Comparing Neoantigen Cancer Vaccines and Immune Checkpoint Therapy Unveils an Effective Vaccine and Anti-TREM2 Macrophage-Targeting Dual Therapy [neoAgSpCD8Tcells]

The goal of therapeutic cancer vaccines and immune checkpoint therapy (ICT) is to eliminate cancer by expanding and/or sustaining T cells with anti-tumor capabilities. Here, we compared effective therapeutic tumor-specific mutant neoantigen (NeoAg) synthetic long peptide (SLP) cancer vaccines with anti-CTLA-4 and/or anti-PD-1 ICT in preclinical models. Effective NeoAg SLP vaccines and ICT required both CD8 and CD4 T cells. Both NeoAg SLP vaccines and ICT induce expansion of intratumoral NeoAg-specific CD8 T cells, though the degree of expansion and acquisition of effector activity was much more substantial following NeoAg SLP vaccination. Further, we found that NeoAg SLP vaccines are particularly adept at inducing proliferating and stem-like NeoAg-specific CD8 T cells. While NeoAg SLP vaccines and anti-PD-1 affected the CD4 T cell compartment, it was to less of an extent than observed with anti-CTLA-4, which notably induced ICOS+Bhlhe40+ Th1-like CD4 T cells. Although effective NeoAg SLP vaccines or ICT expanded intratumoral M1-like iNOS+ macrophages, NeoAg SLP vaccines maintained, rather than suppressed as observed with ICT, M2-like CX3CR1+CD206+ macrophages expressing the TREM2 receptor. While combining NeoAg SLP vaccination with ICT induced superior efficacy compared to either therapy in isolation, we also assessed a novel combination of NeoAg SLP vaccination and anti-TREM2, demonstrating enhanced efficacy of this combination associated with a decrease in intratumoral CX3CR1+CD206+ macrophages and promotion of IFN-g+ NeoAgspecific CD8 T cells. These findings highlight the utility of combining NeoAg SLP vaccines with ICT, as well as novel combinatorial therapy targeting the myeloid compartment via TREM2 blockade to enhance NeoAg SLP vaccine efficacy. Droplet-based 5′ end massively parallel single-cell RNA sequencing was performed by encapsulating sorted mLama4 Tetramer positive CD8 T cells (NeoAg-specific CD8 T cells) into droplets and preparing libraries using Chromium Single Cell 5′ Reagent Kits following the manufacturer’s protocol (10x Genomics). The generated scRNAseq libraries were sequenced using a NovaSeq 6000 Sequencing System (Illumina).

治疗性癌症疫苗与免疫检查点治疗（immune checkpoint therapy, ICT）的目标是通过扩增和/或维持具备抗肿瘤功能的T细胞以清除肿瘤。本研究在临床前模型中，对比了有效的肿瘤特异性突变新抗原（tumor-specific mutant neoantigen, NeoAg）合成长肽（synthetic long peptide, SLP）癌症疫苗与抗CTLA-4和/或抗PD-1免疫检查点治疗的效果。有效的NeoAg SLP疫苗与免疫检查点治疗均需要CD8和CD4 T细胞的参与。NeoAg SLP疫苗与免疫检查点治疗均可诱导瘤内NeoAg特异性CD8 T细胞的扩增，但NeoAg SLP疫苗接种后，该扩增程度以及效应功能的获得均更为显著。进一步研究发现，NeoAg SLP疫苗尤其擅长诱导增殖性及干细胞样的NeoAg特异性CD8 T细胞。尽管NeoAg SLP疫苗与抗PD-1治疗均可对CD4 T细胞亚群产生影响，但其影响程度远低于抗CTLA-4治疗——后者可显著诱导ICOS+Bhlhe40+ Th1样CD4 T细胞。虽然有效的NeoAg SLP疫苗或免疫检查点治疗均可扩增瘤内M1样iNOS+巨噬细胞，但与免疫检查点治疗的作用不同，NeoAg SLP疫苗可维持而非抑制表达TREM2受体的M2样CX3CR1+CD206+巨噬细胞。与单一疗法相比，NeoAg SLP疫苗联合免疫检查点治疗可产生更优异的治疗效果；此外本研究还评估了NeoAg SLP疫苗与抗TREM2的新型联合方案，结果显示该联合疗法可通过减少瘤内CX3CR1+CD206+巨噬细胞，并促进IFN-γ+ NeoAg特异性CD8 T细胞的生成，从而增强治疗效果。本研究结果凸显了将NeoAg SLP疫苗与免疫检查点治疗联合应用的价值，同时也为通过TREM2阻断靶向髓系细胞亚群以强化NeoAg SLP疫苗疗效的新型联合疗法提供了依据。本研究通过将分选得到的mLama4四聚体阳性CD8 T细胞（即NeoAg特异性CD8 T细胞）包裹入微液滴中，并依照制造商方案使用Chromium单细胞5'端试剂试剂盒（Chromium Single Cell 5′ Reagent Kits）构建文库，完成了基于液滴的5'端大规模并行单细胞RNA测序（droplet-based 5′ end massively parallel single-cell RNA sequencing, scRNAseq）。构建得到的scRNAseq文库使用NovaSeq 6000测序系统（NovaSeq 6000 Sequencing System, Illumina）进行测序。