DOI: 10.5061/dryad.q2bvq83t1
title: Payload-delivering engineered γδ T cells display enhanced cytotoxicity, persistence, and efficacy in preclinical models of osteosarcoma

description: 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.
description: # 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](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.](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](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.