1. FVIII proteins with a modified immunodominant T-cell epitope exhibit reduced immunogenicity and normal FVIII activity
by Ruth A. Ettinger, Joseph A. Liberman, Devi Gunasekera, Komal Puranik, Eddie A. James, Arthur R. Thompson, and Kathleen P. Pratt
BloodAdv
Volume 2(4):
February 27, 2018
©2018 by American Society of Hematology

2. Ruth A. Ettinger et al. Blood Adv 2018;2:309-322
©2018 by American Society of Hematology

3. FVIII2194-2205 constitutes the minimal binding peptide to rDRB1*0101.
FVIII constitutes the minimal binding peptide to rDRB1*0101. FVIII and peptides truncated from the C and N termini were tested for binding to rDRB1*01:01 using a competition assay measuring displacement of the high-affinity reference peptide HA The DRB1*01:01 protein was incubated with 0.05, 0.1, 0.5, 1, 5, 10, and 50 μM FVIII peptides plus biotinylated HA and immobilized in wells coated with anti-DR antibody L243. After washing, biotinylated peptide bound to HLA-DR was labeled using europium-conjugated streptavidin and quantified using a time-resolved fluorometer. Sigmoidal binding curves were simulated and IC50 values calculated. Results are expressed as relative affinities (IC50 of FVIII /IC50 of the indicated truncated peptide) ± standard error (SE). The IC50 of FVIII was 0.22 ± 0.03 μM.
Ruth A. Ettinger et al. Blood Adv 2018;2:
©2018 by American Society of Hematology

4. FVIII residues 2196, 2199, 2201, and 2204 contribute to DRB1
FVIII residues 2196, 2199, 2201, and 2204 contribute to DRB1*01:01 binding.
FVIII residues 2196, 2199, 2201, and 2204 contribute to DRB1*01:01 binding. The anchor residues contributing peptide-DRB1*01:01 affinity were determined by competition binding assays with reference peptide HA using FVIII peptides having single Arg (A) or Ala (B) substitutions at every possible position. The Arg substitution was chosen to map the anchor residues as this bulky, charged side chain is rarely accommodated by pockets within the MHCII peptide-binding groove. Ala substitutions are also informative, as they can perturb binding by removing interactions contributing to peptide-MHCII affinity. Results are expressed as relative affinities (IC50 of FVIII /IC50 of the substituted peptide) ± SE. The IC50 of FVIII was 0.26 ± 0.02 μM.
Ruth A. Ettinger et al. Blood Adv 2018;2:
©2018 by American Society of Hematology

5. Effect of systematic alanine substitutions in FVIII2194-2205 on T-cell proliferation of clones.
Effect of systematic alanine substitutions in FVIII on T-cell proliferation of clones. WT and Ala-substituted FVIII peptides were added to 5 T-cell clones isolated from 3 HA subjects, incubated with irradiated PBMCs from a donor with an DRB1*01:01 allele as antigen-presenting cells. For clones 32A-18 (A), 17A-19WK-11 (B), 17A-21MO-5 (C), and 17A-21MO-11 (D) each peptide was added at final concentrations of 0.01, 0.1, 1, 5, 10, 50, and 100 μM, cpm of [3H] thymidine incorporated was measured, and EC50 values were calculated. Clone 56A-C2 (E) was stimulated with peptides at 0.05, 0.1, 0.5, 1, and 10 μM final concentrations. The curves that resulted from these data were not sufficient to calculate accurate EC50 values, therefore, cpm in response to 10 μM peptide was plotted. Results are expressed as relative T-cell proliferation (EC50 or cpm of FVIII /EC50 or cpm of the indicated Ala-substituted peptide) ± SE. The EC50 values for clonal responses to FVIII were 5.7 ± 0.9 μM (A), 48.2 ± 2.1 μM (B), 10.5 ± 0.9 μM (C), and 52.5 ± 3.1 μM (D). Note that the WT peptide already contains an Ala at position These 5 clones had distinct TCR sequences and were previously shown to bind tetramers with high avidity and to proliferate in response to FVIII (Table 2). Although the binding affinities of FVIII and FVIII to rDRB1*01:01 protein were almost identical, the T-cell clones proliferated more robustly in response to FVIII compared with FVIII (supplemental Figure 8). Cytokine analysis of supernatants from stimulated clones is in supplemental Figure 1.
Ruth A. Ettinger et al. Blood Adv 2018;2:
©2018 by American Society of Hematology

6. Effects of all possible amino acid substitutions at FVIII-F2196 on DRB1*01:01 binding and CD4+ T-cell proliferation, and homologous/orthologous residues to human FVIII anchor residues F2196 and M2199.
Effects of all possible amino acid substitutions at FVIII-F2196 on DRB1*01:01 binding and CD4+T-cell proliferation, and homologous/orthologous residues to human FVIII anchor residues F2196 and M2199. (A) All 19 non-Phe amino acids were substituted into FVIII at position F2196. Peptides were tested for binding to rDRB1*01:01 by a competition assay measuring displacement of the DRB1*01:01 reference peptide HA Binding of the F2196C-substituted peptide was tested with 10 mM β-mercaptoethanol in the binding buffer as described.72 Results are expressed as relative binding affinities (IC50 of FVIII /IC50 of the indicated substituted peptide) ± SE. The IC50 of FVIII was 0.34 ± 0.02 μM. (B) Peptides were also tested for presentation on irradiated DRB1*01:01 PBMCs to the T-cell clone 32A-18 by measuring [3H]thymidine incorporation. Results are expressed as relative T-cell proliferation (EC50 of FVIII /EC50 of the indicated substituted peptide) ± SE. The EC50 of FVIII in the proliferation assay was 8.9 ± 0.7 μM. The asterisk (*) indicates that, for those peptides, the relative proliferation was <0.09. (C) FVIII and FV protein sequences aligning with human FVIII residues from different mammalian species were collected from the NCBI reference sequence database including FVIII Homo sapiens, NP_ ; FVIII Canis lupus familiaris, NP_ ; FVIII Bos taurus, NP_ ; FVIII Mus musculus, NP_ ; FVIII Sus scrofa, NP_ ; FVIII Rattus norvegicus, NP_ ; FV M musculus, NP_ ; FV H sapiens, NP_ ; and FV S scrofa, NP_ Amino acids homologous/orthologous to anchor residues FVIII-F2196 and M2199 are highlighted with gray shading. NB, no detectable binding at 50 μM peptide; NP, no detectable proliferation even at the highest peptide concentration, 100 μM.
Ruth A. Ettinger et al. Blood Adv 2018;2:
©2018 by American Society of Hematology

7. CD4+ T-cell clones did not proliferate when stimulated with rFVIII-C2-F2196A.
CD4+T-cell clones did not proliferate when stimulated with rFVIII-C2-F2196A. T-cell clones 32A-18 (A), 17A-19WK-11 (B), 17A-21MO-5 (C), and 17A-21MO-11 (D) were stimulated with purified recombinant rWT-FVIII-C2 and rFVIII-C2-F2196A presented by irradiated PBMCs from an DRB1*01:01 donor. Proteins were added at final concentrations of 1, 10, 50, 100, 500, and 1000 nM. T-cell proliferation was measured by [3H]thymidine incorporation. Results are averages of triplicate determinations ± standard deviation (SD). Stimulation indices were calculated by dividing the mean cpm of [3H]thymidine incorporated into protein-stimulated cells by the mean cpm of [3H]thymidine incorporated into unstimulated cells. Results are also plotted as proliferation (cpm) in supplemental Figure 9.
Ruth A. Ettinger et al. Blood Adv 2018;2:
©2018 by American Society of Hematology

8. Markedly reduced proliferation of CD4+ T-cell clones and polyclonal lines to BDD-FVIII-F2196K and BDD-FVIII-M2199A.
Markedly reduced proliferation of CD4+T-cell clones and polyclonal lines to BDD-FVIII-F2196K and BDD-FVIII-M2199A. FVIII specific T-cell clones 32A-18 (A), 17A-19WK-11 (B), 17A-21MO-5 (C), and polyclonal lines 17A-5YR-L1 (D) and 56A-L2 (E) were stimulated with purified recombinant WT-BDD-FVIII, BDD-FVIII-F2196K, and BDD-FVIII-M2199A presented by iMo-DCs. The iMo-DCs were differentiated from CD14+ cells isolated from an DRB1*01:01 donor and pulsed with FVIII proteins at final concentrations of 1.6, 3.1, 6.2, 12.5, 25, and 50 nM. CD4+ T-cell proliferation was measured by [3H]thymidine incorporation. Stimulation indices were calculated by dividing the cpm of [3H]thymidine incorporated into protein-stimulated cells by the cpm of [3H]thymidine incorporated into unstimulated cells. Results are also plotted as proliferation (cpm) in supplemental Figure 10.
Ruth A. Ettinger et al. Blood Adv 2018;2:
©2018 by American Society of Hematology