Effective antitumor peptide vaccines can induce severe autoimmune pathology

Abstract

Immunotherapy has shown a tremendous success in treating cancer. Unfortunately, this success is frequently associated with severe autoimmune pathology. In this study, we used the transgenic RIP-gp mouse model to assess the antitumor therapeutic benefit of peptide vaccination while evaluating the possible associated autoimmune pathology. We report that palmitoylated gp33-41 peptide and poly-IC adjuvant vaccine (BiVax) generated ∼ 5-10 % of antigen specific T cell responses in wild type and supposedly immune tolerant RIP-gp mice. Boosting with BiVax in combination with αCD40 antibody (TriVax) or BiVax in combination with IL-2/αIL-2 antibody complexes (IL2Cx) significantly increased the immune responses (∼30-50%). Interestingly, although both boosts were equally effective in generating vast T cell responses, BiVax/IL2Cx showed better control of tumor growth than TriVax. However, this effect was associated with high incidence of diabetes in an antigen and CD8 dependent fashion. T cell responses generated by BiVax/IL2Cx, but not those generated by TriVax were highly resistant to PD-1/PD-L1 inhibitory signals. Nevertheless, PD-1 blockade enhanced the ability of TriVax to control tumor growth but increased the incidence of diabetes. Finally, we show that severe autoimmunity by BiVax/IL2Cx was prevented while preserving outstanding antitumor responses by utilizing a tumor antigen not expressed in the pancreas. Our data provides a clear evidence that peptide based vaccines can expand vast endogenous T cell responses which effectively control tumor growth but with high potential of autoimmune pathology.

Keywords: IL-2 complex; anti-CD40; anti-tumor effect; diabetes; peptide vaccine.

Figure 1. Optimization of gp33-41 vaccination strategy in WT mice

(A) WT mice were primed with equimolar amounts of the pam-gp33-41 (Pam2-KMFVTAPDNLGYM) or minimal gp33-41 peptide (KAVYNFATM) and 50 μg poly-IC and 7 days later the immune responses were evaluated by tetramer staining in blood. (B) Diagrammatical representation of the vaccination protocol for panels C and D. (C, D) WT mice were primed with pam-gp33-41 BiVax and 14 days later they were boosted with pam-BiVax, pam-BiVax/IL2Cx or pam-TriVax. The percentages of Kb (B) and Db (C) tetramer+ CD8 T cells in blood after prime and boost are shown. Results are presented for individual mice (each symbol) with the mean ± SD for each group. (*p<0.05, ns: not significant).

Figure 2. TriVax or BiVax/IL2Cx generate vast T cell responses in RIP-gp mice

(A, B) RIP-gp mice were vaccinated with pam-gp33-41 BiVax and 14 days later they were boosted with TriVax or BiVax/IL2Cx. Percentage of Kb (A) and Db (B) tetramer+ CD8 T cells in the blood. (C) Total numbers of Kb and Db specific CD8 T cells in spleens after boost. (D) Splenocytes were stimulated in vitro with the minimal gp33-41 peptide in the presence of Golgiplug for 6 h and the production of IFNγ, TNFα, IFNγ/TNFα, IL-2 and granzyme B was assessed by intracellular staining. Results are presented for individual mice (each symbol) with the mean ± SD for each group. (ns: not significant).

Figure 3. BiVax/IL2Cx but not BiVax alone or TriVax induces diabetes in Rip-gp mice

(A-E) RIP-gp mice were vaccinated as described in Figure 1B. (A) Blood glucose levels in individual mice (each symbol) from at least 3 independent experiments. (B) Insulin staining in formalin fixed pancreatic tissues of WT and RIP-gp mice after TriVax or BiVax/IL2Cx boost was analyzed 8 days after the booster vaccination. (C) RIP-gp mice were primed with BiVax followed by BiVax/IL2Cx. Some mice received 500 μg of αCD8 mAb (i.p.) at days 12 and 14. Blood glucose levels were measured to assess diabetes. (D) Mean fluorescence intensity (MFI) of tetramer stains for Kb and Db specific cells in RIP-gp mice. Each symbol represents an individual mouse. (E) Purified CD8 T cells were incubated with serial dilutions of the minimal Db (KAVYNFATM) or Kb (AVYNFATM) peptides and 48 h later the production of IFNγ in the supernatants was assessed by ELISA. Dashed horizontal lines in A and C represents maximal normal blood glucose level. (*p<0.05, ns: not significant).

Figure 4. BiVax/IL2Cx generates T cell with relatively high Tbet expression

(A-D) RIP-gp mice were vaccinated as in Figure 1B and spleens were collected 7 days after the boost. MFI of PD-1 (A) and LAG3 (B) expression on the surface of Kb and Db specific T cells are shown. MFI of Tbet (C) and Eomes (D) expression in Kb and Db specific T cells. Results are presented for individual mice (each symbol) with the mean ± SD for each group. (*p<0.05, ns: not significant).

Figure 5. BiVax/IL2Cx generates T cell responses resistant to PD-L1 inhibition

(A) Level of PD-L1 expression in B16F10gp33-41 cells (stimulated or not with IFNγ for 48 h). (B) 10⁵ purified CD8 T cells were incubated with untreated or IFNγ treated B16F10gp33-41 cells, in the presence or the absence of 10 μg αPD-L1, for 24 h and the numbers of IFNγ spots were enumerated by EliSpot. Results are presented as mean ± SD. (*p<0.05, ns: not significant). Results are presented for individual mice (each symbol) with the mean ± SD for each group. (*p<0.05, ns: not significant).

Figure 6. TriVax/αPD-L1 combination induces diabetes in RIP-gp mice

RIP-gp mice were primed with pam-gp33-41 BiVax and 14 days later they were boosted with TriVax with or without αPD-L1 mAb (200 μg/mouse i.p. at days 14, 16 and 18). (A) Blood glucose levels in individual mice (each symbol) for each group. Percentages of Db (B) and Kb (C) tetramer+ CD8 T cells in the blood. (D) Absolute numbers of Kb and Db tetramer+ CD8 T cells in spleens. Results are presented for individual mice (each symbol) with the mean ± SD for each group. (ns: not significant).

Figure 7. BiVax/IL2Cx combination show better therapeutic antitumor effects

RIP-gp mice were inoculated s.c. with B16F10gp33-41 melanoma cells (3 × 10⁵ cells/mouse). After 8 days mice received pam-gp33-41 BiVax and 9 days later (17 days after tumor inoculation) they were boosted with either pam-gp33-41 BiVax, pam-gp33-41 TriVax, pam-gp33-41 TriVax+αPD-L1 or pam-gp33-41 BiVax/IL2Cx. αPD-L1 mAb was administered i.p. on days 17, 19 and 21. A control group was primed with pam-Ova257-264 BiVax and 9 days later boosted with pam-Ova257-264 TriVax+αPD-L1 mAb. (A) Mean tumor sizes and (B) overall survival of tumor-bearing mice. (C) Blood glucose levels in individual mice (each symbol) for each group. (D) Percentages of Kb and Db tetramer+ CD8 T cells for each individual mouse in the blood at day 30 after tumor inoculation. Results presented for individual mice (each symbol) with the mean ± SD for each group. (*p<0.05, ****p<0.0001, ns: not significant).

Figure 8. BiVax/IL2Cx utilizing tumor specific antigen controls tumor growth with limited autoimmune pathology

RIP-gp mice were inoculated s.c. with B16F10gp33-41 melanoma cells (3 × 10⁵ cells/mouse) and 8 days later they received either pam-gp33-41 BiVax, pam-Trp1.455-463 BiVax or pam-Ova257-263 BiVax. Nine days later (17 days after tumor inoculation) mice were boosted with the same peptide plus IL2Cx (administered on days 17, 19 and 21). (A) Mean tumor sizes and (B) blood glucose levels in individual mice (each symbol) for each group. (C) Percentages of tetramer+ CD8 T cells in the blood at day 25 after tumor inoculation (Kb tetramer responses for gp33-41). (D) Percentage of gp33-41 Db/Kb+ T cells in spleen and tumor draining lymph nodes (TDLN) of Trp1 and Ova vaccinated mice. Results are presented for individual mice (each symbol) with the mean ± SD for each group. (*p<0.05, ****p<0.0001, ns: not significant).