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Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.

Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J, Arthur CD, Krebber WJ, Mulder GE, Toebes M, Vesely MD, Lam SS, Korman AJ, Allison JP, Freeman GJ, Sharpe AH, Pearce EL, Schumacher TN, Aebersold R, Rammensee HG, Melief CJ, Mardis ER, Gillanders WE, Artyomov MN, Schreiber RD - Nature (2014)

Bottom Line: Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression.Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies.These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, Missouri 63110, USA.

ABSTRACT
The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

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Mutant Lama4 and mAlg8 are therapeutically relevant d42m1-T3 TSMAa, Detection of mLama4 and mAlg8 bound to cellular H-2Kb by mass spectrometry. b, Time dependent tumour infiltration of mLama4- and mAlg8-specific CD8+ T cells (n=5), (top). Data represent means ± s.e.m of 5 independent experiments. Growth kinetics of d42m1-T3 and F244 during αPD-1 immunotherapy (n=5), (bottom). Data represent average tumour diameter ± s.e.m. and are representative of at least three independent experiments. c, IFN-γ ELISPOT analysis of peptide stimulated splenocytes from mice immunized with mLama4 or mAlg8 SLP plus polyI:C (n=3 mice per group). Data are means ± s.e.m. Representative of two independent experiments. Samples were compared using unpaired, two-tailed Student’s t test (*p<0.05, **p<0.01). d, Kaplan-Meier survival curves of d42m1-T3 tumour bearing mice (10 mice per group) therapeutically vaccinated with SLP vaccines plus poly I:C. mLama4 plus mAlg8 compared to HPV control: p=0.0002 [log-rank (Mantel-Cox) test]. Representative of two independent experiments. e, Cumulative data from two independent SLP therapeutic vaccine experiments using mice (7–10 per group) with d42m1-T3 or F244 tumours.
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Figure 2: Mutant Lama4 and mAlg8 are therapeutically relevant d42m1-T3 TSMAa, Detection of mLama4 and mAlg8 bound to cellular H-2Kb by mass spectrometry. b, Time dependent tumour infiltration of mLama4- and mAlg8-specific CD8+ T cells (n=5), (top). Data represent means ± s.e.m of 5 independent experiments. Growth kinetics of d42m1-T3 and F244 during αPD-1 immunotherapy (n=5), (bottom). Data represent average tumour diameter ± s.e.m. and are representative of at least three independent experiments. c, IFN-γ ELISPOT analysis of peptide stimulated splenocytes from mice immunized with mLama4 or mAlg8 SLP plus polyI:C (n=3 mice per group). Data are means ± s.e.m. Representative of two independent experiments. Samples were compared using unpaired, two-tailed Student’s t test (*p<0.05, **p<0.01). d, Kaplan-Meier survival curves of d42m1-T3 tumour bearing mice (10 mice per group) therapeutically vaccinated with SLP vaccines plus poly I:C. mLama4 plus mAlg8 compared to HPV control: p=0.0002 [log-rank (Mantel-Cox) test]. Representative of two independent experiments. e, Cumulative data from two independent SLP therapeutic vaccine experiments using mice (7–10 per group) with d42m1-T3 or F244 tumours.

Mentions: Four subsequent findings supported the conclusion that mLama4 and mAlg8 were the relevant antigens responsible for αPD-1-induced rejection of d42m1-T3. First, mLama4 or mAlg8 epitopes stabilized H-2Kb expression on RMA-S cells, which lack a functional antigen transporter and thus fail to stably express MHC class I proteins on the cell surface (Extended Data Fig. 4b). Second, both epitopes were detected by mass spectrometry (MS) in eluates of affinity purified H-2Kb isolated from d42m1-T3 tumours. Using a discovery MS approach, we identified mLama4 in the H-2Kb eluate (Extended Data Fig. 5a) and verified its identity using an isotope-labelled synthetic mLama4 peptide (Extended Data Fig. 5b). We also found more than 200 WT peptides associated with H-2Kb (Supplementary Table 1), but we have no evidence that any of these function as d42m1-T3 antigens. Mutant Alg8, wtLama4 and wtAlg8 peptides were not detected (Supplementary Table 1). In contrast, using the more sensitive targeted selected reaction monitoring (SRM) MS method, both mLama4 and mAlg8 peptides were identified in the H-2Kb eluate (Fig. 2a, Extended Data Fig. 6a and Supplementary Data 1). Notably, mLama4 and mAlg8 were the only predicted strong-binding mutant epitopes found. Peptides from wtLama4 or wtAlg8 were not detected. Neither mLama4 nor mAlg8 were detected in H-2Kb eluates from F244 cells (Extended Data Fig. 6b). Third, as detected by staining with H-2Kb -mLama4 or -mAlg8 tetramers, CD8+ T cells specific for either antigen accumulated temporally in d42m1-T3 tumours of αPD-1 treated mice, reaching maximal values just prior to tumour rejection (Fig. 2b and Extended Data Fig. 7a). No mLama4- or mAlg8-tetramer positive TILs were observed in F244 sarcomas from αPD-1 treated mice. Fourth, the two H-2Kb-restricted epitopes induced antigen-specific CD8+ T cell responses in naïve mice when injected together with poly I:C (pIC) as assessed by ELISPOT (Fig. 2c).


Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.

Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J, Arthur CD, Krebber WJ, Mulder GE, Toebes M, Vesely MD, Lam SS, Korman AJ, Allison JP, Freeman GJ, Sharpe AH, Pearce EL, Schumacher TN, Aebersold R, Rammensee HG, Melief CJ, Mardis ER, Gillanders WE, Artyomov MN, Schreiber RD - Nature (2014)

Mutant Lama4 and mAlg8 are therapeutically relevant d42m1-T3 TSMAa, Detection of mLama4 and mAlg8 bound to cellular H-2Kb by mass spectrometry. b, Time dependent tumour infiltration of mLama4- and mAlg8-specific CD8+ T cells (n=5), (top). Data represent means ± s.e.m of 5 independent experiments. Growth kinetics of d42m1-T3 and F244 during αPD-1 immunotherapy (n=5), (bottom). Data represent average tumour diameter ± s.e.m. and are representative of at least three independent experiments. c, IFN-γ ELISPOT analysis of peptide stimulated splenocytes from mice immunized with mLama4 or mAlg8 SLP plus polyI:C (n=3 mice per group). Data are means ± s.e.m. Representative of two independent experiments. Samples were compared using unpaired, two-tailed Student’s t test (*p<0.05, **p<0.01). d, Kaplan-Meier survival curves of d42m1-T3 tumour bearing mice (10 mice per group) therapeutically vaccinated with SLP vaccines plus poly I:C. mLama4 plus mAlg8 compared to HPV control: p=0.0002 [log-rank (Mantel-Cox) test]. Representative of two independent experiments. e, Cumulative data from two independent SLP therapeutic vaccine experiments using mice (7–10 per group) with d42m1-T3 or F244 tumours.
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Figure 2: Mutant Lama4 and mAlg8 are therapeutically relevant d42m1-T3 TSMAa, Detection of mLama4 and mAlg8 bound to cellular H-2Kb by mass spectrometry. b, Time dependent tumour infiltration of mLama4- and mAlg8-specific CD8+ T cells (n=5), (top). Data represent means ± s.e.m of 5 independent experiments. Growth kinetics of d42m1-T3 and F244 during αPD-1 immunotherapy (n=5), (bottom). Data represent average tumour diameter ± s.e.m. and are representative of at least three independent experiments. c, IFN-γ ELISPOT analysis of peptide stimulated splenocytes from mice immunized with mLama4 or mAlg8 SLP plus polyI:C (n=3 mice per group). Data are means ± s.e.m. Representative of two independent experiments. Samples were compared using unpaired, two-tailed Student’s t test (*p<0.05, **p<0.01). d, Kaplan-Meier survival curves of d42m1-T3 tumour bearing mice (10 mice per group) therapeutically vaccinated with SLP vaccines plus poly I:C. mLama4 plus mAlg8 compared to HPV control: p=0.0002 [log-rank (Mantel-Cox) test]. Representative of two independent experiments. e, Cumulative data from two independent SLP therapeutic vaccine experiments using mice (7–10 per group) with d42m1-T3 or F244 tumours.
Mentions: Four subsequent findings supported the conclusion that mLama4 and mAlg8 were the relevant antigens responsible for αPD-1-induced rejection of d42m1-T3. First, mLama4 or mAlg8 epitopes stabilized H-2Kb expression on RMA-S cells, which lack a functional antigen transporter and thus fail to stably express MHC class I proteins on the cell surface (Extended Data Fig. 4b). Second, both epitopes were detected by mass spectrometry (MS) in eluates of affinity purified H-2Kb isolated from d42m1-T3 tumours. Using a discovery MS approach, we identified mLama4 in the H-2Kb eluate (Extended Data Fig. 5a) and verified its identity using an isotope-labelled synthetic mLama4 peptide (Extended Data Fig. 5b). We also found more than 200 WT peptides associated with H-2Kb (Supplementary Table 1), but we have no evidence that any of these function as d42m1-T3 antigens. Mutant Alg8, wtLama4 and wtAlg8 peptides were not detected (Supplementary Table 1). In contrast, using the more sensitive targeted selected reaction monitoring (SRM) MS method, both mLama4 and mAlg8 peptides were identified in the H-2Kb eluate (Fig. 2a, Extended Data Fig. 6a and Supplementary Data 1). Notably, mLama4 and mAlg8 were the only predicted strong-binding mutant epitopes found. Peptides from wtLama4 or wtAlg8 were not detected. Neither mLama4 nor mAlg8 were detected in H-2Kb eluates from F244 cells (Extended Data Fig. 6b). Third, as detected by staining with H-2Kb -mLama4 or -mAlg8 tetramers, CD8+ T cells specific for either antigen accumulated temporally in d42m1-T3 tumours of αPD-1 treated mice, reaching maximal values just prior to tumour rejection (Fig. 2b and Extended Data Fig. 7a). No mLama4- or mAlg8-tetramer positive TILs were observed in F244 sarcomas from αPD-1 treated mice. Fourth, the two H-2Kb-restricted epitopes induced antigen-specific CD8+ T cell responses in naïve mice when injected together with poly I:C (pIC) as assessed by ELISPOT (Fig. 2c).

Bottom Line: Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression.Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies.These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, Missouri 63110, USA.

ABSTRACT
The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

Show MeSH
Related in: MedlinePlus