肿瘤特异性蛋白B7-H4纳米抗体的筛选和鉴定

羊驼体内存在天然缺少轻链的重链抗体,克隆重链抗体可变区(VHH),即可构建单域抗体(single-domain antibodies,sdAbs),又称纳米抗体(nanobody,Nb)。利用非免疫羊驼噬菌体文库筛选肿瘤特异性蛋白B7-H4的纳米抗体,经过4轮淘选,ELASE鉴定阳性克隆噬菌体,测序获得其DNA序列后体外转录为mRNA,将修饰纯化后的mRNA转染到肿瘤细胞,利用细胞免疫荧光检测mRNA在肿瘤细胞内是否表达,Western印迹进一步验证其表达情况;通过CCK-8法鉴定其对肿瘤细胞的增殖抑制能力;划痕实验鉴定其对肿瘤细胞迁移能力的影响;Transwell法鉴定其对肿瘤细胞的侵袭能力的影响;裸鼠荷瘤模型瘤旁注射mRNA,鉴定其在体内实验对肿瘤组织的作用。结果显示,通过淘选获得1个高亲和性的噬菌体株菌H6;DNA测序并导出的氨基酸序列符合羊驼纳米抗体结构;将其mRNA导入肿瘤细胞,能有效表达出纳米抗体H6;转染H6-mRNA的肿瘤细胞(0.84±0.08)与未转染H6-mRNA的对照组(1.83±0.04)相比,其增殖能力明显受到抑制,P<0.01,其迁移和侵袭能力(78.60±5.36)明显低于空白对照组(197.80±21.04),效果优于B7-H4 mRNA的siRNA(95.40±16.56);在裸鼠乳腺癌模型中能有效抑制肿瘤生长,效果优于紫杉醇和B7-H4 mRNA的siRNA。这说明筛选所得抗B7-H4纳米抗体H6能特异结合B7-H4蛋白并封闭其功能,导致肿瘤细胞的增殖、迁移和侵袭受到抑制。该结果为利用B7-H4作为治疗癌症的靶点提供了实验基础。     

Pharmacologic modulation of RNA splicing enhances anti-tumor immunity

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High-dimensional Cytometry (ExCYT) and Mass Spectrometry of Myeloid Infiltrate in Clinically Localized Clear Cell Renal Cell Carcinoma Identifies Novel Potential Myeloid Targets for Immunotherapy

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Highlights

• •Using ExCYT, genomics, and Mass Spectrometry, we were able to uncover immune cell marker alterations that provide new insight into the biology of early stage ccRCC.
• •Among the CD45⁺ population, we observed a high level of myeloid cell infiltration in treatment-naïve ccRCC tissues.

     

Advanced Liposomal Vectors as Cancer Vaccines in Melanoma Immunotherapy

Malignant tumors represent a major source of disability and account for more than one of five deaths in Western countries. Among the different cancers, melanoma harbors two distinctive features. First, its has long been recognized as an immunogenic tumor, and second, an unprecedented rise in incidence is currently observed, in face of few therapeutic options. Thus, melanoma represent an ideal target for a cancer immunotherapy program. To date, a number of immunodominant epitopes from tumor associated antigens (TAA) are used as cancer vaccines in clinical trials, in spite of an acknowledged rapid degradation in vivo and low immunogenicity. However, most of the immunotherapy trials reported so far do not achieve consistent clinical results. Hence, there is an urgent need for the development of a carrier system and strong adjuvants suitable for a TAA-based cancer immunotherapy. Liposomes and their further development as virosomes with added adjuvancy may address both these issues. We report here our experience in the tailoring of dedicated advanced liposomal vectors that were developed in the context of an upcoming immunotherapy clinical trial for melanoma.     

Advanced therapeutic modalities in hepatocellular carcinoma: Novel insights

Hepatocellular carcinoma (HCC), the most common type of liver cancer, is usually a latent and asymptomatic malignancy caused by different aetiologies, which is a result of various aberrant molecular heterogeneity and often diagnosed at advanced stages. The incidence and prevalence have significantly increased because of sedentary lifestyle, diabetes, chronic infection with hepatotropic viruses and exposure to aflatoxins. Due to advanced intra- or extrahepatic metastasis, recurrence is very common even after radical resection. In this paper, we highlighted novel therapeutic modalities, such as molecular-targeted therapies, targeted radionuclide therapies and epigenetic modification-based therapies. These topics are trending headlines and their combination with cell-based immunotherapies, and gene therapy has provided promising prospects for the future of HCC treatment. Moreover, a comprehensive overview of current and advanced therapeutic approaches is discussed and the advantages and limitations of each strategy are described. Finally, very recent and approved novel combined therapies and their promising results in HCC treatment have been introduced.     

Effect of intraperitoneal administration of granulocyte/macrophage-colony-stimulating factor in rats on omental milky-spot composition and tumoricidal activity in vivo and in vitro

 Milky spots in the greater omentum are small accumulations of leucocytes that consist mainly of macrophages and have recently shown to be a selective dissemination site of intraperitoneal (i. p.) inoculated tumour cells. However, milky-spot macrophages show tumoricidal activity and may, therefore, be an excellent source of effector cells suited for local immunotherapy. In the present study we first examined whether granulocyte/macrophage- colony-stimulating factor (GM-CSF) treatment of isolated milky-spot macrophages affects the cytotoxicity against syngeneic colon carcinoma cells (CC531) in vitro. Secondly, we studied the influence of intraperitoneal GM-CSF administration on the number and antitumour activity of milky-spot and peritoneal macrophages. All studies were performed in Wag/Rij rats in which a syngeneic colon carcinoma cell line (CC531) is available. The results of the in vitro study showed that GM-CSF treatment of the omental macrophages led to an increased cytotoxicity against the tumour cell line. Intraperitoneal administration of 1000 U GM-CSF daily for 7 consecutive days demonstrated both an enhanced antitumour activity of the milky-spot macrophages and an increase in the milky-spot macrophage population. An increase in the proliferative capacity, according to bromodeoxyuridine incorporation, was shown in the milky-spot macrophages. Taking into account both the enhanced macrophage number and their enhanced activity upon i. p. GM-CSF treatment, the milky-spot macrophages may provide a rationale for local intraperitoneal immunotherapy in the prevention of intra-abdominal tumour growth. Received: 11 April 1996 / Accepted: 21 May 1996     

外泌体在白血病中的重要调控作用

目前,白血病复发是患者死亡的主要原因之一。肿瘤细胞和微环境的相互作用,以及隐匿在骨髓中的肿瘤干细胞,促进了白血病的复发和向淋巴组织的转移,因此白血病的治疗、转移和复发问题受到广泛关注。外泌体是由绝大多数细胞分泌的双层脂质膜囊泡,可以调控细胞间的交流和信息传递。在白血病细胞、基质细胞和内皮细胞之间的相互联系中都涉及到外泌体,白血病细胞来源的外泌体存在于白血病患者的血浆中,能把其携带的白血病相关抗原及微小RNA呈递给靶细胞,促进白血病肿瘤细胞的增殖,有助于肿瘤细胞实现免疫逃避,保护白血病细胞抵抗化疗药物导致的细胞毒性作用,促进血管生成及肿瘤细胞的迁移。因此,外泌体与白血病的转移、治疗及预后密切相关,可以用来检测和监测白血病的进展。本文综述了外泌体的来源、形成与分泌机制,以及外泌体在白血病发生前、发展中、预后和免疫治疗中所扮演的重要角色。     

免疫检查点疗法：战胜癌症的革命性突破 ——写在2018年诺贝尔生理学或医学奖颁布之际

美国免疫学家詹姆斯·艾利森(James P．Allison)与日本免疫学家本庶佑(Tasuku Honjo)因在免疫检查点治疗方面的贡献而获得了2018年诺贝尔生理学或医学奖．这一发现为免疫治疗开启了一扇新的大门．本文回顾了免疫检查点CTLA-4和PD-1的研究历史,免疫检查点药物的研发和应用进展以及免疫检查点疗法在国内的发展现况,提出了免疫检查疗法目前存在的局限性和解决方法．随着近年来我国在免疫治疗领域巨大的资金投入、一流基础研究平台的建设和优秀人才的回国使得我国在这一领域硕果累累,相信在不久的将来我国的免疫检查点抑制剂将会走出国门,为全人类的癌症事业做出贡献．     

A multicomponent approach in the discovery of indoleamine 2,3-dioxygenase 1 inhibitors: Synthesis,biological investigation and docking studies

Indoleamine 2,3-dioxygenase plays a crucial role in immune tolerance and has emerged as an attractive target for cancer immunotherapy. In this study, the Passerini and Ugi multicomponent reactions have been employed to assemble a small library of imidazothiazoles that target IDO1. While the p-bromophenyl and the imidazothiazole moieties have been kept fixed, a full SAR study has been performed on the side-chain, leading to the discovery of nine compounds with sub-micromolar IC₅₀ values in the enzyme-based assay. Compound 7d, displaying a α-acyloxyamide substructure, is the most potent compound, with an IC₅₀ value of 0.20?µM, but a low activity in a cell-based assay. Compound 6o, containing a α-acylaminoamide moiety, shows an IC₅₀ value of 0.81?µM in the IDO1-based assay, a full biocompatibility at 10?µM, together with a modest inhibitory activity in A375 cells. Molecular docking studies show that both 7d and 6o display a unique binding mode in the IDO1 active site, with the side-chain protruding in an additional pocket C, where a crucial hydrogen bond is formed with Lys238. Overall, this work describes an isocyanide based-multicomponent approach as a straightforward and versatile tool to rapidly access IDO1 inhibitors, providing a new direction for their future design and development.