Vaccination with a recombinant Saccharomyces cerevisiae expressing a tumor antigen breaks immune tolerance and elicits therapeutic antitumor responses

Abstract

Purpose: Saccharomyces cerevisiae, a nonpathogenic yeast, has been used previously as a vehicle to elicit immune responses to foreign antigens, and tumor-associated antigens, and has been shown to reduce tumor burden in mice. Studies were designed to determine if vaccination of human carcinoembryonic antigen (CEA)-transgenic (CEA-Tg) mice (where CEA is a self-antigen) with a recombinant S. cerevisiae construct expressing human CEA (yeast-CEA) elicits CEA-specific T-cell responses and antitumor activity.

Experimental design: CEA-Tg mice were vaccinated with yeast-CEA, and CD4(+) and CD8(+) T-cell responses were assessed after one and multiple administrations or vaccinations at multiple sites per administration. Antitumor activity was determined by tumor growth and overall survival in both pulmonary metastasis and s.c. pancreatic tumor models.

Results: These studies demonstrate that recombinant yeast can break tolerance and that (a) yeast-CEA constructs elicit both CEA-specific CD4(+) and CD8(+) T-cell responses; (b) repeated yeast-CEA administration causes increased antigen-specific T-cell responses after each vaccination; (c) vaccination with yeast-CEA at multiple sites induces a greater T-cell response than the same dose given at a single site; and (d) tumor-bearing mice vaccinated with yeast-CEA show a reduction in tumor burden and increased overall survival compared to mock-treated or control yeast-vaccinated mice in both pulmonary metastasis and s.c. pancreatic tumor models.

Conclusions: Vaccination with a heat-killed recombinant yeast expressing the tumor-associated antigen CEA induces CEA-specific immune responses, reduces tumor burden, and extends overall survival in CEA-Tg mice. These studies thus form the rationale for the incorporation of recombinant yeast-CEA and other recombinant yeast constructs in cancer immunotherapy protocols.

Fig. 1

Vaccination with yeast-CEA induces antigen-specific T-cell responses. CEA-Tg mice were vaccinated with 0.1 YU control yeast or yeast-CEA on days 0 and 7. On day 21, mice were sacrificed, spleens were harvested, and splenocytes were used for assays. A, CD4⁺ cell proliferation. Purified CD4⁺ T cells were cultured with irradiated APCs and CEA protein for 5 days. ³H-thymidine (1 µCi/well) was added to the wells for the last 24 h, and proliferation was assayed by measuring incorporated radioactivity. Open squares, control yeast. Closed circles, yeast-CEA. B, CD8⁺ CTL activity after vaccination with yeast-CEA. Splenocytes were stimulated with CEA_572–579 peptide for 6 days before assays. Lymphocytes were incubated for 5 h with ⁵¹Cr-labeled target EL-4 cells pulsed with CEA or VSV-NP control peptide. Radioactivity in the supernatant was measured and specific lysis calculated. SD is based on the mean of triplicate wells. Open circles, CTL activity directed against EL-4 cells pulsed with VSV-NP peptide. Closed circles, CTL activity directed against EL-4 cells pulsed with CEA peptide. C, CD8⁺ CTL activity after vaccination with control yeast. Open squares, CTL activity directed against EL-4 cells pulsed with VSV-NP peptide. Closed squares, CTL activity directed against EL-4 cells pulsed with CEA peptide. C (Insert), CD8⁺ CTL activity against tumor cells after vaccination with yeast-CEA. Splenocytes were stimulated with CEA_572–579 peptide as above. Lymphocytes were incubated for 5 h with ⁵¹Cr-labeled target MC38-CEA⁺ (closed bars), at an E:T ratio of 100:1. As a negative control, splenocytes from mice receiving PBS (vehicle control) were stimulated with CEA peptide as above and CTL activity directed against MC38-CEA⁺ tumor cells (open bars).

Fig. 2

Multiple vaccinations with yeast-CEA continuously increase T-cell response. CEA-Tg mice were vaccinated with 0.1 YU control yeast or yeast-CEA 1, 2, 3, or 4 times at 7-day intervals. Fourteen days after the last vaccination, mice were sacrificed, spleens were harvested, and splenocytes were used for assays. For comparison, mice were vaccinated with CEA-TRICOM. Open squares, control yeast; filled circles, yeast-CEA; filled squares, DEA-TRICOM. A, CD4⁺ T-cell proliferation after vaccination with control yeast. Purified CD4⁺ T cells were cultured with irradiated APCs and CEA protein for 5 days. ³H-thymidine (1 µCi/well) was added to the wells for the last 24 h, and proliferation was assayed by measuring incorporated radioactivity. B, CD4⁺ T-cell proliferation after vaccination with yeast-CEA. C, CD4⁺ T-cell proliferation after vaccination with rV/rF-CEA-TRICOM vaccines. D, CD8⁺ CTL activity after vaccination with control yeast. Splenocytes were stimulated with CEA peptide for 6 days before assays. Lymphocytes were incubated for 5 h with ⁵¹Cr-labeled target EL-4 cells pulsed with CEA or VSV-NP control peptide. Radioactivity in the supernatant was measured and specific lysis calculated. SD is based on the mean of triplicate wells. E, CD8⁺ CTL activity after vaccination with yeast-CEA. F, CD8⁺ CTL activity after vaccination with rV/rF-CEA-TRICOM vaccines. Data are presented as % lysis after subtraction of VSV-NP control.

Fig. 3

CD8⁺ CTL responses after dose escalation of yeast-CEA. CEA-Tg mice were vaccinated with 0, 0.01, 0.1, 1, or 10 YU yeast-CEA twice at 7-day intervals. Fourteen days after the last vaccination, mice were sacrificed, spleens were harvested, and splenocytes were stimulated with CEA peptide for 6 days. Lymphocytes were incubated for 5 h with ⁵¹Cr-labeled target EL-4 cells pulsed with CEA or VSV-NP control peptide. Radioactivity in the supernatant was measured and specific lysis calculated. Open circles, EL-4 cells pulsed with VSV-NP peptide. Closed circles, EL-4 cells pulsed with CEA peptide.

Fig. 4

Vaccination with yeast-CEA reduces tumor growth and increases overall survival in tumor-bearing mice. A, survival in an experimental CEA⁺ lung metastasis model. CEA-Tg mice (n = 7/group) were injected with 1 × 10⁶ MC38-CEA⁺ tumor cells i.v. in the tail on day 0, and mock-treated or injected with 1 YU control yeast or yeast-CEA s.c. on days 4, 11, 18, and 25 (arrows). Mice were monitored and survival was recorded. Open squares, no treatment. Open triangles, control yeast. Closed circles, yeast-CEA. B, Survival in a lung metastasis model with continuous weekly vaccination (arrows). CEA-Tg mice (n = 14/group) were injected with 1 × 10⁶ MC38-CEA⁺ tumor cells i.v. in the tail on day 0, and were mock-treated or injected with 1 YU yeast-CEA s.c. starting on day 4, then weekly for the duration of the experiment. Mice were monitored and survival was recorded. Open squares, no treatment. Closed circles, yeast-CEA. C–D, Vaccination with yeast-CEA in a s.c. pancreatic cancer model. CEA-Tg mice were injected with 1 × 10⁶ Panc02.CEA cells s.c. on day 0, and vaccinated with 1 YU yeast-CEA starting on day 7, then weekly for the duration of the experiment. Tumor volume was measured twice a week and recorded. C, no treatment (n = 18). D, yeast-CEA (n = 10). Bar indicates average tumor volume ± SD.

Fig. 5

CD4⁺ T-cell responses increase when vaccine is distributed to multiple sites. CEA-Tg mice were vaccinated with a total of 1 or 16 YU yeast-CEA s.c. in 1 or 4 sites on days 0 and 7. Fourteen days later, mice were sacrificed, spleens were harvested, and CD4⁺ T cells were purified. Cells were cultured with irradiated APCs and CEA protein for 5 days. ³H-thymidine (1 µCi/well) was added to the wells for the last 24 h, and proliferation was assayed by measuring incorporated radioactivity. SD is based on the mean of triplicate wells. Open bar, no treatment. Black bar, 1 YU in 1 site. Gray bars, 16 total YU.

Fig. 6

Vaccination in multiple sites increases antitumor efficacy. CEA-Tg mice were implanted with 1 × 10⁶ Panc02.CEA cells s.c. on day 0 and vaccinated in 0, 1, 2, 4, or 6 sites with 1 YU yeast-CEA/site starting on day 7, then weekly for the duration of the experiment. Tumor volume was measured twice a week and recorded. A, no treatment (n = 10). B, 1YU in 1 site (n = 9). C, 1 YU in 2 sites (n = 10). D, 1 YU in 4 sites (n = 10). E, 1 YU in 6 sites (n = 10). Bars indicate average tumor volume ± SD. Open squares, no treatment. Closed circles, yeast-CEA.