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1222 Antigen-specific delivery of TCR and costimulatory signals by a protein scaffold selectively activates and markedly expands naïve MART-1-specific CD8+ T cells
  1. April L Mueller,
  2. Scott Garforth,
  3. Steven Almo and
  4. Harris Goldstein
  1. Albert Einstein College of Medicine, Bronx, NY, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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.


  1. 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

Ethics Approval This study was approved by Albert Einstein College of Medicine institution’s Ethics Board; approval number 2017–8116.

Abstract 1222 Figure 1

SynTac Construct Schematic and Validation. (A) SynTac design comprises a light chain (LC) and a heavy chain (HC). The LC encompasses an easily modifiable antigenic peptide linked to human ß2M through a (G4S)5 peptide linker, connected to an anti-CD28 costimulatory signaling molecule. The HC consists of an HLA-A*02:01 MHC heavy chain with a Y84C mutation to facilitate disulfide bridge linkage to the LC. It is fused with a modified human IgG1 Fc domain with an N297Q mutation. The inclusion of the human IgG1 Fc domain enables dimerization between two heavy chains, resulting in synTac molecules with dual MHC-1 and costimulatory ligands. (B) SynTac constructs are validated for binding and signaling. Binding is validated through incubation with a MART-1 TCR cell clone and staining with an anti-His tag to detect synTac bound to the cell surface. Signaling is validated through incubation with MART-1 TCR cell clone containing an NFAT-luciferase reporter system. Upon synTac binding and downstream TCR signaling, NFAT will be activated and luciferase will be expressed which can be detected with a luminometer. (C) MART-1 anti-CD28 and modless synTac binding can be detected with anti-His Tag alexafluor 647 at both 10nM and 100nM. No signal is detected without the addition of syntac. No signal is detected when using a TCRneg cell line indicating no nonspecific binding. (D) Significant luciferase signal (****p=p<0.0001) is detected upon incubation with both 10nM and 100nM synTac. No luciferase is detected in TCRneg indicating low background luciferase signal

Abstract 1222 Figure 2

Expansion and differentiation of naive MART-1 reactive cells. (A) Schematic of naive isolation using immunomagnetic sorting followed by synTac-based stimulation. Cells were incubated at 500K cells/well in a 48 well plate at 10nM synTac or unstimulated control. Initial media contains 30ng/uI IL-21, and at day 3 is supplemented with 10nM IL-7 and 10nM IL-15. Media is exchanged every 2–3 days until day 18 where cells are stained for flow cytometric analysis. Cells at day 18 are saved for further downstream assays. (B) Representative plots are shown of significant expansion of MART-1 specific CD8+ T cells detected with tetramer staining at day 18 when incubated with MART-1 synTac but not unstimulated control. (C) Tetramer positive cells are analyzed for expression of surface markers CD62L and CD45RO. Cells incubated with synTac show increased expression of CD45RO and decreased expression of CD62L. (D) IFN-y ELISPOT assay with unstimulated cells or cells stimulated with modless or anti-CD28 MART-1 synTac using peptide-loaded with MART-1 peptide at 10uM or unloaded T2 cells as targets. Anti-CD28 expanded cells show significant expression of IFN-y when cocultured with peptide loaded T2 cells but not unloaded T2 cells (**** = p<0.0001). (E) 50K Effectors cells were cocultured with 50K 1uM MART1 peptide-loaded or unloaded T2 target cells for 24hr before flow cytometric detection of remaining live T2 cells. Cells stimulated with both anti-CD28 and modless MART-1 synTac resulted in significant killing of peptide-loaded T2 cells compared to unloaded T2 cells (**=p<0.01, ***=p<0.001). Statistical significance determined by 1-way ANOVA, analysis done in GraphPad Prism Version 9.4.1.

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