Rapid Assessment of Functional Avidity of Tumor-Specific T Cell Receptors Using an Antigen-Presenting Tumor Cell Line Electroporated with Full-Length Tumor Antigen mRNA

Abstract

The functional avidity of T-cell receptor (TCR)-engineered T cells towards their cognate epitope plays a crucial role in successfully targeting and killing tumor cells expressing the tumor-associated antigen (TAA). When evaluating in vitro functional T-cell avidity, an important aspect that is often neglected is the antigen-presenting cell (APC) used in the assay. Cell-based models for antigen-presentation, such as tumor cell lines, represent a valid alternative to autologous APCs due to their availability, off-the-shelf capabilities, and the broad range of possibilities for modification via DNA or messenger RNA (mRNA) transfection. To find a valuable model APC for in vitro validation of TAA Wilms’ tumor 1 (WT1)-specific TCRs, we tested four different WT1 peptide-pulsed HLA-A2+ tumor cell lines commonly used in T-cell stimulation assays. We found the multiple myeloma cell line U266 to be a suitable model APC to evaluate differences in mean functional avidity (EC50) values of transgenic TCRs following transfection in 2D3 Jurkat T cells. Next, to assess the dose-dependent antigen-specific responsiveness of WT1 TCR-engineered 2D3 T cells to endogenously processed epitopes, we electroporated U266 cells with different amounts of full-length antigen WT1 mRNA. Finally, we analyzed the functional avidity of WT1 TCR-transfected primary CD8 T cells towards WT1 mRNA-electroporated U266 cells. In this study, we demonstrate that both the APC and the antigen loading method (peptide pulsing versus full-length mRNA transfection) to analyze T-cell functional avidity have a significant impact on the EC50 values of a given TCR. For rapid assessment of the functional avidity of a cloned TCR towards its endogenously processed MHC I-restricted epitope, we showcase that the TAA mRNA-transfected U266 cell line is a suitable and versatile model APC.

Human T cells engineered with a leukemia lipid-specific TCR enables donor-unrestricted recognition of CD1c-expressing leukemia

“…When we compared the reactivity of the DN4.99, PZP8A6 and DN4.2 TCR-JK cells against different doses of mLPA presented on K562-CD1c cells we found that, despite very similar levels of surface expression, the DN4.99 TCR exhibited a lower EC 50 (half-maximal effective concentration) than the other two TCRs, and the highest CD69 expression (Fig. 2e ), suggesting stronger functional avidity 14 , 15 upon antigen engagement.…”

Anti-Tumor Potency of Short-Term Interleukin-15 Dendritic Cells Is Potentiated by In Situ Silencing of Programmed-Death Ligands

“…Fresh 5-10 x 10 6 IL-15 DCs were electroporated with WT1 mRNA in 250 µL OptiMEM (ThermoFisher Scientific) with an exponential decay pulse (300V, 150 µF) ( 7 ). U266, used as control antigen presenting cells (APCs), were treated identically but electroporated using a time constant pulse (300V, 8 ms) ( 23 ). WT1 mRNA transfection efficiency was determined 4 hours post electroporation by means of intracellular staining employing the eBioscience FoxP3/transcription factor intracellular staining buffer set (Invitrogen) and an anti-WT1 primary antibody (clone 6F-H2, Dako, Agilent, CA, US) as described previously ( 23 ).…”

mRNA vaccine: a potential therapeutic strategy

“…Therefore, the down-regulation of MHC-I on tumor cell surfaces helps tumor escape from the immunologic surveillance [ 68 ]. To improve the tumor’s immunogenicity, introducing mRNA encoding MHC-I and TAA [ 69 , 70 ] into tumor cells enabled the up-regulation of MHC-I. Thus, immune cells recognized tumor cells and pathogens rapidly, which improved the therapeutic efficacy of mRNA vaccines by enhancing viral/tumoral immunogenicity.…”