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. 2018 Jun 1;13(6):e0197694.
doi: 10.1371/journal.pone.0197694. eCollection 2018.

Synthetic vaccine particles for durable cytolytic T lymphocyte responses and anti-tumor immunotherapy

Petr O Ilyinskii  1 , Grigoriy I Kovalev  2 , Conlin P O'Neil  1 , Christopher J Roy  1 , Alicia M Michaud  1 , Natalia M Drefs  2 , Mikhail A Pechenkin  2 , Fen-Ni Fu  1 , Lloyd P M Johnston  1 , Dmitry A Ovchinnikov  2 , Takashi Kei Kishimoto  1
Affiliations

Synthetic vaccine particles for durable cytolytic T lymphocyte responses and anti-tumor immunotherapy

Petr O Ilyinskii et al. PLoS One. .

Abstract

We previously reported that synthetic vaccine particles (SVP) encapsulating antigens and TLR agonists resulted in augmentation of immune responses with minimal production of systemic inflammatory cytokines. Here we evaluated two different polymer formulations of SVP-encapsulated antigens and tested their ability to induce cytolytic T lymphocytes (CTL) in combination with SVP-encapsulated adjuvants. One formulation led to efficient antigen processing and cross-presentation, rapid and sustained CTL activity, and expansion of CD8+ T cell effector memory cells locally and centrally, which persisted for at least 1-2 years after a single immunization. SVP therapeutic dosing resulted in suppression of tumor growth and a substantial delay in mortality in several syngeneic mouse cancer models. Treatment with checkpoint inhibitors and/or cytotoxic drugs, while suboptimal on their own, showed considerable synergy with SVP immunization. SVP encapsulation of endosomal TLR agonists provided superior CTL induction, therapeutic benefit and/or improved safety profile compared to free adjuvants. SVP vaccines encapsulating mutated HPV-16 E7 and E6/E7 recombinant proteins led to induction of broad CTL activity and strong inhibition of TC-1 tumor growth, even when administered therapeutically 13-14 days after tumor inoculation in animals bearing palpable tumors. A pilot study in non-human primates showed that SVP-encapsulated E7/E6 adjuvanted with SVP-encapsulated poly(I:C) led to robust induction of antigen-specific T and B cell responses.

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Conflict of interest statement

All authors are employees and shareholders of Selecta Biosciences and SelectaRUS. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. CTL induction and anti-tumor activity of two SVP formulations.
A-D. Analysis of T lymphocyte populations after immunization with SVP. Animals (4 mice/group/time-point) have been injected with SVP and antigen-specific T cells evaluated. A, B–cells from draining lymph nodes (A) or spleens (B) stained for CD8 and SIINFEKL-specific TCR (CD19+ cells gated out). C, D. CD8+SIINFEKL-specifc cells are stained for CD62L and CD44 T markers. C–popliteal lymph nodes; D–spleens. Summary of two independent experiments is shown. E. SVP induction of antigen-specific cytotoxicity. Animals (3–6 per time-point in each group) were injected with SVP[OVA]-PLA or SVP[OVA]-PLGA combined with SVP[R848] and CTL activity measured in vivo at times indicated. F-H. Anti-tumor effect of SVP immunization. Animals inoculated with EG.7-OVA cells were treated with SVP[OVA]-PLA or SVP[OVA]-PLGA combined with SVP[R848] at days 1, 4, 11, and 18 (F) or 3, 7, 14, and 21 (G) by s.c. administration at a tumor-distant site. H. SVP-treated animals surviving EG.7-OVA challenge were re-challenged with the same cells without additional treatment. Summary of two (F, H) or five (G) independent experiments is shown. * p <0.05, ** p <0.01, *** p<0.001, **** p<0.0001.
Fig 2
Fig 2. SVP-induced CTL activity against dominant CTL epitope of HPV-16 E7 protein.
Mice were immunized with free or SVP-entrapped E7.I.49 peptide (3 μg) and TLR agonists and in vivo CTL activity measured. A. R848 used as adjuvant (doses indicated), assay conducted at 7 days after vaccination. B. PS- or PO- forms of CpG-1826 (4 μg) used as adjuvant. Assay dates shown. * p<0.05, ** p <0.01, *** p<0.001.
Fig 3
Fig 3. Treatment of TC-1 tumors by SVP[E7.I.49] combined with different adjuvants.
Survival proportions are shown on each graph. A, B. SVP-entrapped R848 vs. PS-1826 CpG. Treatments administered on days 3, 7, 14 and 21 (A) or days 6, 10, 17 and 24 (B) after tumor inoculation. Summary of three (A) or four (B) independent experiments shown. C, D. SVP-entrapped or free CpG ODN; PS-1826 (C) or PO-1826 (D). Treatments administered on days 6, 10, 17 and 24 after tumor inoculation. Summary of three (C) or two (D) independent experiments is shown. ** p <0.01, *** p<0.001, **** p < 0.0001.
Fig 4
Fig 4. Immunogenicity of SVP-entrapped HPV-16 antigens and their therapeutic efficacy in vivo.
A, B. Antigen-specific cytotoxicity at 7 days after immunization with SVP[E7.I.49], SVP[E7*] or SVP[E7/E6*]. Target cells were pulsed by E7.I.49 peptide (A) or by a pool of subdominant E7 peptides (B). C. Treatment of TC-1 tumors by SVP-entrapped E.I.49, E7* or E7/E6* combined with SVP[R848], SVP administered on days 10, 14, 21 and 28 after tumor inoculation. Number of mice in each group is shown in parentheses. Summary of four independent experiments is shown. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 5
Fig 5. CTL induction and treatment of TC-1 tumors by SVP-encapsulated antigens and adjuvants.
A. Dynamics of CTL activity after a single immunization with SVP[E6/E7*] and SVP-encapsulated adjuvants. 7–8 mice per each time-point in each group with the exception of 3 week time-point (4 mice/group). B-F. Survival (B, C) and tumor growth (D-F) in TC-1-inoculated mice treated with SVP. B, C. SVP[E7/E6*] combined with SVP-entrapped R848, CpG or poly(I:C); treatments on days 10, 14, 21 and 28 (B) or days 13/14, 17, 24 and 31 (C) after tumor inoculation. Each curve is a summary of 2 or 4 independent experiments. D-F. SVP[E7/E6*] combined with SVP[R848] (D), SVP[CpG] (E) or SVP[poly(I:C)] (F); treatments as in C (shown by arrows). The total number of mice (summary of 3 independent experiments run in parallel) is 20 in D, E and 22 in F. Percentages indicate (top to bottom) shares of mice with detectable tumors prior to treatment, those of early tumor breakthroughs and of immediate post-treatment breakthroughs.
Fig 6
Fig 6. Long-term immune memory in SVP-treated TC-1 survivors.
Mice treated with SVP[E7/6*] and SVP[poly(I:C)] as shown in C were (A) re-challenged at day 158 after the first TC-1 inoculation and once again 154 days later (shown by arrows) or (B) sacrificed on day 265 after initial TC-1 inoculation, and their splenocytes inoculated into naïve mice (108/mouse, i.v.), which 8 days later were inoculated with TC-1 cells (8 mice/each group). No further treatment was applied.
Fig 7
Fig 7. Synergy of SVP and other treatment modalities.
A. SVP[Trp2.180–188] and SVP[PO-1826] were injected on d3, 7, 14 and 21 after B16-F10 tumor inoculation combined with aPD-L1 or isotype control (d6, 13, 20). Overall survival is shown; summary of 4 independent experiments. B. Synergy of cisplatin (CPL) and SVP against TC-1. CPL was administered on d5 and 12 and SVP were administered either on d14, 17, 24 and 31 (d14) or d21, 24, 31 and 38 (d21); 6–7 mice/group. * p<0.05; ** p <0.01; *** p < 0.001, **** p < 0.0001.
Fig 8
Fig 8. Experimental scheme for NHP immunization and sample analysis.
Times of SVP vaccinations (thick arrows) are shown above the general timeline, analysis time-points (serum and PBMC isolation) are shown by thin arrows below the timeline.
Fig 9
Fig 9. Induction of T and B cell responses to HPV-16 oncogenic proteins by SVP in non-human primates.
A. IFNγ ELISPOT in PBMC. B. IFNγ+Grnzb+ share of CD8+ cells in PBMC after peptide stimulation followed by FACS analysis. Peptide pools used for PBMC stimulation are indicated. C. Induction of antibodies to HPV-16 E6 and E7 proteins. Animals in A-C are grouped according to an adjuvant used with every group containing 4 animals immunized either with high or low doses of E7/E6* (2 each, 200 and 50 μg).
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