新生抗原疫苗或原位疫苗對肝癌治療效果的研究

Abstract

在我們的研究中，我們正努力尋找新的治療策略，以提升對肝細胞癌（Hepatocellular carcinoma, HCC） 現有治療方法的效果。我們評估了新抗生原疫苗的效果，無論是單獨使用或與抗PD-1 (anti-programmed cell death protein-1，anti-PD-1) 一起使用。首先以全外顯子定序 (whole exome sequencing) 在癌細胞與正常小鼠肝臟進行了比對找出變異序列，重點是頻率較高的非同義變體 （non-synonymous mutations，NSMs）。通過RNA-seq確認了它們的表達，並分析它們的MHC-II結合親和力。排名前20的新生抗原胜肽被合成並在小鼠上進行了 IFN-γ酶聯免疫斑點 （ELIspot） 和流式細胞儀 (flow cytometry) 的測試。挑出最具免疫原性的胜肽 (immunogenic peptides) 作為疫苗，並在皮下 (subcutaneous) 、原位 (orthotopic) 和轉移性肝 (metastatic liver) 三種小鼠HCC模型進行測試。我們比較了對照組、抗PD-1組、肝癌特異性新抗原疫苗組和疫苗與抗PD-1組合組的結果。 在HCC模型中，只有通過新抗原疫苗接種和抗PD-1療法的組合才能實現腫瘤的明顯縮小，這也建立了免疫記憶。雖然新抗原疫苗接種引發了腫瘤特異性免疫反應，但它作為一種單獨的治療方法並不有效。聯合治療和疫苗接種都重塑了腫瘤微環境，增加了CD8+ T細胞和顆粒酶B的表達，同時減少了CD8+T細胞的耗竭。 NanoString分析顯示兩組的免疫細胞數量得分與免疫相關基因表達趨勢相似。然而，聯合療法獨特地減少了Treg和MDSC細胞，活化了更多的免疫途徑。此外，疫苗組在CD8+ T細胞上有最高的PD-1表達。我們假設，抗PD-1組缺乏反應可能是由於CD8+ T細胞不足，而疫苗組的無效可能是由於PD-1的高表達、MDSC細胞的抑制或其他因素阻礙了CD8+ T細胞對腫瘤的細胞毒性。 我們的新抗原疫苗是基於在Hep-55.1C和Dt81 Hepa1-6腫瘤細胞中發現的NSMs來設計的。為了降低後續分析的成本，我們對大量樣本進行了篩選和過濾。不幸的是，NSMs僅佔腫瘤細胞所有序列變異的一部分，這讓我們不得不擴大考慮範疇，考慮其他的免疫原變異，如插入和刪除 （insertions and deletions，INDELs） 以及基因融合 （gene fusions） 。由於抗原的免疫原性無法預測，蛋白質合成的高成本，以及分析過程的耗時，使得這個過程相當具有挑戰性。 在我們尋找解決方案的過程中，我們探索了各種治療策略，並認為原位免疫接種 （in Situ Vaccination，ISV） 具有前景。我們使用一種稱為poly-ICLC的疫苗佐劑 (vaccine adjuvant)，這是一種TLR激動劑 (Toll-like receptor agonists)，直接注射入腫瘤以啟動"免疫週期" (immune cycle) 。這種方法通過poly-ICLC刺激腫瘤部位，可能導致腫瘤細胞溶解，抗原釋放，以及抗原呈現細胞功能的增強，從而引發隨後的後天性免疫反應來攻擊腫瘤細胞。此外，這種方法的一個顯著優點是，它不需要時間來識別潛在的腫瘤抗原。 我們在Hep-55.1C細胞誘導的皮下腫瘤小鼠中測試了瘤內注射 (Intratumoral，IT) 、肌肉注射 (Intramuscular，IM) 和序列性注射 (sequential injection，先IT接著IM) poly-ICLC。聯合注射顯示出最大的腫瘤抑制作用，腫瘤浸潤的CD8+ T細胞增加了四倍，產生了強大的腫瘤特異性和全身免疫力，從而產生了遠端效應 (abscopal effect)。這一結果依賴於CD8+ T細胞，因為去除它們會導致腫瘤不受控制地生長。 總結本論文研究，新生抗原疫苗和抗PD-1治療的結合使用在治療三種不同模型的小鼠肝癌中展現出相當大的有效性。這主要是通過增強腫瘤特異性CD8+ T細胞和減少免疫抑制細胞來實現的。此外，本研究中使用的序列性poly-ICLC注射治療能明顯增加了腫瘤特異性CD8+ T細胞的數量，增強其浸潤HCC腫瘤組織中的能力並引發遠端效應。這些發現有助於推動將新生抗原疫苗與抗PD-1或採用ISV的方式將poly-ICLC用於治療HCC的臨床試驗。

Our research aimed to identify novel therapeutic strategies to improve the efficacy of existing treatments for hepatocellular carcinoma (HCC). We evaluated the efficacy of neoantigen vaccines, both individually and in conjunction with anti-PD-1, in mouse liver cancer models of Hep-55.1C and Dt81 Hepa1-6. Firstly, whole exome sequencing (WES) was carried out in cancer cells and normal mouse liver to identify variant sequences, focusing on the higher frequency of non-synonymous mutations (NSMs). Their expression was confirmed by RNA-seq, and their MHC-II binding affinity was analyzed. Top 20 neoantigen peptides were synthesized and tested on mice using IFN-γ enzyme-linked immunospot (ELIspot) and flow cytometry. Selected the most immunogenic peptides for vaccines and tested on three HCC mouse models (subcutaneous, orthotopic, and metastatic liver tumor). We compared results among control, anti-PD-1, vaccine, and a combination group. In HCC models, only a combination of neoantigen vaccination and anti-PD-1 therapy achieved notable tumor reduction and built immune memory. Solo vaccination-initiated tumor-specific immunity but was insufficient. Both the combined therapy and vaccination reshaped the tumor microenvironment, increasing CD8+ T cells and granzyme B expression while decreasing CD8+ T cell exhaustion. NanoString analysis revealed similar immune cell score and inflammation trends in both groups. However, the combined therapy uniquely reduced Treg and MDSC cells and activated more immune pathways. Moreover, the vaccine group had the highest PD-1 expression on CD8+ T cells. We hypothesized that the lack of response in the anti-PD-1 group might be due to inadequate CD8+ T cells, and ineffectiveness in the vaccine group could be due to high PD-1 expression, MDSC cell inhibition, or other factors hindering CD8+ T cell cytotoxicity against tumors. Our neoantigen vaccine was designed based on the unique NSMs found in Hep-55.1C and Dt81 Hepa1-6 tumor cells. To reduce subsequent analysis costs, we filtered and screened a multitude of samples. Unfortunately, the fact that NSMs made up a fraction of all sequence variations in tumor cells necessitated us to look beyond, considering other immunogenic variants like base insertions and deletions (INDELs) and gene fusions. However, the process was quite challenging due to the unpredictable immunogenicity of antigens, the high cost of protein synthesis, and the time-consuming nature of the analysis process. In our quest for a solution, we explored various therapeutic strategies and identified in situ vaccination (ISV) as promising. We used a vaccine adjuvant called poly-ICLC, which was a Toll-like receptor agonist, injecting it directly into the tumor to initiate the "immune cycle ". This approach stimulated the tumor site with poly-ICLC, potentially leading to tumor cell dissolution, antigen release, and the enhancement of antigen-presenting cell function, thereby instigating a subsequent adaptive immune response to attack the tumor cells. Moreover, a significant advantage of this method was that it did not require taking time to identify potential tumor neoantigens. We further tested if the administration method would affect poly-ICLC’s efficacy, and applied intratumoral (IT), intramuscular (IM), and sequential (IT and IM) injections in subcutaneous tumor mice induced by Hep-55.1C cells. The combined injection group showed the greatest tumor inhibition and a fourfold increase in tumor-infiltrating CD8+ T cells, generating a strong tumor-specific and systemic immunity that triggered an abscopal effect. This result relied on CD8+ T cells, as their removal led to uncontrolled tumor growth. In conclusion, the combination of neoantigen vaccination and anti-PD-1 treatment has demonstrated significant effectiveness in curing three different models of mouse liver cancer. This was primarily achieved by enhancing the presence of tumor-specific CD8+ T cells and reducing immunosuppressive cells. Furthermore, the sequential poly-ICLC treatment utilized in this study markedly increased the number of tumor-specific CD8+ T cells, thereby improving their ability to infiltrate HCC tumor tissues and triggering an abscopal effect. These findings advocated for clinical trials that combined neoantigen vaccines with anti-PD-1, or ISV with poly-ICLC, for HCC treatment.