Supplementary material

Targeting the PD-1/PD-L1 pathway potentiates immunoediting to counterbalance and changes the dynamics of neutral evolution in a mouse model of colorectal cancer

Mirjana Efremova1, Victoria Klepsch2, Pornpimol Charoentong1, Francesca Finotello1, Dietmar Rieder1, Hubert Hackl1, Natascha Hermann-Kleiter2, Martin Löwer3, Gottfried Baier2, Anne Krogsdam1, Zlatko Trajanoski1*

1Biocenter, Division of Bioinformatics, Medical University of Innsbruck, Innsbruck, Austria 2Division of Translational Cell Genetics, Medical University of Innsbruck, Innsbruck, Austria 3TRON –Translational Oncology at the University Medical Center of the Johannes Gutenberg University gGmbH, Mainz, Germany

The supplementary material contains:

Figure S1. The contributions of mutational signatures to individual MC38, wild type and RAG1-/- samples.………………………………………………………………………………………………...……3 Figure S2. Enriched pathways of the significantly upregulated genes in the MC38 cell line vs normal skin samples………………………………………………………………………………………………………4 Figure S3. z-score heatmap of the log2 transformed normalized counts of chemokines and chemokine receptors in MC38, wild type and RAG1-/- samples………………………………………………………...5 Figure S4. z-score heatmap of the log2 transformed normalized counts of selected genes in MC38, wild type and RAG1-/- samples…………………………………………………………………………………...6 Figure S5. Ratio between the number of expressed neoantigens and nonsynonymous mutations and LOH events between samples……………………………………………………………………………………..8 Figure S6. Enriched pathways of the significantly upregulated and downregulated genes in the wild type day 23 vs RAG1-/- day 23 tumors…………………………………………………………………………...7 Figure S7. Number of clonal / subclonal shared nonsynonymous mutations between MC38 and wild type samples during progression………………………………………………………………………………….9 Figure S8. Number of clonal / subclonal shared nonsynonymous mutations between MC38 and RAG1-/- samples during progression………………………………………………………………………………...10 Figure S9. Distribution of the variant allele frequency (VAF)…………………………………………….11 Figure S10. z-score heatmap of the log2 transformed normalized counts of selected genes in the anti-PD- L1 and the IgG2b samples…………………………………………………………………………………12

1 Figure S11. Enriched pathways of the significantly upregulated and downregulated genes in the antiPD-L1 vs the IgG2b tumors………………………………………………………………………………………..13 Figure S12. Ratio between the number of expressed neoantigens and nonsynonymous mutations in MC38, anti-PD-L1 and IgG2b samples……………………………………………………………………………14 Figure S13. Number of clonal / subclonal shared nonsynonymous mutations between MC38, anti-PD-L1 and IgG2b samples...... 15 Figure S14. Genomic and immunogenomic impact of targeting the PD-1/PD-L1 axis using CT26 model………………………………………………………………………………………………………16 Figure S15. Number of clonal / subclonal shared nonsynonymous mutations between CT26, anti-PD-L1 and IgG2b samples...... 17 Table S1. Driver genes in MC38 and mutational hotspots in human CRC………………………………..18 Table S2. Number of mutations that changed their variant allele frequencies (VAF) and cancer cell fractions (CCF) between time points in the three wild type and RAG1-/- samples………………………...22 Table S3. List of expressed predicted neoantigens present in the MC38 cell line and the RAG1-/- samples from day 23, and absent from the wild type samples from day 23………………………………………...23 Table S4. List of expressed predicted neoantigens present in the MC38 cell line and the IgG2b control samples, and absent from the anti-PD-L1 samples………………………………………………………...24 Table S5. List of predicted neoantigens present in the CT26 cell line and the IgG2b control samples, and absent from the anti-PD-L1 samples…………………………………………………………………..…...26

2 Figure S1. The contributions of published mutational signatures from Alexandrov et al. Nature 500, 415-421 (2013) to individual MC38, wild type and RAG1-/- samples. The most prevalent signatures across all samples are Signature 3 (BRCA1/2 mutations), Signature 4 (Smoking) and Signature 6 (DNA MMR 3 deficiency). Figure S2. Enriched pathways of the significantly upregulated genes in the MC38 cell line vs normal skin samples. The pie chart is created using ClueGO and shows the enriched groups (the name of the group is given by the most significant term). The sizes of the sections correlate with the number of terms included in a group. 4 -3 0 3

Figure S3. z-score heatmaps of the log2 transformed normalized counts of chemokines and chemokine receptors in MC38, wild type and RAG1-/- samples. 5 -3.7 0 3.7 Immunoinhibitors Immunostimulators MHC Cytotoxicity

Figure S4. z-score heatmaps of the log2 transformed normalized counts of selected genes in MC38, skin, wild type and RAG1-/- samples. 6 A

B

Figure S5. A) Ratio between the number of expressed neoantigens and nonsynonymous mutations in MC38, Rag 13d, Rag 23d, wt 23d, wt 46d: B) Shared LOH events between MC38, wild type and RAG1-/- samples. LOH events 7 found in at least one sample from the same type are considered. A

B

Figure S6. Enriched pathways of the significantly A) upregulated and B) downregulated genes in wild type day 23 vs RAG1-/- day 23 tumors. The pie chart is created using ClueGO and shows the enriched groups (the name of the group is given by the most significant term). The sizes of the sections correlate with the number of terms included in a 8 group. 475 clonal subclonal not shared S1 19 284 172

S2 28 338 109

S3 13 309 153

S4 12 335 128

S5 12 240 223

s6 37 324 114

sub sub clonal not shared clonal not shared 386 clonal 307 clonal

S1 2 240 144 S1 3 108 196

S2 3 91 292 S2 2 125 180

S3 1 78 307 S3 1 88 218

sub sub clonal not shared clonal not shared 50 clonal 55 clonal S1 1 32 17 S1 0 11 44

S2 0 14 36 S2 1 40 14

S3 0 21 29 S3 1 13 41

S4 0 49 1 S4 1 52 2

Figure S7. Number of clonal / subclonal shared nonsynonymous mutations between MC38 and wild type samples during progression. Each circle represents union of mutations across three replicates. 9 501 clonal subclonal not shared S1 11 284 206

S2 8 323 170

S3 13 311 177

S4 10 339 152

S5 10 281 210

s6 21 291 189

sub sub clonal not shared clonal not shared 501 clonal 473 clonal

S1 2 195 304 S1 3 160 310

S2 2 146 353 S2 0 234 239

S3 1 205 295 S3 1 141 331

sub sub clonal not shared clonal not shared 49 clonal 45 clonal S1 1 26 22 S1 1 22 22

S2 3 28 18 S2 1 19 25

S3 0 24 25 S3 1 25 19

S4 0 48 1 S4 0 44 1

Figure S8. Number of clonal / subclonal shared nonsynonymous mutations between MC38 and RAG1-/- samples during progression. Each circle represents union of mutations across three replicates. 10 A B

wt 23d wt 46d C D

Rag 13d Rag 23d E

MC38

Figure S9. Distribution of the variant allele frequency (VAF) and VAF multiplied by 2 of all mutations in diploid regions in different replicates from wt 23d, wt 46d, Rag 13d, Rag 23d and MC38. 11 -2 0 2

Immunoinhibitors Immunostimulators MHC Cytotoxicity

Figure S10. z-score heatmaps of the log2 transformed normalized counts of selected genes in the anti-PD-L1 and the IgG2b control samples. 12 A

B

Figure S11. Enriched pathways of the significantly A) upregulated and B) downregulated genes in the antiPD-L1 vs the IgG2b tumors. The pie chart is created using ClueGO and shows the enriched groups (the name of the group is given by the most significant term). The sizes of the sections correlate with the number of terms included in a group. 13 Figure S12. Ratio between the number of expressed neoantigens and nonsynonymous mutations in MC38, anti-PD-L1 and IgG2b samples. 14 451 clonal subclonal not shared S1 30 260 161

S2 47 249 155

S3 33 272 146

S4 66 37 348

S5 60 152 239 sub sub clonal not shared s6 40 259 152 clonal not shared 190 clonal 231 clonal S1 7 2 181 S1 0 22 209 S2 3 17 170 S2 2 31 198 S3 1 27 162 S3 1 26 204

sub sub 52 clonal not shared clonal not shared clonal 305 clonal S1 1 0 51 S1 4 60 241 S2 0 5 47 S2 7 78 220 S3 1 14 37 S3 3 71 231 S4 1 44 7 S4 9 265 31

filtered or clonal subclonal 187 no CNA S1 3 126 58

2555 clonal subclonal not shared S1 183 1680 692

S2 275 1619 661

S3 203 1688 664

S4 378 152 2025

S5 334 915 1306

s6 184 1529 842

s7 256 2164 135

Figure S13. Number of clonal / subclonal shared nonsynonymous mutations between MC38, anti-PD-L1 and IgG2b samples during progression. Each circle represents union of mutations across three replicates. 15 A B Nonsynonymous mutations Neoantigens

C D

E F

Figure S14. Genomic and immunogenomic impact of targeting the PD-1/PD-L1 axis. A) Tumor growth curve in anti-PD-L1 treated mice (n=10) compared to tumor growth curve in mice (n=4) injected with IgG2b control. Presented is the mean ± SEM, statistically analyzed by Student’s t test. B) Shared nonsynonymous mutations and neoantigens between CT26, and anti-PD-L1 treated and control samples. Mutations/neoantigens found in at least one sample from the same type are considered. C) Fractions of clonal and subclonal neoantigens in MC38 and all tumor samples. D). Ratio between the number of neoantigens and nonsynonymous mutations in CT26, anti-PD-L1 and IgG2b samples. E) Violin plots showing tumor heterogeneity estimated from the cancer cell fractions. F) Two-dimensional density plots 16 showing the clustering of the cancer cell fractions of all mutations shared between two samples 448 clonal subclonal not shared S1 18 262 168

S2 14 238 196

S3 10 221 217

S4 12 241 195

S5 14 228 206 sub sub clonal not shared s6 12 209 227 clonal not shared 250 clonal 299 clonal S1 0 35 215 S1 2 37 260 S2 0 17 233 S2 2 21 276 S3 0 26 224 S3 1 17 281

sub sub 33 clonal not shared clonal not shared clonal 41 clonal S1 0 3 30 S1 0 13 28 S2 0 4 29 S2 0 11 30 S3 1 6 26 S3 0 4 37 S4 1 16 16 S4 0 21 20

filtered or clonal subclonal 1086 no CNA S1 5 434 647

1745 clonal subclonal not shared S1 115 1283 347

S2 132 1141 472

S3 122 1132 491

S4 117 1255 373

S5 123 1125 497

s6 120 1127 498

s7 141 1373 231

Figure S15. Number of clonal / subclonal shared nonsynonymous mutations between CT26, anti-PD-L1 and IgG2b samples during progression. Each circle represents union of mutations across three replicates. 17 Table S1. Driver genes in MC38 and mutational hotspots in human CRC Mouse CRC mutations Gene AAPos Type MutPosition Braf W487C missense_variant 6:39643182 Trp53 G242V missense_variant 11:69589202 Smad4 G351R missense_variant 18:73649811 Smad2 V319L missense_variant 18:76299953 Acvr2a A4G missense_variant 2:48814782 Tgfb2 L154F missense_variant 1:186649783 Axin2 E366 stop_gained 11:108941939 Sox9 T379R missense_variant 11:112785122 Arid1a Q602H missense_variant&splice_region_variant 4:133720550 Pold1 R209L missense_variant 7:44541780 Pold1 P110L missense_variant 7:44542232 Msh3 D389N missense_variant 13:92342295 Brca2 E2940K missense_variant 5:150560535 Human Colon cancer hotspot mutations (extracted from Chen et al. 2016) Gene AAPos MutSubtypes MutPositions AATK G703 Missense_CpG_CGtv(14) 17:79095629-79095629G/T(14) ABCA7 A2045 Missense_CpG_CGtv(18) 19:1065018-1065018G/T(18) ABCF1 K76 Deletion(10) 6:30545854-30545854A/-(9),6:30545854-30545855AA/-(1) ACTR5 F6 Silent_CpG_CGts(14) 20:37377139-37377139C/T(14) ADAD2 G44 Missense_CpG_CGts(16) 16:84224967-84224967G/A(16) ADAM22 P81 Missense_CpG_CGtv(21) 7:87564497-87564497C/G(21) ADAM29 T746 Missense_ATts(1),Missense_NoCpG_CGts(19) 4:175898913-175898913C/T(19),4:175898912-175898912A/G(1) ALDH2 L189 Missense_ATts(12) 12:112228251-112228251T/C(12) ALOX12 P41 Silent_CpG_CGtv(16) 17:6899559-6899559C/G(16) ANKRD30A P818 Missense_NoCpG_CGts(1),Missense_NoCpG_C 10:37486216-37486216C/T(6),10:37486216- 37486216C/A(5),10:37486215- Gtv(2),Silent_NoCpG_CGts(6),Silent_NoCpG_C 37486215C/A(2),10:37486215-37486215C/T(1) Gtv(5) ANTXR2 A357 Deletion(12) 4:80905990-80905990G/-(12) ANXA2 C8 Silent_ATts(14) 15:60690089-60690089T/C(14) APC R1114 Nonsense_NoCpG_CGts(20),Silent_ATts(2) 5:112174631-112174631C/T(20),5:112174633-112174633A/G(2) APC R1450 Nonsense_NoCpG_CGts(40),Silent_ATts(1) 5:112175639-112175639C/T(40),5:112175641-112175641A/G(1) APC R876 Missense_NoCpG_CGts(1),Nonsense_NoCpG_ 5:112173917-112173917C/T(28),5:112173919-112173919A/G(1),5:112173918- CGts(28),Silent_ATts(1) 112173918G/A(1) ARHGAP5 V474 Missense_ATts(27) 14:32561296-32561296T/C(27) ARHGEF33 S582 Missense_ATts(24) 2:39187190-39187190T/C(24) ATM R337 Missense_NoCpG_CGts(16),Silent_ATtv(2) 11:108117798-108117798C/T(12),11:108117799-108117799G/A(4),11:108117800- 108117800T/A(2) ATR I774 Deletion(17) 3:142274740-142274740A/-(17) ATXN7 T781 Silent_NoCpG_CGts(2),Deletion(8) 3:63981832-63981832C/-(8),3:63981841-63981841C/T(2) BAX E41 Deletion(26) 19:49458971-49458971G/-(26) BCAS4 E56 Missense_CpG_CGtv(27) 20:49411698-49411698G/T(27) BCL9L P1128 Insertion(12) 11:118770651-118770652-/C(12) BIRC6 K4503 Missense_ATts(2),Nonsense_ATtv(2),Silent_AT 2:32820108-32820108A/G(10),2:32820106-32820106A/T(2),2:32820106-32820106A/G(2) ts(10) BMPR1A R486 Missense_NoCpG_CGts(4),Missense_NoCpG_C 10:88683247-88683247G/T(12),10:88683246-88683246C/T(4) Gtv(12) BMPR2 N583 Deletion(17) 2:203420130-203420130A/-(17) BRAF V600 Missense_ATtv(54) 7:140453136-140453136T/A(54) BRAT1 R644 Missense_CpG_CGts(7),Silent_ATts(11) 7:2578237-2578237A/G(11),7:2578238-2578238G/A(7) BRCA2 S1682 Missense_ATts(12),Silent_ATts(4) 13:32913536-32913536A/G(12),13:32913538-32913538T/C(4) C17orf82 L186 Missense_ATts(16) 17:59489893-59489893T/C(16) C1orf106 R538 Missense_CpG_CGts(12) 1:200880978-200880978C/T(12) C8orf55 L63 Silent_CpG_CGts(13) 8:143808951-143808951C/T(13) C8orf80 N631 Missense_ATtv(1),Deletion(18) 8:27888776-27888776A/-(18),8:27888777-27888777A/C(1) CACNA1H D2133 Silent_ATts(20) 16:1270349-1270349T/C(20) CASP5 R23 Deletion(22) 11:104879687-104879687A/-(22) CBWD6 L260 Insertion(10) 9:69218548-69218549-/A(10) CCDC43 R216 Insertion(2),Deletion(14) 17:42756253-42756253A/-(14),17:42756252-42756253-/A(2) CD3G K71 Deletion(13) 11:118220583-118220583A/-(13) CD8B L9 Silent_ATts(24) 2:87088964-87088964T/C(24) CDC42BPA S344 Missense_ATts(9),Silent_ATts(6) 1:227333303-227333303A/G(9),1:227333301-227333301T/C(6) CDKN2D R30 Silent_CpG_CGtv(10) 19:10679240-10679240G/C(10) CEBPB A147 Silent_CpG_CGts(14) 20:48808011-48808011C/T(14) CHEK2 S372 Missense_ATts(1),Missense_NoCpG_CGtv(1),Si 22:29091841-29091841C/T(25),22:29091843-29091843T/C(1),22:29091842- lent_NoCpG_CGts(25) 29091842C/A(1) CHEK2 K373 Missense_ATts(22) 22:29091840-29091840A/G(22) CHML S514 Missense_ATts(5),Missense_NoCpG_CGts(1), 1:241797527-241797527T/C(6),1:241797529-241797529T/C(5),1:241797528- Missense_NoCpG_CGtv(1),Silent_ATts(6) 241797528C/A(1),1:241797528-241797528C/T(1) CLK4 R2 Missense_NoCpG_CGts(18),Missense_NoCpG_ 5:178050414-178050414C/T(16),5:178050413-178050413G/T(4),5:178050413- CGtv(4) 178050413G/A(2) CLOCK L123 Insertion(3),Deletion(13) 4:56336954-56336954T/-(12),4:56336953-56336954-/T(3),4:56336954-56336955TT/-(1) CSNK1D S97 Missense_ATts(12) 17:80213352-80213352T/C(12) CSNK1E N172 Missense_ATts(12) 22:38696780-38696780A/G(9),22:38696779-38696779A/G(3) CYP21A2 L10 Deletion(10) 6:32006215-32006217CTG/-(10) D2HGDH R55 Missense_CpG_CGts(10) 2:242674803-242674803G/A(10) DDHD1 G112 Insertion(94) 14:53619480-53619481-/GGCGGC(92),14:53619494-53619495-/GCGGCG(2)

18 DDX26B L120 Insertion(4),Deletion(14) 23:134680317-134680317T/-(14),23:134680316-134680317-/T(4) DDX4 P48 Missense_NoCpG_CGts(2),Silent_ATts(10),Sile 5:55056044-55056044T/C(10),5:55056042-55056042C/T(2),5:55056044-55056044T/G(2) nt_ATtv(2) DEFB126 P106 Deletion(33) 20:126311-126312CC/-(33) DLC1 K237 Missense_ATts(9),Silent_ATts(6) 8:13356872-13356872A/G(6),8:13356870-13356870A/G(6),8:13356871-13356871A/G(3) DLC1 R347 Silent_ATts(15),Silent_ATtv(3) 8:13259111-13259111A/G(15),8:13259111-13259111A/T(3) DNAH3 S1608 Missense_ATts(4),Silent_ATts(10) 16:21049209-21049209T/C(10),16:21049211-21049211T/C(4) DNTTIP1 P13 Silent_ATts(16) 20:44420682-44420682T/C(16) DOCK3 P1852 Deletion(13) 3:51417604-51417604C/-(11),3:51417604-51417605CC/-(2) DOT1L G1386 Missense_CpG_CGts(28) 19:2226676-2226676G/A(28) DSC2 K270 Missense_ATts(10),Missense_ATtv(2),Silent_A 18:28666673-28666673A/G(8),18:28666671-28666671A/G(4),18:28666673- Tts(4) 28666673A/C(2),18:28666672-28666672A/G(2) DSG4 A601 Missense_NoCpG_CGts(10),Silent_NoCpG_CGt 18:28986205-28986205C/T(6),18:28986204-28986204G/A(4),18:28986206- s(2),Silent_NoCpG_CGtv(2) 28986206G/A(2),18:28986206-28986206G/T(2) DSPP S879 Silent_NoCpG_CGts(20) 4:88536451-88536451C/T(20) EBPL F172 Deletion(12) 13:50235209-50235209T/-(12) EEF1D D189 Missense_NoCpG_CGtv(12) 8:144671685-144671685C/G(12) ENSG00000G98 Silent_ATts(12) 2:39187190-39187190A/G(12) 269210 ERI1 L16 Missense_ATts(10) 8:8860620-8860620T/C(10) ESRP1 N512 Insertion(2),Deletion(20) 8:95686611-95686611A/-(18),8:95686610-95686611-/A(2),8:95686611-95686612AA/-(2) ETHE1 A2 Silent_CpG_CGts(8) 19:44031324-44031324G/A(8) ETNK2 P10 Missense_CpG_CGtv(24) 1:204120952-204120952C/A(24) FADS3 A18 Silent_CpG_CGts(26) 11:61658800-61658800G/A(26) FAM18A F140 Deletion(12) 16:10867203-10867203T/-(12) FAM190A D515 Missense_ATts(12),Missense_NoCpG_CGts(3) 4:91321221-91321221A/G(12),4:91321220-91321220G/A(3) FAM190A E611 Missense_ATts(3),Missense_NoCpG_CGts(3),Si 4:91549284-91549284A/G(9),4:91549282-91549282G/A(3),4:91549283-91549283A/G(3) lent_ATts(9) FAM194B E135 Missense_ATts(18) 13:46170737-46170737A/G(18) FAM194B E136 Missense_NoCpG_CGts(30) 13:46170735-46170735G/A(30) FAM194B E138 Missense_ATts(21) 13:46170728-46170728A/G(21) FAM194B Y139 Missense_ATts(25) 13:46170726-46170726T/C(25) FBXW7 R465 Missense_NoCpG_CGts(44),Silent_ATts(8) 4:153249384-153249384G/A(22),4:153249385-153249385C/T(22),4:153249383- 153249383T/C(8) FEZ2 P50 Missense_CpG_CGts(11) 2:36825137-36825137C/T(11) FGFR1 T141 Missense_ATts(10),Silent_ATts(4) 8:38285891-38285891A/G(10),8:38285889-38285889A/G(4) GLTPD2 D209 Missense_CpG_CGtv(14) 17:4693342-4693342C/A(14) GOT1L1 T415 Insertion(4),Deletion(28) 8:37791834-37791834A/-(28),8:37791833-37791834-/A(4) GPHB5 R53 Missense_NoCpG_CGts(12) 14:63784405-63784405C/T(12) GPRIN2 G237 Insertion(33) 10:46999591-46999592-/ATGAGGGAG(33) GRID2IP A221 Silent_ATtv(30) 7:6566322-6566322A/C(30) GRIK2 N849 Insertion(4),Deletion(18) 6:102503432-102503432A/-(18),6:102503431-102503432-/A(4) GSG2 R82 Missense_CpG_CGts(28) 17:3627473-3627473C/T(28) GSX1 L78 Silent_ATts(14) 13:28367061-28367061A/G(14) GTPBP3 T66 Silent_ATtv(11) 19:17448961-17448961A/C(11) HAP1 K4 Missense_ATts(14) 17:39890876-39890876A/G(14) HCLS1 P368 Silent_NoCpG_CGts(15) 3:121351315-121351315C/T(15) HECW1 R1502 Missense_NoCpG_CGts(12),Missense_NoCpG_ 7:43591930-43591930G/A(12),7:43591929-43591929C/A(6),7:43591930-43591930G/C(2) CGtv(2),Silent_NoCpG_CGtv(6) HLA-DQA1 G79 Deletion(22) 6:32609239-32609241GGT/-(22) HSD17B1 G313 Missense_CpG_CGts(12) 17:40706906-40706906G/A(12) HSPA12B C627 Silent_CpG_CGts(17) 20:3732633-3732633C/T(17) IDH3A S8 Silent_ATts(13) 15:78441769-78441769T/C(13) IFI27 A43 Insertion(18) 14:94582130-94582131-/GGCCATGGC(15),14:94582133-94582134-/CATGGCGGC(3) IFITM3 P55 Silent_NoCpG_CGts(21) 11:320649-320649C/T(21) IGF2R D1317 Missense_ATts(1),Insertion(4),Deletion(9) 6:160485488-160485488G/-(9),6:160485487-160485488-/G(4),6:160485496- 160485496A/G(1) IRF5 P183 Silent_ATts(13) 7:128587381-128587381T/C(13) JPH4 A502 Insertion(4),Deletion(16) 14:24040436-24040436G/-(15),14:24040435-24040436-/G(4),14:24040444-24040444G/- (1) KDM5A G1200 Deletion(14) 12:416953-416953A/-(14) KIAA1024 S702 Deletion(12) 15:79750586-79750586A/-(11),15:79750586-79750587AA/-(1) KIF17 R13 Silent_ATts(16) 1:21044161-21044161T/C(16) KIF25 W3 Missense_ATtv(12) 6:168430272-168430272T/A(12) KLF14 S112 Silent_CpG_CGts(17) 7:130418525-130418525C/T(17) KLHL30 A213 Missense_CpG_CGts(32) 2:239050033-239050033C/T(32) KNDC1 A432 Silent_ATts(20) 10:135000148-135000148T/C(20) KNDC1 V806 Missense_ATtv(16),Silent_ATts(4) 10:135012429-135012429T/A(16),10:135012430-135012430T/C(4) KRAS G12 Missense_NoCpG_CGts(128),Missense_NoCpG 12:25398284-25398284G/A(107),12:25398284-25398284G/T(103),12:25398285- _CGtv(158),Insertion(2) 25398285G/T(24),12:25398285-25398285G/A(21),12:25398284- 25398284G/C(21),12:25398285-25398285G/C(9),12:25398283-25398284- /AGCTGG(2),12:25398284-25398285GG/TT(1) KRAS G13 Missense_NoCpG_CGts(77),Missense_NoCpG_ 12:25398281-25398281G/A(75),12:25398282-25398282G/T(2),12:25398282- CGtv(2) 25398282G/A(2) KRT4 G155 Insertion(27) 12:53207602-53207603-/C(27) KRTAP4-3 R26 Missense_NoCpG_CGts(8),Missense_NoCpG_C 17:39324348-39324348G/A(7),17:39324349-39324349C/A(6),17:39324347- Gtv(6),Silent_NoCpG_CGtv(4),Insertion(2) 39324347C/A(4),17:39324346-39324347-/AAA(2),17:39324349-39324349C/T(1) KRTAP4-3 Q31 Missense_ATtv(18) 17:39324333-39324333A/T(18) KRTAP9-1 C153 Missense_ATtv(24) 17:39346595-39346595T/A(24) KSR1 P291 Missense_NoCpG_CGtv(2),Silent_ATtv(2),Delet17:25910016-25910016C/-(16),17:25910022-25910022C/A(2),17:25910024- ion(16) 25910024A/C(2)

19 LATS2 A324 Missense_CpG_CGts(32) 13:21562948-21562948C/T(32) LIG1 K152 Missense_ATts(51) 19:48657141-48657141A/G(41),19:48657140-48657140A/G(10) LMAN1 E305 Deletion(13) 18:57013194-57013194A/-(13) LRRIQ1 R329 Missense_NoCpG_CGtv(2),Nonsense_NoCpG_ 12:85449558-85449558A/G(10),12:85449557-85449557G/T(2),12:85449556- CGts(2),Silent_ATts(10) 85449556C/T(2) MAGEB2 R23 Missense_NoCpG_CGts(12),Missense_NoCpG_ 23:30236765-30236765G/A(12),23:30236765-30236765G/T(4) CGtv(4) MAML2 Q596 Silent_NoCpG_CGts(13) 11:95825407-95825407G/A(13) MAPK9 K56 Missense_ATts(16),Silent_ATts(4) 5:179696366-179696366A/G(8),5:179696365-179696365A/G(8),5:179696364- 179696364A/G(4) MEGF11 A102 Silent_ATts(16) 15:66274690-66274690A/G(16) MEGF11 A177 Silent_ATts(32) 15:66274690-66274690A/G(32) MLL3 Y366 Missense_ATts(10),Missense_ATtv(2),Silent_A 7:151962211-151962211T/C(8),7:151962209-151962209T/C(4),7:151962211- Tts(4) 151962211T/A(2),7:151962210-151962210A/G(2) MLL3 T3698 Missense_ATts(14),Missense_ATtv(2),Missens 7:151859570-151859570A/G(14),7:151859569-151859569C/T(2),7:151859568- e_NoCpG_CGts(2),Silent_NoCpG_CGts(2) 151859568G/A(2),7:151859570-151859570A/C(2) MRE11A F237 Missense_ATts(12),Silent_NoCpG_CGts(2) 11:94204875-94204875T/C(6),11:94204876-94204876T/C(6),11:94204874- 94204874C/T(2) MSH3 K374 Deletion(14) 5:79970915-79970915A/-(13),5:79970915-79970916AA/-(1) MTX1 T63 Missense_ATtv(14) 1:155178782-155178782A/T(14) MUC2 T1541 Deletion(43) 11:1092802-1092802A/-(43) MUC2 T1542 Deletion(43) 11:1092802-1092802A/-(43) MUC4 T113 Silent_ATtv(30) 3:195518112-195518112A/T(30) MUC6 P1570 Insertion(26) 11:1018093-1018094-/A(26) MUC6 P1571 Deletion(28) 11:1018089-1018089C/-(28) MUC6 T1911 Silent_NoCpG_CGts(44) 11:1017068-1017068G/A(44) MYOM1 R212 Missense_ATts(1),Missense_NoCpG_CGts(23), 18:3188882-3188882G/A(23),18:3188881-3188881G/A(1),18:3188883-3188883A/G(1) Silent_NoCpG_CGts(1) NEFH E645 Missense_NoCpG_CGts(22),Silent_ATts(26) 22:29885564-29885564A/G(26),22:29885562-29885562G/A(22) NEFH A646 Silent_ATtv(32) 22:29885567-29885567A/C(32) NFATC3 K474 Missense_ATts(12) 16:68191791-68191791A/G(10),16:68191790-68191790A/G(2) NFKB2 P423 Silent_ATts(14) 10:104159196-104159196A/G(14) NIN E1559 Nonsense_NoCpG_CGtv(3),Silent_ATts(9) 14:51221338-51221338A/G(9),14:51221340-51221340G/T(3) NIPBL K603 Deletion(14) 5:36985083-36985083A/-(14) NOTCH3 P1521 Silent_ATts(20) 19:15285052-15285052A/G(20) NRAS G12 Missense_NoCpG_CGts(8),Missense_NoCpG_C 1:115258747-115258747G/A(8),1:115258748-115258748G/T(5),1:115258747- Gtv(9) 115258747G/T(2),1:115258747-115258747G/C(2) NRAS Q61 Missense_ATts(3),Missense_ATtv(4),Missense 1:115256530-115256530C/A(10),1:115256529-115256529A/T(3),1:115256529- _NoCpG_CGtv(10),Silent_ATts(1) 115256529A/G(3),1:115256528-115256528A/C(1),1:115256528-115256528A/G(1) NTSR2 A54 Missense_CpG_CGts(22) 2:11810095-11810095C/T(22) OBSCN A94 Silent_ATts(24) 1:228399766-228399766T/C(24) OPRD1 C27 Missense_CpG_CGtv(23) 1:29138975-29138975G/T(23) OR5K4 I301 Deletion(17) 3:98073592-98073592A/-(16),3:98073598-98073598A/-(1) OR6C76 K311 Deletion(56) 12:55820959-55820959A/-(51),12:55820959-55820960AA/-(5) ORAI1 P43 Deletion(41) 12:122064774-122064779CCGCCA/-(41) PANX2 P505 Silent_ATts(20) 22:50616656-50616656T/C(20) PANX2 L555 Silent_ATts(16) 22:50616806-50616806A/G(16) PCDHA7 L352 Missense_NoCpG_CGtv(44) 5:140215022-140215022C/A(44) PCDHGC3 N689 Deletion(16) 5:140857742-140857742A/-(16) PCSK5 E562 Missense_ATts(12),Nonsense_NoCpG_CGtv(3) 9:78784685-78784685A/G(12),9:78784684-78784684G/T(3) PDXDC1 A384 Silent_NoCpG_CGts(12) 16:15122751-15122751C/T(12) PDXDC1 A407 Silent_NoCpG_CGts(13) 16:15122751-15122751C/T(13) PHF2 P988 Silent_NoCpG_CGtv(14) 9:96439007-96439007G/T(14) PIK3CA H1047 Missense_ATts(29),Missense_ATtv(4),Missens 3:178952085-178952085A/G(29),3:178952084-178952084C/T(6),3:178952086- e_NoCpG_CGts(6),Silent_ATts(2) 178952086T/G(2),3:178952086-178952086T/C(2),3:178952085-178952085A/T(2) PIK3CA E542 Missense_ATtv(2),Missense_NoCpG_CGts(22) 3:178936082-178936082G/A(22),3:178936083-178936083A/C(2) PIK3CA E545 Missense_ATts(4),Missense_ATtv(4),Missense 3:178936091-178936091G/A(66),3:178936092-178936092A/C(4),3:178936092- _NoCpG_CGts(66),Missense_NoCpG_CGtv(4) 178936092A/G(4),3:178936093-178936093G/T(2),3:178936091-178936091G/C(2) PIK3CA R88 Missense_NoCpG_CGts(24),Nonsense_NoCpG 3:178916876-178916876G/A(24),3:178916875-178916875C/T(2),3:178916877- _CGts(2),Silent_ATts(2) 178916877A/G(2) PKD1L2 N236 Deletion(12) 16:81242150-81242151AA/-(10),16:81242149-81242150AA/-(2) PKDCC G17 Silent_ATts(12) 2:42275819-42275819T/C(12) PLEC L1184 Silent_ATts(20) 8:145001784-145001784T/C(20) PLEC A1976 Silent_CpG_CGts(20) 8:144998169-144998169G/A(20) POLE V1394 Missense_ATts(8),Missense_NoCpG_CGts(2) 12:133220532-133220532T/C(8),12:133220533-133220533G/A(2) PPP2R3B P533 Silent_ATts(14) 23:295231-295231T/C(14) PRRT4 R391 Silent_CpG_CGtv(12) 7:127992437-127992437G/C(12) PRSS36 S423 Silent_CpG_CGts(15) 16:31154146-31154146C/T(15) PTPLA E64 Missense_CpG_CGts(21) 10:17659149-17659149G/A(21) PTPRD R584 Missense_NoCpG_CGtv(4),Silent_ATts(16) 9:8504331-8504331T/C(16),9:8504333-8504333C/A(4) PURB A107 Silent_ATtv(12) 7:44924627-44924627T/G(12) RAPGEF6 S640 Insertion(3),Deletion(18) 5:130815369-130815369A/-(18),5:130815368-130815369-/A(3) RASA2 E759 Missense_ATts(10),Silent_ATts(6) 3:141328312-141328312A/G(10),3:141328313-141328313A/G(6) RASSF5 L16 Silent_ATtv(25) 1:206680983-206680983A/C(25) RBBP8 K357 Deletion(16) 18:20572853-20572853A/-(16) RBP1 P18 Missense_CpG_CGts(18) 3:139258508-139258508C/T(18) RHPN2 A353 Missense_NoCpG_CGts(15) 19:33493201-33493201G/A(8),19:33493200-33493200C/T(7) RIC8A P210 Deletion(11) 11:209895-209897CCC/-(11) RLIM S501 Missense_ATts(2),Missense_NoCpG_CGts(12), 23:73811648-73811648C/T(12),23:73811649-73811649T/C(2),23:73811647- Silent_ATts(1) 73811647A/G(1)

20 RNF145 K23 Missense_ATts(1),Silent_NoCpG_CGts(1),Delet 5:158630642-158630642A/-(15),5:158630642-158630642A/G(1),5:158630641- ion(15) 158630641G/A(1) RNF145 N27 Deletion(19) 5:158630630-158630630A/-(18),5:158630630-158630631AA/-(1) SBK2 A298 Missense_CpG_CGtv(19) 19:56041255-56041255G/C(19) SCARF2 R727 Silent_CpG_CGtv(12) 22:20780097-20780097C/G(12) SCRIB P1450 Missense_NoCpG_CGts(2),Silent_ATts(18) 8:144874554-144874554A/G(18),8:144874556-144874556C/T(2) SF1 E28 Missense_ATts(14),Silent_ATts(6) 11:64544047-64544047A/G(14),11:64544046-64544046A/G(6) SLAMF1 S277 Deletion(13) 1:160589601-160589601A/-(13) SLC39A3 V236 Silent_ATts(18) 19:2732986-2732986A/G(18) SLC3A2 K331 Deletion(14) 11:62649529-62649529A/-(14) SMAD4 R361 Missense_NoCpG_CGts(22) 18:48591919-48591919G/A(14),18:48591918-48591918C/T(8) SNX13 L652 Missense_ATtv(23) 7:17854565-17854565A/T(22),7:17854565-17854565A/C(1) SNX18 G204 Silent_ATts(26) 5:53814394-53814394T/C(26) SOLH A840 Silent_NoCpG_CGtv(15) 16:602313-602313C/A(15) SP5 A75 Missense_CpG_CGts(11) 2:171572940-171572940G/A(11) SPHK1 A34 Missense_CpG_CGts(13) 17:74381567-74381567G/A(13) SRPR K170 Missense_ATts(1),Deletion(10) 11:126137087-126137087A/-(10),11:126137087-126137087A/G(1) STAMBPL1 K405 Deletion(14) 10:90682146-90682146A/-(14) STARD3NL T130 Missense_ATts(4),Missense_ATtv(1),Missense 7:38256633-38256633C/T(8),7:38256632-38256632A/G(4),7:38256632- _NoCpG_CGts(8),Silent_NoCpG_CGts(1) 38256632A/T(1),7:38256634-38256634G/A(1) SVIL M1863 Insertion(1),Deletion(16) 10:29760116-29760116G/-(16),10:29760115-29760116-/G(1) SgK069 A298 Missense_CpG_CGtv(19) 19:56041255-56041255G/C(19) SgK223 G350 Insertion(16) 8:8234868-8234869-/AGCGGC(16) SgK493 G160 Silent_ATts(12) 2:42275819-42275819T/C(12) TAF1B N66 Deletion(14) 2:9989571-9989571A/-(13),2:9989571-9989572AA/-(1) TBC1D8B K118 Missense_ATts(6),Silent_ATts(8) 23:106064219-106064219A/G(8),23:106064218-106064218A/G(6) TCERG1 K957 Deletion(18) 5:145886723-145886723A/-(18) TCF15 T113 Silent_CpG_CGtv(12) 20:590543-590543C/G(12) TCF15 V114 Missense_CpG_CGtv(12) 20:590542-590542G/C(12) TEAD2 H295 Insertion(2),Deletion(17) 19:49850473-49850473C/-(16),19:49850472-49850473-/C(2),19:49850473-49850474CC/- (1) TFAM E148 Deletion(27) 10:60148570-60148570A/-(27) TMBIM4 Y174 Deletion(18) 12:66531937-66531937T/-(18) TMEM131 G44 Silent_CpG_CGtv(15) 2:98611994-98611994C/A(15) TMEM151B L332 Silent_ATts(15) 6:44243559-44243559A/G(15) TMEM60 A78 Deletion(11) 7:77423460-77423460A/-(11) TMPRSS13 A77 Missense_NoCpG_CGtv(57) 11:117789345-117789345C/G(57) TMPRSS13 Q78 Missense_ATts(15) 11:117789342-117789342A/G(15) TNRC6C N615 Deletion(16) 17:76046980-76046980A/-(16) TP53 R175 Missense_NoCpG_CGts(170) 17:7578406-7578406G/A(162),17:7578407-7578407C/T(8) TP53 R213 Missense_NoCpG_CGtv(3),Nonsense_NoCpG_ 17:7578212-7578212C/T(56),17:7578211-7578211G/T(3) CGts(56) TP53 R248 Missense_NoCpG_CGts(131),Missense_NoCpG 17:7577539-7577539C/T(70),17:7577538-7577538G/A(61),17:7577538-7577538G/T(3) _CGtv(3) TP53 R273 Missense_NoCpG_CGts(92),Deletion(2) 17:7577120-7577120G/A(66),17:7577121-7577121C/T(26),17:7577120-7577121CG/-(2) TRRAP K159 Missense_ATts(16),Silent_ATts(4) 7:98493411-98493411A/G(14),7:98493413-98493413A/G(4),7:98493412-98493412A/G(2) TTK R854 Missense_ATts(2),Insertion(2),Deletion(22) 6:80751897-80751897A/-(20),6:80751905-80751905A/G(2),6:80751897-80751898AA/- (2),6:80751896-80751897-/A(2) TTLL11 A163 Missense_CpG_CGts(24) 9:124855211-124855211G/A(24) TTN I2725 Missense_ATts(15),Missense_NoCpG_CGtv(3), 2:179635207-179635207T/C(9),2:179635208-179635208A/G(6),2:179635206- Silent_NoCpG_CGts(6) 179635206C/T(6),2:179635206-179635206C/G(3) TTN I2771 Missense_ATts(15),Missense_NoCpG_CGtv(3), 2:179635207-179635207T/C(9),2:179635208-179635208A/G(6),2:179635206- Silent_NoCpG_CGts(6) 179635206C/T(6),2:179635206-179635206C/G(3) TYSND1 L258 Silent_CpG_CGtv(12) 10:71905569-71905569G/T(12) UBR5 E2121 Deletion(15) 8:103289349-103289349A/-(15) UHRF1 P674 Insertion(13) 19:4954679-4954680-/C(12),19:4954680-4954681-/T(1) USP35 T411 Insertion(3),Deletion(13) 11:77920856-77920856C/-(13),11:77920855-77920856-/C(3) USP35 T655 Insertion(3),Deletion(13) 11:77920856-77920856C/-(13),11:77920855-77920856-/C(3) USP36 K959 Deletion(12) 17:76798549-76798554GAAAAA/-(12) USP6 V148 Silent_NoCpG_CGts(14) 17:5037241-5037241G/A(14) VEZT S74 Deletion(20) 12:95650969-95650969T/-(20) VPS13A R161 Missense_NoCpG_CGts(3),Missense_NoCpG_C 9:79824436-79824436T/C(12),9:79824434-79824434C/T(3),9:79824434-79824434C/A(3) Gtv(3),Silent_ATts(12) VPS13A K372 Deletion(12) 9:79836219-79836219A/-(12) VSIG10L M356 Missense_CpG_CGts(16) 19:51843808-51843808G/A(16) WDTC1 E290 Insertion(1),Deletion(11) 1:27621108-27621108G/-(11),1:27621107-27621108-/G(1) ZBTB42 R15 Silent_ATts(28) 14:105267579-105267579A/G(28) ZDBF2 K1728 Deletion(14) 2:207174428-207174428A/-(12),2:207174428-207174429AA/-(2) ZNF233 L662 Nonsense_ATtv(1),Deletion(13) 19:44778796-44778796G/-(8),19:44778797-44778797T/-(4),19:44778798- 44778798T/A(1),19:44778799-44778799G/-(1) ZNF365 K399 Deletion(16) 10:64159513-64159513A/-(15),10:64159513-64159514AA/-(1) ZNF516 V1037 Deletion(23) 18:74090958-74090958C/-(23) ZNF518A T929 Deletion(14) 10:97918856-97918856A/-(14) ZNF696 R341 Silent_ATts(14) 8:144378868-144378868A/G(14) ZNF717 E818 Deletion(43) 3:75786315-75786323AGAATGTAG/-(43) ZNF814 G320 Missense_NoCpG_CGts(19),Silent_NoCpG_CGt 19:58385798-58385798G/A(20),19:58385799-58385799G/A(19) s(20) ZNF837 A242 Missense_CpG_CGts(14) 19:58879976-58879976G/A(14) ZSCAN1 L54 Silent_CpG_CGtv(28) 19:58549366-58549366C/G(28)

21 Table S2. Number of mutations that changed their variant allele frequencies (VAF) and cancer cell fractions (CCF) between time points in the three wt and RAG1-/- samples out of the total 3394 (shared between wt23 and wt46) and 3443 (shared between RAG1-/-13 and RAG1-/-23) mutations. wt23 vs wt46 diff in wt23 vs wt46 diff wt23 vs wt46 not RAG1-/-13 vs RAG1-/-23 RAG1-/- vs RAG1-/-23 diff RAG1-/- vs RAG1-/-23 not VAF > 0.2 in CCF > 0.3 shared mutations diff in VAF > 0.2 in CCF > 0.3 shared mutations 138 496 1057 113 446 1087 177 542 1031 105 394 1130 144 614 1015 125 675 1004

22 Table S3. List of expressed predicted neoantigens present in the MC38 cell line and the RAG1-/- samples from day 23, and absent from the wild type samples from day 23

Peptide_mut Chr Start Ref Alt Gene Protein Amino_acids Db_nM Db_Rank Kb_nM Kb_Rank YLVANDGEI chr15 6658562 A C Fyb ENSMUSP00000087947 D/A 285.48 0.125 NA NA ASFHSGNI chr3 145314878 A G Col24a1 ENSMUSP00000029848 T/A NA NA 145.5 0.5 LTLLAGLL chr7 43468715 G A Vsig10l ENSMUSP00000103611 A/T NA NA 482.14 1.3 LGLTLLAGL chr7 43468715 G A Vsig10l ENSMUSP00000103611 A/T NA NA 310.16 0.9 KGTLYYYTL chr10 80322726 G T Pcsk4 ENSMUSP00000020340 T/K NA NA 39.498 0.125 IIPEYVSAF chr11 82987611 T C Slfn9 ENSMUSP00000044435 I/V NA NA 190.61 0.6 IGDLRLATL chr11 101263944 G A Wnk4 ENSMUSP00000099397 G/R NA NA 301 0.9 KGYYFNKGTL chr10 80322726 G T Pcsk4 ENSMUSP00000020340 T/K NA NA 150.84 0.5 KGTLYYYTLL chr10 80322726 G T Pcsk4 ENSMUSP00000020340 T/K NA NA 50.742 0.175 LSFLCRYVAV chr17 56769541 C G Dus3l ENSMUSP00000007747 P/A NA NA 109.83 0.4 NKGYYFNKGTL chr10 80322726 G T Pcsk4 ENSMUSP00000020340 T/K NA NA 482.24 1.3 FNKGTLYYYTL chr10 80322726 G T Pcsk4 ENSMUSP00000020340 T/K NA NA 353.97 1 NKGTLYYYTLL chr10 80322726 G T Pcsk4 ENSMUSP00000020340 T/K NA NA 134.84 0.5 KGTLYYYTLLL chr10 80322726 G T Pcsk4 ENSMUSP00000020340 T/K NA NA 115.6 0.4 IMRKCFPQNRL chr13 59512429 T G Agtpbp1 ENSMUSP00000022040 K/Q NA NA 361.08 1 WLSFLCRYVAV chr17 56769541 C G Dus3l ENSMUSP00000007747 P/A NA NA 326.53 0.9 SSLLAGPRL chr9 108448377 T A Klhdc8b ENSMUSP00000142325 Q/L NA NA 378.61 1.1

23 Table S4. List of expressed predicted neoantigens present in the MC38 cell line and the IgG2b control samples, and absent from the anti-PD-L1 samples

Peptide_mut Chr Start Ref Alt Gene Protein Amino_acid Db_nM Db_Rank Kb_nM Kb_Rank AQLANDVVL chr10 18056095 C G Reps1 ENSMUSP00000123238 P/A 39.083 0.015 NA NA ISFVNAIPW chr11 75489864 A C Prpf8 ENSMUSP00000018449 E/A 407.2091 0.15 206.0801 0.6 YFLDNLDTL chr11 109392383 A C Gna13 ENSMUSP00000020930 K/T 186.9848 0.08 NA NA YLVANDGEI chr15 6658562 A C Fyb ENSMUSP00000087947 D/A 285.4786 0.125 NA NA YSLLLDTYI chr15 37945070 T G Rrm2b ENSMUSP00000022901 I/L 51.0251 0.02 NA NA AAQLANDVVL chr10 18056095 C G Reps1 ENSMUSP00000123238 P/A 102.2957 0.05 NA NA APSLLNWTRV chr10 82699172 G C Hcfc2 ENSMUSP00000020478 R/T 465.7171 0.175 NA NA ISFVNAIPWV chr11 75489864 A C Prpf8 ENSMUSP00000018449 E/A 119.3035 0.05 NA NA KALLNGDGAI chr14 24164486 C A Dlg5 ENSMUSP00000087879 E/D 34.5384 0.015 NA NA RAAQLANDVVL chr10 18056095 C G Reps1 ENSMUSP00000123238 P/A 241.6992 0.09 NA NA AQLANDVVLQI chr10 18056095 C G Reps1 ENSMUSP00000123238 P/A 496.5346 0.175 NA NA AAPSLLNWTRV chr10 82699172 G C Hcfc2 ENSMUSP00000020478 R/T 439.9904 0.15 NA NA ISFVNAIPWVI chr11 75489864 A C Prpf8 ENSMUSP00000018449 E/A 111.3205 0.05 NA NA IKALLNGDGAI chr14 24164486 C A Dlg5 ENSMUSP00000087879 E/D 434.6625 0.15 NA NA IATVQAFL chr2 12410009 G T Fam188a ENSMUSP00000028105 P/T NA NA 440.4284 1.2 LSYLRATL chr2 120030296 G C Jmjd7 ENSMUSP00000041220 V/L NA NA 9.8735 0.02 ICYGSAPL chr3 110250130 C A Prmt6 ENSMUSP00000140836 S/I NA NA 109.7087 0.4 YAFAFSQV chr6 28885053 T G Snd1 ENSMUSP00000001460 I/S NA NA 11.6314 0.025 KARKYFEL chr7 28185387 G T Dyrk1b ENSMUSP00000083064 R/L NA NA 138.8444 0.5 LTLLAGLL chr7 43468715 G A Vsig10l ENSMUSP00000103611 A/T NA NA 482.1449 1.3 KILTFDRL chr7 45720370 A G Rpl18 ENSMUSP00000103365 Q/R NA NA 102.6694 0.4 LTFDRLAL chr7 45720370 A G Rpl18 ENSMUSP00000103365 Q/R NA NA 71.8819 0.25 AMFLGANV chr9 57546426 T C Mpi ENSMUSP00000034856 D/G NA NA 126.6132 0.4 TSSGYPTL chr9 102885994 G C Ryk ENSMUSP00000135858 S/T NA NA 290.6021 0.9 FVMDYIPV chr10 7705809 G T Lats1 ENSMUSP00000132078 G/V NA NA 205.7593 0.6 SSYAHGGF chr11 55487518 G T G3bp1 ENSMUSP00000018727 L/F NA NA 46.7578 0.15 VSRHHRAL chr11 72796528 G C Zzef1 ENSMUSP00000068790 G/R NA NA 285.9487 0.8 VIATVQAFL chr2 12410009 G T Fam188a ENSMUSP00000028105 P/T NA NA 468.1571 1.3 SVYGEVSRL chr2 129066190 C G Ttl ENSMUSP00000046883 A/G NA NA 96.1962 0.3 KSFRQKPNL chr6 48619619 C G Zfp775 ENSMUSP00000056290 S/R NA NA 93.9273 0.3 AGLRRLLLM chr7 25203598 C A Dedd2 ENSMUSP00000049763 R/L NA NA 430.6135 1.2 KARKYFELL chr7 28185387 G T Dyrk1b ENSMUSP00000083064 R/L NA NA 77.7964 0.25 LGLTLLAGL chr7 43468715 G A Vsig10l ENSMUSP00000103611 A/T NA NA 310.1638 0.9 ISPTVRATL chr9 21981403 C A Rgl3 ENSMUSP00000035726 V/L NA NA 162.2901 0.5 LNWTRVSSF chr10 82699172 G C Hcfc2 ENSMUSP00000020478 R/T NA NA 290.533 0.9 SAWVPFGGL chr10 128802236 C A Tmem198b ENSMUSP00000050451 V/F NA NA 42.9606 0.15 VSRHHRALL chr11 72796528 G C Zzef1 ENSMUSP00000068790 G/R NA NA 149.4984 0.5 IIPEYVSAF chr11 82987611 T C Slfn9 ENSMUSP00000044435 I/V NA NA 190.6065 0.6

24 IGDLRLATL chr11 101263944 G A Wnk4 ENSMUSP00000099397 G/R NA NA 300.9963 0.9 VSLKGLCQL chr11 116069391 T C Unc13d ENSMUSP00000074549 E/G NA NA 325.2974 0.9 AGFTKLAPV chr13 89704367 G C Vcan ENSMUSP00000105173 P/A NA NA 53.1282 0.175 SIQDFLLRM chr14 65648256 G C Nuggc ENSMUSP00000078434 M/I NA NA 205.8907 0.6 WSLSYLRATL chr2 120030296 G C Jmjd7 ENSMUSP00000041220 V/L NA NA 415.0376 1.2 SAWVPFGGLM chr10 128802236 C A Tmem198b ENSMUSP00000050451 V/F NA NA 481.1183 1.3 VHSEMYSLLL chr15 37945070 T G Rrm2b ENSMUSP00000022901 I/L NA NA 377.1472 1.1 LSFLCRYVAV chr17 56769541 C G Dus3l ENSMUSP00000007747 P/A NA NA 109.8274 0.4 IMRKCFPQNRL chr13 59512429 T G Agtpbp1 ENSMUSP00000022040 K/Q NA NA 361.0831 1 AGFTKLAPVPL chr13 89704367 G C Vcan ENSMUSP00000105173 P/A NA NA 221.6772 0.7 ISWVKEGFTFL chr16 73978491 G C Robo2 ENSMUSP00000112776 L/V NA NA 63.4355 0.2 WLSFLCRYVAV chr17 56769541 C G Dus3l ENSMUSP00000007747 P/A NA NA 326.5283 0.9 ALYINLAAI chr18 61989909 C A Sh3tc2 ENSMUSP00000055094 Y/* 308.6139 0.125 NA NA AALYINLAAI chr18 61989909 C A Sh3tc2 ENSMUSP00000055094 Y/* 52.0202 0.025 NA NA VAALYINLAAI chr18 61989909 C A Sh3tc2 ENSMUSP00000055094 Y/* 286.6858 0.125 415.9277 1.2

25 Table S5. List of predicted neoantigens present in the CT26 cell line and the IgG2b control samples, and absent from the anti-PD-L1 samples

Peptide_mut Chr Start Ref Alt Gene Protein Amino_acid Db_nM Db_Rank Kb_nM Kb_Rank

VTMVNLFLF chr7 103564442 G A Olfr616 ENSMUSP00000102499 C/R 315.4096 0.125 NA NA

VTMVNLFLFL chr7 103564442 G A Olfr616 ENSMUSP00000102499 C/R 249.8463 0.1 254.267 0.8

VAIRNLFSRI chr9 19585461 C T Olfr855 ENSMUSP00000054790 V/A 41.0658 0.02 199.4939 0.6

KVAIRNLFSRI chr9 19585461 C T Olfr855 ENSMUSP00000054790 V/A 397.6562 0.15 NA NA

VAIRNLFSRII chr9 19585461 C T Olfr855 ENSMUSP00000054790 V/A 87.8645 0.04 NA NA

CTYKRWVM chr7 43815409 A C Klk7 ENSMUSP00000032955 D/N NA NA 315.1298 0.9

VTMVNLFL chr7 103564442 G A Olfr616 ENSMUSP00000102499 C/R NA NA 277.9622 0.8

MVNLFLFL chr7 103564442 G A Olfr616 ENSMUSP00000102499 C/R NA NA 247.3288 0.7

LTLACMNL chr7 106956384 T G Olfr6 ENSMUSP00000048740 K/R NA NA 240.1351 0.7

SMYFFLSH chr7 108255956 G A Olfr488 ENSMUSP00000072735 G/S NA NA 221.9892 0.7

STATFSGV chr11 50951151 A C Prop1 ENSMUSP00000057231 P/L NA NA 171.2505 0.5

VMYCRVYVA chr2 131561348 A G Adra1d ENSMUSP00000099473 A/T NA NA 366.8441 1

TMVNLFLFL chr7 103564442 G A Olfr616 ENSMUSP00000102499 C/R NA NA 178.4518 0.6

LHHSMYFFL chr7 108255956 G A Olfr488 ENSMUSP00000072735 G/S NA NA 304.3168 0.9

SMYFFLSHL chr7 108255956 G A Olfr488 ENSMUSP00000072735 G/S NA NA 3.5083 0.01

MNLTLFSKV chr8 66703937 C T Npy1r ENSMUSP00000045530 G/E NA NA 191.6944 0.6

AIRNLFSRI chr9 19585461 C T Olfr855 ENSMUSP00000054790 V/A NA NA 340.1817 1

NLFSRIISL chr9 19585461 C T Olfr855 ENSMUSP00000054790 V/A NA NA 324.8719 0.9

STATFSGVL chr11 50951151 A C Prop1 ENSMUSP00000057231 P/L NA NA 236.479 0.7

VMYCRVYVAA chr2 131561348 A G Adra1d ENSMUSP00000099473 A/T NA NA 443.6137 1.2

VNLFLFLTPV chr7 103564442 G A Olfr616 ENSMUSP00000102499 C/R NA NA 22.8529 0.06

HSMYFFLSHL chr7 108255956 G A Olfr488 ENSMUSP00000072735 G/S NA NA 14.0948 0.04

SMYFFLSHLA chr7 108255956 G A Olfr488 ENSMUSP00000072735 G/S NA NA 36.728 0.125

RNLFSRIISL chr9 19585461 C T Olfr855 ENSMUSP00000054790 V/A NA NA 312.9349 0.9

DMAYIFLIQL chr16 43969363 C T Zdhhc23 ENSMUSP00000128650 T/A NA NA 397.6562 1.1

MVNLFLFLTPV chr7 103564442 G A Olfr616 ENSMUSP00000102499 C/R NA NA 55.8412 0.175

VNLFLFLTPVL chr7 103564442 G A Olfr616 ENSMUSP00000102499 C/R NA NA 77.5191 0.25

SQLHHSMYFFL chr7 108255956 G A Olfr488 ENSMUSP00000072735 G/S NA NA 498.0195 1.3

HHSMYFFLSHL chr7 108255956 G A Olfr488 ENSMUSP00000072735 G/S NA NA 24.7783 0.07

HSMYFFLSHLA chr7 108255956 G A Olfr488 ENSMUSP00000072735 G/S NA NA 69.3816 0.25

SMYFFLSHLAT chr7 108255956 G A Olfr488 ENSMUSP00000072735 G/S NA NA 83.6292 0.3

IRNLFSRIISL chr9 19585461 C T Olfr855 ENSMUSP00000054790 V/A NA NA 430.064 1.2

ADMAYIFLIQL chr16 43969363 C T Zdhhc23 ENSMUSP00000128650 T/A NA NA 480.5775 1.3

PSTSFLHQNML chr16 14589245 A C A630010A05Rik ENSMUSP00000122979 R/G NA NA 415.1948 1.2

26