METHODS FOR THE PRODUCTION OF TCR GAMMA DELTA + T CELLS
20210361708 · 2021-11-25
Inventors
- Diogo Antonio Remechido Anjos (Cantanhede, PT)
- Daniel Vargas Correia (Cantanhede, PT)
- Afonso Rocha MARTINS DE ALMEIDA (Cantanhede, PT)
Cpc classification
A61P31/00
HUMAN NECESSITIES
A61K35/17
HUMAN NECESSITIES
C12N5/0638
CHEMISTRY; METALLURGY
C12N2501/599
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to novel methods for the isolation and the selective ex vivo expansion of V32′ TCRy6+ T cells and to their clinical application.
Claims
1-41. (canceled)
42. A cell preparation enriched in TCRγδ.sup.+ T cells wherein greater than 80% of the total cells are TCRγδ.sup.+ T cells.
43. A cell preparation according to claim 42 wherein greater than 90% of the total cells are TCRγδ.sup.+ T cells.
44. A cell preparation according to claim 42 wherein greater than 95% of the total cells are TCRγδ.sup.+ T cells.
45. A cell preparation according to claim 42 which comprises both Vδ1.sup.+ TCRγδ.sup.+ T cells and Vδ2.sup.+ TCRγδ.sup.+ T cells.
46. A cell preparation according to claim 45 which comprises about 55-90% Vδ1.sup.+ TCRγδ.sup.+ T cells and about 1-10% Vδ2.sup.+ TCRγδ.sup.+ T cells, of the total TCRγδ.sup.+ T cells in the preparation.
47. A cell preparation according to claim 45 which comprises about 60-80% Vδ1.sup.+ TCRγδ.sup.+ T cells and about 1-5% Vδ2.sup.+ TCRγδ.sup.+ T cells, of the total TCRγδ.sup.+ T cells in the preparation.
48-58. (canceled)
59. A method of modulating an immune response comprising administering an effective amount of TCRγδ.sup.+ T cells obtained by a method for expanding Vδ2− TCRγδ.sup.+ T cells in a sample comprising: (1) culturing cells in the sample in a first culture medium comprising a T cell mitogen and interleukin-4, in the absence of interleukin-15, interleukin-2, or interleukin-7, and (2) culturing the cells obtained in step (1) in a second culture medium comprising a T cell mitogen and interleukin-15, interleukin-2, or interleukin-7, in the absence of interleukin-4, to an animal in need thereof.
60. A method for treating an infection comprising administering an effective amount of TCRγδ.sup.+ T cells obtained by a method for expanding Vδ2− TCRγδ.sup.+ T cells in a sample comprising: (1) culturing cells in the sample in a first culture medium comprising a T cell mitogen and interleukin-4, in the absence of interleukin-15, interleukin-2, or interleukin-7, and (2) culturing the cells obtained in step (1) in a second culture medium comprising a T cell mitogen and interleukin-15, interleukin-2, or interleukin-7, in the absence of interleukin-4, to an animal in need thereof.
61. A method for treating cancer comprising administering an effective amount of TCRγδ.sup.+ T cells obtained by a method for expanding Vδ2− TCRγδ.sup.+ T cells in a sample comprising: (1) culturing cells in the sample in a first culture medium comprising a T cell mitogen and interleukin-4, in the absence of interleukin-15, interleukin-2, or interleukin-7, and (2) culturing the cells obtained in step (1) in a second culture medium comprising a T cell mitogen and interleukin-15, interleukin-2, or interleukin-7, in the absence of interleukin-4, to an animal in need thereof.
62. The method according to claim 60, wherein the cancer is chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, and T cell and B cell leukemias, lymphomas (Hodgkin's and non-Hodgkins), lymphoproliferative disorders, plasmacytomas, histiocytomas, melanomas, adenomas, sarcomas, carcinomas of solid tissues, hypoxic tumors, squamous cell carcinomas, genitourinary cancers such as cervical and bladder cancer, hematopoietic cancers, head and neck cancers, and nervous system cancers.
63. A method for vaccinating an animal comprising administering an effective amount of TCRγδ.sup.+ T cells obtained by a method for expanding Vδ2− TCRγδ.sup.+ T cells in a sample comprising: (1) culturing cells in the sample in a first culture medium comprising a T cell mitogen and interleukin-4; in the absence of interleukin-15, interleukin-2, or interleukin-7; and (2) culturing the cells obtained in step (1) in a second culture medium comprising a T cell mitogen and interleukin-15, interleukin-2, or interleukin-7, in the absence of interleukin-4, to an animal in need thereof.
64. A method according to claim 59, wherein the first or second culture medium, or both culture media, further comprise a second, a third and a fourth growth factor.
65. A method according to claim 64, wherein said growth factors are interferon-γ, interleukin-21 and interleukin-1β or a mimetic or functional equivalent thereof.
66. A method according to claim 59, wherein the first and second culture media further contain serum or plasma.
67. A method according to claim 59, wherein prior to step (1) the cells in the sample are enriched for T cells; enriched for TCRγδ.sup.+ T cells; depleted of TCRαβ+ T cells; first depleted of TCRαβ+ T cells, and then enriched for CD3+ cells; or depleted of non-TCRγδ.sup.+ T cells.
68. A method according to claim 59, wherein the sample is blood or tissue or fractions thereof.
69. A method according to claim 68, wherein the sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, epithelia, thymus, bone marrow, spleen, liver, cancerous tissue, infected tissue, lymph node tissue or fractions thereof.
70. A method according to claim 59, wherein in the first culture medium the T cell mitogen is present in an amount from about 10 to about 5000 ng/ml and interleukin-4 is present in an amount from about 1 to about 1000 ng/ml.
71. A method according to claim 59, wherein in the second culture medium the T cell mitogen is present in an amount from about 0.1 to about 50 μg/ml and interleukin-15 is present in an amount from about 1 to about 1000 ng/ml.
72. A method according to claim 59, wherein the T cell mitogen is an antibody or a fragment thereof.
Description
FIGURES
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[0127] Detailed methods: The MEC-1 CLL cell line.sup.46 was obtained from the German Resource Center for Biologic Material (DSMZ). MEC-1 tumor cells were cultured in T25 flasks in complete 10% RPMI 1640 with 10% Fetal Bovine Serum, 2 mM L-Glutamine and maintained at 10.sup.5 up to 10.sup.6 cells/mL by dilution and splitting in a 1:3 ratio every 3-4 days. For cytotoxicity assays, in vitro expanded TCRγδ.sup.+ T cells were plated in 96-well round-bottom plates. Tumor cell lines or leukemia primary samples were stained with CellTrace Far Red DDAO-SE (1 μM; Molecular Probes, Invitrogen) and incubated at the indicated target:effector ratio with TCRγδ.sup.+ T cells in RPMI 1640 medium for 3 hours at 37° C. and 5% CO.sub.2, in the presence of 70 ng/ml IL-15. All cells were then stained with Annexin V-FITC (BD Biosciences) and analyzed by flow cytometry.
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DETAILED METHODS OF THE IN VIVO STUDIES
[0136] Balb/c Rag.sup.−/− γc.sup.−/−47 animals were obtained from Taconic (USA); NOD-SCIDγc.sup.−/−48 mice were obtained from the Jackson Laboratories (USA). BRG or NSG mice were injected subcutaneously with MEC-1 cells and treated after 6 and 11 days with two intravenous transfers of 10.sup.7 or 2×10.sup.7 DOT cells, and then analyzed (tumor size, histology, flow cytometry of tumor or organ infiltrates, and blood biochemistry) as detailed. All animal procedures were performed in accordance to national guidelines from the Direcao Geral de Veterinaria and approved by the relevant Ethics Committee. For phenotyping after in vivo DOT-cell transfer, animals were euthanized using Eutasil in order for blood collection via cardiac puncture; and quickly perfused with PBS+Heparin. Organs were homogenized and washed in 70 μM cell strainers. Femurs were flushed and then filtered. Cells were then stained with the following antibodies from ebioscience, Biolegend, Myltenyi Biotec or Beckton Dickinson: anti-mouse CD45 (30-F11), and anti-Human: CD45 (HI30). Other antibodies used are common with the in vitro studies. Antibodies were coupled to FITC, PE, PerCP, PerCP-Cy5, PE-Cy7, APC, APC-Cy7, Pacific Blue, Brilliant Violet 421 and Brilliant Violet 510 fluorochromes. Statistical analysis was performed using Graphpad-Prism software. Sample means were compared using the unpaired Student's t-test. In case variances of the two samples were found different using F-test, the data was log transformed and if variances were then found not to be different, the unpaired t-test was applied to the log-transformed data. For Survival data, log-rank (Mantel-Cox) test was used.
[0137] In vivo experimental design: We used a previously described model of xenografted human CLL upon sub-cutaneous adoptive transfer of CLL/SLL-derived MEC-1 cells into Balb/cRag.sup.−/−γc.sup.−/− (BRG) animals, which we further adapted using NOD-SCIDγc.sup.−/− (NSG) animals as hosts. In order to ensure that animals receiving treatment or PBS control were tumor-bearing animals, we transduced MEC-1 CLL cells with firefly-luciferase in order to detect and measure tumor engraftment at early time-points before ascribing treatment cells. After 7 or 4 days (in different studies) we injected luciferin i.p. to determine tumour load as a function of luminescence, before ascribing treatment (or PBS control) to the animals. Animals were distributed randomly in cages and assigned to each treatment (PBS or DOT-Cells) according to luminescence measured at day 7, in such a way that animal with highest luminescence received treatment, second highest received PBS, third highest received treatment, etc. This resulted in a non-randomized distribution into groups but randomized distribution in the different cages. 2 additional animals received DOT-Cells in the indicated experiments for initial homing analysis. We performed two 10.sup.7 or 2×10.sup.7 DOT-Cells transfers (within 5 days), using cells from one different donor per experiment. Tumor was measured using a Caliper and taking three perpendicular measurements. The formula used was ½×L×W×H..sup.49 Animals were sacrificed when tumor measurements reached 1000 mm.sup.3.
[0138] Luminescence Analysis: After transduction of MEC-1 Cell line with GFP-firefly luciferase, growing cells were screened and sorted according to GFP expression using a FACS-Aria (Becton Dickinson, USA), up to >95% GFP positive cells. These cells were then kept in culture until transferred subcutaneous into host animals (in 50 μl PBS). At the indicated time points after transfer, animals were anesthetized (Ketamin/Medetomidine) and Luciferin was injected (i.p.). 4 min later luciferase activity was detected and acquired using IVIS Lumina (Calliper LifeSciences) at the IMM bioimaging facilities. Anesthesia was then reverted and animals returned to previous housing.
[0139] Lymphocyte counts: Cell counts were performed with a hemocytometer or using Accuri Flow cytometer (Becton Dickinson, USA). Counts per organ were estimated when parts of the organ were sampled for histological analysis by weighting organs before and after the samples were split. Numbers presented are then corrected for the whole organ. In Bone Marrow data, absolute numbers were calculated and are displayed for one femur. Histopathology and Immunohistochemistry: Mice were sacrificed with anesthetic overdose, necropsies were performed and selected organs (lung, heart, intestine, spleen, liver, kidney, reproductive tract, brain, cerebellum, spinal cord, and femur) were harvested, fixed in 10% neutral-buffered formalin, embedded in paraffin and 3 μm sections were stained with hematoxylin and eosin (H&E). Bones were further decalcified in Calci-Clear™ (Fisher Scientific) prior to embedding. Tissue sections were examined by a pathologist, blinded to experimental groups, in a Leica DM2500 microscope coupled to a Leica MC170 HD microscope camera. Immunohistochemical staining for CD3 (Dako, cat. no. A0452) was performed by the Histology and Comparative Pathology Laboratory at the IMM, using standard protocols, with a Dako Autostainer Link 48. Antigen heat-retrieval was performed in DAKO PT link with low pH solution (pH 6), and incubation with ENVISION kit (Peroxidase/DAB detection system, DAKO, Santa Barbara, Calif.) was followed by Harri's hematoxylin counterstaining (Bio Otica, Milan, IT). Negative control included the absence of primary antibodies; and CD3 staining was not observed in the negative controls. Images were acquired in a Leica DM2500 microscope, coupled with a Leica MC170 HD microscope camera.
[0140] Mouse Blood Biochemistry: Mice were deeply anaesthetised and blood was collected from the heart to heparin-coated tubes, sent for analysis of biochemical parameters shown at an independent laboratory. Biochemical parameters were measured in serum, in a RX monaco clinical chemistry analyser (RANDOX).
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[0143] (A) Flow cytometry comparison of the cell surface phenotype of Vδ1.sup.+ T cells at day 21 of culture (using the previously described 2-step culture protocol); (full lines) with freshly-isolated Vδ1.sup.+ T cells (dotted lines), as analyzed using the LEGENDScreen kit (Biolegend). Shown are histogram overlays for several markers related to lymphocyte activation and differentiation, and markers implicated in adhesion and migration. Cells from one healthy donor are shown. (B) Heatmap representing percentages of positive cells for each surface marker across cultured Vδ1.sup.+ T cells (at day 21 of culture) produced from 4 different healthy donors (Cultur. 1-4), compared to freshly-isolated Vδ1.sup.+ T cells (from donors 1 and 2). The color code is presented on the right. For phenotyping after cell production: cells were stained with anti-CD3-APC (clone UCHT1), anti-TCRVδ1-FITC and a panel of receptors using the LegendScreen kit (Biolegend).
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[0145] Expanded and differentiated TCRγδ.sup.+ T cells produced from two healthy donors (using the previously described 2-step culture protocol), were tested in different experiments against MEC-1 (CLL) target cells at increasing effector/target ratios (left plot, gray bars) and also in presence of blocking antibodies for (α, anti-) the indicated molecules, either individually (Expt 1) or in combinations (Expt 2). The highest Effector/Target ratio (10:1) was used in blocking experiments and gray bar at this ratio (with IgG isotype antibody) serves as control. Shown are the percentages of dead (Annexin-V.sup.+) MEC-1 target cells. * and # indicate significant differences relative to IgG isotype control or α-TCRVδ1, respectively (Mean+SD; *,#p<0.05; **, ##p<0.01; Student's t-test). For cytotoxicity assays, MEC-1 tumor cells were cultured in T25 flasks in complete 10% RPMI 1640 with 10% Fetal Bovine Serum, 2 mM L-Glutamine and maintained at 10.sup.5 up to 10.sup.6 cells/mL by dilution and splitting in a 1:3 ratio every 3-4 days. In vitro expanded TCRγδ.sup.+ T cells were plated in 96-well round-bottom plates. Tumor cells were stained with CellTrace Far Red DDAO-SE (1 μM; Molecular Probes, Invitrogen) and incubated at the indicated target:effector ratio with TCRγδ.sup.+ T cells in RPMI 1640 medium for 3 hours at 37° C. and 5% CO.sub.2, in the presence of 70 ng/ml IL-15. For receptor blocking, γδ PBLs were pre-incubated for 1 hour with blocking antibodies: human anti-TCRγδ (clone B1); human anti-NKG2D (clone 1D11); human anti-CD2 (clone RPA-2.10); human anti-CD3 (clone OKT-3); human anti-NKp30 (clone P30-15); human anti-NKp44 (clone P44-8), mouse IgG1, k (clone MOPC-21), mouse IgG2b (clone MPC-11), mouse IgG3k (clone MG3-35), all from Biolegend. Human anti-CD226 (clone DX11) was from BD Biosciences. Human anti-Vδ1 TCR (clones TCS-1 or TS8.2) were from Fisher Scientific, and human anti-TCRγδ (clone IMMU510) was from BD Biosciences. The blocking antibodies were maintained in the culture medium during the killing assays.
[0146] Finally, all cells were then stained with Annexin V-FITC (BD Biosciences) and analyzed by flow cytometry.
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[0149] Expanded and differentiated TCRγδ.sup.+ T cells produced from one healthy donor (using the previously described 2-step culture protocol) were stained for CD3, Vδ1 and Vδ2 T cell markers with monoclonal antibodies conjugated to fluorochromes, and the CD3.sup.+ Vδ1.sup.−Vδ2.sup.− T cell population was isolated by flow cytometry. Isolated cells were then tested in a killing assay in vitro against acute myeloid leukemia (AML) target cell lines (KG-1, THP-1, HL-60 and NB-4) at an effector/target ratio of 10:1. The killing assay was performed as previously described.
Tables
[0150] Table 1 describes the molecules used to stimulate the proliferation of Vδ1.sup.+ T cells during the optimization stage. Reagents were used at a concentration range from 0.1 ng/ml to 80 μg/ml. The column on the right shows reagent distributor or manufacturer.
[0151] Table 2 shows a summary of tested culture conditions. TCRγδ.sup.+ PBLs were isolated by MACS from a healthy donor and cultured at 1 million cells/ml in 96 well plates, at 37° C. and 5% CO.sub.2. Cells were expanded in complete medium (Optmizer CTS, GIBCO) supplemented with 5% autologous plasma, 1 mM L-glutamine and with the described growth factors. At the end of the culture period, cells were counted and cell phenotype was analyzed by flow cytometry. Shows selected results of 4 consecutive experiments. The best culture conditions in each experiment are ranked by fold increase. Fold expansion rate of Vδ1.sup.+ T cells was calculated as: (absolute number of Vδ1.sup.+ T cells at the end of the culture)/(absolute number of Vδ1.sup.+ T cells at day 0 of culture). Shows representative results of 2 independent experiments.
[0152] Table 3 shows a summary of tested culture conditions. TCRγδ.sup.+ PBLs were isolated by MACS from a healthy donor and cultured for 14 days in the presence of the described growth factors. At day 14 of culture, cells were split: one fraction of cells was cultured as before, while the other fraction of cells was cultured in the absence of IL-4 and in the presence of the indicated growth factors. At day 21, cells were counted and cell phenotype was analyzed by FACS. Shows representative results from 2 independent experiments. A cytotoxicity assay was also performed at day 21 using the generated TCRγδ.sup.+ cells against MOLT-4 leukemia targets (method is described in
[0153] Table 4 shows a summary of tested culture conditions. TCRγδ.sup.+ PBLs were isolated by MACS from a healthy donor and cultured for 15 days in a two-step and three-step culture protocols, in the presence of the described growth factors. Cells were counted and cell phenotype was analyzed by FACS at the end of the culture period. Shows representative results of 2 independent experiments.
[0154] Table 5 shows a summary of tested culture conditions. TCRγδ.sup.+ PBLs were isolated by MACS from a healthy donor in a two-step protocol and cultured for 21 days in the presence of the described growth factors. Cells were counted and cell phenotype was analyzed by FACS at the end of the culture period. Shows representative results of 2 independent experiments
[0155] Table 6 shows the purity and phenotype of TCRγδ.sup.+ PBLs isolated via a two-step MACS protocol. PBMCs were obtained by density gradient centrifugation in ficoll from Buffy Coat products collected from 8 healthy donors. TCRγδ.sup.+ T cells were further isolated by a two-step MACS protocol as described in
[0156] Table 7 shows the purity and phenotype of in vitro expanded TCRγδ.sup.+ T cells. MACS-sorted TCRγδ.sup.+ T cells from healthy donors (same as in Table 6) were cultured for 21 days in cell culture bags, according to the previously described two-step culture protocol. Cell populations were characterized by flow cytometry. Indicates percentages of TCRγδ.sup.+ T cells and contaminant cells, relative to total live cells present in the cultures.
[0157] Table 8 shows the expression of NCRs and NKG2D in freshly-isolated versus cultured Vδ1.sup.+ T cells. Expression of the activating receptors NKp30, NKp44 and NKG2D in CD3.sup.+Vδ1.sup.+ T cells after 16 or 21 days of cytokine and anti-CD3 mAb treatment. Data in this table are representative of data obtained from 10 independent donors, noting that NKp30 and NKp44 expression varied between around 10% and 70% among different donors, while NKG2D was expressed by more than 80% of Vδ1.sup.+ PBLs of all tested donors.
[0158] Table 9 shows the purity and phenotype of pre- and post-MACS-sorted TCRγδ.sup.+ PBLs from CLL/SLL patients. B-cell chronic lymphocytic leukemia (CLL) cells were obtained from the peripheral blood of patients at first presentation, after informed consent and institutional review board approval. TCRγδ.sup.+ T cells were MACS-sorted from the peripheral blood of 3 CLL patients (CLL-1-3) and cell population phenotype was characterized by flow cytometry analysis of cell surface antigens. Shows percentages of TCRγδ.sup.+ T cells and contaminant cells, obtained immediately before and after the 2-step magnetic isolation procedure. Each cell subset was gated on total live cells.
[0159] Table 10 shows that contaminant autologous B-CLL cells become a residual population in culture. TCRγδ.sup.+ T cells were MACS-sorted from the peripheral blood of 3 CLL/SLL patients (CLL-1-3; as in Table 9) and cultured in vitro for 16 days as previously described. Cell population phenotype was characterized by flow cytometry analysis of cell surface antigens. Shows percentages of TCRγδ.sup.+ T cells and contaminant cells. Each cell subset is gated on total live cells, except NKp30 and NKG2D expression that were gated on Vδ1.sup.+ T cells.
[0160] Table 11 shows in more detail the tested culture conditions presented in Table 2 of a previous application. TCRγδ.sup.+ PBLs were isolated by MACS from a healthy donor and cultured at 1 million cells/ml in 96 well plates, at 37° C. and 5% CO.sub.2 Cells were expanded in complete medium (Optmizer CTS, GIBCO) supplemented with 5% autologous plasma, 1 mM L-glutamine and with the described growth factors. At the end of the culture period, cells were counted and cell phenotype was analyzed by flow cytometry. Shows selected results of 4 consecutive experiments (the same experiments described in Table 2 of a previous application, but further discloses results of parallel control culture conditions, marked with an asterisk, for a more complete understanding of the results). It also shows the percentage of NKp30.sup.+ Vδ1.sup.+ T cells obtained with each condition. Culture conditions in each experiment were ranked by fold increase. Fold expansion rate of Vδ1.sup.+ T cells was calculated as: (absolute number of Vδ1.sup.+ T cells at the end of the culture)/(absolute number of Vδ1.sup.+ T cells at day 0 of culture). Shows representative results of 2 independent experiments.
[0161] Table 12 shows a summary of tested culture conditions.
[0162] TCRγδ.sup.+ PBLs were isolated and expanded from a healthy donor, as described previously, in the presence of the indicated growth factors. Shows selected results of one experiment with multiple culture conditions. To better understand the effects of 1L15/IL-2/IL-7 and IFN-γ on cultured TCRγδ.sup.+ cells, TCRγδ.sup.+ cells were cultured in culture medium and three different concentrations of IL-4 and anti-CD3 mAb, in the presence or absence of 1L15/IL-2/IL-7 and IFN-γ. Shows the detrimental effect of IL-15, IL-2 and IL-7 on TCRγδ.sup.+ T cell expansion, when these cells were cultured in the presence of IL-4 and IFN-γ. Shows representative results of 2 independent experiments.
[0163] Table 13 shows the total absolute number of TCRγδ.sup.+ cells obtained before and after the large-scale 2-step cell culture protocol. MACS-sorted peripheral blood TCRγδ.sup.+ cells obtained from healthy donors (represented in
[0164] Table 14 shows reagents and materials used to produce pharmaceutical grade TCRγδ.sup.+ T cells.
EXAMPLES
[0165] Optimization of the ex-vivo expansion of human Vδ1.sup.+ TCRγδ.sup.+ T cells
[0166] The inventors performed a series of experiments aiming to improve the expansion and purity levels of in vitro cultured Vδ2.sup.− γδ T cells. Since there was no commercially available antibody against the Vδ3.sup.+ chain of the TCR, an anti-TCRVδ1 mAb was used to identify Vδ1.sup.+ T cells in cell samples, during the culture optimization stage. TCRγδ.sup.+ T PBLs from a panel of healthy donors were isolated by MACS and tested for their reactivity to in vitro stimulation with IL-2 and PHA (i.e., detectable changes in cell activation and proliferation). One donor with reactive Vδ1.sup.+ PBLs was selected to provide blood samples for the rest of the optimization study. The preference for a fixed healthy donor was important, since a more reliable comparison could be performed between results obtained in different experiments. The selected donor had a normal (but high) percentage of TCRγδ.sup.+ T cells in the peripheral blood (10%-12% of total PBLs), although a very low percentage of Vδ1.sup.+ PBLs (0.3% of total PBLs, or 3.0% of total TCRγδ.sup.+ T PBLs;
[0167] The single-step MACS protocol used to isolate TCRγδ.sup.+ T cells from PBMCs was very efficient, generating highly pure cell populations (
[0168] The inventors then tested multiple combinations of clinical-grade agonist antibodies and cytokines for their capacity to expand and differentiate (over 2-3 weeks) Vδ1.sup.+ T cells from the peripheral blood. MACS-sorted TCRγδ.sup.+ PBLs collected from the previously selected healthy donor were incubated in culture medium for 2-3 weeks in 96-well plates, at 37° C. and 5% CO.sub.2, in the presence of 58 different T/NK cell activating molecules (Table 1). These included 13 different TCR agonists, 23 different co-receptor agonists, and 22 different cytokines, which were tested in 2,488 different combinations and concentrations. Antibodies were used in both soluble and plastic-bound presentations. Cytokines were tested at a concentration range from 0.1 ng/ml to 1000 ng/ml, TCR agonists were used at 0.1 ng/ml to 40 μg/ml, and co-receptor agonists were used at final concentration 0.5 μg/ml to 80 μg/ml.
[0169] Several sequential cell isolation and cell culture expansion experiments were performed from the same donor; each experiment testing the effect of about 100-400 different combinations of activating molecules. The optimization started from the basic, non-optimized cocktail (i.e., culturing TCRγδ.sup.+ T cells in the presence of IL-2 and PHA). Fresh medium containing the same chosen cocktail of activating molecules was added every 5 days. After 14 days, cells were collected and their phenotype was analyzed by flow cytometry. The best culture condition of each experiment was identified (for the highest fold expansion of Vδ1.sup.+ T cells), and selected for further optimization, combined to all available reagents, tested at various concentrations. Fold expansion and purity levels of Vδ1.sup.+ T cells gradually increased during the optimization stage, after each superior culture condition was obtained.
[0170] Results of experiments 1-4 are summarized in Table 2. Experiment no.1 confirmed previous observations that IL-4 is a key growth factor in promoting Vδ1.sup.+ T cell proliferation and enrichment in culture..sup.27, 42 In this experiment, the inventors tested the activity of 22 different cytokines on cultured TCRγδ.sup.+ T cells, in the presence of a T cell mitogen and IL-2. Clearly, IL-4 was unique in the ability to induce a strong enrichment and expansion of these cells. In contrast, the use of increasing concentrations of IL-2, or the combination of IL-2 with different T cell mitogens, did not produce an equivalent effect, most probably because of increased activation-induced-cell-death (AICD) of cultured cells (conditions 2-3; Table 2).
TABLE-US-00001 TABLE 1 TCR Monoclonal anti-human TCR Vδ1 mAb (Clone TS8.2); purified Thermo Fisher Sci. agonists antibodies anti-human TCR δTCS-1 mAb (Clone TS-1); purified Thermo Fisher Sci. (soluble and anti-human TCR PAN γδ mAb (Clone IMMU510); purified Beckman Coulter plate-bound) anti-human CD3 mAb (Clone OKT3); purified BioXcell/ Biolegend Plant lectins Lectin from Phaseolus vulgaris (red bean; PHA-P), pur. Sigma-Aldrich, Co. (soluble) Concanavalin A (from Canavalia ensiformis; Con-A), pur. Lectin from Phytolacca americana; purified Lectin from Triticum vulgaris (wheat); purified Lectin from Lens culinaris (lentil); purified Lectin from Glycine max (soybean); purified Lectin from Maackia amurensis; purified Lectin from Pisum sativum (pea); purified Lectin from Sambucus nigra (elder); purified Co- Monoclonal anti-human CD2 mAb (Clone S5.2); purified BD Biosciences receptor antibodies anti-human CD6 mAb (Clone UMCD6/3F7B5); purified Ancell Corporation agonists (soluble and anti-human CD9 mAb (Clone MEM-61); purified Exbio Praha, a.s. plate-bound) anti-human CD28 mAb (Clone CD28.2); purified Biolegend anti-human CD43 mAb (Clone MEM-59); purified Exbio Praha, a.s. anti-human CD94 mAb (Clone HP-3B1); purified Santa Cruz Biotech anti-human CD160 mAb (Clone CL1-R2); purified Novus Biologicals anti-human SLAM mAb [Clone A12(7D4)]; purified Biolegend anti-human NKG2D mAb (Clone 1D11); purified Exbio Praha, a.s. anti-human 2B4 mAb (Clone C1.7); purified Biolegend anti-human HLA-A, B, C mAb (Clone W6/32); purified Biolegend anti-human ICAM-3 mAb (Clone MEM-171); purified Exbio Praha, a.s. anti-human ICOS mAb (Clone C398.4A); purified Biolegend Recombinant Human SECTM-1/Fc Chimera (CD7 ligand) R&D Systems proteins Human CD26 (Dipeptidyl Peptidase IV) Sigma-Aldrich, Co. (soluble) Human CD27L (CD27 ligand); PeproTech, inc. Human CD30L (CD30 ligand); PeproTech, inc. Human CD40L (CD40 ligand); PeproTech, inc. Human OX40L (OX40 ligand); PeproTech, inc. Human 4-1BBL (4-1BB ligand) PeproTech, inc. Human ICAM-1 PeproTech, inc. Human Fibronectin Sigma-Aldrich, Co. Human Hydrocortisone Sigma-Aldrich, Co. Cytokines Recombinant Human IFN-γ (Interferon-γ); PeproTech, inc. proteins Human TGF-β (Transforming growth factor beta); PeproTech, inc. (soluble) Human IL-1-β (interleukin-1β); PeproTech, inc. Human IL-2 (interleukin-2); PeproTech, inc. Human IL-3 (interleukin-3); PeproTech, inc. Human IL-4 (interleukin-4); PeproTech, inc. Human IL-6 (interleukin-6); Biolegend Human IL-7 (interleukin-7); PeproTech, inc. Human IL-9 (interleukin-9); PeproTech, inc. Human IL-10 (interleukin-10); PeproTech, inc. Human IL-12 (interleukin-12); PeproTech, inc. Human IL-13 (interleukin-13); PeproTech, inc. Human IL-15 (interleukin-15); PeproTech, inc Human IL-18 (interleukin-18); Southern Biotech Human IL-21 (interleukin-21); PeproTech, inc. Human IL-22 (interleukin-22); PeproTech, inc. Human IL-23 (interleukin-23); PeproTech, inc. Human IL-27 (interleukin-27); Biolegend Human IL-31 (interleukin-31); PeproTech, inc. Human IL-33 (interleukin-33); PeproTech, inc. Human GM-CSF (Granul.-macroph. col. stimul. factor); PeproTech, inc. Human FLT3-L (FMS-like tyrosine kinase 3 ligand); PeproTech, inc.
TABLE-US-00002 TABLE 2 Total Fold Live Vδ1.sup.+ increase Cond. Condition: cells T cells of Vδ1.sup.+ Exp. No (cultured 1 million cells/ml for 14 days in 96-well plates) (%) (%) T cells 1 1 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 68.9 31.6 77 2 500 ng/ml IL-2 + 1 μg/ml PHA 63.3 10.5 4 3 20 ng/ml IL-2 + 1 μg/ml PHA (control) 68.8 1.90 1 2 1 20 ng/ml IL-2 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 90.0 51.7 75 2 20 ng/ml IL-2 + 1 μg/ml α-Vδ1 TCR mAb + 20 ng/ml IL-4 85.2 55.9 69 3 5 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 84.0 61.9 62 4 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 (previous best) 72.0 45.3 27 5 100 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 71.3 55.7 22 6 300 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 71.3 57.0 21 3 1 5 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 91.7 61.4 138 2 5 ng/ml IL-2 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 81.4 59.4 124 3 20 ng/ml IL-2 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 (prev. best) 84.2 45.4 105 4 5 ng/ml IL-15 + 1 μg/ml PHA + 20 ng/ml IL-4 68.0 76.2 21 5 5 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 60.1 69.1 19 6 20 ng/ml IL-15 + 1 μg/ml PHA + 20 ng/ml IL-4 68.6 69.9 13 7 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 62.9 67.7 11 4 1 20 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 87.1 79.5 1 349 2 3 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 85.5 67.4 1 014 3 2 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 87.9 81.6 909 4 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 85.9 67.8 804 5 5 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 (prev. best) 78.9 69.4 624
[0171] Results of experiment no.2 demonstrated that IL-2 (when in the presence of IL-4 and PHA) must be used at low concentrations, for increased proliferation and enrichment of Vδ1.sup.+ T cells in culture (conditions 3-7, Table 2). This effect was not observed in the previous experiment (exp. no. 1), in which fold expansion was higher in the presence of PHA and high levels of IL-2, in the absence of IL-4. Furthermore, the anti-CD3 mAb was shown to be the most effective mitogen promoting survival and proliferation of Vδ.sup.+ T cells in culture (see condition 1 versus condition 2, Table 2).
[0172] Results of experiment no. 3 (Table 2) confirmed previous observations and showed that even lower (than previously used) concentration levels of IL-2 (in the presence of IL-4 and a T cell mitogen), promoted even higher Vδ1.sup.+ T cell proliferation, survival and enrichment in culture. Also, when used at the same concentration, IL-15 was more effective than IL-2 in promoting Vδ1.sup.+ T cell survival, enrichment and proliferation. Again, the anti-CD3 mAb was shown to be the most effective mitogen of our tests.
[0173] Results of experiment no. 4 further indicated that the presence of high levels of IL-15 in the culture medium was detrimental for the proliferation of Vδ1.sup.+ T cells. Indeed, even lower (than previously used) concentration levels of IL-15 promoted even higher expansion levels of Vδ1.sup.+ T cells, when in the presence of IL-4 and α-CD3 (Conditions 3-5, Table 2). Finally, in a totally unexpected finding, the replacement of IL-15 by INF-γ (i.e., the absence of IL-15 and the presence of IFN-γ in the culture medium), was shown to generate increased expansion levels of cultured Vδ.sup.+ T cells.
[0174] Although many different concentrations and combinations of IL-2, IL-7 and IL-15 were tested in parallel, IFN-γ was consistently found to be a much more effective reagent for promoting the selective expansion of Vδ1.sup.+ T cells in culture (when used in the presence of IL-4 and a T cell mitogen, such as anti-CD3 mAb;
[0175] IFN-γ is a dimerized soluble cytokine and is the only member of the type II class of interferons..sup.50 It is structurally and functionally different from common γ-chain cytokines such as IL-2, IL-4, IL-7 or IL-15 and is serologically distinct from Type I interferons: it is acid-labile, while the type I variants are acid-stable.
[0176] Although we have obtained a new and improved method for expanding and enriching populations of Vδ1.sup.+ T cells in culture, we also sought to analyze the anti-tumor function of the generated cells, in vitro. We found that the presence of IL-4 in the culture medium strongly inhibited or decreased the expression of activating receptors such as Natural Cytotoxicity Receptors (NCRs, namely NKp30 and NKp44), and NKG2D on expanded Vδ1.sup.+ T-cells (Table 3).
[0177] Activating Natural Killer (NK) receptors (such as NKp30, NKp44 and NKG2D) are known to play a critical role in the anti-tumor and anti-viral function of Vδ.sup.+ T cells, through binding to their molecular ligands expressed on the surface of tumor or infected cells. Receptor-ligand binding triggers the production and release of granzymes and perforines by Vδ1.sup.+ T cells, leading to the death of target cells..sup.29 In our study, we found that the presence of IL-4 in the culture medium induced a decrease in the levels of activating NK receptors located at the surface of cultured TCRγδ.sup.+ T cells, thereby decreasing their cytotoxic function against MOLT-4 leukemia cells (Table 3). Of note, the inhibition induced by IL-4 seemed to affect all TCRγδ.sup.+ T cell subsets present in the final cellular product, including both Vδ1.sup.− and Vδ1.sup.+ TCRγδ.sup.+ T cells (Table 3).
TABLE-US-00003 TABLE 3 Fold Vδ1.sup.+ Vδ1.sup.+ Vδ1.sup.+ Culture Culture Live Vδ1.sup.+ increase NKp30.sup.+ NKp44.sup.+ KG2D.sup.+ Dead tumor Condition condition condition cells T cells of Vδ1.sup.+ T cells T cells T cells target cells No (days 1-14) (days 15-21) (%) (%) T cells (%) (%) (%) (%) 1 100 ng/ml IL-15 61.4 50.3 .sup. 108 45.0 38.0 99.8 62.6 1 μg/ml a-CD3 mAb (control) 2 100 ng/ml IL-4 81.0 92.6 8 064 0.9 0.2 48.2 14.0 70 ng/ml IFN-γ 1 μg/ml a-CD3 mAb 3 100 ng/ml IL-4 83.1 83.1 2 185 0.1 0.3 46.4 17.5 1 μg/ml a-CD3 mAb 4 100 ng/ml IL-4 68.4 92.8 1 263 1.6 1.1 38.4 13.3 1 μg/ml a-TCRVδ1 mAb 5 100 ng/ml IL-4 69.6 73.8 .sup. 464 1.5 2.2 58.7 9.0 1 μg/ml PHA 6 100 ng/ml IL-4 100 ng/ml IL-15 87.3 85.9 24 152 30.1 14.7 98.9 68.5 70 ng/ml IFN-γ 1 μg/ml a-CD3 mAb 7 1 μg/ml a-CD3 mAb 100 ng/ml IL-2 80.1 88.1 16 374 29.0 18.2 99.4 70.2 1 μg/ml a-CD3 mAb 8 100 ng/ml IL-7 78.3 85.4 18 366 15.6 15.4 99.2 67.6 1 μg/ml a-CD3 mAb 9 100 ng/ml IL-4 100 ng/ml IL-15 83.1 80.6 9 636 19.5 17.7 99.0 64.6 1 μg/ml a-CD3 mAb 1 μg/ml a-CD3 mAb 10 100 ng/ml IL-2 84.0 85.5 7 747 23.0 12.9 98.4 54.4 1 μg/ml a-CD3 mAb 11 100 ng/ml IL-7 76.5 83.0 10 567 10.1 4.4 99.2 50.6 1 μg/ml a-CD3 mAb 12 100 ng/ml IL-4 100 ng/ml IL-15 69.4 72.6 5 564 23.2 6.6 95.4 54.5 1 μg/ml a-Vδ1 mAb 1 μg/ml a-Vδ1 mAb 13 100 ng/ml IL-4 100 ng/ml IL-15 67.8 65.6 2 454 17.6 13.4 95.6 46.0 1 μg/ml PHA 1 μg/ml PHA
[0178] This was in line with a recent study showing that IL-4 promotes the generation of regulatory Vδ1.sup.+ T cells via IL-10 production. IL-4-treated Vδ.sup.+ T cells secreted significantly less IFN-γ and more IL-10 relative to Vδ2.sup.+ T cells. Furthermore, Vδ1.sup.+ T cells showed relatively low levels of NKG2D expression in the presence of IL-4, suggesting that Vδ1.sup.+ T cells weaken the TCRγδ.sup.+ T cell-mediated anti-tumor immune response..sup.42
[0179] Since we were looking for novel culture methods with the objective of improving the anti-tumor effector functions of Vδ1.sup.+ T cells, we attempted to recover the expression of activating NK receptors on IL-4treated cells, also recovering the cytotoxic phenotype of these cells, something that was never tried before.
[0180] TCRγδ.sup.+ T cells were cultured in a two-step protocol. The first step consisted of treating cells in culture medium in the presence of a T cell mitogen (such as anti-CD3 mAb or PHA) and IL-4, and in the absence of IL-2, IL-15 and IL-7, to promote the selective expansion of Vδ1.sup.+ T-cells. The second culture step consisted of treating cells in a culture medium in the absence of IL-4, and in the presence of a T cell mitogen and IL-2, or IL-7 or IL-15, to promote cell differentiation, and NKR expression (Table 3 and Table 4).
[0181] Conditions 1-5 (Table 3) confirmed previous results, showing that Vδ1.sup.+ T cells cultured in the presence of IL-4 could expand in culture several thousand fold, but could not differentiate, becoming inefficient killers of tumor cells. In contrast, as seen in conditions 6-13, when cells were subcultured in a second culture medium in the absence of IL-4 and in the presence of a T cell mitogen and IL-2, or IL-7, or IL-15, the ability to eliminate tumor cells increased radically. The presence of each one of these three cytokines (IL-2, IL-7 and IL-15), alone or in combination, was able to revert the phenotype of cultured Vδ1.sup.+ T cells and thus can be used for this purpose.
[0182] Results of experiments no. 6 and 7 (Table 4) showed that 2-step protocols and even 3-step protocols could be more efficiently used to stimulate the proliferation and differentiation of Vδ1.sup.+ T-cells. In the case of 3-step protocols, where cells were cultured in 3 different culture media (see for example condition 2 of experiment n° 7 of Table 4), it was very important to separate the IL-4 containing medium from medium containing IL-2 or IL-7 or IL-15. From these results it could also be concluded that a fraction of old culture medium should be removed during each subculture step for improved cell expansion; and that in the second culture medium, IL-15 is slightly more efficient than IL-2 in promoting Vδ1.sup.+ T-cell proliferation.
[0183] Additional experiments using 3-step and 4-step culture protocols further demonstrated that other growth factors can be added to the first and/or second culture medium (Table 3 and Table 4) for increased expansion levels of Vδ1.sup.+ T cells and expression of NK receptors on these cells. INF-γ, IL-21 and IL-16 were identified as efficient inducers of Vδ1.sup.+ T cell expansion and survival (Table 5). These growth factors could be used in the first or in the second culture media.
[0184] Finally, the addition of a soluble ligand of the CD27 receptor, or a soluble ligand of the CD7 receptor or a soluble ligand of SLAM receptor resulted in enhanced expansion of Vδ1.sup.+ T cells (Conditions 3-6 of Table 5). CD27 receptor is typically required for the generation and long-term maintenance of T cell immunity. It binds to its ligand CD70, and plays a key role in regulating B-cell activation and immunoglobulin synthesis. CD7 receptor is a member of the immunoglobulin superfamily. This protein is found on thymocytes and mature T cells. It plays an essential role in T-cell interactions and also in T-cell/B-cell interaction during early lymphoid development. SLAM receptor is a member of the signaling lymphocytic activation molecule family of immunomodulatory receptors.
TABLE-US-00004 TABLE 4 Fold Vδ1.sup.+ Vδ1.sup.+ increase NKp30.sup.+ Cond. Condition: T cells of Vδ1.sup.+ T cells Exp. No (cultured 5 × 10.sup.5 million cells/ml for 15 days in 96-well plates): (%) T cells (%) 6 1 Days 0-5: 100 ng/ml IL-4 + 1 μg/ml α-CD3 + 70 ng/ml IFN-γ 72.4 7 635 39.4 Days 6-15: 100 ng/ml IL-15 + 2 μg/ml α-CD3 2 Days 0-5: 100 ng/ml IL-4 + 1 μg/ml α-CD3 + 70 ng/ml IFN-γ 60.1 5 100 37.9 Days 6-15: 100 ng/ml IL-7 + 2 μg/ml α-CD3 3 Days 0-5: 100 ng/ml IL-4 + 1 μg/ml α-CD3 + 70 ng/ml IFN-γ 68.2 4 135 36.5 Days 6-15: 100 ng/ml IL-2 + 2 μg/ml α-CD3 7 1 Days 0-5: 100 ng/ml IL-4 + 1 μg/ml α-CD3 + 70 ng/ml IFN-γ 65.0 4 468 45.4 Days 6-10: 100 ng/ml IL-15 + 2 μg/ml α-CD3 Days 11-15: remove medium, 100 ng/ml IL-15 + 2 μg/ml α-CD3 2 Days 0-5: 30 ng/ml IL-4 + 1 μg/ml α-CD3 + 70 ng/ml IFN-γ 80.5 3 987 36.0 Days 6-10: 100 ng/ml IL-15 + 1 μg/ml α-CD3 + 2 ng/ml IL-21 Days 11-15: 100 ng/ml IL-15 + 1 μg/ml α-CD3 + 5 ng/ml IL-21 3 Days 0-5: 100 ng/ml IL-4 + 1 μg/ml α-CD3 + 70 ng/ml IFN-γ 64.0 3 683 41.0 Days 6-10: remove medium, 100 ng/ml IL-15 + 2 μg/ml α-CD3 Days 11-15: 100 ng/ml IL-15 + 2 μg/ml α-CD3
[0185] Of note, several different culture media were tested (
TABLE-US-00005 TABLE 5 Fold Vδ1.sup.+ Culture Culture Culture Culture Vδ1.sup.+ increase NKp30.sup.+ Condition condition condition condition condition T cells of Vδ1.sup.+ T cells number: (days 1-6) (days 7-11) (days 12-16) (days 17-21) (%) T cells (%) 1 100 ng/ml IL-4 70 ng/ml IFN-γ 75.0 61 417 54.1 70 ng/ml IFN-γ 2 μg/ml α-CD3 mAb 70 ng/ml α-CD3 mAb 100 ng/ml IL-15 70 ng/ml IL-21 15 ng/ml IL-1β 2 100 ng/ml IL-4 70 ng/ml IFN-γ 80.1 37 457 38.5 70 ng/ml IFN-γ 2 μg/ml α-CD3 mAb 70 ng/ml α-CD3 mAb 100 ng/ml IL-15 70 ng/ml IL-21 3 100 ng/ml IL-4 1 μg/ml α-CD3 mAb 72.4 10 535 22.5 70 ng/ml IFN-γ 100 ng/ml IL-15 70 ng/ml α-CD3 mAb 1 μg/ml sCD27L 4 100 ng/ml IL-4 69.6 9 566 25.4 70 ng/ml IFN-γ 70 ng/ml α-CD3 mAb 1 μg/ml α-SLAM mAb 5 100 ng/ml IL-4 70.7 7 764 24.5 70 ng/ml IFN-γ 70 ng/ml α-CD3 mAb 1 μg/ml SCD7L 6 100 ng/ml IL-4 72.8 5 594 21.4 70 ng/ml IFN-γ 70 ng/ml α-CD3 mAb
[0186] In vitro characterization of large-scale expanded TCRγδ.sup.+ T cells
[0187] Having established an effective protocol for the isolation and expansion of Vδ1.sup.+ T cells in culture, we sought to test it with blood samples collected from a larger number of healthy donors and also from cancer patients. This was necessary to test the robustness and general applicability of the new culture method. Moreover, instead of plastic plates or flasks, cells were cultured in closed, large-scale, gas-permeable cell bags developed for clinical applications.
[0188] The adopted two-step method of magnetic-associated cell sorting (MACS) produced viable cell populations enriched in TCRγδ.sup.+ T cells from 8 different donors (Table 6). Vδ1.sup.+ TCRγ.sup.+ T cells comprised only about 1% to 44% of the total viable cells initially present after MACS. However, within 11-21 days of treatment following the optimized 2-step culture method and in the presence of the described cocktail of cytokines and T cell mitogen, Vδ1.sup.+ T cells became the dominant cell subset in culture, varying between 60-80% of total cells between donors (
[0189] The expression of activating Natural Cytotoxicity Receptors (NCRs; including NKp30 and NKp44), and NKG2D was robustly induced in long-term cultured Vδ1.sup.+ T cells, of all tested donors (Table 8 and
[0190] Finally, Vδ2.sup.− TCRγδ.sup.+ T cells could be efficiently isolated and expanded from the PBLs of elderly CLL patients with very high tumour burden (Table 9 and Table 10 and
[0191] This collection of data fully demonstrates the unique ability of the invention to generate functional Vδ2.sup.− γδ T cells (namely from cancer patients) for autologous or allogeneic adoptive cell therapy. The method is robust enough to enrich (>60%) and expand (up to 2,000-fold) Vδ1.sup.+ T cells from highly unpurified samples obtained from CLL patients, differentiating them into NKR-expressing and highly cytotoxic TCRγδ.sup.+ T cells.
[0192] Importantly, preliminary tests also demonstrated in vitro reactivity of cultured TCRγδ.sup.+ T cells against tumor cells of other tissue origins (
TABLE-US-00006 TABLE 6 Cell lineage: TCRαβ.sup.+ TCRγδ.sup.+ Vδ1.sup.+ Vδ2.sup.+ Total cell B cells NK cells T cells T cells T cells T cells viability (CD19.sup.+ (CD56.sup.+ T cells (TCRαβ.sup.+ (TCRγδ.sup.+ (TCRVδ1.sup.+ (TCRVδ2.sup.+ (Trypan Donor CD20.sup.+ cells) CD3.sup.− cells) (CD3.sup.+ cells) CD3.sup.+ cells) CD3.sup.+ cells) CD3.sup.+ cells) CD3.sup.+ cells) Blue.sup.− cell A 24.6 9.92 43.5 0.01 41.9 13.5 23.6 79.4 B 6.69 0.07 75.0 0.71 63.2 21.6 30.0 80.1 C 1.36 0.36 95.3 0.37 94.5 1.25 92.6 95.2 D 13.9 6.79 41.2 0.76 40.2 18.3 21.5 89.1 E 17.0 1.84 65.8 2.80 59.6 17.7 40.9 89.9 F 0.28 8.14 91.4 0.81 90.0 2.05 86.1 93.7 G 7.32 0.25 87.5 0.58 81.8 24.0 45.0 84.0 H 3.51 0.10 88.8 0.80 84.8 44.0 27.0 86.0
TABLE-US-00007 TABLE 7 Cell lineage: TCRαβ.sup.+ TCRγδ.sup.+ Vδ1.sup.+ Vδ2.sup.+ Total cell B cells NK cells T cells T cells T cells T cells viability (CD19.sup.+ (CD56.sup.+ T cells (TCRαβ.sup.+ (TCRγδ.sup.+ (TCRVδ1.sup.+ (TCRVδ2.sup.+ (Trypan Donor CD20.sup.+ cells) CD3.sup.− cells) (CD3.sup.+ cells) CD3.sup.+ cells) CD3.sup.+ cells) CD3.sup.+ cells) CD3.sup.+ cells) Blue.sup.− cell A 0 0.50 99.5 0.01 99.3 82.6 3.9 89.0 B 0 0.02 99.7 0.06 99.5 80.8 3.7 93.3 C 0 0.50 96.3 0.03 92.8 69.9 4.2 90.3 D 0 0.03 99.6 0.02 99.1 62.2 2.3 94.5 E 0 0.11 99.5 0.01 99.2 63.3 3.3 95.9 F 0 0 99.9 0.02 98.0 73.3 4.3 93.2 G 0 0.10 99.4 0.01 98.4 71.7 3.5 90.0 H 0 0.40 97.5 0 98.2 72.0 1.6 89.0
TABLE-US-00008 TABLE 8 Activating Donor receptor Day 0 Day 16 Day 21 A NKp30 0.51 66.8 65.0 NKp44 0.30 18.3 23.3 NKG2D 46.0 96.5 98.0 B NKp30 0.56 71.6 68.0 NKp44 0 37.2 38.7 NKG2D 55.0 90.7 95.1
TABLE-US-00009 TABLE 9 Cell lineage: TCRαβ.sup.+ TCRγδ.sup.+ Vδ1.sup.+ Cell B cells NK cells T cells T cells T cells viability (CD19.sup.+ (CD56.sup.+ T cells (TCRαβ.sup.+ (TCRγδ.sup.+ (TCRVδ1.sup.+ (Trypan Donor CD20.sup.+ cells) CD3.sup.− cells) (CD3.sup.+ cells) CD3.sup.+ cells) CD3.sup.+ cells) CD3.sup.+ cells) Blue.sup.− cells) Before MACS (Day 0) CLL-1 63.4 1.22 30.4 27.5 0.66 0.22 92.0 CLL-2 85.7 0.92 8.35 6.97 0.43 0.03 90.0 CLL-3 90.4 0.15 3.74 3.31 0.35 1.9 × 10.sup.−3 87.0 After MACS (Day 0) CLL-1 38.0 0.72 37.2 0.19 7.32 4.00 88.0 CLL-2 35.4 0.26 61.3 0.05 36.7 1.70 83.0 CLL-3 57.0 0.45 39.5 0.02 10.4 0.28 80.0
TABLE-US-00010 TABLE 10 Cell phenotype after in vitro culture (Day 21) Cell lineage: TCRαβ.sup.+ TCRγδ.sup.+ Vδ1.sup.+ NKp30.sup.+ NKG2D.sup.+ B cells NK cells T cells T cells T cells Vδ1.sup.+ Vδ1.sup.+ (CD19.sup.+ (CD56.sup.+ T cells (TCRαβ.sup.+ (TCRγδ.sup.+ (TCRVδ1.sup.+ T cells T cells Donor CD20.sup.+ cells) CD3.sup.− cells) (CD3.sup.+ cells) CD3.sup.+ cells) CD3.sup.+ cells) CD3.sup.+ cells) (pre-gated) (pre-gated) CLL-1 0.04 0.11 96.8 0.08 94.1 60.1 23.0 95.6 CLL-2 0.07 0.01 99.5 0.02 97.4 80.0 11.0 98.9 CLL-3 0.05 0.01 99.8 0.01 99.6 70.1 13.4 97.2
[0193] In vivo studies of expanded TCRγδ.sup.+ T cells
[0194] Having successfully developed a method to generate large numbers of functional TCRγδ.sup.+ T cells, which we called “DOT-cells”, we next investigated their homing and anti-leukaemia activity in vivo. We took advantage of a xenograft model of human CLL previously shown to reproduce several aspects of the disease and used to test the efficacy of other cellular therapies, including CAR-T cells..sup.51, 52 The model relies on the adoptive transfer of CLL/SLL-derived MEC-1 cells into Balb/c Rag.sup.−/−γc.sup.−/− (BRG) animals, which lack all lymphocytes and thus do not immediately reject the human cells. However, some myeloid lineage-mediated rejection of human xenografts still occurs. This rejection varies in its magnitude according to the mouse strain used, due to different alleles encoding for SIRP-α.sup.53. We have thus further adapted the model in order to characterize TCRγδ.sup.+ T cells at late time points after transfer, using NOD-SCID γc.sup.−/− (NSG) animals as hosts. Indeed, upon transfer into tumour-bearing NSG hosts we were able to recover TCRγδ.sup.+ T cells in all tissues analysed, 30 days after transfer, with a strong enrichment for CD3.sup.+Vδ.sup.+ T cells (
[0195] In order to dynamically follow tumour growth using bioluminescence, we transduced MEC-1 cells with firefly luciferase-GFP, and transferred 10.sup.7 MEC-1 cells sub-cutaneously into BRG animals. After 7 days we injected luciferin i.p. to determine tumour load as a function of luminescence, before ascribing treatment (or PBS control) to the animals. We performed two transfers of TCRγδ.sup.+ T cells within 5 days. We then measured tumour size as a function of time using a Caliper; importantly, we detected a clear reduction in primary tumour size in treated animals when compared to controls (
[0196] Tumour progression growth was faster in NSG hosts, which seemingly prevented TCRγδ.sup.+ T cells from interfering with primary tumour growth (
[0197] Examination of the TCRγδ.sup.+ T cell progeny at the end of the experiment in the NSG model confirmed robust infiltration into the tumour tissue (
[0198] Collectively, these in vivo data provide great confidence in the safety and efficacy of the generated TCRγδ.sup.+ T cells for CLL treatment, thus inspiring their clinical application.
[0199] In conclusion, we have developed a new and robust (highly reproducible) clinical-grade method, devoid of feeder cells, for selective and large-scale expansion and differentiation of cytotoxic Vδ2.sup.− γδ T cells; and tested their therapeutic potential in pre-clinical models of chronic lymphocytic leukemia (CLL). Our cellular product, named DOT-cells, does not involve any genetic manipulation; and specifically targets leukemic but not healthy cells in vitro; and prevents wide-scale tumor dissemination to peripheral organs in vivo, without any signs of healthy tissue damage. Our results provide new means and the proof-of-principle for clinical application of DOT-cells in adoptive immunotherapy of cancer.
[0200] Supplementary Data
[0201] The following section discloses additional data generated with the use of the previously described invention. The data contained herein confirmed previous results and expanded on previous observations and should be used as supporting information for a better understanding of the subject matter.
[0202] As previously explained, the combination of interleukin-2 (IL-2) and interleukin-4 (IL-4) has been used with some success to expand Vδ1.sup.+ T cells in vitro. However, we found that the presence of IL-4 in the culture medium induced a strong downregulation of natural killer (NK) activating receptors (such as NKG2D, NKp30 and NKp44) on cultured TCRγδ.sup.+ T cells, weakening their anti-tumor responses.
[0203] Our previous results of experiments 1-4 are presented here again in more detail (see Table 11). Additional results obtained in parallel culture conditions (marked with an asterisk) are shown, for a more complete understanding of the results. It is also disclosed herein the percentage of NKp30.sup.+Vδ1.sup.+ T cells observed after cell culture with each condition. The observed downregulation of expression of NKp30 on cultured cells further confirmed that the potent inhibitory effects of IL-4 on Vδ2.sup.− γδ T cells also occurred when IL-2 was present in the culture medium (i.e., when the culture medium contained both IL-2 and IL-4). These data confirmed that the inhibitory effects of IL-4 are dominant over the activating effects of IL-2 on cultured TCRγδ.sup.+ T cells, and highlighted the importance of removing IL-4 on the second culture step.
[0204] As previously explained, although many different concentrations and combinations of IL-2, IL-7 and IL-15 were tested in parallel, IFN-γ was consistently found to be a much more effective reagent for promoting the selective expansion of Vδ.sup.+ T cells in culture (when used in the presence of IL-4 and a T cell mitogen, such as anti-CD3 mAb). As it was previously suggested (but not formally shown), the use of IFN-γ alone was more effective (in promoting cell expansion in culture), than the combination of IFN-γ with either IL-2, IL-7 or IL-15, or than the combination of IL-2 with either IL-15 or IL-7 (Table 12). These data confirmed that IL-15, IL-2 and IL-7 have a detrimental effect on TCRγδ.sup.+ T cell expansion, when cells are cultured in the presence of IL-4 and IFN-γ.
[0205] As previously explained, fold expansions in large cell culture bags were, as expected, of lower magnitude than those from 96-well plates, but still generated relevant numbers for clinical translation. Total absolute cell numbers obtained after large-scale cell culture in clinical grade cell bags are now detailed in Table 13.
[0206] As previously explained, the cell culture protocol obtained with the previously described method is appropriate for use in clinical applications. In fact, several materials and reagents have been approved by at least one regulatory agency (such as the European Medicines Agency or the Food and Drug Administration) for use in clinical applications. The full list is detailed in Table 14.
[0207] As previously described, the 2-step method of cell isolation proposed by the described invention generates cell samples enriched in TCRγδ.sup.+ T cells that are viable and can be further cultured.
[0208] As previously explained, a very reproducible expansion was achieved with the culture method of the present invention, and the composition of the final cellular product was remarkably similar across multiple donors.
[0209] For a more complete characterization of cells obtained with the previously described invention, and given the novelty of the method and resulting cellular product, we performed large-spectrum phenotyping of 332 different cell surface markers (
[0210] As previously explained, preliminary experiments with the use of blocking antibodies against activating receptors expressed on TCRγδ.sup.+ T cells showed that anti-tumor cytotoxicity was partially reliant on NKG2D and NKp30 receptors expressed by the expanded TCRγδ.sup.+ T cells. Additional experiments presented herein also revealed a role for the γδTCR in tumor cell recognition (
[0211] As previously explained, the expression of activating Natural Cytotoxicity Receptors (NCRs, including NKp30 and NKp44), and NKG2D was robustly induced in long-term cultured Vδ1.sup.+ T cells. Here we show that the same effect was observed in the Vδ1.sup.−Vδ2.sup.− cell subset. When we applied a gate (in FACS plot analysis) to the expanded (and differentiated) CD3.sup.+Vδ1.sup.− Vδ2.sup.− cell subset in the same cultures, we observed that these cells expressed around the same levels of NCRs as expressed by differentiated Vδ1.sup.+ cells (
[0212] As previously explained, expanded and differentiated Vδ1.sup.+ cells obtained with the method of the present invention were highly cytotoxic against leukemia cells in vitro. Here we show in more detail that the expanded and differentiated Vδ1.sup.−Vδ2.sup.− cell subset is also highly cytotoxic against tumor targets. We sorted CD3.sup.+ Vδ1.sup.+ cells and CD3.sup.+ Vδ1.sup.−Vδ2.sup.− cells from the same cultured cell samples by flow cytometry and co-cultured each subset with target tumor cells, in vitro. We observed that both subsets could efficiently eliminate target cells. (
TABLE-US-00011 TABLE 11 Fold NKp30+ Vδ1.sup.+ increase Vδ1.sup.+ Cond. Condition: T cells of Vδ1.sup.+ T cells Exp. No (cultured 1 million cells/ml for 14 days in 96-well plates) (%) T cells (%) 1 1 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 31.6 77 0.5 2* 20 ng/ml IL-2 + 1 μg/ml α-Vδ1 TCR mAb 4.7 8 6.2 3 500 ng/ml IL-2 + 1 μg/ml PHA 10.5 4 13.4 4* 20 ng/ml IL-2 + 1 μg/ml α-CD3 mAb 5.3 2 9.1 5 20 ng/ml IL-2 + 1 μg/ml PHA (control) 1.9 1 10.6 2 1 20 ng/ml IL-2 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 51.7 75 0.0 2 20 ng/ml IL-2 + 1 μg/ml α-Vδ1 TCR mAb + 20 ng/ml IL-4 55.9 69 0.2 3 5 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 61.9 62 0.0 4* 1 μg/ml PHA + 20 ng/ml IL-4 79.6 38 0.3 5 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 (previous best) 45.3 27 0.2 6 100 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 55.7 22 0.4 7 300 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 57.0 21 1.6 8* 20 ng/ml IL-2 + 20 ng/ml IL-4 2.4 2 0.0 3 1 5 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 61.4 138 0.3 2 5 ng/ml IL-2 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 59.4 124 1.2 3 20 ng/ml IL-2 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 (prev. best) 45.4 105 1.0 4 5 ng/ml IL-15 + 1 μg/ml PHA + 20 ng/ml IL-4 76.2 21 1.2 5 5 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 69.1 19 1.6 6 20 ng/ml IL-15 + 1 μg/ml PHA + 20 ng/ml IL-4 69.9 13 1.3 7 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 67.7 11 1.0 4 1 20 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 79.5 1 349 0.8 2 3 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 67.4 1 014 0.4 3 2 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 81.6 909 1.8 4 5 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 (prev. best) 69.4 624 1.9
TABLE-US-00012 TABLE 12 Total Fold Live Vδ1.sup.+ increase Cond. Condition: cells T cells of Vδ1.sup.+ No (cultured 1 million cells/ml for 14 days in 96-well plates) (%) (%) T cells 1 20 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 100 ng/ml IL-4 85.9 80.1 12 166 2 7 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 100 ng/ml IL-4 89.9 93.0 10 757 3 2 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 100 ng/ml IL-4 87.3 75.2 9 394 4 0.3 ng/ml IL-15 + 2 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 100 ng/ml IL-4 77.1 59.7 4 361 5 2 ng/ml IL-15 + 2 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 100 ng/ml IL-4 90.0 67.0 811 6 0.3 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 100 ng/ml IL-4 89.7 75.5 614 7 7 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 85.7 83.2 10 083 8 2 ng/ml IL-2 + 2 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 87.5 59.4 7 208 9 2 ng/ml IL-2 + 7 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 80.4 71.6 7 151 10 7 ng/ml IL-7 + 2 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 91.1 65.6 6 193 11 2 ng/ml IL-2 + 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 88.7 70.5 5 192 12 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 89.0 68.3 1 890 13 0.3 ng/ml IFN-γ + 2 μg/ml α-CD3 mAb + 100 ng/ml IL-4 82.7 59.9 6 139 14 2 ng/ml IL-7 + 0.3 ng/ml IFN-γ + 2 μg/ml α-CD3 mAb + 100 ng/ml IL-4 87.6 72.9 5 290 15 0.3 ng/ml IL-15 + 0.3 ng/ml IFN-γ + 2 μg/ml α-CD3 mAb + 100 ng/ml IL-4 85.9 80.1 4 840 16 0.3 ng/ml IL-15 + 2 μg/ml α-CD3 mAb + 100 ng/ml IL-4 90.0 64.2 2 943 17 2 μg/ml α-CD3 mAb + 100 ng/ml IL-4 85.8 73.0 1 826
TABLE-US-00013 TABLE 13 Total live cells generated from 1 Buffy Coat unit: (millions of cells) Donor: Day 0 Day 21 A 2.4 .sup. 968.0 B 4.8 1 004.0 C 83.3 .sup. 440.0 D 5.7 1 152.0 E 9.2 1 024.0 F 25.0 1 564.0 G 4.0 1 604.0 H 2.0 1 276.0
TABLE-US-00014 TABLE 14 Product Manufacturing Reagent/Material Manufacturer reference quality system* For magnetic depletion of TCRα/β.sup.+ cells: CliniMACS ® Plus Instrument Miltenyi Biotec, GmbH 151-01 cGMP, ISO 13485 CliniMACS ® TCRα/β Kit 200-070-407 compliant CliniMACS ® Depletion Tubing Set 261-01 CliniMACS ® PBS/EDTA Buffer 700-25 For magnetic enrichment of CD3.sup.+ cells: CliniMACS ® CD3 reagent Miltenyi Biotec, GmbH 273-01 cGMP, ISO 13485 CliniMACS ® Tubing Set TS 161-01 compliant For cell culture: Cell culture cassettes Saint-Gobain CC-0500 cGMP, 21 CFR Clamps Saint-Gobain 1C-0022 820 compliant VueLife ® cell culture FEP bag Saint-Gobain 750-C1 OpTmizer ™ T-cell expansion medium Thermo Fisher Scientific A10485-01 cGMP, ISO L-Glutamine Thermo Fisher Scientific 25030-032 13485: 2003 Human anti-CD3 mAb (clone OKT-3) Miltenyi Biotec, GmbH 170-076-116 or ISO Recombinant Human IL-4 CellGenix GmbH 1003-050 9001: 2008 Recombinant Human IL-21 CellGenix GmbH 1019-050 compliant Recombinant Human IFN-γ R&D Systems 285-GMP Recombinant Human IL-1β CellGenix GmbH 1011-050 Recombinant Human IL-15 CellGenix GmbH 1013-050 *Note: Some products are sold as certified medical devices for use in the EU and/or US. All other products are sold for the manufacturing of cell-based products for clinical research. They can be used in clinical trials under Investigational New Drug (IND) or Investigational Device Exemption (IDE) applications.
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