A Novel Paradigm for the Development of a Peptide Vaccine to Treat KRAS Mutant Cancers

摘要 RAS突变在许多人类肿瘤中非常普遍，但将这一重大发现转化为 有效的治疗方法一直很难。免疫疗法已经成为最有希望的治疗方法。 到目前为止，肿瘤学领域。抗癌疫苗提供了一种简单而直接的癌症治疗方法 免疫疗法。然而，可以追溯到30年前的抗RAS疫苗的早期努力并不成功。显然， 基于RAS的疫苗将受益于最近对肿瘤的大量了解 免疫学。我们开发了一种抗EGFRvIII多肽疫苗，在临床试验中取得了一些成功 胶质母细胞瘤。为了改进序列，我们发现加强蛋白酶体的处理显著增强了 抗肿瘤作用和结果与抗PD-1治疗有更大的协同作用。此外，这种增强的蛋白酶体 催化的多肽剪接(PCPs)导致大量抗原肽的产生，从而增加 功效。当新冠肺炎大流行爆发时，我们将所学应用于SARS-CoV-2。我们 开发了加速从大蛋白中鉴定这些表位的方法和寻找 来自长序列的PCP片段。将这一点应用于KrasG12D与wt KRAS，我们惊讶地发现 虽然这两个蛋白质有18个共同的片段，但有89个片段是KrasG12D所独有的，但wt RAS 只有13个独特的多肽与人类白细胞抗原A2结合。这表明了一个意想不到的发现：有许多 在突变的RAS中存在更多的CD8+表位，这些表位位于第12密码子附近，可以被利用为 疫苗的基础。 具体目标#1.鉴定KrasG12D产生的MHC I类结合肽 突变。在这一目标中，我们将扩大我们的工作，以确定那些与小鼠MHC H-2kb结合的多肽。 将评估蛋白酶体与免疫蛋白酶体生成的片段，以及酪氨酸是否 密码子12处的替换进一步增强了突变特异性片段的产生。我们预计将有许多多肽 因此，我们将进一步评估在MHC-I膜稳定实验中哪些多肽是高度阳性的。 通过评估哪些多肽是内源性的，将对多肽集合进行进一步的细化 由携带KrasG12D的GL261细胞提出。特异性靶点2.突变体的抗肿瘤活性和CTL活性 KRAS特异性多肽。从特定目标#1中选择的多肽将用于肿瘤疫苗接种 使用GL261细胞的实验和肿瘤的退化和存活率将被评估。免疫学研究 检测血清滴度，测定CD8+、CD4+、树突状细胞和NK细胞的数量。我们还将 分析几个检查点分子的上调。如有需要，我们会进行疫苗接种。 实验加上检查点抑制剂，看看这是否增强了抗肿瘤的效果。CTL检测将是 以确认CD8+T细胞的诱导。最后，将进行毒理学研究，以证实 这些多肽不能诱导对正常组织的自身免疫。

Abstract Ras mutations are highly prevalent in many human tumors but translating this major discovery into effective therapeutics has been difficult. Immunotherapy has emerged as the most promising approach in the field of oncology thus far. Anti-cancer vaccines offer the promise of a simple yet directed approach to cancer immunotherapy. Yet early efforts at anti-Ras vaccines, dating from 30 years ago, were not successful. Clearly, Ras based vaccines would benefit from the significant amount that has recently been learned about tumor immunology. We developed an anti-EGFRvIII peptide vaccine that showed some success in clinical trials for glioblastoma. To improve the sequence, we found enhancing proteasomal processing significantly enhances the anti-tumor effect and results in greater synergy with anti-PD-1 therapy. Moreover, this enhanced proteasome catalyzed peptide splicing (PCPS) leading to the creation of numerous antigenic peptides that increases the efficacy. When the COVID-19 pandemic struck, we applied what we learned towards SARS-CoV-2. We developed methods to accelerate the identification of these epitopes from large proteins and methods to find the PCPS fragments from lengthy sequences. Applying this to KRasG12D vs. wt KRAS, we were surprised to find that while the two proteins had 18 fragments in common, 89 fragments were unique to KRasG12D but wt Ras only had 13 unique peptides that bound to HLA-A2. This indicates the unexpected finding that there are many more CD8+ epitopes present in mutant Ras that are outside the vicinity of codon 12 that could be exploited as the basis for vaccines. Specific Aim #1. Identifying the MHC Class I binding peptides that arise from the KRasG12D mutation. In this aim, we will extend our work to identify those peptides that bind to murine MHC H-2Kb. Proteasome vs. immunoproteasome generation of fragments will be evaluated, as well as whether a tyrosine substitution at codon 12 further enhances mutant specific fragment generation. We expect many peptides will be found, so we will further evaluate which peptides are highly positive in a MHC-I membrane stabilization assay. Further refinement of the peptide set will be done by evaluating which peptides are actively endogenously presented by GL261 cells which bear KRasG12D. Specific Aim #2. Anti-tumor and CTL activity of mutant KRAS specific peptides. Selected peptides from Specific Aim #1 will be used in tumor vaccination experiments using GL261 cells and tumor regression and survival will be assessed. Immunologic studies to assay serum titers, and the number of CD8+, CD4+, dendritic and NK cells will be determined. We will also analyze for the upregulation of several checkpoint molecules. If indicated, we will perform vaccination experiments plus checkpoint inhibitors to see if this potentiates the anti-tumor effect. CTL assays will be performed to confirm induction of CD8+ T cells. Finally, toxicology studies will be performed to verify that these peptides do not induce autoimmunity against normal tissues.