1. Sample details
Number of non-synonymous mutations
Total number of peptides in sample
Number of predicted neo-antigens
Neo-antigens (HLA peptidomics)
Neo-antigens (predictions)
Cancer/melanoma antigens
Patient name
Metastasis name
Type of sample
Class I
Class II
12T -
Cell line
1019
11205
4093 3 7 65 42 A
Tumor
224
2749
657
1179
Not tested 48 10 B
221
1216
709
1050 17 8 51
1055
2735
1511
4157 11 54 16
1072
3387
3190
4186 74 64 C
3680
1790 76 26 55
457
2032
1262
1680 20
302
5626
3717
1047 59
105
4122
3155 1 36 61 D
3951
2377
4126
8221 93
300 60
1106
2327
1826
4031 22 86
327
6252
6339
1136 98
387
4540
5207
1339 47
2544
3361 13 23 92
162
3832
1948
523 25
172
4325
1635
575 53
Supplementary Table 3: Samples information, number of identified HLA peptides, neo-antigens, TAAs and number of predicted neo-antigens

2. % similar peptides between at least two metastases
Patient sample
Number of peptides
% similar peptides between at least two metastases
Total peptides
TAAs
HLA-I
HLA-II 42
42A
2749
657 48 10
30%
36%
35%
25%
42B
1216
709 17 8 51
51A
2735
1511 54 16
80%
50%
73%
41%
51B
3387
3190 74 64
51C
3680
1790 76 26 55
55A
2032
1262 20
56%
70.50%
66%
82.50%
55B
5626
3717 59
105
55C
4122
3155 36 61
55D
3951
2377 86
86A
6252
6339 98
53%
54%
45.50%
86B
4540
5207 47
86C
2544
3361 13 23 92
92A
3832
1948 25
77%
65%
72%
92B
4325
1635 53
Supplementary Table 6: Similarity between the peptides presented by the different tumor metastases

3. Patient
Sample name
Number of unique nucleotide reads
Number of unique amino acids sequences 42
42A
1326
1280
42B
789
764 51
51A
1377
1345
51B
3449
3378 55
55A
2598
2574
55B
6487
6428 60
60A
2790
2532 86
86A
31468
30200
86B
4692
4624 92
92A
7465
7278
92B
3059
2985
Supplementary Table 7: Number of identified TCR sequences in the different tumor metastases

4. HLA
Score
Aff(nM)
%Rank
BindLevel
12T - MED15 - Mutant - DANSFLQSV
12T - MED15 - WT - DANPFLQSV
HLA-A*02:01
15119 18 15
HLA-A*03:01 55
34269 49
HLA-B*08:01
8690.6
9.5 12
HLA-B*51:01
0.1879
6547 1 WB
4035.5
0.6
HLA-C*01:02 9 6
HLA-C*07:01
8885 4
6824.3 3
12T - TPD52L2 - Mutant - KLFEDRVGTIK
12T - TPD52L2 - WT - KSFEDRVGTIK
5563.3 47
75.8
0.3 SB
535.1
1.4 38
33568 60 41 26
0.0187
40843 35 37
30054 28 23
12T - NCAPH2 - Mutant - GVYPMPGTQK
12T - NCAPH2 - WT - GVSPMPGTQK
0.0465
23.7
0.08
147.5 85 90
0.0101 65 32 39
55C - OSBPL8 - Mutant - KLFELNPLTGEWHY
55C - OSBPL8 - WT - KLFELDPLTGEWHY
HLA-A*02:05
0.106
604.1
1.5
1602.4
HLA-B*07:02 42 43
HLA-B*40:32 31
HLA-C*06:02 30
HLA-C*07:02 24
86B - TAS2R43 - Mutant - LFIMISVWSF
86B - TAS2R43 - WT - LSIMISVWSF
HLA-A*24:02
112.2
0.25
1114.3
1.2
HLA-A*30:01
9857.9
HLA-B*13:02 14
0.1701
7937
HLA-B*15:01
242.6
1.3
47.5
0.4
HLA-C*03:03
0.1923
6242.6 27
Supplementary Table 9: Binding affinity of the identified neo-antigens according to the NetMHCpan algorithm. The binding affinity of the identified neo-antigens was predicted, using NetMHCpan, for the alleles expressed in the cells that underwent HLA-peptidomics. The binding affinity of the wild-type version of the peptide was also predicted.

5. Supplementary Table 11: Binding affinity of the predicted neo-antigens is not a predictor for their reactivity. The table describe the binding affinity of the predicted minimal epitopes (as calculated by NetMHCpan) and the reactivity of each epitope. It can be seen that the most reactive minimal epitopes in most cases were not the ones which predicted to be the strongest binders to the patients’ HLA alleles.

6. Neo-antigens that were identified by HLA peptidomics:
Neo-antigens that were identified by prediction:
TAAs that were identified by HLA peptidomics:
Estimation for all other TAAs that were identified by HLA peptidomics:
Total:
Supplementary Table 12: 87.4% of the 12TIL composition can be estimated with the neo-antigens and TAAs that were identified. We estimated the percentage of reactive and non-reactive T cells against the neo-antigens and reactive T cells against TAAs using IFNγ secretion values and tetramer staining. The percentage of reactive and non-reactive T cells against the MED15 and NCAPH2 was calculated by their IFNγ secretion values and tetramer staining. The percentage of reactive T cells against the predicted neo-antigens was calculated by their IFNγ secretion values compare to the MED15 IFNγ secretion values and tetramer staining. The percentage of reactive T cells against the TAAs was calculated by their IFNγ secretion values (they were divided to different ranges) compare to the GPNMB and CDK4 IFNγ secretion values and tetramer staining.