1. 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

2. Supplementary Figure 2
Liu et al.

3. Supplementary Figure 3
Liu et al. A B

4. 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

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

6. 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

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

8. 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

9. Supplementary Figure 9
Liu et al. A B

10. 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