(I-331) Self-assembly peptide neoantigen for personalized cancer vaccines

Introduction::

Cancer vaccines are one of the most important immunotherapies against tumor. In the past decades, the development neoantigen has greatly enhanced the specificity of tumor antigens and enable the design of “personalized” cancer vaccine for individual patients. However, the clinical success of neoantigen cancer vaccines is still limited by their poor in vivo distribution behavior. In this project, we aim to develop a novel nanoscale vaccine delivery system based on the self-assembly of synthetic long peptide (SLP) containing neoantigen epitopes. Through decorating neoantigen peptides with pH-sensitive flanking sequence of optimized length, we can easily obtain the amphiphilic synthetic long peptides which are ready to co-assembly with molecular adjuvants in mild conditions. The self-assembly neoantigens could greatly enhance the extracellular delivery including the localization of neoantigen and adjuvants in peripheral lymphoid organs, such as lymph nodes, as well as the endocytosis with antigen-presenting cells, such as dendritic cells. For intercellular delivery, the pH-responsive nanoparticle can rapidly disassemble in the acidic environment of the endosome to enhance the escaping of antigens to cytoplasm, thus enhancing the antigen cross-presentation to CD8 T cells. Additionally, Self-assembly design maximizes the loading capacity of antigens and adjuvants, and the size of the particle can be optimized by adjusting the flanking peptide sequence and self-assembly conditions to meet the specific requirements of vaccine delivery. In summary, the self-assembly peptide neoantigen is potent candidate to address the challenge in neoantigen vaccine delivery.

Materials and Methods:: The self-assembly synthetic long peptides containing neoepitops were purchased from Genscript and used without forthur modification. The adjuvants including DMXAA, Pam3CSK4, and Poly(I:C) was purchased from Invivogen. The cancer celline including B16-OVA, B16F10, and MC38 were purchased from Sigma-Aldrich. The C57BL/6 mice and OT-1 mice were purchased from Jackson Lab. All chemicals were purchased from Sigma-Aldrich.
For self-assembly, the peptides were first dissolved in DMSO and added slowly to the aqueous solution of adjuvant with a syring pump. The obtained self-assembly nanoparticles were measured by DLS for quality control before use.
For therapeuty study. The C57BL/6 mices were first inoculated with tumor subcutaneously, once the tumor was able to measure (D3-D7), the vaccine nanoparticles were administrated intradermally every 3 days with totaly 3 times together with i.p. treatment of anti-PD-1. The tumor sizes were measured every 3 days. The results were analized with one-way ANOVA test and Long-rank test.

Results, Conclusions, and Discussions::

The self-assembly of amphiphilic neoantigen peptides generates regular nanoparticle of 50~150 nm with narrow size distribution as measured by DLS, TEM, and SEM. The particles remain stable under 4°C for more than one week. Once exposed to acidic condition mimicking the endosome microenvironment (pH 5-6), the nanoparticles show the behavior of disassembly.

For the in vitro assays, the DC uptake assay showed greatly enhanced antigen and adjuvants endocytosis of self-assembly neoantigen vaccines comparing with soluble neoantigens and adjuvants. The self-assembly neoantigens exhibited equal DC maturation and enhanced antigen cross-presentation capability with soluble antigens.

For the in vivo assessments, the self-assembly neoantigen vaccine showed greatly enhanced lymph node localization comparing with the simply mixed neoantigens and adjuvants. In the therapeutic study with mice bearing B16-OVA and MC38 tumor, the treatment of self-assembly neoantigen vaccine showed significantly suppressed tumor growth as well as greatly increased survival.

In summary, the self-assembly neoantigen vaccine is an efficient delivery system for neoantigen and adjuvants through enhancing both extracellular and intracellular travelling. The safe and simple design has great potential for clinical application.  

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