Co-delivery of Peptide Neoantigens and Stimulator of Interferon Genes Agonists Enhances Response to Cancer Vaccines

Abstract

Cancer vaccines targeting patient-specific neoantigens have emerged as a promising strategy for improving responses to immune checkpoint blockade. However, neoantigenic peptides are poorly immunogenic and inept at stimulating CD8⁺ T cell responses, motivating a need for new vaccine technologies that enhance their immunogenicity. The stimulator of interferon genes (STING) pathway is an endogenous mechanism by which the innate immune system generates an immunological context for priming and mobilizing neoantigen-specific T cells. Owing to this critical role in tumor immune surveillance, a synthetic cancer nanovaccine platform (nanoSTING-vax) was developed that mimics immunogenic cancer cells in its capacity to efficiently promote co-delivery of peptide antigens and the STING agonist, cGAMP. The co-loading of cGAMP and peptides into pH-responsive, endosomolytic polymersomes promoted the coordinated delivery of both cGAMP and peptide antigens to the cytosol, thereby eliciting inflammatory cytokine production, co-stimulatory marker expression, and antigen cross-presentation. Consequently, nanoSTING-vax significantly enhanced CD8⁺ T cell responses to a range of peptide antigens. Therapeutic immunization with nanoSTING-vax, in combination with immune checkpoint blockade, inhibited tumor growth in multiple murine tumor models, even leading to complete tumor rejection and generation of durable antitumor immune memory. Collectively, this work establishes nanoSTING-vax as a versatile platform for enhancing immune responses to neoantigen-targeted cancer vaccines.

Keywords: cancer vaccine; immune checkpoint blockade; immunotherapy; neoantigen; polymer nanoparticle.

Figure 1.. NanoSTING-vax – a platform for dual-delivery of peptide antigens and cyclic dinucleotide STING agonists to enhance responses to neoantigen-targeted cancer vaccines.

(a) Schematic of nanoSTING-vax structure. Peptide antigens and cyclic dinucleotide STING agonists (e.g., cGAMP) are co-loaded into pH-responsive polymersomes comprised of endosomolytic diblock polymers. (b) NanoSTING-vax enables uptake of peptides and cGAMP by antigen presenting cells and facilitates cytosolic co-delivery of neoantigenic peptides and cGAMP via endosomal escape. Cytosolic delivery of antigen promotes MHC class I presentation while cytosolic cGAMP delivery enhances its immunostimulatory adjuvant capacity, collectively resulting in enhanced CD8⁺ T cell priming and activation. Credit: ©Fairman Studios, LLC, 2020.

Figure 2.. Endosomolytic nanoparticles enhance dual-delivery of cGAMP and peptide antigens to the cytosol.

(a) Dynamic light scattering analysis (intensity and number average size distributions) of cGAMP-loaded polymersomes (NP-cGAMP) and polymersomes loaded with both cGAMP and the Ova-derived peptide SGLEQLESIINFEKL (nanoSTING-vax). (b) Representative transmission electron micrograph of nanoSTING-vax, here loaded with cGAMP and the peptide SGLEQLESIINFEKL. (c) Dose–response curves of the IFN-I response elicited by indicated cGAMP-containing formulations in RAW 264.7 cells with an IFN regulatory factor (IRF)-inducible reporter construct (n = 3 biologically independent samples). (d) B3Z T cell response to DC2.4 dendritic cells treated with the indicated formulation (n = 4 biologically independent samples). (e) Flow cytometric analysis of median fluorescent intensity (MFI) of BMDCs treated with the indicated formulation and stained with an antibody (25-D1.16) specific to the SIINFEKL/H-2K^b complex (n = 3 biologically independent samples). (f) Flow cytometric quantification (MFI) of MHC-II, CD86, and CD40 expression by BMDCs treated with indicated formulation (n=3 biologically independent samples). Statistical data are presented as mean ± s.d. Statistical significance between nanoSTING-vax and all other formulations are shown; **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA with Tukey post-hoc test.

Figure 3.. NanoSTING-vax improves delivery of cGAMP and peptide antigens to vaccine site draining lymph nodes.

(a) Representative images (left) and IVIS quantification of fluorescence (right) of the vaccine site draining inguinal LN 18 h following subcutaneous administration of nanoSTING-vax containing an Alexa Fluor 700-labeled peptide or a soluble mixture of Alexa Fluor 700-peptide and cGAMP (mean +/− s.e.m; n = 8–10 mice/group; *P<0.05; one-way ANOVA with Tukey post-hoc test). (b) Ifnb1 expression in the inguinal LN 4 h following administration of indicated vaccine formulation (mean +/− s.e.m; n = 4–5 mice/group; ****P<0.001; one-way ANOVA with Tukey post-hoc test). (c) Percentage of NP-Cy5⁺ cells among cell populations in the inguinal LN following administration of the labeled nanoparticle (n = 5 mice/group). MΦ, macrophage; DC, dendritic cell; NK, natural killer cell. (d) Percentage of Alexa Fluor 700-peptide⁺ cells among cell populations in the inguinal LN following administration of the indicated formulation (n = 5 mice/group; two-tailed Student’s t-test; *P<0.05, **P<0.01). MΦ, macrophage; DC, dendritic cell; NK, natural killer cell. (e) Flow cytometric quantification (MFI) of CD86 and CD40 expression by CD11c⁺ dendritic cells in the inguinal LN in response to immunization with the indicated formulation (n = 5 mice/group; ***P<0.001, ****P<0.0001; one-way ANOVA with Tukey post-hoc test).

Figure 4.. NanoSTING-vax enhances CD8⁺ T cell responses to peptide antigens.

(a) Administration, analysis, and tumor challenge scheme for mice immunized with nanoSTING-vax or indicated control formulations containing Ova peptide. (b) Quantification of the frequency of SIINFEKL-specific CD8⁺ T cells in peripheral blood via peptide/MHC tetramer staining (n = 15 mice/group. Statistical significance between nanoSTING-vax and all other formulations are shown; ***P<0.001, ****P<0.0001; one-way ANOVA with Tukey post-hoc test). (c) Average tumor volume following challenge of mice immunized with indicated vaccine formulations with B16-Ova cells (n = 8–15; mice/group; **P<0.01, ****P<0.0001; one-way ANOVA with Tukey post-hoc test on day 20). (d) Administration scheme for mice immunized with nanoSTING-vax containing Reps1 and Adpgk neoantigenic peptides or indicated control formulations. (e) Percentage of IFN-γ⁺TNF-α⁺ CD8α⁺ T cells in peripheral blood after ex vivo restimulation with Reps1 and Adpgk epitopes and intracellular cytokine staining following by flow cytometric analysis (n = 7–8 mice/group; *P<0.05, ***P<0.001, ****P<0.0001; one-way ANOVA with Tukey post-hoc test). Statistical data are presented as mean ± s.d unless otherwise indicated.

Figure 5.. NanoSTING-vax enhances response to immune checkpoint blockade.

(a) Tumor inoculation, therapeutic vaccination, immune checkpoint blockade regimen, and re-challenge scheme for mice with subcutaneous MC38 tumors. (b) Spider plots of individual tumor growth curves, with the numbers of complete responders denoted. (c) Average MC38 tumor volume in response to indicated treatment (n = 6–7 mice/group; *P<0.05; unpaired t-test of nanoSTING-vax + αPD-1 vs. NP-cGAMP + αPD-1 on day 27). (d) Kaplan–Meier survival curves of mice growing MC38 tumors treated with the indicated formulation using a 1,500 mm³ tumor volume as the endpoint criteria (n = 6–7 mice/group; **P<0.01; two-tailed Mantel–Cox test). (e) Kaplan–Meier survival curves for treatment-naïve and mice demonstrating complete responses to nanoSTING-vax + αPD-1 after challenge with MC38 cells on the contralateral flank after 200 d without any further treatment (n = 5 mice; **P<0.01; two-tailed Mantel–Cox test). (f) Tumor inoculation, therapeutic vaccination, immune checkpoint blockade regimen for mice with subcutaneous B16.F10 tumors. (g) Average B16.F10 tumor volume in response to indicated treatment (n = 6–7 mice/group; *P<0.05; unpaired t-test of nanoSTING-vax + αPD-1 + αCTLA-4. cGAMP + peptides on day 21). (h) Kaplan–Meier survival curves of mice growing B16.F10 tumors treated with the indicated formulation using a 1,500 mm³ tumor volume as the endpoint criteria (n = 6–7 mice/group; ***P<0.001; two-tailed Mantel–Cox test). All statistical data are presented as mean ± s.e.m.