CD63：早期診斷和精準(zhǔn)治療的冉冉新星

前  言

CD63也被稱為溶酶體相關(guān)膜蛋白3（LAMP3），屬于四次跨膜蛋白超家族成員，與細(xì)胞的活化、黏附、變異及腫瘤的侵襲、轉(zhuǎn)移等有關(guān)。CD63是外泌體特異性標(biāo)志蛋白，為新型藥物遞送及靶向外泌體提供契機(jī)，還可作為腫瘤的潛在標(biāo)志物（如卵巢癌、肺癌等）。研究人員利用重組CD63蛋白開發(fā)多種適配體，用于非侵入型外泌體的檢測(cè)。CD63靶點(diǎn)的多面性為治療、診斷和生物醫(yī)學(xué)研究帶來(lái)新希望。

01 CD63生理學(xué)和病理學(xué)意義

CD63參與多種細(xì)胞進(jìn)程，在晚期內(nèi)體和多泡體的細(xì)胞蛋白分選中發(fā)揮關(guān)鍵作用，促進(jìn)外泌體形成。通過(guò)靶向CD63介導(dǎo)外泌體藥物的精準(zhǔn)遞送有助于實(shí)現(xiàn)靶向治療。在免疫反應(yīng)中，表達(dá)CD63的外泌體有助于抗原呈遞并激活CD4+T細(xì)胞進(jìn)行免疫調(diào)控。這凸顯了CD63在免疫治療中的重要性及其作為小分子抑制劑靶點(diǎn)的潛力。此外，它與整合素的相互作用控制細(xì)胞粘附和遷移。

除了在細(xì)胞穩(wěn)態(tài)中的發(fā)揮基本功能外，CD63在多種腫瘤中均有表達(dá)，可促進(jìn)腫瘤生長(zhǎng)并調(diào)節(jié)腫瘤微環(huán)境，可作為癌癥治療的潛在靶標(biāo)。此外，研究證明CD63外泌體在卵巢癌和肺癌等特定癌癥中差異表達(dá)，表明其作為診斷生物標(biāo)志物的潛在用途，對(duì)早期診斷和個(gè)性化治療方案的制定產(chǎn)生影響。

 CD63 作為診斷及治療一體化靶點(diǎn)

（源自：doi.org/10.1002/adbi.202300078）

02 CD63從實(shí)驗(yàn)室到臨床應(yīng)用

CD63靶點(diǎn)的多樣性為治療干預(yù)提供了廣闊的應(yīng)用前景。在個(gè)性化醫(yī)療和靶向治療中，直接或間接地將治療藥物添加到外泌體中已成為一種新的策略。這一創(chuàng)新的方案為精確治療和療效改善奠定了基礎(chǔ)。值得一提的是，作為創(chuàng)新藥物遞送載體--細(xì)胞來(lái)源的胞外囊泡（EVs）已在癌癥治療的研究中如火如荼地開展，其表面表達(dá)CD9、CD63、CD81和CD82標(biāo)記物。這些源自植物細(xì)胞、間充質(zhì)細(xì)胞、T細(xì)胞和樹突狀細(xì)胞的外泌體具有作為有效藥物載體的潛力，對(duì)多種疾病的治療發(fā)揮作用。除了其治療潛力外，CD63存在于外泌體和循環(huán)囊泡中，使其適用于從生物體液中進(jìn)行非侵入性檢測(cè)。這一先天優(yōu)勢(shì)為疾病早期診斷和持續(xù)監(jiān)測(cè)開辟了新的途徑。截至2023年8月，已有2項(xiàng)針對(duì)CD63的臨床前研究。

臨床試驗(yàn)中CD63靶向藥物列表

來(lái)源：data.pharnexcloud。com/

03 CD63在研究中的應(yīng)用

CD63重組蛋白可用于新型核酸適體的開發(fā)。張振團(tuán)隊(duì)開發(fā)了一種無(wú)需分離純化、基于適體的熒光偏振檢測(cè)外泌體的方法（AFPExo）。AFPExo通過(guò)熒光偏振檢測(cè)外泌體，為臨床研究分析外泌體提供了一種可靠的方案。劉靜團(tuán)隊(duì)進(jìn)行的另一項(xiàng)研究，鑒定出一種新的核酸適體LL4A，用于CD63高表達(dá)的PLX4032耐藥黑色素瘤患者的預(yù)后及靶向治療。通過(guò)抗體或抑制劑阻斷CD63的表達(dá)，并使用LL4A作為CD63 siRNA的遞送工具，有望成為威羅菲尼（Vemurafenib）的新型聯(lián)合療法一個(gè)潛在新選擇。

A. AFPExo法測(cè)定外泌體的選擇性。使用5.0 μg/mL的CD63和BSA進(jìn)行檢測(cè)，結(jié)果顯示外泌體特異性結(jié)合CD63。B. 熒光偏振隨CD63濃度變化的曲線圖（插圖為熒光偏振對(duì)ΔFP的線性圖），結(jié)果顯示外泌體對(duì)CD63表現(xiàn)出高靈敏度，隨CD63濃度的增加而增加。（源自：doi.org/10.1039/C9NR01589B）

通過(guò)一系列實(shí)驗(yàn)證實(shí)CD63是LL4A的結(jié)合靶點(diǎn)，圖為酶聯(lián)寡核苷酸測(cè)定法（ELONA）測(cè)定純化的CD63重組蛋白（貨號(hào)：11271-H08H，His標(biāo)簽，義翹神州）與LL4A的解離常數(shù)。（源自：doi.org/10.1016/j.omtn.2019.10.005）

?義翹神州CD63明星產(chǎn)品

高純度：

CD63 Protein, Mouse, Recombinant (His Tag), HPLC-verified, Cat: 50557-M08H

Purity ≥ 95 % as determined by SDS-PAGE, ≥ 95 % as determined by SEC-HPLC.

CD63 Protein, Human, Recombinant ( hFc Tag), HPLC-verified, Cat: 11271-H02H

Purity ≥ 95 % as determined by SDS-PAGE, ≥ 95 % as determined by SEC-HPLC.

相關(guān)應(yīng)用: FCM, ICC/IF

Anti-CD63 Antibody, Mouse MAb,  Cat: 11271-MM03

Immunofluorescence staining of CD63 in MCF7 cells.

相關(guān)應(yīng)用: IHC-P

Anti-CD63 Antibody, Mouse MAb,  Cat: 11271-MM01

Immunochemical staining of human CD63 in human malignant melanoma.

【參考文獻(xiàn)】

1. Cheerathodi, M, et al. Epstein-Barr Virus LMP1 Modulates the CD63 Interactome. Viruses, 2021. doi.org/10.3390/v13040675 

2. Yu, D, et al. Exosomes as a new frontier of cancer liquid biopsy. Molecular cancer, 2022. doi.org/10.1186/s12943-022-01509-9

3. Mosquera-Heredia, et al. Exosomes: Potential Disease Biomarkers and New Therapeutic Targets. Biomedicines, 2021. doi.org/10.3390/biomedicines9081061

4. Z. Song, et al. Development of a CD63 aptamer for efficient cancer immunochemistry and Immunoaffinity-Based exosome Isolation. Molecules, 2020. doi: 10.3390/molecules25235585.

5. Zhang, Z., et al. Aptamer-based fluorescence polarization assay for separation-free exosome quantification. Nanoscale, 2019. doi.org/10.1039/c9nr01589b 

6. Li, H., et al. A Novel Aptamer LL4A Specifically Targets Vemurafenib-Resistant Melanoma through Binding to the CD63 Protein. Molecular therapy. Nucleic acids, 2019. doi.org/10.1016/j.omtn.2019.10.005

7. S. Tugues et al. Tetraspanin CD63 promotes vascular endothelial growth factor receptor 2-Β1 integrin complex formation, thereby regulating activation and downstream signaling in endothelial cells in vitro and in vivo. Journal of Biological Chemistry, 2013. doi: 10.1074/jbc.m113.468199. 

8. Lema, D. A., & Burlingham, W. J. Role of exosomes in tumour and transplant immune regulation. Scandinavian journal of immunology, 2019. doi.org/10.1111/sji.12807

9. Valencia, J. C., Egbukichi, N., & Erwin-Cohen, R. A. Autoimmunity and Cancer, the Paradox Comorbidities Challenging Therapy in the Context of Preexisting Autoimmunity. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 2019. doi.org/10.1089/jir.2018.0060

10. W. Sun, J. Luo, H. Jiang, and D. Duan, Tumor exosomes: a double-edged sword in cancer therapy. Acta Pharmacologica Sinica, 2018. doi: 10.1038/aps.2018.17.

11. Akbari, A.,et al. Engineered Exosomes for Tumor-Targeted Drug Delivery: A Focus on Genetic and Chemical Functionalization. Pharmaceutics, 2022. doi.org/10.3390/pharmaceutics15010066

12. B. De Goeij et al. Efficient payload delivery by a bispecific Antibody–Drug conjugate targeting HER2 and CD63. Molecular Cancer Therapeutics, 2016. doi: 10.1158/1535-7163.mct-16-0364.

13. Dai, X., Ye, Y., & He, F. Emerging innovations on exosome-based onco-therapeutics. Frontiers in immunology, 2022. doi.org/10.3389/fimmu.2022.865245

14. Kim, J. H., Lee, C. H., & Baek, M. C. Dissecting exosome inhibitors: therapeutic insights into small-molecule chemicals against cancer. Experimental & molecular medicine, 2022. doi.org/10.1038/s12276-022-00898-7

15. Herrmann, I. K., Wood, M. J. A., & Fuhrmann, G. Extracellular vesicles as a next-generation drug delivery platform. Nature nanotechnology, 2021. doi.org/10.1038/s41565-021-00931-2

16. Rezaie, J., Feghhi, M., & Etemadi, T. A review on exosomes application in clinical trials: perspective, questions, and challenges. Cell communication and signaling : CCS, 2022. doi.org/10.1186/s12964-022-00959-4

17. H. Odaka, et al. CD63-positive extracellular vesicles are potential diagnostic biomarkers of pancreatic ductal adenocarcinoma. BMC Gastroenterology, 2022. doi: 10.1186/s12876-022-02228-7.