TCRs were cocultured with MAGE-A3 or TITIN
peptide–pulsed antigen-presenting cells. Although
20a-18 or 94a-14 showed enhanced cytotoxicity,
degranulation, and cytokine secretion (Fig. 5, K
to M) after coculturing with MAGE-A3–pulsed
cells, none of these TCR clones responded to
the presented TITIN peptide (Fig. 5, N to P).
Similarly, the 20a-5 and 27a-5 clones medi-
ated potent cytotoxic responses to MAGE-A3
(fig. S11, N to P) but only minimal cross-
reactivity to TITIN at high concentrations of
peptide (fig. S11, Q to S).Profiling the cross-reactivity of engineered
MAGE-A3 TCRs
Although the engineered TCRs lacked substan-
tial reactivity with the TITIN peptide, we asked
whether the engineered TCRs had acquired new,
other off-target peptide reactivities. We turnedZhaoet al.,Science 376 , eabl5282 (2022) 8 April 2022 7 of 14
Fig. 5. Cytotoxicity and specificity of engineered MAGE-A3Ðspecific TCR.
(AandB) Killing of A375 melanoma cell line by different MAGE-A3–specific
TCR transduced human primary T cells. (CtoE) IFN-g, TNF, and cytotoxic
granule release (CD107a staining) by different MAGE-A3–specific TCR
transduced human primary T cells, induced by the A375 melanoma cell line.
(FandG) Killing of HCT-116 colon cancer cell line by different MAGE-A3–
specific TCR transduced human primary T cells. (HtoJ) IFN-g, TNF, and
cytotoxic granule release (CD107a staining) by different MAGE-A3–specific
TCR transduced human primary T cells, induced by the HCT-116 colon cancer
cell line. (KtoM) Cytotoxic granule release (CD107a staining), TNF, and IFN-g
by different MAGE-A3–specific TCR transduced human primary T cells,
induced by HLA-A1+293T cells pulsed with a titration of MAGE-3 peptide.
(NtoP) Cytotoxic granule release (CD107a staining), TNF, and IFN-gby
different MAGE-A3–specific TCR transduced human primary T cells, induced
by HLA-A1+293T cells pulsed with a titration of TITIN peptide. [(A) to (P)]
Data are representative of three independent experiments. Data are shown as
means ± SDs of technical duplicates. ns, not significant; *P< 0.05; **P<
0.01; ***P< 0.001; ****P< 0.0001.RESEARCH | RESEARCH ARTICLE