Joshua Shelton, Xue Bai, Brian J. Thomas, Agustin T. Barcellona, Donald H. Burke, Bret D. Ulery
{"title":"B049:增强伯基特淋巴瘤结合、内化和治疗递送的双特异性适配体","authors":"Joshua Shelton, Xue Bai, Brian J. Thomas, Agustin T. Barcellona, Donald H. Burke, Bret D. Ulery","doi":"10.1158/1538-7445.pediatric25-b049","DOIUrl":null,"url":null,"abstract":"Burkitt lymphoma is an aggressive cancer that accounts for 30% of all pediatric lymphomas. Due to its aggressive clinical course, patients are immediately started on a high-intensity chemo-immunotherapy protocol with central nervous system (CNS) prophylaxis to prevent relapse or CNS involvement. The Magrath regimen (CODOX-M/IVAC – cyclophosphamide, vincristine, doxorubicin, high-dose methotrexate, ifosfamide, cytarabine, etoposide, and intrathecal methotrexate) is most commonly used for medical management, yielding two-year event-free survival rates as high as 90%. However, this drug regimen consists of nonspecific, broadly toxic medications and has frequently led to off-target toxicity. To prevent these unwanted effects, it is necessary to design a more targeted therapeutic that utilizes tumor-specific markers. Burkitt lymphoma cells (Ramos) have been shown to inappropriately display a component of the spliceosomal complex (hnRNP U) on their cell surface and many human tumors have been shown to overexpress the transferrin receptor (TfR) relative to native tissues, making these exciting targets. We aimed to produce dual-targeting aptamers and to characterize their binding to, internalization by, and therapeutic deliver to Ramos cells. Anti-hnRNP U DNA aptamer (c10.36) and an anti-TfR RNA aptamer (E3) each containing a 3’-terminal 21-nucleotide antitail or tail, respectively, were synthesized and annealed together using sense:antisense complexation. For fluorescent experiments, monospecific aptamers were annealed to a cyanine 3 (Cy3)-labelled antitail and bispecific aptamers were constructed using DNA aptamers with 3’ cy3 modification. Ramos cells were treated with bispecific, monospecific, or control aptamers for 1 hour prior to flow cytometric analysis or fixation, staining, and imaging by a confocal microscope. Ramos cells treated with c10.36-E3 bispecific aptamers demonstrated 9-fold higher fluorescence by flow cytometry relative to monospecific controls. Confocal microscopy showed greater fluorescent intensity and diffuse punctate staining pattern in Ramos cells treated with c10.36-E3 bispecific aptamers relative to their monospecific controls. In Ramos cells stained with LysoTracker dye, co-localization of c10.36-E3 bispecific aptamers with lysosomes was observed whereas this was not observed with control aptamers. When Ramos cells were pre-treated with endocytic inhibitors or transferrin, internalization of c10.36-E3 was found to be reduced by treatment with sucrose, low temperature, or human transferrin. Finally, Ramos cells were treated with on- or off-target bispecific aptamers containing 3’-Val-Cit-PABA-MMAF and viability was assessed using live-dead stain HelixNP via flow cytometry. Over 48 hours, c10.36-E3 led to dramatic reductions in viability relative to appropriate control products. In conclusion, the c10.36-E3 bispecific aptamer is internalized through TfR-mediated endocytosis with superior binding and trafficking relative to controls making it an exciting vehicle capable of targeted therapeutic delivery to Ramos cells. Citation Format: Joshua Shelton, Xue Bai, Brian J. Thomas, Agustin T. Barcellona, Donald H. Burke, Bret D. Ulery. Bispecific Aptamers for Enhanced Binding, Internalization, and Therapeutic Delivery in Burkitt Lymphoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2): nr B049.","PeriodicalId":9441,"journal":{"name":"Cancer research","volume":"19 1","pages":""},"PeriodicalIF":16.6000,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Abstract B049: Bispecific Aptamers for Enhanced Binding, Internalization, and Therapeutic Delivery in Burkitt Lymphoma\",\"authors\":\"Joshua Shelton, Xue Bai, Brian J. Thomas, Agustin T. Barcellona, Donald H. Burke, Bret D. Ulery\",\"doi\":\"10.1158/1538-7445.pediatric25-b049\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Burkitt lymphoma is an aggressive cancer that accounts for 30% of all pediatric lymphomas. Due to its aggressive clinical course, patients are immediately started on a high-intensity chemo-immunotherapy protocol with central nervous system (CNS) prophylaxis to prevent relapse or CNS involvement. The Magrath regimen (CODOX-M/IVAC – cyclophosphamide, vincristine, doxorubicin, high-dose methotrexate, ifosfamide, cytarabine, etoposide, and intrathecal methotrexate) is most commonly used for medical management, yielding two-year event-free survival rates as high as 90%. However, this drug regimen consists of nonspecific, broadly toxic medications and has frequently led to off-target toxicity. To prevent these unwanted effects, it is necessary to design a more targeted therapeutic that utilizes tumor-specific markers. Burkitt lymphoma cells (Ramos) have been shown to inappropriately display a component of the spliceosomal complex (hnRNP U) on their cell surface and many human tumors have been shown to overexpress the transferrin receptor (TfR) relative to native tissues, making these exciting targets. We aimed to produce dual-targeting aptamers and to characterize their binding to, internalization by, and therapeutic deliver to Ramos cells. Anti-hnRNP U DNA aptamer (c10.36) and an anti-TfR RNA aptamer (E3) each containing a 3’-terminal 21-nucleotide antitail or tail, respectively, were synthesized and annealed together using sense:antisense complexation. For fluorescent experiments, monospecific aptamers were annealed to a cyanine 3 (Cy3)-labelled antitail and bispecific aptamers were constructed using DNA aptamers with 3’ cy3 modification. Ramos cells were treated with bispecific, monospecific, or control aptamers for 1 hour prior to flow cytometric analysis or fixation, staining, and imaging by a confocal microscope. Ramos cells treated with c10.36-E3 bispecific aptamers demonstrated 9-fold higher fluorescence by flow cytometry relative to monospecific controls. Confocal microscopy showed greater fluorescent intensity and diffuse punctate staining pattern in Ramos cells treated with c10.36-E3 bispecific aptamers relative to their monospecific controls. In Ramos cells stained with LysoTracker dye, co-localization of c10.36-E3 bispecific aptamers with lysosomes was observed whereas this was not observed with control aptamers. When Ramos cells were pre-treated with endocytic inhibitors or transferrin, internalization of c10.36-E3 was found to be reduced by treatment with sucrose, low temperature, or human transferrin. Finally, Ramos cells were treated with on- or off-target bispecific aptamers containing 3’-Val-Cit-PABA-MMAF and viability was assessed using live-dead stain HelixNP via flow cytometry. Over 48 hours, c10.36-E3 led to dramatic reductions in viability relative to appropriate control products. In conclusion, the c10.36-E3 bispecific aptamer is internalized through TfR-mediated endocytosis with superior binding and trafficking relative to controls making it an exciting vehicle capable of targeted therapeutic delivery to Ramos cells. Citation Format: Joshua Shelton, Xue Bai, Brian J. Thomas, Agustin T. Barcellona, Donald H. Burke, Bret D. Ulery. Bispecific Aptamers for Enhanced Binding, Internalization, and Therapeutic Delivery in Burkitt Lymphoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2): nr B049.\",\"PeriodicalId\":9441,\"journal\":{\"name\":\"Cancer research\",\"volume\":\"19 1\",\"pages\":\"\"},\"PeriodicalIF\":16.6000,\"publicationDate\":\"2025-09-25\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cancer research\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1158/1538-7445.pediatric25-b049\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"ONCOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cancer research","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1158/1538-7445.pediatric25-b049","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ONCOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
伯基特淋巴瘤是一种侵袭性癌症,占所有儿童淋巴瘤的30%。由于其侵袭性的临床病程,患者立即开始高强度的化学免疫治疗方案与中枢神经系统(CNS)预防,以防止复发或累及中枢神经系统。Magrath方案(CODOX-M/IVAC -环磷酰胺、长春新碱、阿霉素、大剂量甲氨蝶呤、异环磷酰胺、阿糖胞苷、乙糖苷和鞘内甲氨蝶呤)最常用于医疗管理,两年无事件生存率高达90%。然而,这种药物方案包括非特异性、广泛毒性的药物,并经常导致脱靶毒性。为了防止这些不良影响,有必要设计一种利用肿瘤特异性标记物的更有针对性的治疗方法。伯基特淋巴瘤细胞(Ramos)已被证明在其细胞表面不恰当地显示剪接体复合体(hnRNP U)的一个成分,许多人类肿瘤已被证明相对于天然组织过度表达转铁蛋白受体(TfR),使这些令人兴奋的靶标。我们的目标是生产双靶向适配体,并表征它们与拉莫斯细胞的结合、内化和治疗递送。合成了抗hnrnp U DNA适体(c10.36)和抗tfr RNA适体(E3),每个适体分别含有一个3 '端21个核苷酸的反尾或尾,并使用正反义络合一起退火。在荧光实验中,将单特异性适配体退火为氰氨酸3 (Cy3)标记的抗尾体,用3 ' Cy3修饰的DNA适配体构建双特异性适配体。Ramos细胞分别用双特异性、单特异性或对照适体处理1小时,然后进行流式细胞分析或固定、染色和共聚焦显微镜成像。用c10.36-E3双特异性适配体处理的Ramos细胞,通过流式细胞术显示出比单特异性对照高9倍的荧光。共聚焦显微镜显示,与单特异性对照相比,c10.36-E3双特异性适配体处理的Ramos细胞的荧光强度和弥漫性点状染色模式更大。在用LysoTracker染料染色的Ramos细胞中,可以观察到c10.36-E3双特异性适配体与溶酶体的共定位,而在对照适配体中则没有观察到这种情况。当用内吞抑制剂或转铁蛋白预处理Ramos细胞时,发现蔗糖、低温或人转铁蛋白处理可减少c10.36-E3的内化。最后,用含有3 ' - val - cte - paba - mmaf的靶向或脱靶双特异性适配体处理Ramos细胞,并通过流式细胞术使用HelixNP活死染色剂评估细胞活力。48小时后,相对于适当的对照产品,c10.36-E3导致存活能力急剧下降。综上所述,c10.36-E3双特异性适配体通过tfr介导的内吞作用被内化,与对照相比具有更好的结合和运输能力,使其成为一种令人兴奋的载体,能够靶向治疗拉莫斯细胞。引用格式:Joshua Shelton, Xue Bai, Brian J. Thomas, Agustin T. Barcellona, Donald H. Burke, Bret D. Ulery。增强伯基特淋巴瘤结合、内化和治疗递送的双特异性适配体[摘要]。AACR癌症研究特别会议论文集:儿童癌症的发现和创新-从生物学到突破性疗法;2025年9月25日至28日;波士顿,MA。费城(PA): AACR;癌症研究2025;85(18_Suppl_2): nr B049。
Abstract B049: Bispecific Aptamers for Enhanced Binding, Internalization, and Therapeutic Delivery in Burkitt Lymphoma
Burkitt lymphoma is an aggressive cancer that accounts for 30% of all pediatric lymphomas. Due to its aggressive clinical course, patients are immediately started on a high-intensity chemo-immunotherapy protocol with central nervous system (CNS) prophylaxis to prevent relapse or CNS involvement. The Magrath regimen (CODOX-M/IVAC – cyclophosphamide, vincristine, doxorubicin, high-dose methotrexate, ifosfamide, cytarabine, etoposide, and intrathecal methotrexate) is most commonly used for medical management, yielding two-year event-free survival rates as high as 90%. However, this drug regimen consists of nonspecific, broadly toxic medications and has frequently led to off-target toxicity. To prevent these unwanted effects, it is necessary to design a more targeted therapeutic that utilizes tumor-specific markers. Burkitt lymphoma cells (Ramos) have been shown to inappropriately display a component of the spliceosomal complex (hnRNP U) on their cell surface and many human tumors have been shown to overexpress the transferrin receptor (TfR) relative to native tissues, making these exciting targets. We aimed to produce dual-targeting aptamers and to characterize their binding to, internalization by, and therapeutic deliver to Ramos cells. Anti-hnRNP U DNA aptamer (c10.36) and an anti-TfR RNA aptamer (E3) each containing a 3’-terminal 21-nucleotide antitail or tail, respectively, were synthesized and annealed together using sense:antisense complexation. For fluorescent experiments, monospecific aptamers were annealed to a cyanine 3 (Cy3)-labelled antitail and bispecific aptamers were constructed using DNA aptamers with 3’ cy3 modification. Ramos cells were treated with bispecific, monospecific, or control aptamers for 1 hour prior to flow cytometric analysis or fixation, staining, and imaging by a confocal microscope. Ramos cells treated with c10.36-E3 bispecific aptamers demonstrated 9-fold higher fluorescence by flow cytometry relative to monospecific controls. Confocal microscopy showed greater fluorescent intensity and diffuse punctate staining pattern in Ramos cells treated with c10.36-E3 bispecific aptamers relative to their monospecific controls. In Ramos cells stained with LysoTracker dye, co-localization of c10.36-E3 bispecific aptamers with lysosomes was observed whereas this was not observed with control aptamers. When Ramos cells were pre-treated with endocytic inhibitors or transferrin, internalization of c10.36-E3 was found to be reduced by treatment with sucrose, low temperature, or human transferrin. Finally, Ramos cells were treated with on- or off-target bispecific aptamers containing 3’-Val-Cit-PABA-MMAF and viability was assessed using live-dead stain HelixNP via flow cytometry. Over 48 hours, c10.36-E3 led to dramatic reductions in viability relative to appropriate control products. In conclusion, the c10.36-E3 bispecific aptamer is internalized through TfR-mediated endocytosis with superior binding and trafficking relative to controls making it an exciting vehicle capable of targeted therapeutic delivery to Ramos cells. Citation Format: Joshua Shelton, Xue Bai, Brian J. Thomas, Agustin T. Barcellona, Donald H. Burke, Bret D. Ulery. Bispecific Aptamers for Enhanced Binding, Internalization, and Therapeutic Delivery in Burkitt Lymphoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2): nr B049.
期刊介绍:
Cancer Research, published by the American Association for Cancer Research (AACR), is a journal that focuses on impactful original studies, reviews, and opinion pieces relevant to the broad cancer research community. Manuscripts that present conceptual or technological advances leading to insights into cancer biology are particularly sought after. The journal also places emphasis on convergence science, which involves bridging multiple distinct areas of cancer research.
With primary subsections including Cancer Biology, Cancer Immunology, Cancer Metabolism and Molecular Mechanisms, Translational Cancer Biology, Cancer Landscapes, and Convergence Science, Cancer Research has a comprehensive scope. It is published twice a month and has one volume per year, with a print ISSN of 0008-5472 and an online ISSN of 1538-7445.
Cancer Research is abstracted and/or indexed in various databases and platforms, including BIOSIS Previews (R) Database, MEDLINE, Current Contents/Life Sciences, Current Contents/Clinical Medicine, Science Citation Index, Scopus, and Web of Science.