Immunogenic wt-1 peptides and methods of use thereof
A technology for immune regulation and vaccines, applied in chemical instruments and methods, peptides, specific peptides, etc., can solve the unsatisfied problems of WT1-derived peptides
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Embodiment 1
[0278] Example 1. Materials and methods
[0279] Peptide design. Using three computer-based predictive algorithms BIMAS (http: / / www-bimas.cit.nih.gov / cgi-bin / molbio / ken_parker_combo form), SYFPEITHI (http: / / www.syfpeithi.de / ) and RANKPEP (http: / / bio.dfci.harvard.edu / Tools / rankpep.html), select epitopes against CD8 and CD4 T cells by starting with the native WT1 protein sequence capable of inducing an immune response in normal donors. Metamorphic peptides were designed by changing a single amino acid in the anchor residue of a class I native peptide, which resulted in higher predicted binding than its native sequence. Class II peptides were designed to simultaneously stimulate CD4 and CD8 T cells by adding flanking residues to class I peptides. While many sequences can be predicted by the algorithms, in 30% of cases these models do not predict binding to MHC when tested on hepatocytes (Gomez-Nunez et al., Leuk Res. 2006;30 (10):1293-8), so in vitro testing is necessary. In a...
Embodiment 2
[0286] Example 2. Binding of natural and analog peptides to HLA-A0201 and HLA-A2402
[0287] The use of pools of 15mer overlapping peptides spanning the human WT1 protein to sensitize human T cells in vitro, in the case of HLA-A2402, the sequence 239-248 (NQMNLGATL; SEQ ID NO:5; abbreviated herein as NQM or ) has recently been described. Identified as an immunogenic CD8 T cell epitope (Doubrovina et al., Blood 2012;123(8):1633-46). To generate similar peptides that are more immunogenic, native peptides and those with various amino acid substitutions in positions 2 and 9 (class I anchor residues) were screened using three online available databases (BIMAS, RANKPEP, and SYFPEITHI). Predictive scores for possible analogs. Predicted binding scores from all three databases showed that the native NQMNLGATL (SEQ ID NO:5) peptide bound better to HLA-A0201 than to the HLA-A2402 molecule (Table I). When glutamine at position 2 was replaced by leucine, binding scores to HLA-A2402 remai...
Embodiment 3
[0291] Example 3. Binding of peptides to HLA-A0201 and HLA-A2402 molecules
[0292] Immunogenicity of MHC class I-restricted peptides requires the ability to bind and stabilize MHC class I molecules on the surface of living cells. Furthermore, in silico predictions are only at best 70% accurate; therefore, the interaction between peptides and HLA-A0201 molecules is limited by conventional binding and stabilization assays using antigen transport deficient (TAP2-negative) HLA-A0201 human T2 cells. The strength of seeks a direct measurement. T2 cells lack TAP function and are therefore defective in proper loading of class I molecules with antigenic peptides produced in the cytosol. Association of exogenously added peptides with thermolabile empty HLA-A0201 molecules stabilizes them and leads to increased levels of surface HLA-A0201 that can be recognized by specific anti-HLA-A0201 mAbs such as BB7.2.
[0293] T2 binding assays showed that the native NQMNLGATL (SEQ ID NO:5) pept...
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