Analysis of HPV E6 and E7 Peptide Sequences and Antigen-Specific Binders

Alignment of HPV 16 and 18 E6 and E7 protein sequences, highlighting specific peptide positions. The study focuses on isolating HPV antigen-specific binders with HuTARGTM, optimizing library construction for HPV E6 and E7 binders, and examining the sensitivity and near off-target activity of light-chain optimized binders. The figures illustrate these processes and provide valuable insights into HPV-related research.

• HPV
• E6
• E7
• Antigen
• Binders

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1. Supplementary information Supplementary information Figures

2. Supplementary Figure 1: Alignment of HPV 16 and 18 E6 and E7 protein sequences, showing positions of HPV E6 29-38 and HPV E711-19 peptides. HPV E6 29-38 HPV E659-67 HPV E693-101 HPV16 E6: 1 MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNY 101 | |.|:||..||.|||.|||||.|::.||.:.|||||..|...||::|||:||.:||||..|:|.|.||:.|||:|.|.|||..|:||.|||:..|. HPV18 E6: 1 M-----ARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNT 96 HPV16 E6 102 NKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSR-----TRRETQL 158 |..|.:|||||:.|||||.|.||.|||::|:|||||.|.:.|:|.|||..:| .|||||: HPV18 E6 97 NTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV 158 HPV E77-15 HPV E711-19 HPV E782-90 HPV16 E7: 1 MHGDTPTLHEYMLDLQPET---TDLYCYEQLNDSSEEEDEIDG------PAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP 98 |||...||.:.:|.|:|:. .||.|:|||:||.||.||||| ||.:|||.| :.::..||||::.::|.|:|:..|:|..:.|.:.||..|||.|:.:. HPV18 E7: 1 MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQR--HTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWCASQQ 105

3. Supplementary Figure 2: Isolating HPV antigen-specific binders with HuTARGTM. Identify CARs (parents) Identify lead CARs Optimized CARs Primary HC-library screen LC-library HC-library binder optimization screen Go/No-Go Checkpoint binder optimization screen Jurkat and Primary T Functional Characterization Jurkat and Primary T Functional Characterization Jurkat Functional Characterization 3

4. Supplementary Figure 3: Optimization library construction for HPV E6 29-38 and HPV E711-19 binders. Primary binders (common light chain) Light-chain variants of each primary binder (pooled) Selected AM binders HuTARG HuTARG HuTARG HuTARG HuTARG HuTARG HuTARG HuTARG HuTARG HuTARG IgK-C P2A IgH-V Barcode CDR3 junction + IgK-V w/ RSS IgH-Constant-TM AM IgK IgK-C P2A IgH-V IgH IgK-C P2A IgH-V PCR primers introduce unique barcode with CDR3 junction from primary HC Barcoded fragments are cloned into acceptor pool with 24 LC bipartites in CDRs 1, 2 and 3 AM binders are selected by flow cytometry by comparison to top primary binders 4

5. Supplementary Figure 4: Examples of sensitivity and near off-target activity of HPV E711-19 light-chain optimized binders (EC50s of cross-reacting peptides are shown in supplementary Table 3). HPV E711-19 PSME4782-790 PSME4787-795 SBK3281-289 AGL538-546 HPV E711-19 ZFN236818-827 ZFN236819-288 SH3GLB1242-252 SH3GLB1243-252 HPV E711-19 TNS1713-722 TNS1714-723 TNS1715-724 HPV E711-19 TNS1716-725 TNS1714-722 TNS1716-724 36000 36000 36000 36000 32000 32000 32000 32000 28000 28000 28000 28000 C1800 24000 24000 24000 24000 20000 20000 20000 20000 16000 16000 16000 16000 12000 12000 12000 12000 8000 8000 8000 8000 1x10-8 1x10-5 1x10-8 1x10-5 1x10-8 1x10-5 1x10-8 1x10-5 0.01 10 0.01 10 0.01 10 0.01 10 HPV E711-19 PSME4782-790 PSME4787-795 SBK3281-289 AGL538-546 HPV E711-19 ZFN236818-827 ZFN236819-288 SH3GLB1242-252 SH3GLB1243-252 HPV E711-19 TNS1713-722 TNS1714-723 TNS1715-724 HPV E711-19 TNS1716-725 TNS1714-722 TNS1716-724 50000 50000 50000 50000 46000 46000 46000 46000 42000 42000 42000 42000 C1806 38000 38000 38000 38000 34000 34000 34000 34000 30000 30000 30000 30000 26000 26000 26000 26000 22000 22000 22000 22000 18000 18000 18000 18000 1x10-8 1x10-5 1x10-8 1x10-5 1x10-8 1x10-5 1x10-8 1x10-5 0.01 10 0.01 10 0.01 10 0.01 10

6. Supplementary Figure 5: Light-chain optimized HPV E629-38 CARs show minimal near off-target cross reactivity. Selectivity of cross-reactive peptides was determined by peptide dose-response and is summarized in Table 3. C2388 90 100 80 70 60 POC 50 40 Cut-off = 10% 30 20 10 0 HPV E6 29-38 GLT12_HUMAN-161 IMA4_HUMAN-447 COMD9_HUMAN-96 OBSL1_HUMAN-997 RNF43_HUMAN-207 RNBP6_HUMAN-1077 PTPRD_HUMAN-1603 RBM45_HUMAN-445 O10X1_HUMAN-225 CFA47_HUMAN-2773 ZN862_HUMAN-608 O4C45_HUMAN-108 LMBD1_HUMAN-102 RTKN2_HUMAN-150 SI1L2_HUMAN-164 CSF3R_HUMAN-168 ITA8_HUMAN-833 RLA0_HUMAN-209 PHLP2_HUMAN-833 TCPG_HUMAN-31 VP33A_HUMAN-87 GRID2_HUMAN-150 RTL5_HUMAN-295 PTPRG_HUMAN-1293 PYR1_HUMAN-347 MACF1_HUMAN-3420 COPG1_HUMAN-667 COPG2_HUMAN-664 FEM1B_HUMAN-550 PGAP3_HUMAN-175 LMBR1_HUMAN-382 PRR5_HUMAN-346 HINFP_HUMAN-422 FEZ2_HUMAN-144 GNTK_HUMAN-63 DNM1L_HUMAN-458 STA5B_HUMAN-318 SRGP1_HUMAN-115 DYHC2_HUMAN-2545 TMCO3_HUMAN-177 -10 C1987 100 90 80 70 POC 60 50 40 30 Cut-off = 10% 20 10 0 HPV E6 29-38 GLT12_HUMAN-161 RBM45_HUMAN-445 RNF43_HUMAN-207 O10X1_HUMAN-225 LMBD1_HUMAN-102 RTKN2_HUMAN-150 CSF3R_HUMAN-168 IMA4_HUMAN-447 ZN862_HUMAN-608 TCPG_HUMAN-31 OBSL1_HUMAN-997 O4C45_HUMAN-108 RTL5_HUMAN-295 PHLP2_HUMAN-833 RNBP6_HUMAN-1077 PTPRD_HUMAN-1603 RLA0_HUMAN-209 COPG1_HUMAN-667 CFA47_HUMAN-2773 COPG2_HUMAN-664 SI1L2_HUMAN-164 ITA8_HUMAN-833 PYR1_HUMAN-347 MACF1_HUMAN-3420 COMD9_HUMAN-96 VP33A_HUMAN-87 FEM1B_HUMAN-550 PTPRG_HUMAN-1293 GRID2_HUMAN-150 DNM1L_HUMAN-458 STA5B_HUMAN-318 LMBR1_HUMAN-382 PRR5_HUMAN-346 FEZ2_HUMAN-144 PGAP3_HUMAN-175 DYHC2_HUMAN-2545 TMCO3_HUMAN-177 HINFP_HUMAN-422 GNTK_HUMAN-63 SRGP1_HUMAN-115 -10 6

7. Supplementary Figure 6: Representative flow cytometry data for the EPV TCR (C882) using mTCR antibody (anti mouse) and E711-19/HLA-A*02 pMHC probe. C882 stained using probe and anti- mTCR separately UTD C882 Anti-mTCR E711-19 probe

8. Supplementary Figure 7: Standard curve for T2 loading of peptide to estimate pMHCs/cell (original binding data from Purbhoo et al. (2006). Molecules/cell listed in table to right of 1E-8 are estimated by scaling from the flow cytometry MFI by a factor of 550/20, the values at 1E-8 M measurements where the most reliable overlap lies between the single-molecule fluorescence counting and population mean from flow cytometry (MFI). Peptide (M) 1E-11 1E-10 1E-9 1E-8 1E-7 1E-6 1E-5 MFI (FACS) ND 5 10 20 60 130 180 Molecules/cell (microscopy) 5 10 50 550 1650 3575 4950 Extrapolated from MFI 200 6000 MFI (FACS) Molecules/cell (microscopy) 150 Molecules/cell MFI (FACS) 4000 100 2000 50 0 0 1 10-12 1 10-11 1 10-10 1 10-9 1 10-8 1 10-7 1 10-6 1 10-5 Peptide (M) 8

9. Supplementary information Supplementary information Tables

10. Supplementary Table 1. Summary of incidence of HPV-associated cancers in US and contributions of main subtypes. Total US/yr Incidence (deaths) HPV+ Cervical Cancer HPV+ Non-cervical 11,000 (3,500) ~28,000 (~6,200) ~39,000 (~9,700) All 6,100 (1,900) 26,000 (6,000) 32,100 (7,900) HPV16 Subtype 1,200 (400) ~2,000 (~200) ~3,200 (~600) HPV18 Estimated, rounded number of new US cases in 2018 (deaths); compiled from combination of databases 1. 2. 3. https://seer.cancer.gov/statfacts/ https://www.cdc.gov/cancer/hpv/statistics/cases.htm Doorbar et al. Rev. Med. Virol. 2016; 25: 2 23. 10

11. Supplementary Table 2: Near off-target peptides tested with E629-38 binders in this study. Peptide sequence Peptide sequence Peptide sequence Name Name Name Name Peptide sequence HPV E629-38 HPV E629-38 TIHDIILECV TIHDIILECV TIHDIILECV TIHDIILECV HPV E629-38 HPV E629-38 TITDIISALV TLHSRFLEGV SMLDEILEDV KLHDAIVEVV STA5A RLA0L TMCO3 DNM1L TITDICFENV TLHGKILNGV NLVDQILESV IIIHDFILEA RTKN2 RBM45 PRR5 PTPRG1292-1302 TIFVIILASV LLFDIFLETV TLIAVTLECV IIHDFILEA RNF43 PYR1 OBSL1 PTPRG1293-1302 TISDIDAEDV ILHSIYLCCV TLTEIILLSV FIHDALLEA SI1L2 PGAP3 O4C45 PTPRG1111-1119 TSEDLWLECV TLLLIILTDV TLYSVILFCV FIHDALLEAI RNBP6 O10X1 LMBD1 PTPRG1111-1120 TSPDILLEEV TLVSQELECV SLQGIILETV YIFIHDALL GLT12 MACF1 HINFP PTPRG1109-1117 TQGDSILDCV TMTKIIGNCV YLHDVILRVV FIHDAILEA CSF3R LMBR1 GRID2 PTPRM1146-1154 FIHDALLEAV HLFDIILTSV DMHSIIVSCV YVFIHDAIL PTPRD DYHC2 FEZ2 PTPRM1144-1152 IIHDFILEAT TLNDQTLENV TLHSIIISLV YIFIHDAIL PTPRG1293-1303 COPG1 FEM1B PTPRU/M/K TISDTILEVG LLTKIILEHV TLNDQLLEKV ITA8 COMD9 COPG2 TIADIIRTCL VLIDIILTSV DLDELILECV TCPG CFA47 RTL5 TIAEIIEECG TLKREILEDV TMADVLLEEV IMA4 ZN862 PHLP2 TLSDIYLNNV ELMDIIAENV NLHDILLRDV SRGP1 VP33A GNTK

12. Supplementary Table 3: Summary of EC50 of binders shown in Supplementary Figure 4. EC50 (nM) Emax (RLU) Selectivity Constructs Construct format/source Test peptide name Test peptide sequence Test peptide name HPV E711-19 16 44000 N/A YMLDLQPET on PSME off 6 32000 0.4 YLLDSFLQP SBK3 off 19000 17000 1188 ALLQGLLDL ZFN236818-827 off 5500 19000 344 AMLDLEPQHV ZFN236819-827 off 3200 19000 200 C1800 LC opt CAR MLDLEPQHV SH3GLB1242-252 off 11700 16000 731 QYMLDLQKQL SH3GLB1243-252 off 3000 27000 188 YMLDLQKQL TNS1714-724 off 20000 23000 1250 MLMLDLEPAS TNS1715-724 off 7800 19000 491 LMLDLEPASA YMLDLQPET HPV E711-19 on 5.8 51000 N/A PSME off 12000 35000 2069 YLLDSFLQP SBK3 off 3520 49000 607 ALLQGLLDL ZFN236818-827 off 1600 45000 276 AMLDLEPQHV ZFN236819-827 C1806 LC opt CAR off 2300 45000 397 MLDLEPQHV SH3GLB1242-252 off 19000 37000 3276 QYMLDLQKQL SH3GLB1243-252 off 4000 45000 690 YMLDLQKQL TNS1714-724 off 17000 42000 2931 MLMLDLEPAS TNS1715-724 off 4000 33000 690 LMLDLEPASA LLMGTLGIV C1342 Primary CAR on 3.4 43000 N/A HPV E782-90 LLMGTLGIV C1346 Primary CAR on 4 77000 N/A HPV E782-90 LLMGTLGIV C1369 Primary CAR on 13.6 20000 N/A HPV E782-90 LLMGTLGIV C1373 Primary CAR on 4.9 32000 N/A HPV E782-90 C2008 Primary CAR on 9600 15000 N/A HPV E77-15 TLHEYMLDL C2016 Primary CAR on >100 N/A N/A HPV E77-15 TLHEYMLDL

13. Supplementary Table 4: Summary of EC50 of ICD and LBD variants of HPV E629-38 CAR as shown in Figure 5. EC50 (nM) Emax (RLU) Constructs Parent construct LBD ICD 0.5 30000 CT503 C1987 HL-scFv CD28-41BB-CD3zeta CT803 HL-scFv 41BB-CD3zeta 7 34000 CT503 CD28-41BB-CD3zeta- CD3zeta CT568 HL-scFv 6.6 11000 CT503 CT574 LH-scFv CD28-41BB-CD3zeta 7.6 21000 CT503 CT580 HL/LH-scFv CD28-41BB-CD3zeta 4.8 20000 CT503 CT512 C1992 HL-scFv CD28-41BB-CD3zeta 0.2 30000 CT804 CT512 HL-scFv 41BB-CD3zeta 12 20000 CD28-41BB-CD3zeta- CD3zeta CT570 CT512 HL-scFv 2.7 13000 CT575 CT512 LH-scFv CD28-41BB-CD3zeta 4.6 27000 CT582 CT512 HL/LH-scFv CD28-41BB-CD3zeta 9.2 24000