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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.

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Immunogenicity of the wild-type and enhanced POTE323 epitopes and anti-tumor cytotoxicity induced by enhanced epitopes.AAD mice were immunized s.c. with 50 nmol of peptide in 100 µl of emulsion as described in the Methods section. (A) Direct ex vivo IFN-γ ELISPOT assay using target cells pulsed with 1000 nM peptide. Figures show numbers of spots per million cells. (B) CTL cross-reactivity on POTE 323 and POTE 323-3F peptides. (C) Anti-tumor cytotoxicity against a POTE-expressing human lung cancer cell line NCI-H522 induced by POTE 553-1Y, 323-3F and 252-9V. HHD-2 mice were immunized with 50 nmol of the peptide indicated in 100 µl of emulsion and restimulated for 7 days with 1000 nM peptide before being tested for ability to lyse the tumor cells. (D) Summary of the anti-tumor cytotoxicity in POTE-expressing and non-POTE-expressing human tumor cells. NCI-H522 is a human lung cancer line expressing both POTE and HLA-A2, whereas HTB-19 is a human mammary carcinoma that expresses POTE but not HLA-A2, and MDA-MB-231 is a human mammary carcinoma cell line expressing HLA-A2 but not POTE. Killing of only the first of these shows the specificity for POTE in combination with HLA-A2. (Negative values are due to experimental 51Cr release slightly below spontaneous release with no effector cells, within experimental error, and so should be viewed as equivalent to zero).
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pone-0064365-g004: Immunogenicity of the wild-type and enhanced POTE323 epitopes and anti-tumor cytotoxicity induced by enhanced epitopes.AAD mice were immunized s.c. with 50 nmol of peptide in 100 µl of emulsion as described in the Methods section. (A) Direct ex vivo IFN-γ ELISPOT assay using target cells pulsed with 1000 nM peptide. Figures show numbers of spots per million cells. (B) CTL cross-reactivity on POTE 323 and POTE 323-3F peptides. (C) Anti-tumor cytotoxicity against a POTE-expressing human lung cancer cell line NCI-H522 induced by POTE 553-1Y, 323-3F and 252-9V. HHD-2 mice were immunized with 50 nmol of the peptide indicated in 100 µl of emulsion and restimulated for 7 days with 1000 nM peptide before being tested for ability to lyse the tumor cells. (D) Summary of the anti-tumor cytotoxicity in POTE-expressing and non-POTE-expressing human tumor cells. NCI-H522 is a human lung cancer line expressing both POTE and HLA-A2, whereas HTB-19 is a human mammary carcinoma that expresses POTE but not HLA-A2, and MDA-MB-231 is a human mammary carcinoma cell line expressing HLA-A2 but not POTE. Killing of only the first of these shows the specificity for POTE in combination with HLA-A2. (Negative values are due to experimental 51Cr release slightly below spontaneous release with no effector cells, within experimental error, and so should be viewed as equivalent to zero).

Mentions: POTE 323-3F was the only modified peptide with better HLA-A2 binding affinity than wild type POTE 323 (Figure 1D). As shown in Figure 4A, both POTE 323 and POTE 323-3F induced some IFN-γ responses after stimulation with the identical peptide. Even though the POTE 323 peptide was the best HLA-A2 binder within the POTE sequence, only marginal IFN-γ responses (less than 2-fold over background level) were detected by the ELISPOT assay. After one week in vitro stimulation, CTLs induced by POTE 323 failed to lyse target cells pulsed with POTE 323. This negative CTL response might be due to the loss of a substantial portion of responding cells with high avidity that could have been killed during the incubation with a relative high concentration of peptide (1.0 µM). POTE 323-3F induced significant IFN-γ spots under POTE 323-3F stimulation, and some degree of IFN-γ response was also detected after stimulation with wild type POTE 323 epitope. After in vitro stimulation with the cognate peptide, CD8+ CTLs induced with the enhanced epitope POTE 323-3F lysed target cells pulsed with the POTE 323-3F, as well as wild type POTE 323, suggesting that the TCRs of the CTLs could recognize the wild type peptide (POTE 323)/MHC class I complex (Figure 4B). POTE 323-3F was more immunogenic in inducing CTLs specific for the wild type epitope than was the wild type epitope itself. Therefore, POTE 323-3F might be an excellent vaccine candidate to target tumor cells expressing POTE.


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 POTE323 epitopes and anti-tumor cytotoxicity induced by enhanced epitopes.AAD mice were immunized s.c. with 50 nmol of peptide in 100 µl of emulsion as described in the Methods section. (A) Direct ex vivo IFN-γ ELISPOT assay using target cells pulsed with 1000 nM peptide. Figures show numbers of spots per million cells. (B) CTL cross-reactivity on POTE 323 and POTE 323-3F peptides. (C) Anti-tumor cytotoxicity against a POTE-expressing human lung cancer cell line NCI-H522 induced by POTE 553-1Y, 323-3F and 252-9V. HHD-2 mice were immunized with 50 nmol of the peptide indicated in 100 µl of emulsion and restimulated for 7 days with 1000 nM peptide before being tested for ability to lyse the tumor cells. (D) Summary of the anti-tumor cytotoxicity in POTE-expressing and non-POTE-expressing human tumor cells. NCI-H522 is a human lung cancer line expressing both POTE and HLA-A2, whereas HTB-19 is a human mammary carcinoma that expresses POTE but not HLA-A2, and MDA-MB-231 is a human mammary carcinoma cell line expressing HLA-A2 but not POTE. Killing of only the first of these shows the specificity for POTE in combination with HLA-A2. (Negative values are due to experimental 51Cr release slightly below spontaneous release with no effector cells, within experimental error, and so should be viewed as equivalent to zero).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672105&req=5

pone-0064365-g004: Immunogenicity of the wild-type and enhanced POTE323 epitopes and anti-tumor cytotoxicity induced by enhanced epitopes.AAD mice were immunized s.c. with 50 nmol of peptide in 100 µl of emulsion as described in the Methods section. (A) Direct ex vivo IFN-γ ELISPOT assay using target cells pulsed with 1000 nM peptide. Figures show numbers of spots per million cells. (B) CTL cross-reactivity on POTE 323 and POTE 323-3F peptides. (C) Anti-tumor cytotoxicity against a POTE-expressing human lung cancer cell line NCI-H522 induced by POTE 553-1Y, 323-3F and 252-9V. HHD-2 mice were immunized with 50 nmol of the peptide indicated in 100 µl of emulsion and restimulated for 7 days with 1000 nM peptide before being tested for ability to lyse the tumor cells. (D) Summary of the anti-tumor cytotoxicity in POTE-expressing and non-POTE-expressing human tumor cells. NCI-H522 is a human lung cancer line expressing both POTE and HLA-A2, whereas HTB-19 is a human mammary carcinoma that expresses POTE but not HLA-A2, and MDA-MB-231 is a human mammary carcinoma cell line expressing HLA-A2 but not POTE. Killing of only the first of these shows the specificity for POTE in combination with HLA-A2. (Negative values are due to experimental 51Cr release slightly below spontaneous release with no effector cells, within experimental error, and so should be viewed as equivalent to zero).
Mentions: POTE 323-3F was the only modified peptide with better HLA-A2 binding affinity than wild type POTE 323 (Figure 1D). As shown in Figure 4A, both POTE 323 and POTE 323-3F induced some IFN-γ responses after stimulation with the identical peptide. Even though the POTE 323 peptide was the best HLA-A2 binder within the POTE sequence, only marginal IFN-γ responses (less than 2-fold over background level) were detected by the ELISPOT assay. After one week in vitro stimulation, CTLs induced by POTE 323 failed to lyse target cells pulsed with POTE 323. This negative CTL response might be due to the loss of a substantial portion of responding cells with high avidity that could have been killed during the incubation with a relative high concentration of peptide (1.0 µM). POTE 323-3F induced significant IFN-γ spots under POTE 323-3F stimulation, and some degree of IFN-γ response was also detected after stimulation with wild type POTE 323 epitope. After in vitro stimulation with the cognate peptide, CD8+ CTLs induced with the enhanced epitope POTE 323-3F lysed target cells pulsed with the POTE 323-3F, as well as wild type POTE 323, suggesting that the TCRs of the CTLs could recognize the wild type peptide (POTE 323)/MHC class I complex (Figure 4B). POTE 323-3F was more immunogenic in inducing CTLs specific for the wild type epitope than was the wild type epitope itself. Therefore, POTE 323-3F might be an excellent vaccine candidate to target tumor cells expressing POTE.

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