Figure 1

Clone KVA11-01 from tumor infiltrating lymphocytes and construct its corresponding expression vector A, the flow chat to acquire KVA11-01 TCR from tumor-infiltrating lymphocytes of colorectal cancer; B, select anti-CD8 and anti-MHC tetramer double positive cells by single cell sorting through flow cytometry; C, schematic diagram of expression vector of KVA11-01 TCR; D, flow fluorescence signal was detected after transfer of lentivirus candidate TCR into Jurkat reporter cell containing NFAT reporter gene, as measured by flow cytometry. MHC, major histocompatibility complex; RACE, 5′-rapid amplification of cDNA ends; PCR, polymerase chain reaction; TCR, T cell receptor; NFAT, nuclear factor of activated T-cells; GFP, green fluorescent protein; PBMC, peripheral blood mononuclear cell; TRBV, T-cell receptor beta variable; mTRBC, TCR beta chain constant of mouse; TRAV, T-cell receptor alpha variable; mTRAC, TCR alpha chain constant of mouse; CRC, colorectal cancer; TILs, tumor-infiltrating lymphocytes; FITC, fluorescein isothiocyanate; SSC, side scatter; APC, allophycocyanin."

Table 1

Comparison between KVA11-01 and TK34 TCRs for KRAS G12V mutation"

Figure 2

Comparison of affinity and expression patterns between KAV11-01 and TK34 TCR A, relative activation level of TCR+ Jurkat cells after co-incubation with T2 cells loaded with peptides, which were tested from 10-5 to 10-10mol/L and measured by flow cytometry; B, the affinity of KVA11-01 and TK34 for targeting antigen was calculated, based on the above results; C, transduction efficiencies of KAV11-01 and TK34 TCRs were analyzed by multi-staining cells with anti-CD3, anti-CD4, anti-CD8, and anti-murine TCRβ chain constant region antibody. Abbreviations as in Figure 1."

Figure 3

Specific killing of KVA11-01 TCR targeting for antigen-positive tumor cell lines A, comparison of the killing effect of KVA11-01 and TK34 TCR-T on T2KO-A1101 cell lines loaded with mutant KV1 peptides. B, comparison of IFN-γ production which were genetically engineered with KVA11-01 and TK34 TCR coculture with T2 cell lines loaded with different concentration of peptides. KC1 peptides represented for G12C, KD2 for G12D, KV1 for G12V, KWT5 for wild KRAS peptides (WT). C, the cytotoxicity capacity of KVA11-01 and TK34 TCR-T against T2KO-A1101 cell lines loaded with mutant KV1 peptides under different effect/target ratios. D, cytotoxic effect of KVA11-01 and TK34 TCR-T against SW480-1101 cells overexpressing HLA-A*11:01 and SW480-1101-G12V cells overexpressing HLA-A*11:01 and G12V. E, IFN-γ ELISA measurement of KVA11-01 and TK34 TCR-T against SW480-1101 cells overexpressing HLA-A*11:01 and SW480-1101-G12V cells overexpressing HLA-A*11:01 and G12V. F, the cytotoxicity capacity of KVA11-01 and TK34 TCR-T against SW480-1101-G12V cells overexpressing HLA-A*11:01 and G12V under different effect/target ratios. G, IFN-γ ELISA measurement of KVA11-01 TCR-T against PANC-1, UM-UC-3, ASPC-1, NCI-2030 cell lines separately or simultaneously overexpressing HLA-A*11:01 and G12V. H, detection of the proliferation of KVA11-01 TCR-Ts and PBMC coculture with T2 cell lines loaded with mutant KV1 peptides. ** P < 0.001, TK34 vs. PBMCTK34, KVA11-01 vs. PBMCKVA11-01; *** P < 0.000 1, KVA11-01 vs. PBMCKVA11-01. WT, wild type; OE, overexpressing; IFN-γ, interferon-γ; Other abbreviations as in Figure 1."

Figure 4

Evaluation of the off-target cross and allogeneic reactivity of KVA11-01 A, IFN-γ ELISA measurement of KVA11-01 TCR-T and PBMC against 23 cell lines from various human tissues and organs (n=3, two-way ANOVA or mixed model, ${\bar x}$±s). B, no allogeneic reactivity with HLA-A*11:01 when KVA11-01 TCR-T cocultured with T2 cell lines expressing 66 HLA subtypes (n=3, nonparametric tests, ${\bar x}$±s). Abbreviations as in Figure 1; HLA, human leukocyte antigen; IFN-γ, interferon-γ."

Figure 5

Antitumor activity of KVA11-01 TCR-T against cell lines overexpressing HLA-A*11:01 and G12V derived xenograft models A, therapeutic effect of KVA11-01 TCR-T against PANC-1 cells overexpressing HLA-A*11:01 and G12V bearing mice, measured with fluorescence value (n=6 in each group, Wilcoxon test, mean with range). B, fluorescence image displayed therapeutic effect after injection of 1×107 KVA11-01 TCR-Ts 7 days later (n=6 in each group, Wilcoxon test, mean with range). C, therapeutic effect of KVA11-01 TCR-T against HeLa cells overexpressing HLA-A*11:01 and G12V bearing mice, measured with tumor volume. D, no destruction was observed in brain, spleen and liver (HE ×10; upper right corner, HE ×4; lower right corner, HE ×20). E, KVA11-01 TCR-T cells were extensively infiltrated into the xenograft tumor tissues (scale bar: 100 μm). Abbreviations as in Figure 1."