Payload-delivering engineered γδ T cells display enhanced cytotoxicity, persistence, and efficacy in preclinical models of osteosarcoma
Data files
May 15, 2024 version files 170.98 KB
-
Data_File_S2_supp_figs.xlsx
72.67 KB
-
Excel_data_compilation_main_figs.xlsx
88.39 KB
-
README.md
9.92 KB
Abstract
T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous αβ T cells. These approaches are limited by tonic signaling of synthetic constructs and costs associated with manufacturing. γδ T cells are an emerging alternative for cellular therapy, possessing innate anti-tumor activity, potent antibody-dependent cellular cytotoxicity, and minimal alloreactivity. We present an immunotherapeutic platform technology built around the innate properties of the Vγ9Vδ2 T cell, harnessing specific characteristics of this cell type and offering an allo-compatible cellular therapy that recruits bystander immunity. We engineered γδ T cells to secrete synthetic tumor-targeting opsonins in the form of an scFv-Fc fusion protein and a mitogenic IL-15Ra–IL-15 fusion protein (stIL15). Using GD2 as a model antigen, we show that GD2-specific opsonin-secreting Vγ9Vδ2 T cells (stIL15-OPS-γδ T cells) have enhanced cytotoxicity and promote bystander activity of other lymphoid and myeloid cells. Secretion of stIL-15 abrogated the need for exogenous cytokine supplementation and further mediated activation of bystander natural killer cells. Compared to unmodified γδ T cells, stIL15-OPS-γδ T cells exhibited superior in vivo control of subcutaneous tumors and persistence in the blood. Moreover, stIL15-OPS-γδ T cells were efficacious against patient-derived osteosarcomas in animal models and in vitro, where efficacy could be boosted with the addition of zoledronic acid. Together the data identify stIL15-OPS-γδ T cells as a candidate allogeneic cell therapy platform combining direct cytolysis with bystander activation to promote tumor control.
README: γδ T cells engineered to secrete immune-active payloads display enhanced persistence and efficacy in preclinical models of osteosarcoma
https://doi.org/10.5061/dryad.q2bvq83t1
2 datasets are provided, one pertaining to the main figures (Dataset 1) and one to the supplementary. figures (Dataset 2). The datasets comprise tabulated data for all of the figures shown.
Description of the data and file structure
Data is shown in tabulated Excel files, with each tab pertaining to a panel in the figures
Data File S1
1B: Dead cell % for SUP-T1-wt targets or SUP-T1-GD2 targets co-cultured with Unmodified gdT cells or OPS-gdT cells, as determined by flow cytometry
1C: Absolute numbers of gdT cells ± engineering at different days of expansion in the conditions indicated in the columns
1D-E: Differences in expression or indicated markers between unmodified-gdT cells or 14G2a stIL15-OPS-gdT cells expressed as Earth Movers Distance values, derived from mass cytometric analysis. Calculation of Earth Movers Distance for this and subsequent figures was done using a custom Python script which is available at: https://doi.org/10.5281/zenodo.10993245.
1F: Fold change of SUP-T1-wt or SUP-T1-GD2 tumour cell numbers in co-culture with gdT cells ± engineering. Numbers shown are fold changes in target cell number, relative to baseline.
2B: % human Vd2 gdT cells in mouse blood as determined by flow cytometry
2C: Bioluminescence of subcutaneous SUP-T1-GD2 tumours in NSG mice treated with gdT cells ± engineering, over a 15 day period
2D: Survival of mice bearing subcutaneous SUP-T1-GD2 tumours treated with gdT cells ± engineering
3C: DREMI scores for the interaction between transgene and pERK in CAR-abT cells and 14G2a-stIL15-OPS-gdT cells in the presence or absence of antigen-postivie targets. DREMI scores derived from mass cytometric signalling analysis and calculated using simpledremi (https://dpeerlab.github.io/dpeerlab-website/dremi-download.html)
3E: DREMI scores for the interaction between transgene and key signalling markers in CAR-abT cells, calculated from mass cytometric data
3F: DREMI scores for the influence of transgene expression on key markers in 14G2a-stIL15-OPS-gdT cells, calculated from mass cytometric data
3H: Earth movers distance for the differences between transduced and non-transduced abT or gdT cells, with corresponding DREMI scores for the influence of transgene expression on key markers in 14G2a-stIL15-OPS-gdT or CAR-abT cells, calculated from mass cytometric data
4A: Dead cell % for SUP-T1-wt targets or SUP-T1-GD2 targets co-cultured with Unmodified gdT cells in the presence of supernatant from unmodified gdT supernatant or 14G2a-OPS-gdT supernatant, as determined by flow cytometry
4B: Dead cell % for SUP-T1-wt targets or SUP-T1-GD2 targets co-cultured with fresh NK cells in the presence of supernatant from unmodified gdT supernatant, 14G2a-OPS-gdT supernatant, 14G2a-stIL15-OPS-gdT supernatant or 1ug/ml ch14.18 as determined by flow cytometry
4C: Dead cell % for SUP-T1-wt targets or SUP-T1-GD2 targets co-cultured with macrophages in the presence of supernatant from unmodified gdT supernatant, 14G2a-OPS-gdT supernatant, 14G2a-stIL15-OPS-gdT supernatant or 1ug/ml ch14.18 as determined by flow cytometry and expressed as % death relative to the mean of the tumour alone control.
5B: Dead cell % for SUP-T1-GD2 targets co-cultured with unmodified gdT cells in the presence of different concentrations of ch14.18, as determined by flow cytometry
5C: Dead cell % for SUP-T1-GD2 targets co-cultured with unmodified gdT cells in the presence of 1 ng/ml ch14.18, as determined by flow cytometry
5D: Relative fluorescence intensity for chemokine receptor expression in gdT cells ± engineering, as determined by flow cytometry and compared to FMO controls.
6B: Differences in marker expression between unmodified gdT cell monocultures and 14G2a-stIL15-OPS-gdT cells ± co-culture with osteosarcoma cells, expressed as earth movers distance derived from mass cytometric data
6C: Differences in marker expression between osteosarcoma monocultures and those in co-culture with unmodified gdT or 14G2a-stIL15-OPS-gdT cells, expressed as earth movers distance derived from mass cytometric data
6D: Killing of luciferase-transduced osteosarcoma cells by gdT cells ± engineering ± ch14.18, expressed as luminescence relative to targets alone
6E: Killing of luciferase-transduced osteosarcoma cells by eingeered gdT cells ± zoledronic acid, expressed as luminescence relative to targets alone
7A: Tumour growth of osteosarcoma in NSG mice treated with the indicated interventions, expressed as total bioluminescent flux over time
7B: Event free survival of NSG mice treated with the indicated interventions. An event is defined as tumour total flux doubling.
Data file S2
S1C: Transduction efficiency of gdT cells as determined by % GFP expression using flow cytometry. Vd2 % in the specified preparations (also detemrined by flow cytometry) is also shown
S1E: Dead cell % for SUP-T1-wt targets or SUP-T1-GD2 targets co-cultured with Unmodified gdT cells, as determined by flow cytometry
S1F: Median Fluorescence Intensity Standard curve generation for determination of SFP concentration by flow cytometry, with curresponding SFP yield from engineered gdT cells
S2B: Dead cell % for SUP-T1-wt targets or SUP-T1-GD2 targets co-cultured with Unmodified gdT cells, 14G2a-OPS gdT cells or 14G2a CAR-gdT cells, as determined by flow cytometry
S2C: Relative tumour cell count for SUP-T1-wt targets or SUP-T1-GD2 targets co-cultured with Unmodified gdT cells, 14G2a-OPS gdT cells or 14G2a CAR-gdT cells, as determined by flow cytometry and relative to baseline cell numbers
S2D: Median fluorecence intensity of 4 markers associated with exhaustion on 14G2a-OPS gdT cells or 14G2a CAR-gdT cells, determined by flow cytometry
S3B: % GFP expression over time on unmodified or engineered Vd2+CD3+ cells in presence or absence of IL-2 as determined by flow cytometry
S3D: Concentration of IL-15 in supernatants from unmodified or engineered gdT cells, as determined by IL-15 ELISA
S3E-I: Differences in expression or indicated markers between unmodified-gdT cells or stIL15-gdT cells expressed as Earth Movers Distance values, derived from mass cytometric analysis
S4A: Frequency of CD3+Vd2+ cells within live cell populations in the blood of NSG mice at 7 and 14 days after injection of indicated cell type
S4B: Concentration of IL-15 in supernatants from unmodified or engineered gdT cells, as determined by IL-15 ELISA
S4D: Concentration of SFP in supernatants from unmodified or engineered gdT cells, as determined by flow cytometry
S4F: Fold change in unmodfiied or engineered Vd2 numbers during expansion
S4G: Percentage of Vd2+CD3+ cells at D0 and D12 of expansion, comparin unmodified and engineered gdT cells
S4H: Absolute Vd2+CD3+ cell counts for cultures of engineered gdT cells
S5A-C: Differences in expression or indicated markers between unmodified-gdT cells or 14G2a-stIL15-OPS-gdT cells expressed as Earth Movers Distance values, derived from mass cytometric analysis
S5E: Numbers of engineered or unmodfieid Vd2+ cells in co-cultures with SUP-T1-WT or SUP-T1-GD2, expressed relative to cell numbers at D0
S5G: Killing of Kelly neuroblastoma cells by unmodified or engineered gdT cells, expressed relative to target only
S6C-D: Subcutaneous tumour volumes (mm3) for SUP-T1-GD2 tumours in NSG mice treated with unmodified or engineered gdT cells
S8C: Dead cell % for SUP-T1-wt targets or SUP-T1-GD2 targets co-cultured with stem-cell derived neutrophils, in the presence of supernatant from 14G2a-OPS gdT cells or unmodified gdT cells, as determined by flow cytometry
S9B: Dead cell % for SUP-T1-wt targets or SUP-T1-GD2 targets co-cultured with unmodified gdT cells with varying CD16 expression (expressed as % of cells CD16+) in the presence of 1ug/ml ch14.18 antibody
S9C: % Expression of CD16 on CD3+Vd2+ cells ± engineering on D12-14 of expansion, as determined by flow cytometry
S9D: CD45RA/CD27 expression on CD3+Vd2+ cells ± engineering on D12 of expansion, as determined by flow cytometry
S10: Dead cell % for SUP-T1-GD2 targets co-cultured with unmodified gdT cells or engineered gdT cells which were prepared in a G-REX culture system
S12A: Differences in marker expression between unmodified gdT cell monocultures and 14G2a-stIL15-OPS-gdT cells ± co-culture with PDOS25 osteosarcoma cells, expressed as earth movers distance derived from mass cytometric data
S12A: Differences in marker expression between unmodified gdT cell monocultures and 14G2a-stIL15-OPS-gdT cells ± co-culture with PDOS19 osteosarcoma cells, expressed as earth movers distance derived from mass cytometric data
S13: Killing of luciferase-transduced osteosarcoma cells by NK cells ± supernatasnt from engineered gdT cells, expressed as luminescence relative to targets alone
S14A: Pre-treatment bioluminescent total flux for NSG mice with intratibial xenografts of luciferase expressing PDOS25, divided by treatment group
S15B: Transduction efficiency of 14G2a-CAR abT cells as determined by flow cytometry
S15C: Killing of luciferase-transduced PDOS25 osteosarcoma cells or Kelly neuroblastoma cells by 14G2a CAR abT cells or unmodified abT cells, expressed as luminescence relative to targets alone
S15D: Expression of GD2 on surviving cells after co-culture with 14G2a CAR abT cells or unmodified abT cells, expressed relative to starting levels of GD2 expression
S16A: Event free survival of NSG mice treated with the indicated interventions. An event is defined as tumour total flux doubling.