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 553 epitopes in AAD mice.AAD mice were immunized s.c. with a mixture of peptide and cytokines in adjuvant described in the Methods section. (A,B) Two weeks after the second boost, splenocytes pooled from three mice were restimulated with splenocytes of naïve AAD mice pulsed with 10 nM to 1000 nM of each peptide at different E/T ratios (1000 nM was used in panel A). Spots were counted by an ELISPOT reader (AID ELISPOT reader system). Figures show numbers of spots per million cells. (C) CTL cross-reactivity on each peptide. In a 4 hour 51Cr release assay, CIR.AAD cells were pulsed with 1.0 µM each peptide and labeled with 51Cr. After washing three times, target cells were mixed with different numbers of effector cells and then cultured for 4 hours before harvesting.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672105&req=5

pone-0064365-g003: Immunogenicity of the wild type and enhanced POTE 553 epitopes in AAD mice.AAD mice were immunized s.c. with a mixture of peptide and cytokines in adjuvant described in the Methods section. (A,B) Two weeks after the second boost, splenocytes pooled from three mice were restimulated with splenocytes of naïve AAD mice pulsed with 10 nM to 1000 nM of each peptide at different E/T ratios (1000 nM was used in panel A). Spots were counted by an ELISPOT reader (AID ELISPOT reader system). Figures show numbers of spots per million cells. (C) CTL cross-reactivity on each peptide. In a 4 hour 51Cr release assay, CIR.AAD cells were pulsed with 1.0 µM each peptide and labeled with 51Cr. After washing three times, target cells were mixed with different numbers of effector cells and then cultured for 4 hours before harvesting.

Mentions: All the substituted POTE 553 peptides showed better binding affinity to HLA-A2 molecules than the POTE 553 (Figure 1C). We chose the two best binders among these enhanced peptides, along with the wild type peptide, to test the immunogenicities and cross-reactive CTL responses. As shown in Figure 3A and B, POTE 553 did not induce any significant IFN-γ responses at any peptide concentration used for stimulation. After one week in vitro stimulation, there was likewise not any significant detectable target cell lysis (Figure 3C). Therefore POTE 553, as an intermediate HLA-A2 binder, is not immunogenic. Both POTE 553-1Y and POTE 553-2L induced some level of IFN-γ responses only after stimulation with the same peptide (Figure 3A, B). In Figure 3B, the target cells were pulsed with different concentrations of peptide to demonstrate that the enhanced peptide could induce high avidity T cells to kill target cells in the presence of low antigen concentrations. According to the ex vivo IFN-γ ELISPOT assay, no cross-reactive response to wild type peptide was detected in the CTLs induced by POTE 553-1Y or POTE 553-2L peptides. However, after one week in vitro stimulation, CD8+ CTLs induced with the enhanced epitope POTE 553-1Y lysed target cells pulsed with not only POTE 553-1Y, but also POTE 553 and POTE 553-2L peptides, suggesting that the TCRs of the CTLs recognized the POTE 553/MHC class I complex to some extent (Figure 3C). From this point of view, POTE-553-1Y might be a possible alternative cancer vaccine against the POTE antigen. Although POTE 553-2L can induce certain IFN-γ responses after stimulation with the same peptide (Figure 3A, B), CD8+ CTLs induced with POTE 553-2L did not lyse target cells pulsed with POTE 553-2L (Figure 3C).


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 553 epitopes in AAD mice.AAD mice were immunized s.c. with a mixture of peptide and cytokines in adjuvant described in the Methods section. (A,B) Two weeks after the second boost, splenocytes pooled from three mice were restimulated with splenocytes of naïve AAD mice pulsed with 10 nM to 1000 nM of each peptide at different E/T ratios (1000 nM was used in panel A). Spots were counted by an ELISPOT reader (AID ELISPOT reader system). Figures show numbers of spots per million cells. (C) CTL cross-reactivity on each peptide. In a 4 hour 51Cr release assay, CIR.AAD cells were pulsed with 1.0 µM each peptide and labeled with 51Cr. After washing three times, target cells were mixed with different numbers of effector cells and then cultured for 4 hours before harvesting.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0064365-g003: Immunogenicity of the wild type and enhanced POTE 553 epitopes in AAD mice.AAD mice were immunized s.c. with a mixture of peptide and cytokines in adjuvant described in the Methods section. (A,B) Two weeks after the second boost, splenocytes pooled from three mice were restimulated with splenocytes of naïve AAD mice pulsed with 10 nM to 1000 nM of each peptide at different E/T ratios (1000 nM was used in panel A). Spots were counted by an ELISPOT reader (AID ELISPOT reader system). Figures show numbers of spots per million cells. (C) CTL cross-reactivity on each peptide. In a 4 hour 51Cr release assay, CIR.AAD cells were pulsed with 1.0 µM each peptide and labeled with 51Cr. After washing three times, target cells were mixed with different numbers of effector cells and then cultured for 4 hours before harvesting.
Mentions: All the substituted POTE 553 peptides showed better binding affinity to HLA-A2 molecules than the POTE 553 (Figure 1C). We chose the two best binders among these enhanced peptides, along with the wild type peptide, to test the immunogenicities and cross-reactive CTL responses. As shown in Figure 3A and B, POTE 553 did not induce any significant IFN-γ responses at any peptide concentration used for stimulation. After one week in vitro stimulation, there was likewise not any significant detectable target cell lysis (Figure 3C). Therefore POTE 553, as an intermediate HLA-A2 binder, is not immunogenic. Both POTE 553-1Y and POTE 553-2L induced some level of IFN-γ responses only after stimulation with the same peptide (Figure 3A, B). In Figure 3B, the target cells were pulsed with different concentrations of peptide to demonstrate that the enhanced peptide could induce high avidity T cells to kill target cells in the presence of low antigen concentrations. According to the ex vivo IFN-γ ELISPOT assay, no cross-reactive response to wild type peptide was detected in the CTLs induced by POTE 553-1Y or POTE 553-2L peptides. However, after one week in vitro stimulation, CD8+ CTLs induced with the enhanced epitope POTE 553-1Y lysed target cells pulsed with not only POTE 553-1Y, but also POTE 553 and POTE 553-2L peptides, suggesting that the TCRs of the CTLs recognized the POTE 553/MHC class I complex to some extent (Figure 3C). From this point of view, POTE-553-1Y might be a possible alternative cancer vaccine against the POTE antigen. Although POTE 553-2L can induce certain IFN-γ responses after stimulation with the same peptide (Figure 3A, B), CD8+ CTLs induced with POTE 553-2L did not lyse target cells pulsed with POTE 553-2L (Figure 3C).

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