Preclinical Toxicology: Safety Evaluation of pNGVL4a-CRT-E6E7L2 (detox) Recombinant DNA Vaccine in Mice

pNGVL 4a-hCRT/E6 E7 L2是针对HPV E6、E7和L2蛋白的裸DNA疫苗，目的是开发能够诱导细胞免疫以消除HPV感染和HPV相关病变的治疗性疫苗。新批准的HPV疫苗Gardasil®和Cervarix®均基于L1病毒样颗粒（VLP），仅对预防疫苗中包含的HPV基因型引起的新生殖器感染有效。不幸的是，L1 VLP疫苗不会改变已建立的HPV感染的过程，因此在几十年内不会降低HPV相关癌症的发病率;此外，它们对所有致癌HPV类型都无效。对广泛保护性HPV疫苗的需求显然未得到满足，该疫苗还能够触发已建立的HPV感染和相关瘤形成的清除，特别是在免疫功能低下的人群中，例如HPV相关癌症发病率较高的人类免疫缺陷病毒（HIV）+患者。pNGVL 4a-hCRT/E6 E7 L2是通过将人钙网蛋白（CRT）与HPV 16 E6和E7基因融合而产生的，每个基因含有多个失活突变和针对高水平表达优化的密码子，以及衣壳L2蛋白氨基酸残基11 - 200。在小鼠中的概念验证（proof-of-concept，CBT）研究中，用pNGVL 4a-hCRT/E6 E7 L2 DNA疫苗接种显示能够产生强的E6/E7特异性CD 8 + T细胞免疫应答，其导致针对表达E6/E7的TC-1肿瘤细胞的有效治疗效果。此外，产生了能够中和HPV 16病毒体的显著的L2特异性抗体。在另一项针对小鼠的研究中，即使在CD 4 + T细胞耗竭的小鼠中，hCRTE 6 E7 L2 DNA疫苗接种也能对表达E7的肿瘤产生强效抗肿瘤作用，这表明该疫苗即使在免疫受损的宿主中也可能具有活性。在临床前模型中进行了编码与HPV 16 E7连接的CRT（pNGVL 4a-CRT/E7）的DNA疫苗递送的各种施用方法的头对头比较，并使用常规肌内（IM）针施用、电穿孔（EP）介导的IM递送和通过基因枪的颗粒介导的表皮递送进行了测试。结果表明，EP介导的IM交付产生更高水平的循环蛋白和更高数量的E7特异性CD 8 + T细胞的脾细胞相比，传统的IM注射。在EP接种的小鼠中检测到的增强的CD 8 + T细胞免疫应答转化为荷瘤小鼠中表达E7的肿瘤中的上级抗肿瘤作用，表明EP介导的递送与常规IM递送疫苗相比可以提供优势。基于这一有前景的临床前数据，合同生产商已经生成了GMP主细胞库并生产了临床级pNGVL 4a-hCRT/E6 E7 L2 DNA。评估pNGVL 4a-hCRT/E6 E7 L2 DNA疫苗在使用TriGrid递送系统（TDS）-IM EP装置（Ichor Medical Systems，Inc.）作为宫颈癌SPORE计划的一部分，需要支持一项计划中的临床试验，评估HPV 16相关宫颈上皮内瘤变（CIN）2/3患者DNA疫苗接种的安全性和可行性。

pNGVL4a-hCRT/E6E7L2, is a naked DNA vaccine directed at HPV E6, E7, and L2 proteins, with the goal to develop a therapeutic vaccine capable of inducing cellular immunity to eliminate HPV infection and HPV-associated lesions. The newly licensed HPV vaccines, Gardasil® and Cervarix®, both based upon L1 virus-like particles (VLPs), are effective only for prevention of new genital infections by the HPV genotypes included in the vaccines. Unfortunately, the L1 VLP vaccines do not alter the course of established HPV infections, and thus will not reduce rates of HPV-related cancer for several decades; additionally, they are not effective against all oncogenic HPV types. There is a clear unmet need for a broadly protective HPV vaccine that is also able to trigger the clearance of established HPV infections and associated neoplasia, particularly in immunocompromised persons such as human immunodeficiency virus (HIV)+ patients who have higher rates of HPV-related cancers. pNGVL4a-hCRT/E6E7L2 was generated by fusing human calreticulin (CRT) to HPV16 E6 and E7 genes, each containing multiple inactivating mutations and codons optimized for high level expression, and capsid L2 protein amino acid residues 11−200. In proof-of-concept (PoC) studies in mice, vaccination with the pNGVL4a-hCRT/E6E7L2 DNA vaccine was shown to be capable of generating a strong E6/E7-specific CD8+ T cell immune response that resulted in a potent therapeutic effect against E6/E7-expressing TC-1 tumor cells. Furthermore, significant L2-specific antibodies were generated that were able to neutralize HPV 16 virions. In another PoC study in mice, vaccination with hCRTE6E7L2 DNA elicited a potent antitumor effect against E7-expressing tumors even in mice depleted for CD4+ T cells, suggesting that this vaccine may be active even in immune-compromised hosts. A head-to-head comparison of various administration methods for delivery of a DNA vaccine encoding CRT linked to HPV16 E7 (pNGVL4a-CRT/E7) was performed in a preclinical model, and tested with conventional intramuscular (IM) needle administration, electroporation (EP)-mediated IM delivery, and particle-mediated epidermal delivery via gene gun. Results showed that EP-mediated IM delivery generated higher levels of circulating protein and higher numbers of E7-specific CD8+ T cells among splenocytes compared to conventional IM injection. The enhanced CD8+ T cell immune response detected in EP-vaccinated mice translated into superior antitumor effects in E7-expressing tumors in tumor-bearing mice suggesting that EP-mediated delivery may provide an advantage compared to conventional IM delivery of the vaccine. Based on this promising preclinical data, a contract manufacturer has generated a GMP Master Cell Bank and manufactured clinical grade pNGVL4a-hCRT/E6E7L2 DNA. A toxicology study to assess the safety of pNGVL4a-hCRT/E6E7L2 DNA vaccine in mice when administered using the TriGrid Delivery System (TDS)-IM EP device (Ichor Medical Systems, Inc.) is required to support a planned clinical trial evaluating the safety and feasibility of DNA vaccination of patients with HPV16-associated cervical intraepithelial neoplasia (CIN) 2/3, as part of the Cervical Cancer SPORE program.