目的：本课题计划构建一种新型纳米药物，用于提升抗肿瘤免疫效应。免疫检查点抑制剂（Immune checkpoint blockade, ICB）是近些年肿瘤治疗中的一个重大突破，对多种恶性肿瘤都产生了显著的临床效果，因此，探索新的技术策略以进一步提高ICB的疗效，具有较为重大的潜在医学价值。除抗体外，核酸适配体（Aptamer, Apt）也能够特异性地结合并阻断免疫检查点，提升抗肿瘤免疫作用。目前已有研究报道了针对CTLA-4的DNA aptamer，在细胞和动物水平上均显示出一定的抗肿瘤疗效。肿瘤免疫微环境（Tumor immune microenvironment, TIME）是影响抗肿瘤免疫效果的重要因素。将ICB疗法与能够调节TIME的药物联用，有希望进一步改善ICB的疗效。非索非那定（Fexofenadine, FEXO）是一种组胺受体1（Histamine receptor H1, H1R）的阻断剂，能够通过调节TIME以增强抗肿瘤免疫反应。基于上述背景，本课题计划构建一种表面连有CTLA-4 aptamer且内部包载FEXO的白蛋白纳米颗粒（Apt-NP-FEXO），并评估该纳米药物在体外及体内的抗肿瘤疗效。

        方法：将巯基修饰的CTLA-4 aptamer与白蛋白通过sulfo-SMCC相连，形成Apt-BSA，利用琼脂糖凝胶电泳、SDS-PAGE和zeta电位测定的方法，评估Apt-BSA是否形成；通过对先前报道的方法加以改进，采用自组装的方式，分别制备白蛋白纳米颗粒（NP）、CTLA-4 aptamer修饰的白蛋白纳米颗粒（Apt-NP）、包载FEXO的白蛋白纳米颗粒（NP-FEXO）、和表面连有CTLA-4 aptamer且内部包载FEXO的白蛋白纳米颗粒（Apt-NP-FEXO），利用动态光散射（Dynamic light scattering, DLS）测定上述纳米颗粒的粒径及zeta电位，利用透射电子显微镜（Transmission electron microscope, TEM）观察纳米颗粒的形态；通过高效液相色谱（High performance liquid chromatography, HPLC）评估NP-FEXO、Apt-NP-FEXO的载药率、包裹率及FEXO的释放情况；通过流式细胞术、荧光显微镜和共聚焦显微镜评估CTLA-4修饰的纳米颗粒能否结合CTLA-4阳性的细胞；通过CFSE标记的混合淋巴细胞反应（CFSE-labelled mixed lymphocyte reaction，CFSE-MLR），评估CTLA-4 aptamer修饰的纳米颗粒对淋巴细胞增殖的影响；通过MTS实验，在体外评估游离aptamer及各纳米颗粒是否影响淋巴细胞对肿瘤细胞的杀伤；通过小鼠肿瘤模型，评估各纳米颗粒在体内的抗肿瘤疗效。

       结果：巯基修饰的CTLA-4 aptamer能够与白蛋白连接形成Apt-BSA，并成功制备出NP、Apt-NP、NP-FEXO和Apt-NP-FEXO，这些纳米颗粒的平均粒径分别为117.8 nm、149.0 nm、139.3 nm和159.0 nm，其zeta电位分别为 -12 mV、-18.5 mV、-0.371 mV 和-5.36 mV。所有纳米颗粒均为球形。NP-FEXO、Apt-NP-FEXO对FEXO的包裹率分别为31.06%、32.75%，且均具有药物缓释作用。通过流式细胞术、荧光显微镜及共聚焦显微镜观察到，CTLA-4 aptamer修饰的纳米颗粒能够与CTLA-4阳性的细胞相结合。CFSE-MLR实验发现，与NP相比，Apt-NP、Apt-NP-FEXO能够在一定程度上促进淋巴细胞的增殖。MTS实验发现，与游离的CTLA-4 aptamer类似，Apt-NP和Apt-NP-FEXO能够显著提高淋巴细胞对肿瘤细胞的杀伤作用。动物实验显示，与游离的aptamer相比，Apt-NP能够显著增强对肿瘤的抑制作用；而与Apt-NP相比，Apt-NP-FEXO能够进一步显著提升抗肿瘤疗效，且未显示出明显的毒副作用。

       结论：本课题将ICB疗法与能够调节TIME的药物联用，构建了一种表面连有CTLA-4 aptamer、内部包载FEXO的白蛋白纳米颗粒（Apt-NP-FEXO），能够显著增强抗肿瘤疗效，在肿瘤的免疫治疗中具有一定的应用潜能。

        Purpose: Immune checkpoint blockade (ICB) is a promising strategy for cancer treatment and has generated remarkable clinical results against multiple malignancies. Exploration of new technical approaches to further boost the therapeutic efficacy of ICB is therefore of potential medical importance. In addition to antibodies, other newly developed ligands, including aptamers, may also serve as alternative ICB agents. A previously developed CTLA-4-antagonizing DNA aptamer can significantly inhibit tumor growth in animal models. It has been recognized that tumor immune microenvironment (TIME) is play an important role in anticancer immunity. Hence, it is reasonable to combine ICB therapeutics with TIME-modulating agents. Fexofenadine, a histamine receptor H1 (H1R) blocker, appears to modulate TIME and facilitate antitumor immunity by inhibiting the polarization of macrophages to M2 phenotype. Based on the above information, in this study, a novel nanotherapeutics (Apt-NP-FEXO) was constructed by encapsulating FEXO into albumin nanoparticles that were functionalized with CTLA-4 aptamers. So far as we know, this is the first report on using nanotechnology with FEXO to enhance the therapeutic function of CTLA-4 blockade.

Methods: Thiol-modified CTLA-4 aptamer was linked to the amino groups of BSA via sulfo-SMCC to form Apt-BSA. To evaluate whether the CTLA-4 aptamer was conjugated with BSA to form Apt-BSA, agarose gel electrophoresis, SDS-PAGE, and DLS were performed. NP, Apt-NP, NP-FEXO, and Apt-NP-FEXO were fabricated with a modified self-assembling method reported previously. The average sizes and zeta potentials of nanoparticles were determined by DLS. The morphology of nanoparticles was observed by TEM. To estimate the drug loading efficiency, encapsulation efficiency, and FEXO release pattern, HPLC was conducted. To evaluate whether the aptamer-modified NP could still recognize its target, the bindings of Apt-NP or NP to CTLA-4 positive cells were evaluated by flow cytometry, fluorescent microscopy and confocal microscopy. CFSE-MLR was applied to evaluate the proliferation of lymphocytes treated by CTLA-4 aptamer and aptamer-modified NPs. MTS assay was used to study whether the CTLA-4 aptamer or aptamer-modified NPs could boost lymphocyte-mediated cytotoxicity.  Animal studies were performed to assess in vivo antitumor efficacy of the nanoparticles.

       Results: CTLA-4 aptamer was conjugated with BSA, forming Apt-BSA. The average sizes of the nanoparticles were as follows: NP (117.8 nm), Apt-NP (149.0 nm), NP-FEXO (139.3 nm), and Apt-NP-FEXO (159.0 nm). The apparent zeta potentials of the nanoparticles were as follows: NP (-12 mV), Apt-NP (-18.5 mV), NP-FEXO (-0.371 mV), and Apt-NP-FEXO (- 5.36 mV). The FEXO encapsulation efficiencies were 31.06% for NP-FEXO and 32.75% for Apt-NP-FEXO. Both FEXO-encapsulating NPs exhibited typical sustained drug release profiles. CTLA-4 aptamer modified NP could bind with CTLA-4 expressing cells, and stimulate the proliferation of lymphocytes to some extent. Similar to free CTLA-4 aptamer, both Apt-NP and Apt-NP-FEXO remarkably enhanced PBMC-mediated cytotoxicity against the tumor cells. Animal studies revealed that although free CTLA-4 aptamers suppressed tumor growth to some extent, Apt-NP generated a more prominent antitumor effect. Notably, Apt-NP-FEXO further enhanced tumor suppression vs. Apt-NP, and did not generate extra systemic toxicity in vivo.

       Conclusion: In this study, we combined ICB therapeutics with TIME-modulating agents and designed a novel nanotherapeutics (Apt-NP-FEXO) for co-delivery of FEXO and CTLA-4 aptamers. Apt-NP-FEXO significantly improved the antitumor efficacy in vivo, and may have application potential in cancer immunotherapy.