Mutant Alanine Position

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**** *** * **** **** *** Shahriary et al.- Supplementary Figure 4 (a)

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Mutant Alanine Position Supplementary Figure 1 Liu et al. A Clone A Clone B Clone C Clone D Events MFI MFI MFI MFI T2-AFP158 T2-AFP158 Ala-1 T2-AFP158 Ala-3 T2-AFP158 Ala-4 T2-AFP158 Ala-5 T2-AFP158 Ala-6 T2-AFP158 Ala-7 T2-AFP158 Ala-8 B Alanine scanning results: mean fluorescence intensity (MFI) Peptide Mutant Alanine Position Clone A Clone B Clone C Clone D FMNKFIYEI 37900 30400 14400 30100 AMNKFIYEI 1 468 12300 5458 21000 FMAKFIYEI 3 4470 38600 38200 786 FMNAFIYEI 4 21 15 38 79 FMNKAIYEI 5 21800 3719 49300 34 FMNKFAYEI 6 18800 169 40300 28 FMNKFIAEI 7 42500 1110 29300 907 FMNKFIYAI 8 38500 1748 53300 16100

Supplementary Figure 2 Liu et al.

Supplementary Figure 3 Liu et al. A B

Supplementary Figure 4 Liu et al. A B IL-6 IL-4 IL-8 GM-CSF IL-10 CD4 SK- HEP-1-MG SK-HEP-1 Hep G2 A IL-4 IL-8 GM-CSF IL-10 Hep G2 B CD4 T Cells Only SK-HEP-1 (A2+/AFP-) SK-HEP-1-MG (A2+/AFP+) Hep G2 (A2+/AFP+) 19.5 45.0 19.6 36.3 18.1 17.4 30.5 18.2 18.6 25.5 1.1 8.1 9.5 41.6 59.0 31.0 7.1 5.1 1.3 31.4 56.2 37.4 0.2 0.0 0.1 59.3 40.5 61.2 38.7 38.8 61.0 30.7 61.1 37.9 61.6 40.2 59.6 41.5 58.3 58.6 43.7 56.3 43.9 55.9 TNFα IFNɣ IL-2 IL-6

Supplementary Figure 5 Liu et al. Hep G2 (A2+/AFP+) SK-HEP-1 (A2+/AFP-) SK-HEP-1-MG (A2+/AFP+) Phase contrast PI

E:T=5:1 Supplementary Figure 6 Liu et al. A B C Mock AFP-CAR Mock AFP- Unstained Mock AFP- CAR C E:T=5:1 Mock AFP-CAR Hep G2 SK-HEP-1 SK-HEP-1-MG

Supplementary Figure 7 Liu et al. AFP-CAR Mock CAR+ CD4+ CAR- CD4+

a′ a b′ c′ b c α-human CD3 α-human CD3 Supplementary Figure 8 Liu et al. A α-human CD3 a′ a b′ c′ b c B α-human CD3

Supplementary Figure 9 Liu et al. A B

Chimeric mouse IgG1 antibody binding affinity to AFP158/HLA-A*02:01 Supplementary Table 1 Liu et al. Chimeric mouse IgG1 antibody binding affinity to AFP158/HLA-A*02:01 Clone # ka [1/Ms] kd [1/s] KD [nM] A 1.17E+06 2.35E-04 0.201 B 5.13E+05 1.31E-05 0.0255 C 6.32E+05 1.10E-04 0.173 D 4.60E+05 5.33E-05 0.116