Novel Complex of PD-L1 Aptamer and Holliday Junction Enhances Antitumor Efficacy in Vivo

Blocking the PD-1/PD-L1 pathway can diminish immunosuppression and enhance anticancer immunity. PD-1/PD-L1 blockade can be realized by aptamers, which have good biocompatibility and can be synthesized in quantity economically. For in vivo applications, aptamers need to evade renal clearance and nuclease digestion. Here we investigated whether DNA nanostructures could be used to enhance the function of PD-L1 aptamers. Four PD-L1 aptamers (Apt) were built into a Holliday Junction (HJ) to form a tetravalent DNA nanostructure (Apt-HJ). The average size of Apt-HJ was 13.22 nm, which was above the threshold for renal clearance. Apt-HJ also underwent partial phosphorothioate modification and had improved nuclease resistance. Compared with the monovalent PD-L1 aptamer, the tetravalent Apt-HJ had stronger affinity to CT26 colon cancer cells. Moreover, Apt-HJ markedly boosted the antitumor efficacy in vivo vs. free PD-L1 aptamers without raising systemic toxicity. The results indicate that multiple aptamers attached to a DNA nanostructure may significantly improve the function of PD-L1 aptamers in vivo.     

In vivo Fluorescence Imaging of Tumors using Molecular Aptamers Generated by Cell‐SELEX

Poor sensitivity and low specificity of current molecular imaging probes limit their application in clinical settings. To address these challenges, we used a process known as cell‐SELEX to develop unique molecular probes termed aptamers with the high binding affinity, sensitivity, and specificity needed for in vivo molecular imaging inside living animals. Importantly, aptamers can be selected by cell‐SELEX to recognize target cells, or even surface membrane proteins, without requiring prior molecular signature information. As a result, we are able to present the first report of aptamers molecularly engineered with signaling molecules and optimized for the fluorescence imaging of specific tumor cells inside a mouse. Using a Cy5‐labeled aptamer TD05 (Cy5‐TD05) as the probe, the in vivo efficacy of aptamer‐based molecular imaging in Ramos (B‐cell lymphoma) xenograft nude mice was tested. After intravenous injection of Cy5‐TD05 into mice bearing grafted tumors, noninvasive, whole‐body fluorescence imaging then allowed the spatial and temporal distribution to be directly monitored. Our results demonstrate that the aptamers could effectively recognize tumors with high sensitivity and specificity, thus establishing the efficacy of these fluorescent aptamers for diagnostic applications and in vivo studies requiring real‐time molecular imaging.     

Label‐ and modification‐free‐based in situ selection of bovine serum albumin specific aptamer

Systematic evolution of ligands by exponential enrichment is a traditional approach to select aptamer, which has a great potential in biosensing field. However, chemical modifications of DNA library or targets before selection might block the real recognition and binding sites between aptamers and their targets. In this study, a label‐ and modification‐free‐based in situ selection strategy was developed to overcome this limitation. The strategy is an attempt to screen bovine serum albumin aptamers according to the principle of electrophoretic mobility shift assay, and allowed single‐stranded DNA sequence to be fully exposed to interact with bovine serum albumin which was mixed with the agarose gel beforehand. After eight rounds of selection, specific aptamer with low dissociation constant (K_d) value of 69.44 ± 7.60 nM was selected and used for subsequent establishment of fluorescence biosensor. After optimization, the optimal aptasensor exhibited a high sensitivity toward bovine serum albumin with a limit of detection of 0.24 ng/mL (linear range from 1 to 120 ng/mL). These results indicated that the label‐ and modification‐free‐based in situ selection strategy proposed in this work could effectively select specific aptamer to develop aptasensor for sensitive detection of bovine serum albumin or other targets in actual complicated samples.     

适配体在癌症诊断与靶向治疗中的研究进展

核酸适配体是利用体外筛选技术,即指数富集的配体系统进化技术(SELEX),从核酸分子文库中得到的寡核苷酸片段。其与靶标物有很高的特异性和亲和力,将适配体作为识别单元的生物传感研究以及适配体偶联成像试剂的生物体内外成像研究在临床诊断中有很大的应用前景,此外,适配体靶向癌细胞或组织的治疗方法相比传统化学治疗副作用更小,在临床上也有极大的应用前景。本文综述了适配体目前在癌症诊断和靶向治疗两个方面的研究进展,并分析现阶段存在的问题以及面临的挑战。     

Equipping Natural Killer Cells with Specific Targeting and Checkpoint Blocking Aptamers for Enhanced Adoptive Immunotherapy in Solid Tumors

Herein, we propose an aptamer‐equipping strategy to generate specific, universal and permeable (SUPER) NK cells for enhanced immunotherapy in solid tumors. NK cells were chemically equipped with TLS11a aptamer targeting HepG2 cells and PDL1‐specific aptamer without genetic alteration. The dual aptamer‐equipped NK cells exhibited high specificity to tumor cells, resulting in higher cytokine secretion and apoptosis/necrosis compared to parental or single aptamer‐equipped NK cells. Interestingly, dual aptamer‐equipped NK cells induced remarkable upregulation of PDL1 expression in HepG2 cells, enhancing checkpoint blockade. Furthermore, in vivo intravital imaging demonstrated high infiltration of aptamer‐equipped NK cells into deep tumor region, leading to enhanced therapeutic efficacy in solid tumors. This work offers a straightforward chemical strategy to equip NK cells with aptamers, holding considerable potential for enhanced adoptive immunotherapy in solid tumors.     

Biphenyl Ether Analogs Containing Pomalidomide as Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction

New biphenyl-based chimeric compounds containing pomalidomide were developed and evaluated for their activity to inhibit and degrade the programmed cell death-1/programmed cell death- ligand 1 (PD-1/PD-L1) complex. Most of the compounds displayed excellent inhibitory activity against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay. Among them, compound 3 is one of the best with an IC₅₀ value of 60 nM. Using an ex vivo PD-1/PD-L1 blockade cell line bioassay that expresses human PD-1 and PD-L1, we show that compounds 4 and 5 significantly restore the repressed immunity in this co-culture model. Western blot data, however, demonstrated that these anti-PD-L1/pomalidomide chimeras could not reduce the protein levels of PD-L1.     

核酸适配体在肿瘤靶向治疗方面的研究进展

传统的抗肿瘤药物大多不具有选择性,在临床治疗中产生了严重的毒副作用。核酸适配体是一种小分子核酸,能够与靶标高亲和性、高特异性地结合。选择与癌症发生发展过程密切相关的生物标记物为靶标进行SELEX过程筛选出的核酸适配体自身可作为药物,也可与药物、siRNA、纳米粒等结合构成靶向给药体系,该体系能靶向作用于特定的肿瘤细胞,降低对正常细胞的毒性,用药量显著降低,药效提高。本文综述了近年来核酸适配体直接作为抗肿瘤药物、药物载体、siRNA载体以及作为纳米材料靶向剂构成多元复合靶向给药体系在肿瘤靶向治疗领域的研究进展。     

Target-Dependent Protection of DNA Aptamers against Nucleolytic Digestion Enables Signal-On Biosensing with Toehold-Mediated Rolling Circle Amplification

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5′-end protects it from 3′-exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration-dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn-on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real-time fluorescence detection of two human proteins: platelet-derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay.     

Cell-specific internalization study of an aptamer from whole cell selection

Nucleic acid aptamers have been shown many unique applications as excellent probes in molecular recognition. However, few examples are reported which show that aptamers can be internalized inside living cells for aptamer functional studies and for targeted intracellular delivery. This is mainly due to the limited number of aptamers available for cell-specific recognition, and the lack of research on their extra- and intracellular functions. One of the major difficulties in aptamers' in vivo application is that most of aptamers, unlike small molecules, cannot be directly taken up by cells without external assistance. In this work, we have studied a newly developed and cell-specific DNA aptamer, sgc8. This aptamer has been selected through a novel cell selection process (cell-SELEX), in which whole intact cells are used as targets while another related cell line is used as a negative control. The cell-SELEX enables generation of multiple aptamers for molecular recognition of the target cells and has significant advantages in discovering cell surface binding molecules for the selected aptamers. We have studied the cellular internalization of one of the selected aptamers. Our results show that sgc8 is internalized efficiently and specifically to the lymphoblastic leukemia cells. The internalized sgc8 aptamers are located inside the endosome. Comparison studies are done with the antibody for the binding protein of sgc8, PTK7 (Human protein tyrosine kinase-7) on cell surface. We also studied the internalization kinetics of both the aptamer and the antibody for the same protein on the living cell surface. We have further evaluated the effects of sgc8 on cell viability, and no cytotoxicity is observed. This study indicates that sgc8 is a promising agent for cell-type specific intracellular delivery.     

Fluorogenic resolution of ligand binding by a nucleic acid aptamer

An ATP-binding nucleic acid aptamer was converted into a solution-phase fluorescence-detected sensor by incorporating a 2'-amine group in a structure that is sensitive to a ligand-induced conformational change in the aptamer. The 2'-amine substitution in the free aptamer reacts efficiently with fluorescamine to yield a fluorescent product detectable by energy transfer to a tethered fluor emitting at 615 nm. In the presence of ATP, the fluorogenic reaction is substantially reduced, both under simplified conditions and in a urine background. Fluorogenic chemistry represents a potentially general approach for creating sensors from aptamers that bind their ligands via induced fit.     

核酸适配体在多肽研究中的应用

多肽在生命体的生理过程中发挥着重要作用,其生理功能一直是生物学、药理学和医学等领域的重要研究内容.核酸适配体是经体外筛选获得的单链DNA或RNA,能与靶标高亲和力、高特异性地结合,有"化学抗体"或"化学家的抗体"之称.以多肽为靶标筛选获得的核酸适配体主要有两大用途:一是基于其识别功能,作为亲和试剂来建立分析检测方法或开展生物成像研究;二是基于它们的生物学活性,作为拮抗剂在活体水平影响靶标多肽的正常功能,阻碍下游信号通路,从而对疾病进行治疗.本文总结了近年来以多肽为靶标筛选的核酸适配体在体内及体外的用途,并探讨了其在筛选、表征及应用中存在的问题,并对其未来的发展趋势进行了展望.     

Discovery of Novel Small-Molecule Inhibitors of PD-1/PD-L1 Interaction via Structural Simplification Strategy

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction is currently the focus in the field of cancer immunotherapy, and so far, several monoclonal antibodies (mAbs) have achieved encouraging outcomes in cancer treatment. Despite this achievement, mAbs-based therapies are struggling with limitations including poor tissue and tumor penetration, long half-life time, poor oral bioavailability, and expensive production costs, which prompted a shift towards the development of the small-molecule inhibitors of PD-1/PD-L1 pathways. Even though many small-molecule inhibitors targeting PD-1/PD-L1 interaction have been reported, their development lags behind the corresponding mAb, partly due to the challenges of developing drug-like small molecules. Herein, we report the discovery of a series of novel inhibitors targeting PD-1/PD-L1 interaction via structural simplification strategy by using BMS-1058 as a starting point. Among them, compound A9 stands out as the most promising candidate with excellent PD-L1 inhibitory activity (IC₅₀ = 0.93 nM, LE = 0.43) and high binding affinity to hPD-L1 (K_D = 3.64 nM, LE = 0.40). Furthermore, A9 can significantly promote the production of IFN-γ in a dose-dependent manner by rescuing PD-L1 mediated T-cell inhibition in Hep3B/OS-8/hPD-L1 and CD3-positive T cells co-culture assay. Taken together, these results suggest that A9 is a promising inhibitor of PD-1/PD-L1 interaction and is worthy for further study.     

N-glycosylation and ubiquitinylation of PD-L1 do not restrict interaction with BMS-202: A molecular modeling study

The Programmed cell Death protein-1/Ligand 1 (PD-1/L1) checkpoint is a major target in oncology. Monoclonal antibodies targeting PD-1 or PD-L1 are used to treat different types of solid tumors and lymphoma. PD-L1-binding small molecules are also actively searched. The lead compound is the biphenyl drug BMS-202 which stabilizes PD-L1 protein dimers and displays a potent antitumor activity in experimental models. Here we have investigated the effect of N-glycosylation (at N35, N192, N200 and N219) and mono-ubiquitination (at K178) of PD-L1 on the interaction with BMS-202 by molecular modeling. Two complementary tridimensional models of PD-L1, based on available crystallographic structures, were constructed with BMS-202 bound. The structures were glycosylated, with a fucosylated bi-antennary N-glycan and ubiquitinated. Model 1 refers to glycoPD-L1 bearing 16 N-glycans, with or without 4 ubiquitin residues. Model 2 presents 8 N-glycans and 2 ubiquitin residues. In both cases, BMS-202 was bound to the protein interface, stabilizing a PD-L1 dimer. The incorporation of the N-glycans or the ubiquitins did not significantly alter the drug-protein recognition. The interface of the drug-stabilized protein dimer is unaffected by the glycosylation or ubiquitination. Calculations of the binding energies indicated that the glycosylation slightly reduces the stability of the drug-protein complexes but does not prevent the drug binding process. Our modeling study suggests that the drug can target efficiently the different forms of PD-L1 in cells, glycosylated, ubiquitinated or not. These models of N-glycosylated and ubiquitinated PD-L1 will be useful to study other PD-L1 protein complexes.     

Cotinine-conjugated aptamer/anti-cotinine antibody complexes as a novel affinity unit for use in biological assays

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5'' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.     

Luminescent Gold Nanoparticles with Discrete DNA Valences for Precisely Controlled Transport at the Subcellular Level

Ultrasmall luminescent gold nanoparticles (AuNPs) with excellent capabilities to cross biological barriers offer great promise in designing intelligent model nanomedicines for investigating structure–property relationships at the subcellular level. However, the strict surface controllability of ultrasmall AuNPs is challenging because of their small size. Herein, we report a facile in situ method for precisely controlling DNA aptamer valences on the surface of luminescent AuNPs with emission in the second near-infrared window using a phosphorothioate-modified DNA aptamer, AS1411, as a template. The discrete DNA aptamer number of AS1411-functionalized AuNPs (AS1411-AuNPs, ≈1.8 nm) with emission at 1030 nm was controlled in one aptamer (V1), two aptamers (V2), and four aptamers (V4). It was then discovered that not only the tumor-targeting efficiencies but also the subcellular transport of AS1411-AuNPs were precisely dependent on valences. A slight increase in valence from V1 to V2 increased tumor-targeting efficiencies and resulted in higher nucleus accumulation, whereas a further increase in valence (e.g., V4) significantly increased tumor-targeting efficiencies and led to higher cytomembrane accumulation. These results provide a basis for the strict surface control of nanomedicines in the precise regulation of in vivo transport at the subcellular level and their translation into clinical practice in the future.     

Aptamer-conjugated nanobubbles for targeted ultrasound molecular imaging

Targeted ultrasound contrast agents can be prepared by some specific bioconjugation techniques. The biotin-avidin complex is an extremely useful noncovalent binding system, but the system might induce immunogenic side effects in human bodies. Previous proposed covalently conjugated systems suffered from low conjugation efficiency and complex procedures. In this study, we propose a covalently conjugated nanobubble coupling with nucleic acid ligands, aptamers, for providing a higher specific affinity for ultrasound targeting studies. The sgc8c aptamer was linked with nanobubbles through thiol-maleimide coupling chemistry for specific targeting to CCRF-CEM cells. Further improvements to reduce the required time and avoid the degradation of nanobubbles during conjugation procedures were also made. Several investigations were used to discuss the performance and consistency of the prepared nanobubbles, such as size distribution, conjugation efficiency analysis, and flow cytometry assay. Further, we applied our conjugated nanobubbles to ex vivo ultrasound targeted imaging and compared the resulting images with optical images. The results indicated the availability of aptamer-conjugated nanobubbles in targeted ultrasound imaging and the practicability of using a highly sensitive ultrasound system in noninvasive biological research.     

Anti-idiotype RNAs that mimic the leucine-rich nuclear export signal and specifically bind to CRM1/exportin 1

BACKGROUND: Anti-idiotype approaches are based on the assumption that an antibody recognising a ligand can be structurally related to the receptor. Recently we have generated anti-idiotype RNA aptamers designed to mimic the human immunodeficiency virus-1 (HIV-1) Rev nuclear export signal (NES). Nuclear injection of either NES-peptide conjugates or aptamer causes the inhibition of Rev-mediated export. This implied that NES mimics and export substrate might compete for binding to the NES receptor. The mechanism of inhibition, however, is unknown. RESULTS: The interaction between the export aptamer and CRM1 was characterised in vitro. The aptamer binds specifically to CRM1 and this interaction is sensitive to competition by Rev NES-peptide conjugates. The recognition domain of CRM1 has been mapped and includes residues found previously to affect binding of leptomycin B, a fungicide interfering with nuclear export. CONCLUSIONS: Inhibition of Rev-mediated export in vivo by export aptamers appears to result from the binding of the aptamers to the NES-recognition domain of CRM1. This observation demonstrates that anti-idiotype RNA can mimic faithfully structural and functional properties of a protein and can be used to map ligand-binding domains of receptors.     

Selection and Characterization of Vimentin-Binding Aptamer Motifs for Ovarian Cancer

The application of aptamers in biomedicine is emerging as an essential technology in the field of cancer research. As small single-stranded DNA or RNA ligands with high specificity and low immunogenicity for their targets, aptamers provide many advantages in cancer therapeutics over protein-based molecules, such as antibodies. Vimentin is an intermediate filament protein that is overexpressed in endothelial cells of cancerous tissue. High expression levels of vimentin have been associated with increased capacity for migration and invasion of the tumor cells. We have selected and identified thioated aptamers with high specificity for vimentin using human ovarian cancer tissues. Tentative binding motifs were chosen for two vimentin aptamers based on predicted secondary structures. Each of these shorter, tentative binding motifs was synthesized, purified, and characterized via cell binding assays. Two vimentin binding motifs with high fidelity binding were selected and further characterized via cell and tissue binding assays, as well as flow cytometric analysis. The equilibrium binding constants of these small thioated aptamer constructs were also determined. Future applications for the vimentin binding aptamer motifs include conjugation of the aptamers to synthetic dyes for use in targeted imaging and therapy, and ultimately more detailed and precise monitoring of treatment response and tumor progression in ovarian pathology.     

Fluorescence anisotropy analysis for mapping aptamer-protein interaction at the single nucleotide level

Structural characterization of aptamer-protein interactions is challenging and limited despite the tremendous applications of aptamers. Here we for the first time report a fluorescence anisotropy (FA) approach for mapping the interaction of an aptamer and its protein target at the single nucleotide level. Nine fluorescently labeled aptamers, each conjugated to a single tetramethylrhodamine at a specified nucleotide in the aptamer, were used to study their interactions with thrombin. Simultaneous monitoring of both fluorescence anisotropy changes and electrophoretic mobility shifts upon binding of the fluorescently modified aptamer to the protein provides unique information on the specific nucleotide site of binding. T25, T20, T7 and the 3'-end were identified as the close contact sites, and T3, C15T, and the 5'-end were identified as the sites distant from the binding. This approach is highly sensitive and does not require cross-linking reactions. Studies of aptamer-protein interactions using this technique are potentially useful for design, evolution, and modification of functional aptamers for a range of bioanalytical, diagnostic, and therapeutic applications.     

核酸适体偶联药物的生物偶联构建技术与应用

核酸适体被称为“化学抗体”, 具有与抗体类似或更加优异的特异性和亲和力, 可以精准地靶向靶蛋白, 与靶蛋白特异性结合. 此外, 核酸适体还具有获取简单、 合成简便、 易于进行化学修饰、 不易变性、 靶标范围广、 免疫原性低及细胞内化快等优点, 已被广泛应用于众多研究领域. 在癌症治疗领域, 核酸适体作为一种优异的靶向识别工具和药物递送载体, 可实现抗肿瘤药物的精准递送. 将核酸适体与药物分子偶联, 可通过核酸适体的靶向作用使药物分子随核酸适体共同进入靶细胞, 实现药物分子在靶细胞内的富集, 进而促进靶细胞的死亡. 近年来, 核酸适体偶联药物已成为癌症靶向治疗的前沿新兴领域, 希望通过该领域的深入研究为癌症靶向治疗领域提供新思路. 本文综合评述了以生物偶联技术构建的核酸适体偶联药物及其应用研究.