PO.CL05.13 · 临床研究
利用表达双特异性巨噬细胞接合器的溶瘤腺病毒促进肿瘤抗原的非特异性交叉呈递
Promoting improved agnostic cross presentation of tumor antigens with an oncolytic adenovirus expressing bispecific macrophage engagers
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摘要 Abstract
中文摘要
背景：肿瘤抗原（TAA）的非特异性（“泛特异性”）交叉呈递对于产生针对癌症的适应性免疫应答至关重要。抗原呈递细胞（APC）通过吞噬癌细胞的蛋白质摄取TAA，并将其呈递在HLA分子上以激活T细胞。为诱导APC对癌细胞的摄取，我们旨在开发一种双特异性巨噬细胞接合器（BiME），它能使癌细胞和APC相互靠近，并假设可利用溶瘤病毒在肿瘤微环境中表达BiME。
方法：我们构建了一种表达BiME的溶瘤腺病毒（Ad-BiME），其设计能够结合癌细胞上的肿瘤相关抗原和APC上的激活性受体，从而促进相互作用并触发APC激活。所表达的双特异性蛋白能高效结合APC和癌细胞上的配体。共培养试验评估了吞噬作用、巨噬细胞激活及抗原交叉呈递。为分析交叉呈递，我们构建了表达NY-ESO-1的HLA-A2阴性癌细胞，并将其与HLA错配的HLA-A2阳性APC共培养。经NY-ESO-1(157-165)肽负载的HLA-A2特异性1G4 TCR原代T细胞检测交叉呈递。最后，使用新鲜解剖的结直肠肿瘤和正常结肠活检组织评估癌症特异性病毒复制、BiME生成及巨噬细胞激活。
结果：BiME显著增强了巨噬细胞对癌细胞的吞噬作用，超过了经临床测试的抗CD47抗体。它进一步激活了巨噬细胞，上调了CD86和CD80的表达。值得注意的是，Ad-BiME诱导了最强的巨噬细胞激活。BiME治疗后巨噬细胞对肿瘤抗原的交叉呈递明显增加。离体实验表明，溶瘤病毒仅在结直肠肿瘤活检组织中复制并产生BiME，在这些组织中诱导了内源性巨噬细胞激活，而在正常组织中则不然。仅在肿瘤样本中检测到IL-1beta、TNF、IL-6、CXCL10和IFN-gamma升高，表明BiME的表达具有靶向性。有趣的是，游离BiME治疗明显增加了CCL17，这是一种吸引调节性T细胞（Treg）的趋化因子。相比之下，Ad-BiME并未升高CCL17，这可能是由于BiME在原位逐步生成。整体RNA分析进一步揭示，Ad-BiME独特地上调了与抗原交叉呈递和T细胞趋化相关的巨噬细胞通路。
结论：我们构建了首个表达BiME的溶瘤病毒，并证明了其作为一种有前景的机制，在开发针对实体瘤的癌症疫苗中实现抗原非特异性交叉呈递的潜力。
查看英文原文 English abstract
Background: Non-specific (‘agnostic') cross-presentation of tumor antigens (TAA) is essential for generating adaptive immune responses against cancer. APCs uptake TAA by phagocytozing proteins from cancer cells and present them on HLA molecules to activate T cells.To induce cancer cell uptake by the APCs, we aimed develop a bispecific macrophage engager (BiME) that can bring cancer cells and APCs into close proximity, and we hypothesized that oncolytic viruses could be used to express BiMEs in the tumor microenvironment.
Methods: We generated an oncolytic adenovirus expressing a BiME (Ad-BiME) designed to bind a tumor-associated antigen on cancer cells and an activating receptor on APCs, thereby promoting interaction and triggering APC activation. The expressed bispecific protein efficiently bound ligands on both APCs and cancer cells. Co-culture assays evaluated phagocytosis, macrophage activation, and antigen cross-presentation. To analyse cross-presentation, we engineered NY-ESO-1-expressing HLA-A2-negative cancer cells and co-cultured them with HLA-mismatched HLA-A2-positive APCs. NY-ESO-1(157-165) peptide-loaded HLA-A2-specific 1G4 TCR primary T cells detected cross-presentation. Finally, freshly dissected colorectal tumor and normal colon biopsies were used to assess cancer-specific virus replication, BiME generation, and macrophage activation.
Results: The BiME significantly enhanced phagocytosis of cancer cells by macrophages, surpassing the clinically tested anti-CD47 antibody. It further activated macrophages, upregulating CD86 and CD80 expression. Notably, Ad-BiME induced the strongest macrophage activation. Tumor antigen cross-presentation by macrophages was markedly increased following BiME treatment. Ex vivo experiments showed that oncolytic viruses replicated and produced BiME exclusively in colorectal tumor biopsies, where they induced endogenous macrophage activation, unlike in normal tissues. Elevated IL-1beta, TNF, IL-6, CXCL10, and IFN-gamma were detected only in tumor samples, indicating targeted BiME expression. Interestingly, free BiME treatment markedly increased CCL17, a chemokine attracting regulatory T cells (Tregs). In contrast, Ad-BiME did not elevate CCL17, likely due to gradual in situ BiME generation. Bulk RNA analysis further revealed that Ad-BiME uniquely upregulated macrophage pathways linked to antigen cross-presentation and T-cell chemotaxis.
Conclusion: We generated the first oncolytic virus expressing a BiME and demonstrated its potential as a promising mechanism for antigen-agnostic cross-presentation in developing cancer vaccines for solid tumors.
利益披露 Disclosure
A. Hazini, None..
M. Rei, None..
N. Cartwright, None..
R. A. Fernandes, None..
K. Fisher, None..
L. Seymour, None.