Department of Surgery and UVM Cancer Center, University of Vermont Larner College of Medicine, Burlington, VT 05405, United States.
Pediatric Oncology Translational Research Program, Helen DeVos Children's Hospital at Spectrum HealthGrand Rapids, MI, United States.
J Immunol Methods. 2018 Sep;460:51-62. doi: 10.1016/j.jim.2018.06.009. Epub 2018 Jun 12.
The aim of this preclinical study was to evaluate T7 bacteriophage as a nanoparticle platform for expression of neoantigens that could allow rapid generation of vaccines for potential studies in human cancer patients. We have generated recombinant T7 phage vaccines carrying neoepitopes derived from mutated proteins of B16-F10 melanoma tumor cells. With the single mutated amino acid (AA) centered, peptides were expressed on the outer coat of T7 phage. All peptides with 11 and 34 AAs were successfully expressed. Trimers of the 11-AA peptides were successfully expressed in only 3 of 8 peptides. The 11-AA peptide was better in stimulating antibodies selective for the mutated region than the longer 34-AA peptide. We observed a dose response for vaccines which provides an initial framework of the minimum phage required for vaccination. A single injection with phage-peptide vaccines in both monomer and trimer formats produced significant immune responses in mice on day 21, as assessed by lymph node cell counts, next generation sequencing (NGS), and plasma titers against T7 phage and vaccine peptides. A trimer provided no additional serum response to the monomer format. Immunization of mice with a mixture of 8 different peptide vaccines resulted in antibodies to most of the peptides. It was encouraging that induced antibodies had higher binding to the mutated peptides compared to the corresponding normal peptides. The NGS of lymph node cells demonstrated a low B cell receptor diversity and clonal hyperpolarization in vaccine-draining lymph nodes in comparison to those in unvaccinated mice nodes. The NGS data also revealed phenomenal increase in IgG and other class-switched antibodies following vaccination. These results agree with the higher plasma titers of IgG antibodies against T7 phage and vaccine peptides. Antibodies bound whole B16-F10 cells, lysates and multiple bands on Western blot. This indicates that these vaccine peptides successfully induced antibodies that bind full proteins from which the vaccine peptides were derived. We demonstrate a preclinical platform for rapid production of vaccines that can deliver mutated peptides and stimulate an appropriate B cell response. We anticipate further research in utilizing the cells from a tumor or vaccine draining lymph node as a resource for therapeutic anticancer reagents.
本临床前研究旨在评估 T7 噬菌体作为表达新抗原的纳米颗粒平台，以便为人类癌症患者的潜在研究快速生成疫苗。我们已经生成了携带源自 B16-F10 黑色素瘤肿瘤细胞突变蛋白的新表位的重组 T7 噬菌体疫苗。以单个突变氨基酸 (AA) 为中心，肽在 T7 噬菌体的外壳上表达。所有带有 11 和 34 个 AA 的肽都成功表达。只有 8 个肽中的 3 个成功表达了 11-AA 肽的三聚体。11-AA 肽在刺激针对突变区域的抗体方面优于更长的 34-AA 肽。我们观察到疫苗的剂量反应，为接种所需的最小噬菌体数量提供了初步框架。在第 21 天，以淋巴结细胞计数、下一代测序 (NGS) 和针对 T7 噬菌体和疫苗肽的血浆滴度评估，在单体和三聚体形式下单次注射噬菌体-肽疫苗在小鼠中产生了显著的免疫反应。三聚体对单体形式没有额外的血清反应。用 8 种不同的肽疫苗混合物免疫小鼠导致针对大多数肽的抗体。令人鼓舞的是，诱导的抗体与相应的正常肽相比，对突变肽的结合更高。与未接种疫苗的小鼠淋巴结相比，疫苗引流淋巴结中的淋巴细胞 B 细胞受体多样性低且克隆极化。NGS 数据还显示，接种后 IgG 和其他同种型转换抗体的数量显著增加。这些结果与针对 T7 噬菌体和疫苗肽的 IgG 抗体的更高血浆滴度一致。抗体结合整个 B16-F10 细胞、裂解物和 Western blot 上的多个条带。这表明这些疫苗肽成功地诱导了结合源自疫苗肽的全长蛋白的抗体。我们展示了一种快速生产疫苗的临床前平台，该平台可以递呈突变肽并刺激适当的 B 细胞反应。我们预计将进一步研究利用肿瘤或疫苗引流淋巴结中的细胞作为治疗性抗癌试剂的资源。
