Adjuvant-free peptide vaccine targeting Clec9a on dendritic cells can induce robust antitumor immune response through Syk/IL-21 axis

Dendritic cells (DCs) can process the antigens of cancer vaccine and thus stimulate the CD8+ T cells to recognize and kill the tumor cells that express these antigens. However, lack of promising carriers for presenting the antigens to DCs is one of the main barriers to the development of clinically effective cancer vaccines. Another limitation is the risk of inflammatory side effects induced by the adjuvants. It is still unclear how we can develop ideal adjuvant-free DC vaccine carriers without adjuvants. Methods: A 12-mer peptide carrier (CBP-12) with high affinity for Clec9a expressed on DCs was developed using an in silico rational optimization method. The therapeutic effects of the adjuvant-free vaccine comprising CBP-12 and exogenous or endogenous antigenic peptides were investigated in terms of antigen cross-presentation efficacy, specific cytotoxic T lymphocyte response, and antitumor activity. We also explored the mechanism involved in the antitumor effects of the adjuvant-free CBP-12 vaccine. Finally, we assessed the effects of the CBP-12 conjugated peptide vaccine combined with radiotherapy. Results: Here, we developed CBP-12 as a vaccine carrier that enhanced the uptake and cross-presentation of the antigens, thus inducing strong CD8+ T cell responses and antitumor effects in both anti-PD-1-responsive (B16-OVA) and -resistant (B16) models, even in adjuvant-free conditions. CBP-12 bound to and activated Clec9a, thereby stimulating Clec9a+ DC to product IL-21, but not IL-12 by activating of Syk. The antitumor effects of the CBP-12 conjugated peptide vaccines could be blocked by an IL-21 neutralizing antibody. We also observed the synergistic antitumor effects of the CBP-12 conjugated peptide vaccine combined with radiotherapy. Conclusions: CBP-12 could serve as an adjuvant-free peptide vaccine carrier for cancer immunotherapy.


Mutant peptide design
Based on our previous study, Asp248 and Trp250 were two key residues for Clec9a to bind with WH peptide. In order to improve the binding of WH peptide to Clec9a, a silico residue scanning approach was applied to design high affinity WH mutants. The mutants were predicted by using the module of Residue Scanning mutates function, MOE (Molecular Operating Environments) software (2018 version, Chemical Computing Group Inc, Montreal, Quebec, Canada). Two hundred and twenty peptides were obtained, among which 15 peptides were selected and synthesized for experimental identification.

In vitro enzyme degradation of peptide
Peripheral blood of mice was stand at room temperature for 1 h, centrifuged for 10 min and then the supernatant was acquired. The peptide (1 mM) was added into 10% (V/V) serum saline solution and incubated for 2 h at 37 ℃. The samples taken at different time points were added 90% (V/V) acetonitrile-glacial acetic acid mixture and centrifuged for 15 min, then the supernatants were collected. The content of the residual peptide in samples was analyzed at 0 h, 0.25 h, 0.5 h, 1 h and 2 h by RP-HPLC, respectively. CBP-12-OVA targeting to Clec9a together with adjuvant treated tumor model C57BL/6 or Clec9a −/− mice were s.c. injected with 2 × 10 5 B16-OVA cells. Three days after the tumor implantation, the mice were treated weekly for total of three times, with either 100 μg peptide or normal saline and 30 μg CpG ODN 1826 as adjuvant.
Then the mice were sacrificed after five days of the last injection.

Animal immunization and ex vitro CTL priming without adjuvant
For animal immunization and ex vivo CTL priming, female C57BL/6 mice of 6-8 weeks were immunized with 20 μg OVA257-264, GA-OVA, CBP-12-OVA, or normal saline s.c. injected once a week for total of three times. Five days after the last 4 injection, mice were sacrificed and ex vivo CTL priming assay was performed.
Splenocytes were incubated with 10 μg/mL OVA257-264 peptide and 1 μL/mL brefeldin A (Becton Dickinson, US) for 6 h to test the ability of CD8 + T cells activation by ICS.
Other spleen cells were re-stimulated with 10 μg/mL OVA257-264 peptide for five days.
The IFN-γ was measured by a Mouse IFN gamma ELISA kit.

MTT assay
The effect of peptide on the proliferation of B16-OVA cells was determined by a MTT assay. Briefly, cells were seeded into a 96-well plate and treated with various concentrations of peptide for 24, 48 and 72 h at 37°C with 5% CO2. Cell viability was detected using 20 μL/well of 5 mg/mL MTT reagents (Sigma, St. Louis, MO, USA).