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DNA疫苗，目的是开发一种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（VLPs），仅对疫苗中包含的HPV基因型预防新的生殖器感染有效。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，它们对所有致癌的HPV类型都无效。对具有广泛保护作用的HPV疫苗的需求显然还没有得到满足，这种疫苗也能够触发清除已建立的HPV感染和相关肿瘤，特别是在免疫功能低下的人群中，如人类免疫缺陷病毒（HIV）阳性患者，他们的HPV相关癌症发病率较高。pNGVL4a-hCRT/E6E7L2是通过将human calreticulin （CRT）与HPV16 E6和E7基因融合产生的，每个基因含有多个灭活突变和高水平表达优化的密码子，衣壳L2蛋白氨基酸残基为11−200个。在小鼠的概念验证（PoC）研究中，接种pNGVL4a-hCRT/E6E7L2 DNA疫苗被证明能够产生强烈的E6/ e7特异性CD8+ T细胞免疫应答，从而对表达E6/ e7的TC-1肿瘤细胞产生有效的治疗效果。此外，产生了能够中和HPV 16病毒粒子的显著的l2特异性抗体。在另一项小鼠PoC研究中，即使在CD4+ T细胞缺失的小鼠中，接种hCRTE6E7L2 DNA也能对表达e7的肿瘤产生有效的抗肿瘤作用，这表明这种疫苗即使在免疫功能低下的宿主中也可能有效。在临床前模型中，对HPV16 E7 （pNGVL4a-CRT/E7）编码CRT的DNA疫苗的不同给药方法进行了头对头的比较，并对传统的肌肉注射（IM）针给药、电穿孔（EP）介导的IM给药和基因枪介导的颗粒介导的表皮给药进行了测试。结果表明，与常规IM注射相比，ep介导的IM递送在脾细胞中产生更高水平的循环蛋白和更多数量的e7特异性CD8+ T细胞。在ep接种小鼠中检测到的增强的CD8+ T细胞免疫应答转化为在荷瘤小鼠中表达e7的肿瘤中具有优越的抗肿瘤作用，这表明ep介导的递送可能比传统的IM递送具有优势。基于这一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，并制造了临床级pNGVL4a-hCRT/E6E7L2 DNA。pNGVL4a-hCRT/E6E7L2 DNA疫苗在使用TriGrid递送系统(TDS)-IM EP设备（Ichor Medical Systems, Inc.）给药时，需要进行毒理学研究，以评估小鼠的安全性，以支持计划中的临床试验，评估hpv16相关宫颈上皮内瘤（CIN） 2/3患者DNA疫苗接种的安全性和可行性，作为宫颈癌SPORE项目的一部分。

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.