Table 1.
Identification of glypican-3 (GPC3)-derived and promiscuous HLA class II-restricted CD4+ T-cell epitopes encompassing cytotoxic T lymphocyte (CTL) epitopes
| Designation of long peptide (LP) | a.a. residue position | Sequence | a.a. length | T-cell donor | aDonors' MHC class II | Immune response | Restriction MHC class II molecule | Natural processingb |
|---|---|---|---|---|---|---|---|---|
| GPC3-LP1 | 92-116 | LLQSASMELKFLIIQNAAVFQEAFE | 25 | HD3 | DRB1*08:02/15:02 | negative | ||
| HD10 | DRB1*07:01/13:02 | positive | DR52b (DRB3*02:02) (L) | positive in vitro | ||||
| HD5 | DRB1*04:05/09:01 | positive | DR9 (DRB1*09:01) (L) | n.t. | ||||
| GPC3-LP2 | 137-161 | LTPQAFEFVGEFFTDVSLYILGSDI | 25 | HD3 | DRB1*08:02/15:02 | positive | ||
| cCTL epitope:A2-GPC3144-152 | HD10 | DRB1*07:01/13:02 | positive | DR52b (DRB3*02:02) (L, allo) | positive in vitro | |||
| HD5 | DPB1*02:01/04:02 | positive | DP2 (DPB1*02:01) (L, allo) | n.t. | ||||
| HD4 | DRB1*08:03/14:05 | positive | DR8 (DRB1*08:03) (L, allo) | n.t. | ||||
| HD11 | DRB1*09:01/14:54 | positive | n.t. | |||||
| dTgm | I-Ab | positive | I-Ab | n.t. | ||||
| GPC3-LP3 | 289-313 | VVEIDKYWREYILSLEELVNGMYRI | 25 | HD3 | DRB1*08:02/15:02 | negative | ||
| cCTL epitope:A24-GPC3298-306 | HD10 | DRB1*07:01/13:02 | positive | n.t. | negative in vitropositive in vivo (in patient) | |||
| HD5 | DRB1*04:05/09:01 | positive | DR9 (DRB1*09:01)(L) | |||||
| HD4 | DRB1*08:03/14:05 | negative | ||||||
| HD11 | DRB1*09:01/14:54 | positive | n.t. | |||||
| GPC3-LP4 | 386-412 | SRRRELIQKLKSFISFYSALPGYICSH | 27 | HD3 | DRB1*08:02/15:02 | positive | DR15 (DRB1*15:02) (allo) | n.t. |
| HD10 | DRB1*07:01/13:02 | positive | DR13 (DRB1*13:02) (L) | positive in vitro | ||||
| HD5 | DRB1*04:05/09:01 | negative | ||||||
| GPC3-LP5 | 556-576 | GNVHSPLKLLTSMAISVVCFF | 21 | HD3 | DRB1*08:02/15:02 | negative | ||
| HD10 | DRB1*07:01/13:02 | positive | DR13 (DRB1*13:02) (L) | positive in vitro | ||||
| HD5 | DRB1*04:05/09:01 | positive | DR9 (DRB1*09:01) (L) | n.t. |
We tested the immunogenicity of GPC3-derived long peptides in these healthy donors and dHLA-A2 transgenic mice with C57BL/6 background (Tgm); details of donors' HLA alleles are shown in Supplementary Table 1.
An in vitro immune response of T helper (Th) cells to dendritic cells pulsed with GPC3 proteins; in vivo, a long peptide (LP)-specific Th cell response was observed in patients with hepatocellular carcinoma.
Underlined and bold sequences are CTL epitopes;9 GPC3-LP1,4,5 sequences were selected due to high-affinity binding to HLA class II molecules predicted by the algorithm; GPC3-LP2 and GPC3-LP3 were selected due to both the prediction of HLA class II binding and the proximity to the known CTL epitopes. a.a.: amino acid, negative: we could not obtain positive data and did not proceed further, n.t.: not tested, LP: long peptide, SP: short peptide, HD: healthy donor, L: restriction HLA class II molecules were confirmed using L-cell lines expressing single allelic product of HLA class II molecules, allo: confirmed by means of allogeneic peripheral blood mononuclear cells (PBMCs) in which at least one of the HLA class II alleles was shared with the donors.