纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞
1Immune Regulation Group,Helmholtz Centre for Infection Research, 2Infection Immunology Group, Institute of Medical Microbiology, Infection Prevention and Control, Health Campus Immunology, Infectiology and Inflammation,Otto-von-Guericke University Magdeburg, 3Institute for Molecular and Clinical Immunology, Health Campus Immunology, Infectiology and Inflammation,Otto-von-Guericke University Magdeburg, 4Cellular Proteome Research,Helmholtz Centre for Infection Research, 5Experimental Pneumology, University Hospital of Pneumology, Health Campus Immunology, Infectiology and Inflammation,Otto-von-Guericke University Magdeburg
Summary
我们描述了一种将模型抗原卵清蛋白化学偶联到内吞作用受体特异性抗体的方案，用于体内树突状细胞靶向。该协议包括抗体的纯化，抗原的化学偶联，以及偶联物的纯化和有效偶联的验证。
Abstract
靶向抗原递送到 体内 交叉呈递树突状细胞（DC）可有效诱导T效应细胞反应，并在疫苗设计中显示出一种有价值的方法。抗原通过内吞作用受体（如 DEC-205）特异性抗体递送到 DC，这些受体可诱导摄取、加工和 MHC I 类和 II 类表现。
将所需抗原高效可靠地偶联到合适的抗体是DC靶向的关键步骤，除其他因素外，还取决于抗原的格式。全长蛋白质与纯化抗体的化学偶联是一种可能的策略。过去，我们已经成功地建立了模型抗原卵清蛋白（OVA）和DEC-205特异性IgG2a抗体（αDEC-205）的交联，用于小鼠 体内 DC靶向研究。该协议的第一步是通过亲和色谱法从NLDC（非淋巴树突状细胞）-145杂交瘤的上清液中纯化抗体。纯化的抗体通过磺基-SMCC（磺基琥珀酰亚胺基4-[N-马来亚胺基]环己烷-1-羧酸盐）激活以进行化学偶联，同时通过与TCEP-HCl（三（2-羧乙基）盐酸膦）孵育暴露于OVA蛋白的巩基。除去过量的TCEP-HCl和磺基-SMCC，并将抗原与活化的抗体混合过夜偶联。所得的αDEC-205/OVA偶联物被浓缩并从未结合的OVA中释放出来。通过蛋白质印迹分析和酶联免疫吸附测定（ELISA）验证OVA与αDEC-205的成功偶联。
我们已经成功地使用化学交联的αDEC-205 / OVA在肝脏中诱导细胞毒性T细胞反应，并比较了不同的佐剂在 体内 靶向DEC-205+ DC后诱导体液和细胞免疫的潜力。除此之外，这种化学偶联的抗体/抗原偶联物为有效诱导疫苗对肿瘤抗原的反应提供了有价值的工具，并且已被证明在预防和治疗各种类型肿瘤方面优于经典免疫方法。
Introduction
树突状细胞（DC）是免疫系统的核心参与者。它们是一组专门用于抗原呈递的多样化细胞，其主要功能是桥接先天性和适应性免疫1，2。重要的是，DC不仅在有效和特异性的病原体导向反应中起重要作用，而且还参与抗肿瘤免疫1，3的许多方面。
由于它们在宿主免疫中的排他性作用，DC成为疫苗接种靶细胞4的焦点。一种方法是在体内将抗原靶向DC以诱导抗原特异性免疫反应，并且在过去几年中，大量研究致力于确定合适的受体和靶向策略1，4。一个例子是C型凝集素受体DEC-205，其可以通过DEC-205特异性抗体靶向诱导内吞作用。重要的是，DEC-205靶向与合适的佐剂组合已被证明可以有效地诱导长寿命和保护性的CD4+和CD8+ T细胞，以及抗体反应，也针对肿瘤抗原3，5，6，7，8，9。
有许多研究表明，靶向DC的偶联抗原优于游离的未偶联抗原3，5，10，11，12。这使得抗原与相应的DC靶向部分的共轭成为DC靶向方法的核心步骤。在通过抗体或抗体片段靶向DC的情况下，抗原可以是化学或遗传连接的，并且任何一种策略都提供了自己的（dis）优势1。一方面，在基因工程抗体 – 抗原构建体中，抗原剂量以及位置的控制在批次1之间提供卓越的可比性。然而，与此同时，化学偶联需要较少的准备工作，并提供更大的灵活性，特别是在尝试在实验和临床前模型中测试和比较不同的抗原和/或疫苗接种策略时。
在这里，我们提出了一种方案，用于将卵清蛋白（OVA）作为模型蛋白抗原与适合小鼠体内DC靶向的DEC-205特异性IgG2a抗体（αDEC-205）进行有效和可靠的化学偶联。首先，从NLDC-145杂交瘤细胞13中纯化αDEC-205。对于化学偶联，使用含有NHS（N-羟基琥珀酰亚胺基）和马来酰亚胺基的异双功能交联剂4-[N-马来酰亚胺甲基]环己烷-1-羧酸酯（磺基-SMCC），允许含胺和巯基分子的共价共轭。具体而言，抗体的伯胺最初与磺基-SMCC反应，所得的马来酰亚胺活化的αDEC-205随后与通过TCEP-HCl（三（2-羧乙基）膦盐酸盐）还原的含巯基OVA蛋白反应。最终产物是化学偶联的αDEC-205 / OVA（图1）。除了化学偶联本身之外，我们的方案还描述了从偶联物中去除过量的OVA，以及通过蛋白质印迹分析和特定的酶联免疫吸附测定来验证成功偶联。我们过去曾成功采用这种方法将OVA和其他蛋白质或免疫原性肽化学偶联至αDEC-205。我们证明了在体外与CD11c+细胞的有效结合以及体内细胞和体液免疫的有效诱导 。
当然，这种方法存在缺点，例如批次间可比性以及最终偶联物中抗原的精确剂量。然而，与基因工程构建体相比，化学偶联在选择抗体和蛋白质抗原方面提供了实验灵活性。因此，我们认为这种方法在评估临床前小鼠模型中不同抗原在DC靶向中的效率方面特别有价值，重要的是在特定的抗肿瘤免疫反应的背景下也是如此。
Protocol
所有描述的动物实验都得到了当地政府机构的批准（Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit;文件编号33.12-42502-04-10/0108），并根据国家和机构指南进行。 1. 从杂交瘤细胞系NLDC-145生产αDEC-205 对于抗体生产，在37°C的水浴中解冻冷冻保存的NLDC-145细胞产生αDEC-205。在37°C和5%CO下扩增细胞2.两个 75 厘米2 需要瓶子才能进行抗体生产。细胞?…
Representative Results
使用该协议将αDEC-205与OVA蛋白的化学偶联通常允许有效地生成αDEC-205 / OVA用于体内DC靶向方法。有不同的策略来验证技术本身并测试所产生共轭物的功能。蛋白质印迹分析和ELISA用于验证成功的偶联，同时检测可能留下的游离OVA（图2）。体外结合研究（图3）和体内免疫（图4）证实了偶…
Discussion
内吞作用受体特异性抗体和蛋白质抗原的化学偶联为临床前小鼠模型 中的体内 DC靶向提供了一种有效且重要的灵活方法。通过我们的方案，我们为模型抗原OVA与DEC-205特异性IgG抗体的成功偶联提供了一种有效的方法。
在我们的方案中，αDEC-205是从杂交瘤细胞系中纯化的，过去，我们已经使用如上所述的蛋白G琼脂糖纯化了抗体。值得注意的是，在后来的研究中，我们还?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢S. Prettin的专家技术援助。这项工作得到了亥姆霍兹德国研究中心协会（HGF）的资助，该资助是亥姆霍兹联盟”癌症免疫疗法”（HCC_WP2b）的一部分。
Materials
| antibody buffer 2 % | 2 % (w/v) Slim-Fast Chocolate powder in TBS-T | ||
| antibody buffer 5 % | 5 % milk powder (w/v) in TBS-T | ||
| blocking buffer (ELISA) | 10 % FBS in PBS | ||
| blocking buffer 4 % | 4 % (w/v) Slim-Fast Chocolate powder in TBS-T | ||
| blocking buffer 10 % | 10 % milk powder (w/v) in TBS-T | ||
| cell culture flask T25 | Greiner Bio-One | 690175 | we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (690195 ) |
| cell culture flask T75 | Greiner Bio-One | 658175 | we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (658195) |
| cell culture flask T175 | Greiner Bio-One | 661175 | we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (661195) |
| centrifugal concentrator MWCO 10 kDa | Sartorius | VS2001 | Vivaspin 20 centrifugal concentrator |
| centrifugal protein concentrator MWCO 100 kDa, 5 – 20 ml | Thermo Fisher Scientific | 88532 | Pierce Protein Concentrator, PES 5 -20 ml; we use the Pierce Concentrator 150K MWCO 20mL (catalog number 89921), which is however no longer available |
| centrifuge bottles | Nalgene | 525-2314 | PPCO (polypropylene copolymer) with PP (polypropylene) screw closure, 500 ml; obtained from VWR, Germany |
| coating buffer (ELISA) | 0.1 M sodium bicarbonate (NaHCO3) in H2O (pH 9.6) | ||
| desalting columns MWCO 7 kDa | Thermo Fisher Scientific | 89891 | Thermo Scientific Zeba Spin Desalting Columns, 7K MWCO, 5 mL |
| detection reagent ELISA (HRPO substrate) | Sigma-Aldrich/Merck | T8665-100ML | 3,3′,5,5′-Tetramethylbenzidine (TMB) liquid substrate system |
| detection reagent western blot (HRPO substrate) | Roche/Merck | 12 015 200 01 | Lumi-Light Western Blotting Substrate (Roche) |
| dialysis tubing MWCO 12 – 14 kDa | SERVA Electrophoresis | 44110 | Visking dialysis tubing, 16 mm diameter |
| ELISA 96-well plate | Thermo Fisher Scientific | 442404 | MaxiSorp Nunc-Immuno Plate |
| fetal calf serum | PAN-BIOtech | P40-47500 | FBS Good forte |
| ISF-1 medium | Biochrom/bioswisstec | F 9061-01 | |
| milk powder | Carl Roth | T145.2 | powdered milk, blotting grade, low in fat; alternatively we have also used conventional skimmed milk powder from the supermarket |
| NLDC-145 hybridoma | ATCC | HB-290 | if not already at hand, the hybridoma cells can be acquired from ATCC |
| non-reducing SDS sample buffer 4 x | for 12 ml: 4 ml of 10 % SDS, 600 µl 0.5 M Tris-HCl (ph 6.8), 3.3 ml sterile H2O, 4 ml glycerine, 100 µl of 5 % Bromphenol Blue | ||
| ovalbumin | Hyglos (via BioVendor) | 321000 | EndoGrade OVA ultrapure with <0.1 EU/mg |
| Penicillin/Streptomycin | Thermo Fisher Scientific | 15140122 | Gibco Penicillin/Streptomycin 10.000 U/ml; alternatively Gibco Penicillin/Streptomycin 5.000 U/ml (15070-063) can be used |
| PETG polyethylene terephthalate glycol cell culture roller bottles | Nunc In Vitro | 734-2394 | standard PDL-coated, vented (1.2X), 1050 cm², 100 – 500 ml volume; obtained from VWR, Germany |
| pH indicator strips | Merck | 109535 | pH indicator strips 0-14 |
| polyclonal goat αrat-IgG(H+L)-HRPO (western blot) | Jackson ImmunoResearch | 112-035-062 | obtained from Dianova, Germany; used at 1:5000 for western blot |
| polyclonal goat αrat-IgG+IgM-HRPO antibody (ELISA) | Jackson ImmunoResearch | 112-035-068 | obtained from Dianova, Germany; used at 1:2000 for ELISA |
| polyclonal goat αrabbit-IgG-HRPO (western blot) | Jackson ImmunoResearch | 111-035-045 | obtained from Dianova, Germany; used at 1:2000 for western blot |
| polyclonal rabbit αOVA (ELISA) | Abcam | ab181688 | used at 3 ng/µl |
| polyclonal rabbit αOVA antibody (western blot) | OriGene | R1101 | used at 1:3,000 for western blot |
| Protein G Sepharose column | Merck/Millipore | P3296 | 5 ml Protein G Sepharose, Fast Flow are packed onto an empty column PD-10 (Merck, GE 17-0435-01) |
| protein standard | Thermo Fisher Scientific | 26616 | PageRuler Prestained Protein ladder 10 – 180 kDa |
| PVDF (polyvinylidene difluoride) membrane | Merck/Millipore | IPVH00010 | immobilon-P PVDF (polyvinylidene difluoride) membrane |
| rubber plug | Omnilab | 5230217 | DEUTSCH & NEUMANN rubber stoppers (lower Φ 17 mm; upper Φ 22 mm) |
| silicone tube | Omnilab | 5430925 | DEUTSCH & NEUMANN (inside Φ 1 mm; outer Φ 3 mm) |
| Slim-Fast | we have used regular Slim-Fast Chocolate freely available at the pharmacy as in this western blot approach it yielded better results than milk powder | ||
| stopping solution (ELISA) | 1M H2SO4 | ||
| sulfo-SMCC | Thermo Fisher Scientific | 22322 | Pierce Sulfo-SMCC Cross-Linker; alternatively use catalog number A39268 (10 x 2 mg) |
| syringe filter unit 0.22 µm | Merck/Millipore | SLGV033RS | Millex-GV Syringe Filter Unit, 0.22 µm, PVDF, 33 mm, gamma sterilized |
| syringe 10 ml | Omnilab | Disposable syringes Injekt® Solo B.Braun | |
| Sterican® cannulas | B. Braun | Sterican® G 20 x 1 1/2""; 0.90 x 40 mm; yellow | |
| TBS-T | Tris-buffered saline containing 0.1 % (v/v) Tween 20 | ||
| TCEP-HCl | Thermo Fisher Scientific | A35349 | |
| tubing connector | Omnilab | Kleinfeld miniature tubing connectors for silicone tube |
References
- Amon, L., Hatscher, L., Heger, L., Dudziak, D., Lehmann, C. H. K. Harnessing the complete repertoire of conventional dendritic cell functions for cancer immunotherapy. Pharmaceutics. 12 (7), 12070663 (2020).
- Banchereau, J., et al. Immunobiology of dendritic cells. Annual Review of Immunology. 18, 767-811 (2000).
- Wculek, S. K., et al. Dendritic cells in cancer immunology and immunotherapy. Nature Reviews: Immunology. 20 (1), 7-24 (2020).
- Kastenmuller, W., Kastenmuller, K., Kurts, C., Seder, R. A. Dendritic cell-targeted vaccines–hope or hype. Nature Reviews: Immunology. 14 (10), 705-711 (2014).
- Bonifaz, L. C., et al. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination. Journal of Experimental Medicine. 199 (6), 815-824 (2004).
- Boscardin, S. B., et al. Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses. Journal of Experimental Medicine. 203 (3), 599-606 (2006).
- Hossain, M. K., Wall, K. A. Use of Dendritic Cell Receptors as Targets for Enhancing Anti-Cancer Immune Responses. Cancers. 11 (3), 11030418 (2019).
- Johnson, T. S., et al. Inhibition of melanoma growth by targeting of antigen to dendritic cells via an anti-DEC-205 single-chain fragment variable molecule. Clinical Cancer Research. 14 (24), 8169-8177 (2008).
- Trumpfheller, C., et al. The microbial mimic poly IC induces durable and protective CD4+ T cell immunity together with a dendritic cell targeted vaccine. Proceedings of the National Academy of Sciences of the United States of America. 105 (7), 2574-2579 (2008).
- Idoyaga, J., et al. Comparable T helper 1 (Th1) and CD8 T-cell immunity by targeting HIV gag p24 to CD8 dendritic cells within antibodies to Langerin, DEC205, and Clec9A. Proceedings of the National Academy of Sciences of the United States of America. 108 (6), 2384-2389 (2011).
- Sancho, D., et al. Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin. Journal of Clinical Investigation. 118 (6), 2098-2110 (2008).
- Volckmar, J., et al. Targeted antigen delivery to dendritic cells elicits robust antiviral T cell-mediated immunity in the liver. Scientific Reports. 7, 43985 (2017).
- Kraal, G., Breel, M., Janse, M., Bruin, G. Langerhans’ cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody. Journal of Experimental Medicine. 163 (4), 981-997 (1986).
- Volckmar, J., et al. The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells. Vaccine. 37 (35), 4963-4974 (2019).
Cite This Article
Volckmar, J., Knop, L., Hirsch, T., Frentzel, S., Erck, C., van Ham, M., Stegemann-Koniszewski, S., Bruder, D. Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo. J. Vis. Exp. (168), e62018, doi:10.3791/62018 (2021).