Limits...
Identification and enhancement of HLA-A2.1-restricted CTL epitopes in a new human cancer antigen-POTE.

Huang YH, Terabe M, Pendleton CD, Stewart Khursigara D, Bera TK, Pastan I, Berzofsky JA - PLoS ONE (2013)

Bottom Line: POTE protein is a newly identified cancer antigen that was found to be expressed in a wide variety of human cancers, including prostate, colon, lung, breast, ovary and pancreas.While POTE 252-9V was the strongest in this respect, POTE 323-3F had the greatest increase in immunogenicity compared to wild type.In conclusion, the immunogenicity of POTE epitopes can be enhanced by peptide modification to induce T cells that kill human cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, Infection and Immunity Research Center, National Yang-Ming University, Division of Gastroenterology, Taipei Veterans General Hospital, Taipei, Taiwan.

ABSTRACT
Identification of CD8(+) T cell epitopes that can induce T cells to kill tumor cells is a fundamental step for development of a peptide cancer vaccine. POTE protein is a newly identified cancer antigen that was found to be expressed in a wide variety of human cancers, including prostate, colon, lung, breast, ovary and pancreas. Here, we determined HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) epitopes in the POTE protein, and also designed enhanced epitopes by amino acid (AA) substitutions. Five 9-mer peptides were first selected and their binding affinity to HLA-A2 molecules was measured by the T2 binding assay. POTE 272-280 and POTE 323-331 showed the strongest HLA-A2 binding affinity. AA substituted peptides POTE 252-9V (with valine at position 9), POTE 553-1Y (with tyrosine at position 1) and POTE 323-3F (with phenylalanine at position 3) conferred higher affinity for HLA-A2, and induced CTL responses cross-reactive with wild type antigens. While POTE 252-9V was the strongest in this respect, POTE 323-3F had the greatest increase in immunogenicity compared to wild type. Importantly, two modified epitopes (POTE-553-1Y and POTE-323-3F) induced CTLs that killed NCI-H522, a POTE-expressing HLA-A2(+) human non-small cell lung cancer cell line, indicating natural endogenous processing of these epitopes. In conclusion, the immunogenicity of POTE epitopes can be enhanced by peptide modification to induce T cells that kill human cancer cells. A combination of POTE 553-1Y and POTE 323-3F epitopes might be an attractive vaccine strategy for HLA-A2 cancer patients to overcome tolerance induced by tumors and prevent escape.

Show MeSH

Related in: MedlinePlus

Immunogenicity of the wild type and enhanced POTE 252 epitopes in AAD mice.(A) Mice were immunized with 50 nmol of POTE 252 in 100 µl of emulsion, and targets were pulsed with 1.0 µM POTE252. Spots were counted by an ELISPOT reader (AID ELISPOT reader system). Figures show numbers of spots per million cells. (B) CTL cross-reactivity on each peptide. Two weeks after the second boost, pooled spleen cells from three mice were restimulated with irradiated splenocytes pulsed with 1.0 µM each peptide for 7 days. Then a 4 hour 51Cr release assay was performed. (C) HLA-A2/peptide tetramer staining and (D) TCR repertoire determination for POTE 252- and POTE 252-9V-induced CTLs in HHD-2 mice.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672105&req=5

pone-0064365-g002: Immunogenicity of the wild type and enhanced POTE 252 epitopes in AAD mice.(A) Mice were immunized with 50 nmol of POTE 252 in 100 µl of emulsion, and targets were pulsed with 1.0 µM POTE252. Spots were counted by an ELISPOT reader (AID ELISPOT reader system). Figures show numbers of spots per million cells. (B) CTL cross-reactivity on each peptide. Two weeks after the second boost, pooled spleen cells from three mice were restimulated with irradiated splenocytes pulsed with 1.0 µM each peptide for 7 days. Then a 4 hour 51Cr release assay was performed. (C) HLA-A2/peptide tetramer staining and (D) TCR repertoire determination for POTE 252- and POTE 252-9V-induced CTLs in HHD-2 mice.

Mentions: Both POTE 252-9V and POTE 252-1Y had better binding affinity to HLA-A2 molecules than the POTE 252 (Figure 1B). As shown in Figure 2A, both POTE 252 and POTE 252-9V induced a significant number of IFN-γ spots with cross-reactivity to both POTE 252 and POTE 252-9V. Although POTE 252-1Y had a better binding affinity to HLA-A2 molecules than POTE 252, no significant IFN-γ responses were detected in the assay even in the presence of POTE 252-1Y peptide. After 1-week of in vitro stimulation with cognate peptide, CD8+ CTLs induced with both wild type POTE 252 and enhanced epitope POTE 252-9V lysed target cells pulsed with the wild type POTE 252 as well as POTE 252-9V. This result suggested that the TCRs of CTLs generated in mice immunized with either wild type or enhanced peptide could recognize the wild type peptide (POTE 252)/MHC class I complex (Figure 2B).


Identification and enhancement of HLA-A2.1-restricted CTL epitopes in a new human cancer antigen-POTE.

Huang YH, Terabe M, Pendleton CD, Stewart Khursigara D, Bera TK, Pastan I, Berzofsky JA - PLoS ONE (2013)

Immunogenicity of the wild type and enhanced POTE 252 epitopes in AAD mice.(A) Mice were immunized with 50 nmol of POTE 252 in 100 µl of emulsion, and targets were pulsed with 1.0 µM POTE252. Spots were counted by an ELISPOT reader (AID ELISPOT reader system). Figures show numbers of spots per million cells. (B) CTL cross-reactivity on each peptide. Two weeks after the second boost, pooled spleen cells from three mice were restimulated with irradiated splenocytes pulsed with 1.0 µM each peptide for 7 days. Then a 4 hour 51Cr release assay was performed. (C) HLA-A2/peptide tetramer staining and (D) TCR repertoire determination for POTE 252- and POTE 252-9V-induced CTLs in HHD-2 mice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672105&req=5

pone-0064365-g002: Immunogenicity of the wild type and enhanced POTE 252 epitopes in AAD mice.(A) Mice were immunized with 50 nmol of POTE 252 in 100 µl of emulsion, and targets were pulsed with 1.0 µM POTE252. Spots were counted by an ELISPOT reader (AID ELISPOT reader system). Figures show numbers of spots per million cells. (B) CTL cross-reactivity on each peptide. Two weeks after the second boost, pooled spleen cells from three mice were restimulated with irradiated splenocytes pulsed with 1.0 µM each peptide for 7 days. Then a 4 hour 51Cr release assay was performed. (C) HLA-A2/peptide tetramer staining and (D) TCR repertoire determination for POTE 252- and POTE 252-9V-induced CTLs in HHD-2 mice.
Mentions: Both POTE 252-9V and POTE 252-1Y had better binding affinity to HLA-A2 molecules than the POTE 252 (Figure 1B). As shown in Figure 2A, both POTE 252 and POTE 252-9V induced a significant number of IFN-γ spots with cross-reactivity to both POTE 252 and POTE 252-9V. Although POTE 252-1Y had a better binding affinity to HLA-A2 molecules than POTE 252, no significant IFN-γ responses were detected in the assay even in the presence of POTE 252-1Y peptide. After 1-week of in vitro stimulation with cognate peptide, CD8+ CTLs induced with both wild type POTE 252 and enhanced epitope POTE 252-9V lysed target cells pulsed with the wild type POTE 252 as well as POTE 252-9V. This result suggested that the TCRs of CTLs generated in mice immunized with either wild type or enhanced peptide could recognize the wild type peptide (POTE 252)/MHC class I complex (Figure 2B).

Bottom Line: POTE protein is a newly identified cancer antigen that was found to be expressed in a wide variety of human cancers, including prostate, colon, lung, breast, ovary and pancreas.While POTE 252-9V was the strongest in this respect, POTE 323-3F had the greatest increase in immunogenicity compared to wild type.In conclusion, the immunogenicity of POTE epitopes can be enhanced by peptide modification to induce T cells that kill human cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, Infection and Immunity Research Center, National Yang-Ming University, Division of Gastroenterology, Taipei Veterans General Hospital, Taipei, Taiwan.

ABSTRACT
Identification of CD8(+) T cell epitopes that can induce T cells to kill tumor cells is a fundamental step for development of a peptide cancer vaccine. POTE protein is a newly identified cancer antigen that was found to be expressed in a wide variety of human cancers, including prostate, colon, lung, breast, ovary and pancreas. Here, we determined HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) epitopes in the POTE protein, and also designed enhanced epitopes by amino acid (AA) substitutions. Five 9-mer peptides were first selected and their binding affinity to HLA-A2 molecules was measured by the T2 binding assay. POTE 272-280 and POTE 323-331 showed the strongest HLA-A2 binding affinity. AA substituted peptides POTE 252-9V (with valine at position 9), POTE 553-1Y (with tyrosine at position 1) and POTE 323-3F (with phenylalanine at position 3) conferred higher affinity for HLA-A2, and induced CTL responses cross-reactive with wild type antigens. While POTE 252-9V was the strongest in this respect, POTE 323-3F had the greatest increase in immunogenicity compared to wild type. Importantly, two modified epitopes (POTE-553-1Y and POTE-323-3F) induced CTLs that killed NCI-H522, a POTE-expressing HLA-A2(+) human non-small cell lung cancer cell line, indicating natural endogenous processing of these epitopes. In conclusion, the immunogenicity of POTE epitopes can be enhanced by peptide modification to induce T cells that kill human cancer cells. A combination of POTE 553-1Y and POTE 323-3F epitopes might be an attractive vaccine strategy for HLA-A2 cancer patients to overcome tolerance induced by tumors and prevent escape.

Show MeSH
Related in: MedlinePlus