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

PNGVL4a-hCRT/E6E7L2是一种针对HPV E6、E7和L2蛋白的裸露DNA疫苗，目的是开发一种能够诱导细胞免疫的治疗性疫苗，以消除HPV感染和HPV相关病变。新批准的HPV疫苗Gardasil®和Cervarx®均基于L1病毒样颗粒(VLP)，仅对预防疫苗中包括的HPV基因型造成的新生殖器感染有效。不幸的是，L1 VLP疫苗不会改变已确立的HPV感染过程，因此在几十年内不会降低HPV相关癌症的发病率；此外，它们并不能有效地对抗所有致癌的HPV类型。显然，对广泛保护性HPV疫苗的需求尚未得到满足，这种疫苗还能够清除已建立的HPV感染和相关的肿瘤，特别是在免疫功能受损的人，如人类免疫缺陷病毒(HIV)+具有较高HPV相关癌症发病率的患者。PNGVL4a-hCRT/E6E7L2是将人钙网蛋白与人乳头瘤病毒16型E6、E7基因和衣壳L2蛋白氨基酸残基11−200融合而成。在小鼠的概念验证(PoC)研究中，pNGVL4a-hCRT/E6E7L2 DNA疫苗被证明能够产生强大的E6/E7特异性CD8+T细胞免疫反应，从而对表达E6/E7的TC-1肿瘤细胞产生强大的治疗作用。此外，产生了显著的L2特异性抗体，能够中和HPV16型病毒粒子。在另一项对小鼠的PoC研究中，接种hCRTE6E7L2 DNA疫苗对表达E7的肿瘤产生了强大的抗肿瘤作用，即使在缺乏CD4+T细胞的小鼠中也是如此，这表明这种疫苗即使在免疫受损的宿主中也可能是有效的。在临床前模型中，对编码HPV16E7基因CRT的DNA疫苗(pNGVL4a-CRT/E7)的不同给药方法进行了面对面的比较，并用传统的肌肉注射(IM)针给药、电穿孔(EP)介导的IM给药和基因枪颗粒介导的表皮给药进行了测试。结果表明，与传统的IM注射相比，EP介导的IM注射在脾细胞中产生了更高水平的循环蛋白和更多的E7特异性CD8+T细胞。在EP疫苗接种的小鼠中检测到的增强的CD8+T细胞免疫反应在荷瘤小鼠的E7表达的肿瘤中转化为更好的抗肿瘤效果，这表明EP介导的递送可能比传统的IM递送疫苗提供优势。基于这些有希望的临床前数据，一家合同制造商已经生成了GMP主细胞库，并生产了临床级别的pNGVL4a-hCRT/E6E7L2 DNA。评估使用TriGrid递送系统(TDS)-IM EP设备(Ichor Medical Systems，Inc.)的pNGVL4a-hCRT/E6E7L2 DNA疫苗在小鼠中的安全性的毒理学研究作为子宫颈癌孢子计划的一部分，需要支持一项计划中的临床试验，评估HPV16相关性宫颈上皮内瘤变(CIN)2/3患者DNA疫苗的安全性和可行性。

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.