Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Kinderspitalgasse 15, 1090, Vienna, Austria.
Institute of Environmental Health, Medical University of Vienna, 1090, Vienna, Austria.
BMC Cancer. 2017 Feb 9;17(1):118. doi: 10.1186/s12885-017-3098-7.
We previously identified three short single peptides (P4, P6 and P7) representing different B-cell epitopes on the extracellular domain of Her-2/neu for a vaccine that was tested in a phase-I clinical trial. Here we describe the improvement of the multi peptide vaccine by fusing the single peptides to a hybrid peptide P467.
After coupling to either virosomes or to diphtheria toxoid CRM197 (CRM), the hybrid peptide was tested in different concentrations in combination with either Montanide or Aluminium hydroxide (Alum) in preclinical studies.
Already low amount (10 μg) of P467 conjugated to CRM led to faster onset of high antibody levels compared to the P467-virosome. The formulation P467-CRM-Montanide induced higher serum IgG antibody titers, compared with P467-CRM-Alum, as examined by ELISA using recombinant Her-2/neu or Her-2/neu natively expressed on the tumor cell line SK-BR-3. Compared to P467-CRM-Alum, higher in vitro production of IL-2 and IFNγ in the Montanide-immunized mice was induced after re-stimulation of splenocytes with CRM but also with P467, indicating a clear Th1-biased response. In contrast to the single B cell peptides, the hybrid peptide led to T cell proliferation and cytokine production as CD4 T cell epitopes were generated in the fusion region of the single peptides P4 and P6 or P6 and P7. Additionally, a significantly higher proportion IFNγ-producing CD8+ T cells was found in the P467-CRM-Montanide immunized mice, probably by Montanide-driven bystander activation. Importantly, anti-P467 IgG antibodies exhibited anti-tumor properties and the combination of anti-P467 specific IgG with Herceptin® was found to inhibit the proliferation of Her-2/neu-overexpressing cell line SK-BR-3 in a significantly higher capacity than Herceptin® alone.
Fusion of the B cell peptides has led to additional generation of CD4 T cell epitopes, and this P467-multi epitope vaccine was found to induce polyclonal antibody responses with anti-proliferative capacity against Her-2/neu. The hybrid vaccine together with Montanide induced higher and long-lasting antibody levels, Th1-biased cellular responses being superior to vaccination with the single B cell peptides. This vaccine formulation is now planned to be evaluated in a phase Ib/II study in Her-2/neu overexpressing cancer patients.
我们之前鉴定了三个短的单肽（P4、P6 和 P7），它们代表 Her-2/neu 细胞外结构域上的不同 B 细胞表位，用于进行 I 期临床试验的疫苗。在此，我们描述了通过将单肽融合到混合肽 P467 上来改进多肽疫苗。
在与病毒体或白喉类毒素 CRM197（CRM）偶联后，在临床前研究中，用不同浓度的混合肽与 Montanide 或氢氧化铝（Alum）联合进行测试。
与 P467-病毒体相比，即使在低浓度（10μg）下与 CRM 偶联的 P467 也能更快地诱导高抗体水平。与 P467-CRM-Alum 相比，P467-CRM-Montanide 诱导的血清 IgG 抗体滴度更高，ELISA 检测使用重组 Her-2/neu 或 SK-BR-3 肿瘤细胞系上天然表达的 Her-2/neu。与 P467-CRM-Alum 相比，CRM 刺激后混合肽免疫小鼠脾细胞中 IL-2 和 IFNγ的体外产生更高，表明明显的 Th1 偏向反应。与单 B 细胞肽不同，融合肽在 P4 和 P6 或 P6 和 P7 的融合区产生 CD4 T 细胞表位，从而导致 T 细胞增殖和细胞因子产生。此外，在 P467-CRM-Montanide 免疫的小鼠中发现了更高比例的 IFNγ 产生 CD8+T 细胞，可能是由于 Montanide 驱动的旁观者激活。重要的是，抗 P467 IgG 抗体具有抗肿瘤特性，并且发现抗 P467 特异性 IgG 与赫赛汀®的组合比单独使用赫赛汀®更能抑制 Her-2/neu 过表达细胞系 SK-BR-3 的增殖。
B 细胞肽的融合导致了额外的 CD4 T 细胞表位的产生，并且发现这种 P467-多表位疫苗可诱导针对 Her-2/neu 的多克隆抗体反应，具有抗增殖能力。与单 B 细胞肽相比，混合疫苗与 Montanide 联合使用可诱导更高和更持久的抗体水平，Th1 偏向的细胞反应更好。目前计划在 Her-2/neu 过表达的癌症患者中进行 Ib/II 期研究评估该疫苗制剂。
