stable disease (SD) or progressive disease (PD) can also be measured using PET-CT and / or CT based assessments. (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin Clin Oncol 4(1):5).

[0293] In some respects, progression-free survival (PFS) is described as the length of time during and after the treatment of a disease, such as cancer, that a subject lives with the disease but it does not get worse. In some aspects, objective response (OR) is described as a measurable response. In some aspects, objective response rate (ORR) is described as the proportion of patients who achieved CR or PR. In some aspects, overall survival (OS) is described as the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, that subjects diagnosed with the disease are still alive. In some aspects, event-free survival (EFS) is described as the length of time after treatment for a cancer ends that the subject remains free of certain complications or events that the treatment was intended to prevent or delay. These events may include the return of the cancer or the onset of certain symptoms, such as bone pain from cancer that has spread to the bone, or death.

[0294] In some instances, the measure of duration of response (DOR) includes the time from documentation of tumor response to disease progression. In some instances, the parameter for assessing response can include durable response, e.g., response that persists after a period of time from initiation of therapy. In some instances, durable response is indicated by the response rate at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months after initiation of therapy. In some instances, the response is durable for greater than 3 months or greater than 6 months.

[0295] In some aspects, the RECIST criteria is used to determine objective tumor response; in some aspects, in solid tumors. (Eisenhauer et al., European Journal of Cancer 45 (2009) 228-247.) In some aspects, the RECIST criteria is used to determine objective tumor response for target lesions. In some respects, a complete response as determined using RECIST criteria is described as the disappearance of all target lesions and any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. In other aspects, a partial response as determined using RECIST criteria is described as at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters. In other aspects, progressive disease (PD) is described as at least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm (in some aspects the appearance of one or more new lesions is also considered progression). In other aspects, stable disease (SD) is described as neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.

[0296] In some aspects, the administration in accord with the disclosed methods, and / or with the disclosed articles of manufacture or compositions, generally reduces or prevents the expansion or burden of the disease or condition in the subject. For example, where the disease or condition is a tumor, the methods generally reduce tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable cancer and / or improve prognosis or survival or other symptom associated with tumor burden.

[0297] Disease burden can encompass a total number of cells of the disease in the subject or in an organ, tissue, or bodily fluid of the subject, such as the organ or tissue of the tumor or another location, e.g., which would indicate metastasis. For exam...

Claims

1. A method of determining a unit dose of engineered T cells for treating a subject, the method comprising: (a) assessing, in a therapeutic composition comprising a plurality of CD8+ and / or CD4+ T cells engineered to express a recombinant receptor, the number, percentage or ratio of T cells that express the recombinant receptor and CCR7 and CD27 (receptor+ / CCR7+ / CD27+); (b) based on the number, percentage or ratio of receptor+ / CCR7+ / CD27+ cells, determining one or more unit doses of cells for administration to a subject having a disease or condition, wherein the unit dose comprises a defined number of recombinant receptor-expressing CD8+ T cells that express CCR7 and CD27 (receptor+ / CD8+ / CCR7+ / CD27+ cells) and / or a defined number of recombinant receptor-expressing CD4+ T cells that express CCR7 and CD27 (receptor+ / CD4+ / CCR7+ / CD27+ cells) and / or a defined ratio of receptor+ / CD8+ / CCR7+ / CD27+ cells to receptor+ / CD4+ / CCR7+ / CD27+ cells and / or a defined ratio of receptor+ / CD8+ / CCR7+ / CD27+ cells and / or receptor+ / CD4+ / CCR7+ / CD27+ cells to another subset of cells in the composition; wherein the recombinant receptor is a chimeric antigen receptor (CAR).

2. A method of producing a composition comprising a unit dose of a T cell composition, the method comprising: (a) assessing, in a therapeutic composition comprising a plurality of CD8+ and / or CD4+ T cells engineered to express a recombinant receptor, the number, percentage or ratio of T cells that express the recombinant receptor and CCR7 and CD27 (receptor+ / CCR7+ / CD27+); and (b) filling a container with all or a portion of the therapeutic composition and optionally another solution to achieve a unit dose of the T cell composition, wherein the unit dose comprises a defined number of recombinant receptor-expressing CD8+ T cells that express CCR7 and CD27 (receptor+ / CD8+ / CCR7+ / CD27+ cells) and / or a defined number of recombinant receptor-expressing CD4+ T cells that express CCR7 and CD27 (receptor+ / CD4+ / CCR7+ / CD27+ cells) and / or a defined ratio of receptor+ / CD8+ / CCR7+ / CD27+ cells to receptor+ / CD4+ / CCR7+ / CD27+ cells and / or a defined ratio of receptor+ / CD8+ / CCR7+ / CD27+ cells and / or receptor+ / CD4+ / CCR7+ / CD27+ cells to another subset of cells in the composition; wherein the recombinant receptor is a chimeric antigen receptor (CAR).

3. A therapeutic composition comprising a plurality of CD8+ and CD4+ T cells engineered to express a recombinant receptor for use in a method of treating a subject having a disease or condition, wherein the method comprises administering one or more unit doses of the therapeutic composition to the subject, and wherein at least 50%, 60%, 70%, 80%, or 90% of the total receptor+ / CD8+ cells in the composition are receptor+ / CD8+ / CCR7+ / CD27+ and at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total receptor+ / CD4+ cells in the composition are receptor+ / CD4+ / CCR7+ / CD27+; wherein the recombinant receptor is a chimeric antigen receptor (CAR).

4. The method of claim 1 or claim 2, or the therapeutic composition for use according to claim 3, wherein: i) the unit dose comprises between 1 x 105 and 1 x 108, between 5 x 105 and 1 x 107, or between 1 x 106 and 1 x 107 total CD8+ cells that express the recombinant receptor (receptor+ / CD8+ cells) or total CD4+ cell that express the recombinant receptor (receptor+ / CD4+ cells), total receptor+ / CD8+ / CCR7+ / CD27+ cells, or total receptor+ / CD4+ / CCR7+ / CD27+ cells, each inclusive; and / or ii) the unit dose comprises no more than 1 x 108, no more than 5 x 107, no more than 1 x 107, no more than 5 x 106, no more than 1 x 106, or no more than 5 x 105 total receptor+ / CD8+ cells or total receptor+ / CD4+ cells, total receptor+ / CD8+ / CCR7+ / CD27+ cells, or total receptor+ / CD4+ / CCR7+ / CD27+ cells; and / or iii) the unit dose comprises at least 3 x 106, 4 x 106, 5 x 106, 6 x 106, 7 x 106, 8 x 106, 9 x 106, or 1 x 107 total receptor+ / CD8+ / CCR7+ / CD27+ cells and / or at least 3 x 106, 4 x 106, 5 x 106, 6 x 106, 7 x 106, 8 x 106, 9 x 106, or 1 x 107 total receptor+ / CD4+ / CCR7+ / CD27+ cells; and / or iv) the unit dose comprises between 3 x 106 and 2.5 x 107, between 4 x 106 and 2 x 107, or between 5 x 106 and 1 x 107 total receptor+ / CD8+ / CCR7+ / CD27+ cells and / or between 3 x 106 and 2.5 x 107, between 4 x 106 and 2 x 107, or between 5 x 106 and 1 x 107 total receptor+ / CD4+ / CCR7+ / CD27+ cells, each inclusive; and / or v) at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the total receptor+ cells in the unit dose, or between 15% and 90%, between 20% and 80%, between 30% and 70%, or between 40% and 60%, each inclusive, of the total receptor+ cells in the unit dose are receptor+ / CD8+ / CCR7+ / CD27+ or receptor+ / CD4+ / CCR7+ / CD27+; and / or vi) the unit dose of cells comprises a defined ratio of receptor+ / CD8+ / CCR7+ / CD27+ cells to receptor+ / CD4+ / CCR7+ / CD27+ cells, which ratio optionally is 1:1 or is between 1:3 and 3:1; and / or vii) the unit dose comprises between 1 x 105 and 5 x 108, between 1 x 105 and 1 x 108, between 5 x 105 and 1 x 107, or between 1 x 106 and 1 x 107 total CD3+ cells that express the recombinant receptor (receptor+ / CD3+ cells) or total CD3+ cells, each inclusive; and / or viii) the unit dose comprises no more than 5 x 108, no more than 1 x 108, no more than 5 x 107, no more than 1 x 107, no more than 5 x 106, no more than 1 x 106, or no more than 5 x 105 total receptor+ / CD3+ cells or total CD3+ cells.

5. A therapeutic composition comprising T cells expressing a recombinant receptor, wherein at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of T cells in the composition, or the total number of T cells in the composition expressing the recombinant receptor, are surface positive for CCR7 and CD27, wherein: i) the T cells in the composition and / or expressing the recombinant receptor comprise or consist of CD8+ and CD4+ T cells; and ii) at least 50%, 60%, 70%, 80%, or 90% of the total receptor+ / CD8+ cells in the composition are receptor+ / CD8+ / CCR7+ / CD27+ and at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total receptor+ / CD4+ cells in the composition are receptor+ / CD4+ / CCR7+ / CD27+; wherein the recombinant receptor is a chimeric antigen receptor (CAR).

6. The therapeutic composition of claim 5, wherein the composition comprises one or more unit doses of cells.

7. The therapeutic composition of claim 5 or claim 6, wherein at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the total receptor+ cells in the unit dose or composition, or between 15% and 90%, between 20% and 80%, between 30% and 70%, or between 40% and 60%, each inclusive, of the total receptor+ cells in the unit dose or composition are receptor+ / CDS+ / CCR7+ / CD27+, or receptor+ / CD4+ / CCR7+ / CD27+.

8. The therapeutic composition of any of claims 5-7, wherein the unit dose or number of cells expressing the recombinant receptor comprises between 1 x 105 and 5 x 108, between 1 x 105 and 1 x 108, between 5 x 105 and 1 x 107, or between 1 x 106 and 1 x 107 total CD3+ cells that express the recombinant receptor (receptor+ / CD3+ cells) or total CD3+ cells, each inclusive; and / or the unit dose or number of cells expressing the recombinant receptor comprises no more than 5 x 108, no more than 1 x 108, no more than 5 x 107, no more than 1 x 107, no more than 5 x 106, no more than 1 x 106, or no more than 5 x 105 total receptor+ / CD3+ cells or total CD3+ cells.

9. The method of any of claims 1, 2 or 4, the therapeutic composition for use according to claim 3 or claim 4, or the therapeutic composition according to any of claims 5-8, wherein: i) the T cells are primary T cells obtained from a subject; and / or ii) the T cells are autologous to the subject.

10. The method of claim 9, the therapeutic composition for use according to claim 9, or the therapeutic composition according to claim 9, wherein the subject has a cancer, optionally wherein the cancer is a leukemia or a lymphoma.

11. The method of claim 10, the therapeutic composition for use according to claim 10, or the therapeutic composition according to claim 10, wherein the subject is a subject that, at the time of obtaining the cells, is identified or known to have a high tumor burden; optionally wherein: i) the subject has high tumor burden if a sum of product dimensions (SPD) of a tumor in the subject is above 30 cm2, 40 cm2, 50 cm2, 60 cm2 or 70 cm2; further optionally wherein: a) the subject has high tumor burden if the SPD of a tumor in the subject is above 50 cm2; and / or b) the subject has high tumor burden if the CRP is above 20 miligrams per liter in the biological sample; and / or ii) the subject has high tumor burden if C reactive protein (CRP) in a biological sample, optionally a serum sample, from the subject is above 5 miligrams per liter, 10 miligrams per liter, 15 miligrams per liter, 20 miligrams per liter, 25 miligrams per liter, 30 miligrams per liter, 40 miligrams per liter or 50 miligrams per liter; optionally wherein: a) the subject has high tumor burden if the SPD of a tumor in the subject is above 50 cm2; and / or b) the subject has high tumor burden if the CRP is above 20 miligrams per liter in the biological sample.

12. The therapeutic composition of claim 5 for use in a method of treating a subject having a disease or condition, the method comprising administering to the subject one or more unit doses of the composition.

13. A method of predicting likelihood of response to a therapeutic T cell composition, the method comprising: (a) assaying an engineered cell composition comprising T cells expressing a recombinant receptor for a factor indicative of the function or phenotype of engineered cells in the composition, the factor being the percentage of T cells expressing the recombinant receptor that are surface positive for CCR7 and CD27; and (b) determining the likelihood of response following administration of a cell therapy comprising a dose of the engineered cells, wherein if the factor is at or above a threshold value identifying the subject as likely to achieve a durable response or progression free survival, optionally for at least 3 months, following administration the therapy; or if the factor is below a threshold value identifying the subject as not likely to exhibit a durable response or progression free survival to the therapy, wherein the threshold value of the factor is 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of T cells in the composition or of the total number of T cells in the composition expressing the recombinant receptor, are surface positive for CCR7 and CD27; and wherein the recombinant receptor is a chimeric antigen receptor (CAR).

14. The method of claim 13, wherein the subject is a subject that, at the time of the assaying, is identified or known to have a high tumor burden; optionally wherein: i) the subject has high tumor burden if a sum of product dimensions (SPD) of a tumor in the subject is above 30 cm2, 40 cm2, 50 cm2, 60 cm2 or 70 cm2; optionally wherein: a) the subject has high tumor burden if the SPD of a tumor in the subject is above 50 cm2; and / or b) the subject has high tumor burden if the CRP is above 20 miligrams per liter in the biological sample; and / or ii) the subject has high tumor burden if C reactive protein (CRP) in a biological sample, optionally a serum sample, from the subject is above 5 miligrams per liter, 10 miligrams per liter, 15 miligrams per liter, 20 miligrams per liter, 25 miligrams per liter, 30 miligrams per liter, 40 miligrams per liter or 50 miligrams per liter; optionally wherein: a) the subject has high tumor burden if the SPD of a tumor in the subject is above 50 cm2; and / or b) the subject has high tumor burden if the CRP is above 20 miligrams per liter in the biological sample.

15. The method of claim 13 or claim 14, wherein the T cells are primary T cells obtained from a subject, optionally wherein the T cells are autologous to the subject.

16. The method of any of claims 1, 2, 4, 9-11, or 13-15, the therapeutic composition for use according to any of claims 3, 4 or 9-12, or the therapeutic composition according to any of claims 5-11, wherein the recombinant receptor is capable of binding to a target antigen that is associated with, specific to, and / or expressed on a cell or tissue of a disease, disorder or condition; optionally wherein: i) the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or a cancer; and / or ii) the target antigen is selected from among αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase 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-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

17. The method of any of claims 1, 2, 4, 9-11 or 13-16, the therapeutic composition for use according to any of claims 3, 4, 9-12 or 16, or the therapeutic composition according to any of claims 5-11 or 16, wherein: the recombinant receptor comprises an extracellular domain comprising an antigen-binding domain and an intracellular signaling region comprising an intracellular signaling domain, and further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region; optionally wherein: a) the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, which optionally is a single chain fragment, further optionally an scFv; and / or b) the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof; and / or c) the intracellular signaling region further comprises a costimulatory signaling domain, optionally wherein the costimulatory signaling domain comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof.

18. The method of any of claims 1, 2, 4, 9-11 or 13-17, the therapeutic composition for use according to any of claims 3, 4, 9-12, 16, or 17, or the therapeutic composition according to any of claims 5-11, 16 or 17, wherein: i) the costimulatory signaling domain comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof; and / or ii) the costimulatory signaling domain is between the transmembrane domain and the intracellular signaling domain.