Background Adoptive cell transfer (ACT) shows promise as an immunotherapy for melanoma and other cancers. However, there are several challenges associated with ACT such as the logistical complexity and inconsistency when using patient-derived antigen-presenting cells for ex vivo expansion of CD8+ T cells. To overcome this limitation, we have developed a modular artificial antigen presenting cell platform called synthetic T cell activation (synTac) capable of antigen-specific TCR activation and costimulatory signaling.1 Here, we describe the construction of a synTac capable of selective activation of TCRs specific for the melanoma-associated MART-1 antigen and demonstrate its capacity to selectively activate and expand MART-1-reactive naive CD8+ T cells from healthy donors which differentiate into functional MART-1-specific cytotoxic T cells.
Methods The synTac protein consists of a light chain (LC) and a heavy chain (HC). The LC includes an antigenic peptide linked to human β2M through a (G4S)5 peptide linker, connected to an anti-CD28 costimulatory signaling molecule. The HC comprises an HLA-A*02:01 MHC heavy chain with a Y84C mutation for disulfide bridge linkage to the LC. It is fused with a modified human IgG1 Fc domain with an N297Q mutation to prevent FcγR binding and antibody-dependent cellular cytotoxicity against synTac-bound T cells. The human IgG1 Fc domain allows dimerization of two heavy chains, resulting in synTac molecules with dual peptide-MHC-I and costimulatory ligands. The MART-1 synTac was produced and its binding to MART-1-specific TCR was demonstrated. TCR-specific signaling was confirmed through NFAT-luciferase reporter assay. Expansion of MART-1-specific naïve T cells was quantified using flow cytometry and their memory phenotype was assessed with surface memory markers. Functional activity of the expanded cells was evaluated using enzyme-linked immunospot assay (ELISPOT) for cytokine production and peptide-loaded T2 cell cytotoxicity assays.
Results We validated MART-1 synTac constructs (figure 1) and confirmed that CD28 costimulation efficiently activated primary naïve MART-1-specific T cells in healthy donors (figure 2A,B). Expanded T cells exhibited differentiation from naïve-like to Teffector and Tcentral memory cells (figure 2C). They demonstrated high functionality through ELISPOT assays (figure 2D) and effectively killed target cells in cytotoxicity assays (figure 2E).
Conclusions SynTac is an innovative biologic that activates and expands naïve CD8+ T cells into functional tumor specific CD8+ T cells ex vivo. It expands immunotherapy options for cancer by providing an off-the-shelf, scalable solution for generating clinical-grade T cells for adoptive immunotherapy. Additionally, it holds potential as an immunotherapeutic for in vivo expansion of tumor-specific T cells.
Li M, Garforth SJ, O’Connor KE, et al. T cell receptor-targeted immunotherapeutics drive selective in vivo HIV- and CMV-specific T cell expansion in humanized mice. J Clin Invest. 2021;131(23):e141051. doi:10.1172/JCI141051
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