Background
Osteosarcoma is the primary malignant bone tumor with peak incidence in both children and young adults. Despite significant progress in treatment, individuals with metastatic or recurrent disease have poor prognosis due to lung metastasis with limited clinical trials. While autologous chimeric antigen receptor (CAR) therapies have shown promise, they face limitations due to their high cost, manufacturing failures, time-consuming production, and often limited efficacy. Therefore, development of cost-effective allogeneic cell therapies is essential as it can provide a readily available product for multiple patients. As such, gamma delta () T-cells are an ideal candidate for allogenic CAR therapy as they combine innate and adaptive immune properties and have been successfully administered in patients without causing graft-versus-host disease (GvHD). Given limitation of expansion methods utilizing Zoledronic acid, we present an enhanced approach for propagating T-cells with a diverse T-cell receptor (TCR) repertoire, yielding clinically significant quantities.
Methods
To provide activation and proliferation signals necessary for the expansion of T-cells, we have modified the K562 cell line, a natural target for T-cells, to express CD32, CD64, CD86, CD137L, and enhanced IL15/IL15Rα fusion protein (membrane-bound IL-15; mIL15). Column enriched TCR T-cells from peripheral blood were co-cultured with irradiated OKT3 loaded K562 AaPC along with addition of exogenous cytokines (IL-2, and IL-21) in a 7-day stimulation cycle, and ex vivo expanded TCR T-cells were phenotypically and functionally characterized.
Results
Peripheral blood-derived TCR T-cells expanded (average) 3×103– fold (n=4) in 29-days upon co-culture with K562-derived feeder cells. T-cells generated were predominantly CD3+TCR + (mean±SD, 99%±2%) with polyclonal repertoire consisting of all three V (V1, V2, V3) and expressed markers of central memory T-cells (CCR7/NKG2D; 20%/50%). Activated TCR T-cells were also able to produce abundant proinflammatory Th1 cytokines (IFN-, TNF-α) and low Th2 cytokines (IL-4, IL-10) in response to activation via TCR (figure 1). Polyclonal T-cells exhibited a range of cytolysis against osteosarcoma tumor cell lines and patient derived xenografts (PDX). Furthermore, when tested in immunocompromised (NSG) mice bearing established SJSA1 osteosarcoma, we observed superior survival (p<0.05) of group receiving the TCR T-cells compared with no treatment control. This was confirmed at necropsy by reduced tumor burden and presence of T-cells in mice treated with TCR T-cells.
Conclusions
Our data demonstrate that K562-derived AaPCs are a superior platform for generation of ‘off the shelf’ TCR T-cell with desired phenotype for enhanced persistence in treatment of osteosarcoma after adoptive transfer.
Abstract 423 Figure 1
Characterization of peripheral blood derived TCR T cells against Osteosarcoma. (A) Phenotypic characterization of TCR T cells isolated from peripheral blood (day 0) and end of coculture (day 24) is shown. Miltenyi column isolated TCR T cells were co-cultured with K562-derived AaPC every 7-days in a stimulation cycle; at the end of the stimulation cycle T-cells were enumerated and evaluated by flow cytometry. Viable cells were gated for CD3+ T cells and analyzed for the presence of CD56, CD3, CD4, CD8, TCR yo and TCRaß using flow cytometry. (B) Expansion kinetics of CD3*, TCR + T cells is shown over time. (C) Killing of osteosarcoma cell lines and patient-derived xenografts (PDX) by ex vivo expanded TCR T cells in a standard 4-hr chromium release assay. Allogeneic CD19* B cells was used as control to depict background lysis and lack of allo-reactivity. (D) In vivo anti-tumor activity of TCR T cells against SJSA1 cell line (modified to express a luciferase) in NSG mice. NSG mice were injected with SJSA1 on day 0 and tumor was allowed to engraft for 5 days, following which T cells were injected day 6. Tumor burden was calculated using serial bioluminescent imaging from SJSA1