Our scientists were among the first to develop custom antigen-specific T cells for research use and have created them against a diverse range of targets, including viral, tumor, and self-antigens.

We’re now developing the next generation of antigen-specific T cells to target some of today’s most prevalent and challenging diseases. More on that below. But first, a primer on antigen-specific T cells.

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The Role of Antigen-Specific T Cells in Disease Research and Treatment

T cells are a major player in the immune response in various diseases, including cancer and pathogenic infections, and are mediators in inflammation and autoimmunity. A large body of research has been conducted over the past few decades to manipulate T cell responses, leading to many clinical strategies including cancer vaccines, T cell adoptive therapy, CAR-T therapy, immune-oncology, and the use of regulatory T cells for autoimmune diseases.

Antigen-specific T cells are an important tool in T cell biology research and potency assays. Investigators can isolate them directly from the disease site (e.g., tumor-infiltrating lymphocytes (TIL) or inflammatory lesion infiltrate populations), but these procedures often yield a limited number of cells due to low cell frequency and lack of robust isolation methods.

Alternatively, antigen-specific T cells can be generated in vitro using antigen-presenting cells pulsed with whole antigens or peptides. However, this process is often time-consuming with a low success rate.

About the gp100 Antigen

gp100 (aka Pmel17) is a 661 amino acid melanocyte glycoprotein that is overexpressed in melanomas. Several gp100-derived peptides have been identified as targets of cytotoxic T cells in melanoma patients. One specific gp100 peptide — wild type gp100^209-217 (ITDQVPFSV) and its analog, which contains a threonine to methionine substitution at position 210, gp100^{209-217, T210M} (IMDQVPFSV) — have been used in several melanoma vaccine trials. The latter is shown to be more immunogenic resulting from increased binding to the HLA-A-02:01 molecule.

Ignyte Bio’s anti-gp100-specific T cells are generated against the analog peptide gp100^{209-217, T210M}.

Figure 1 shows specific IFN-γ secretion when T cells are incubated with HLA-A-02:01⁺ target cell in the presence of gp100^{209-217, T210M} peptide but not a control peptide. These T cells also recognize the wild type gp100^209-217 in association with HLA–A-02:01 as shown in a tetramer binding study in Figure 2.  These T cells specifically lyse HLA-A-02:01⁺ target cells in the presence of both wild type and analog gp100 peptides, as seen in Figure 3.

Figure 1. Recognition of Antigenic Peptide (gp100 ^{209-217, 210M)}) Lead to Specific Secretion of IFN-γ.  Freshly thawed anti-gp100 CD8⁺ T cells were plated at 2 x 10⁴ cells/well in the presence of 2 x 10⁴ T2 cells, an HLA-A-02:01⁺ B-LCL (ATCC, Manassas, VA) and different concentrations of gp100^{209-217, T210M} (IMDQVPFSV) or tyrosinase^369-377 (YMDGTMSQV) control peptide in a 96-well round bottom plate. After 18-24 hours of incubation at 37^oC, 5% CO₂, culture supernatants were harvested and assayed for IFN-g using the Lumit™ IFN-γ Immunoassay (Promega, Madison, WI). IFN-γ concentration is plotted against gp100 and control peptide concentrations.

Figure 2. Recognition of Wild Type gp100 ^209-217 by T Cells Generated Against Mutant gp100 ^{209-217, M210} Peptide Demonstrated by Peptide/MHC Tetramer Binding Analysis. Cryopreserved T cells were thawed and stained with APC-labelled HLA-A-02:01-gp100^209-217 (ITDQVPFSV, gp100 Tetramer APC) or a control tetramer HLA-A-02:01-tyrosinase^369-377 (YMDGTMSQV, TYR Tetramer APC). These cells were counterstained with anti-CD8 Pacific Blue reagent and analyzed by flow cytometry. Non-viable cells were excluded from the analysis using 7-AAD.

Figure 3. gp100-Specific T Cells Recognize and Kill Target Cells in the Presence of Mutant gp100 ^{209-217, M210} and Wild Type gp100^209-217 Peptides.  Freshly thawed anti-gp100 CD8⁺ T cells were plated at 2 x 10⁴ cells/well in the presence of 2 x 10⁴ T2 cells, an HLA-A-02:01⁺ B-LCL (ATCC, Manassas, VA) and different concentrations of analog gp100^{209-217, T210M}(IMDQVPFSV), wild type gp100^209-217 (ITDQVPFSV)  or WT-1 ^126-134 (RMFPNAPYL) control peptide in a 96-well round bottom plate. After 18-24 hours of incubation at 37^oC, 5% CO₂, cells in each well were stained with the viability dye 7-AAD and analyzed using flow cytometry. % Cytotoxicity (=% 7-AAD⁺ target cells) is plotted against the respective peptide concentrations.

Conducting Melanoma Research?