T Cell Receptor Optimized Peptide Skewing of the T-Cell Repertoire Can Enhance Antigen Targeting

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T Cell Receptor Optimized Peptide Skewing of the T-Cell Repertoire Can Enhance Antigen Targeting Julia Ekeruche-Makinde 1*, Mathew Clement 1*, David K Cole 1*, Emily S J Edwards 1, Kristin Ladell 1, John J Miles 1,2, Katherine K Matthews 1, Anna Fuller 1, Katy A Lloyd 1, Florian Madura 1, Garry M Dolton 1, Johanne Pentier 1, Anna Lissina 1, Emma Gostick 1, Tiffany K Baxter 3, Brian M Baker 3, Pierre J Rizkallah 1, David A Price 1, Linda Wooldridge 1* and Andrew K Sewell 1* 1 Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, UK. 2 Australian Centre for Vaccine Development, Human Immunity Laboratory, Queensland Institute of Medical Research, Brisbane 429, Australia. 3 Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA. * These authors contributed equally to this work Running Head: TCR optimized peptide skewing of the T-cell repertoire To whom correspondence should be addressed: Andrew K Sewell. E-mail: SewellAK@cardiff.ac.uk; Tel: (+)44 29 268 755; Fax: (+)44 29 268 77. Keywords: APL (altered peptide ligand), CTCL (cutaneous T-cell lymphoma), pmhc (peptide- MHC), pmhci (peptide-mhc class I), TCR (T-cell receptor), SPR (surface plasmon resonance), TOP (TCR optimized peptide), TOPSORT (TCR optimized peptide skewing of the repertoire of T- cells) Background: Current peptide vaccines may select sub-optimal antigen-specific T-cells from polyclonal populations Results: A combinatorial peptide library screen was used to generate an optimal ligand that could preferentially activate a known effective T-cell clonotype Conclusion: Rationally designed altered peptide ligands may enable the preferential selection of high quality, antigen-sensitive T-cell clonotypes Significance: This proof-of-principle study could facilitate the development of more effective peptide vaccination strategies

SUPPLEMENTARY TABLES Supplementary Table S1. Data collection and refinement statistics (molecular replacement). Data set statistics HLA A*21-GIGIITV HLA A*21-FLTGIGIITV Space Group C 1 2 1 C 1 2 1 a=22.4, b=46.6, c=116.6 a=21.5, b=46.8, c=115.9 Unit Cell parameters (Å) α=9., β=121.5, γ=9. α=9., β=121.7, γ=9. Radiation Source DIAMOND I3 DIAMOND I3 Wavelength (Å).9763.9763 Resolution (Å) 2.75 2.35 Unique reflections 26,5 (1,911) 36,5 (2,72) Completeness (%) 99.5 (99.6) 92.4 (94.2) Multiplicity 4 (4.1) 4 (3.6) I/Sigma(I) 1.4 (2.2) 8.8 (2.2) Rmerge (%) 9.4 (62.4) 9 (7.1) No reflections used 23,24 34,22 Rcryst (no cutoff) (%) 2.7 21.1 Rfree (%) 29.9 28.5 Bond lengths (Å).17.15 Bond Angles ( ) 1.698 1.664 Mean B value (Å 2 ) 68.5 45.7 Outliers Ramachandran plot (%) Overall ESU based on Maximum Likelihood (Å).41.15 One crystal was used for data collection. *Values in parentheses are for the highest-resolution shell.

Supplementary Table S2. Comparison of CD8+ T-cell frequencies primed by ELAGIGILTV and GIGIITV. Donor ID % EAA tetramer +ve cells % ELA tetramer +ve cells ELA primed primed ELA primed primed 1 1.46 4.8 2.5 1.31 2.67 2.4.81 3.56 3 7.21 11.6 21.1 1.58 4 6.96 2.22 1.36.461 5 16.6 1.32 7.56 8.73 6 3.18 3.13 nm nm 7 1.22 12.3 15.9 1.31 8 1.96 1.4 14.2 1.5 9.337 1.37 1.71 4.59 1 27.3.74 2.62 1.8 nm = not measured No statistically significant differences were detected between the ELAGIGILTV-primed and GIGIITV-primed CD8 + T-cell populations with respect to response frequencies.

SUPPLEMENTARY FIGURES Figure S1: Melan A-specific CD8 + T-cell clones exhibit differential recognition of natural tumor epitopes. (A) Relative stabilities of HLA A*21-AAGIGILTV (AAG9), HLA A*21- EAAGIGILTV (EAA1) and HLA A*21-ELAGIGILTV (ELA1) complexes. Thermal stability was monitored by circular dichroism (CD), which reports directly on peptide binding affinity for MHC. Thermal stability (T m ) values for the three complexes are shown on the plot and indicate an affinity ranking of AAGIGILTV < EAAGIGILTV << ELAGIGILTV. (B&C) 3x1 4 MEL5 (B) or MEL187.c5 (C) CD8 + T-cells were incubated overnight with 6x1 4 C1R A2 cells pre-pulsed with the indicated concentrations of the peptides shown in the key. Supernatants were harvested and assayed for MIP-1 by ELISA. (D&E) Binding of soluble MEL5 TCR (D) or MEL187.c5 TCR (E) to the HLA A*21-AAGIGILTV complex (A2-AAGIGILTV) measured by SPR.

A MEL187.c5 B MEL5 % CD17a +ve CD8+ T-cell 3 3 4 6 25 5 3 4 2 2 3 15 2 2 1 1 1 1 5 1-8 1-7 1-6 1-5 1-7 1-6 1-5 1-7 1-6 1-7 1-6 5 4 3 2 IFN-γ [pg/ml] 15 1 5 ELAGIGILTV vs FLTGIGIITV TNF-α [pg/ml] 15 1 5 IL2 [pg/ml] 25 2 15 1 1 5 1-9 1-8 1-7 1-6 1-5 1-9 1-8 1-7 1-6 1-9 1-8 1-7 1-6 1-9 1-8 1-7 1-6 C MEL187.c5 D MEL5 % CD17a +ve CD8+ T-cell 7 6 5 4 3 2 1 1-9 1-8 1-7 1-6 1-5 1-4 9 8 7 6 5 4 3 2 1 1-9 1-8 1-7 1-6 1-5 IFN-γ [pg/ml] 1-4 5 4 3 2 1 25 2 15 1 5 ELAGIGILTV vs GIGIITV 1-9 1-8 1-7 1-6 1-5 1-9 1-8 1-7 1-6 TNF-α [pg/ml] 5 4 3 2 1 15 1 5 1-9 1-8 1-7 1-6 1-9 1-8 1-7 1-6 Peptide concentration [M] IL2 [pg/ml] 5 4 3 2 1 1-9 1-8 1-7 1-6 1-5 25 2 15 1 5 1-9 1-8 1-7 1-6 1-5 Figure S2: Recognition of the GIGIITV and FLTGIGIITV peptides by the MEL5 and MEL187.c5 CD8 + T-cell clones. C1R A2 cells were incubated with the indicated peptides for 1 hr at the concentrations shown and then used as targets for the MEL187.c5 and MEL5 CD8 + T-cell clones. (A&B) Recognition of the ELAGIGILTV and FLTGIGIITV peptides by the MEL187.c5 (A) and MEL5 (B) CD8 + T-cell clones. (C&D) Recognition of the ELAGIGILTV and GIGIITV peptides by the MEL187.c5 (C) and MEL5 (D) CD8 + T-cell clones.

A 4 3 Donor 2 MEL 526 ELA E 6 4 Donor 4 MEL 526 ELA I 4 Donor 6 MEL 526 6 ELA 2 1 2 2 B 6:1 12:1 6:1 3.1:1 3:1 Donor 2 MEL 624.75:1 6 ELA F 12:1 6:1 3.1:1.75:1 Donor 4 MEL 624 6 ELA.75:1 3. 6. J 15:1 7.5:1 3.7:1 1.8:1.9:1.4:1.2:1.1:1 Donor 6 MEL 624 15 ELA 4 4 1 2 2 5 C 6:1 12:1 6:1 3.1:1.75:1 3:1 Donor 3 MEL 526.75:1.375:1 4 ELA 3 G 12:1 6:1 3.1:1.75:1.75:1 Donor 5 MEL 526 3. 6. 8 ELA 6 K 15:1 7.5:1 3.7:1 1.8:1.9:1.4:1.2:1.1:1 4 Donor 7 MEL 526 15:1 7.5:1 3.75:1 1.875:1.9:1.46:1.23:1.12:1 6 ELA 2 1 4 2 2 % specific lysis D 12:1 6:1 3.1:1.75:1.75:1 4 ELA 3 2 1 12:1 6:1 3.1:1.75:1 E:T ratio.75:1 Donor 3 MEL 624 3. 3. 6. 6. H 4 2.75:1 12:1 6:1 3.1:1 3. 6..75:1 6 ELA Donor 5 MEL 624 12:1 6:1 3.1:1.75:1 L 12:1 6:1 3.1:1.75:1.75:1.75:1 Donor 7 MEL 624 3. 3. 6. 6. 8 ELA 6 4 2 12:1 6:1 3.1:1.75:1 Figure S3: The optimized analogue peptide GIGIITV can prime superior Melan A-specific CD8 + T-cells. 6x1 6 PBMCs from seven healthy HLA A*21 + donors were pulsed with 1 M peptide (ELAGIGILTV or GIGIITV) for 1 hr. Lines were grown out for 14 days and then tested for antigen specificity using pmhci tetramer staining. Specific lysis was measured for each primed CD8 + T-cell line after exposure to the melanoma cell lines Mel 526 and Mel624 at a range of effector:target (E:T) ratios (A-L). Error bars are standard deviation from three experiments.

Figure S4: GIGIITV-primed CD8 + T-cells are clonotypically distinct from those primed by the heteroclitic peptide ELAGIGILTV. 5x1 4 cells from HLA A*21-restricted Melan A-specific CD8 + T-cell lines were incubated with LIVE/DEAD Fixable Aqua amine-reactive fluorescent dye for 15 min at room temperature, washed once and stained with APC-conjugated HLA A*21- ELAGIGILTV tetramer. Cells were then stained with PerCP-conjugated anti-human CD8 and a panel of anti-human V antibodies for 3 min at 4 C. Corresponding data for HLA A*21-EAAGIGILTV tetramer + cells from the same CD8 + T-cell lines are shown in Figure 8.

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