Figure 2. In Vivo Persistence and Reactivity Profile of KRAS G12D–Specific T-Cell Clones in the Infusion Product.
Panel A shows the results of deep sequencing of the variable region of the T-cell receptor (TCR) beta chain to measure the frequency of each of the four identified KRAS G12D–reactive T-cell clones in the infusion product (Rx1), in three metastatic lung samples before cell transfer (designated Tu-1, Tu-2A, and Tu-2B), in the one progressing lesion after cell therapy (designated Tu-Pro), and in the peripheral blood of Patient 4095 before and at various times after cell therapy. T-cell receptors are identified according to their gene names (TRBV5-6 and TRBV10-02, with A, B, and C denoting different T-cell receptor clonotypes that share the same T-cell receptor beta variable gene family). The numbers in parentheses indicate the rank of the T-cell receptor sequence in the given sample. A circle with an X and ND denote not detected (<0.0002%). Autologous peripheral-blood T cells were genetically engineered to express each of the indicated KRAS G12D–reactive T-cell receptors that were present in the infusion product. Panel B shows the expression of the T-cell activation marker 4-1BB on T cells engineered with the indicated T-cell receptor after overnight coculture with autologous peripheral-blood mononuclear cells that were incubated with titrated amounts of wild-type KRAS or G12D mutant peptides consisting of either 9 or 10 amino acids (9mer or 10mer). Panel C shows interferon-γ (IFN-γ) production (left) and 4-1BB expression (right) of T cells that were genetically engineered with the indicated T-cell receptor after overnight coculture with two KRAS G12D–positive pancreatic-cancer cell lines not expressing the HLA-C*08:02 allele (mock) or expressing the HLA-C*08:02 allele with the use of enzyme-linked immunosorbent spot (ELISPOT) assay and flow cytometry, respectively. MDA denotes MDA Panc48 cell line, and HP HPAC cell line. Flow cytometry data are gated on CD8+ KRAS G12D–reactive cells. Values of more than 500 on the ELISPOT assay were not considered to be accurate.