Name: chemical conjugation of a purified dec-205-directed antibody with full-length protein for targeting mouse dendritic cells in vitro and in vivo
Uploaded: 2021-02-05T14:00:00
Description: Watch this Scientific Journal Video about Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo at JoVE.com

The authors thank S. Prettin for expert technical assistance. This work was supported by a grant of the Helmholtz Association of German Research Centers (HGF) that was provided as part of the Helmholtz Alliance &#39;&#39;Immunotherapy of Cancers&#34; (HCC_WP2b).

All of the described animal experiments were approved by the local government agency (Nieders&#228;chsisches Landesamt f&#252;r Verbraucherschutz und Lebensmittelsicherheit; file number 33.12-42502-04-10/0108) and were performed according to the national and institutional guidelines.
1. Production of &#945;DEC-205 from the hybridoma cell line NLDC-145
<ol>
	<li>For antibody production, thaw cryopreserved NLDC-145 cells producing &#945;DEC-205 at 37 &#176;C in a water bath. Expand the cells a...

<ol>
	<li>Amon, L., Hatscher, L., Heger, L., Dudziak, D., Lehmann, C. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+complete+repertoire+of+conventional+dendritic+cell+functions+for+cancer+immunotherapy.">Harnessing the complete repertoire of conventional dendritic cell functions for cancer immunotherapy.</a> Pharmaceutics. 12 (7), 12070663 (2020).</li><li>Banchereau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunobiology+of+dendritic+cells.">Immunobiology of dendritic cells.</a> Annual Review of Immunology. 18, 767-811 (2000).</li><li>Wculek, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+in+cancer+immunology+and+immunotherapy.">Dendritic cells in cancer immunology and immunotherapy.</a> Nature Reviews: Immunology. 20 (1), 7-24 (2020).</li><li>Kastenmuller, W., Kastenmuller, K., Kurts, C., Seder, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell-targeted+vaccines--hope+or+hype.">Dendritic cell-targeted vaccines--hope or hype.</a> Nature Reviews: Immunology. 14 (10), 705-711 (2014).</li><li>Bonifaz, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+targeting+of+antigens+to+maturing+dendritic+cells+via+the+DEC-205+receptor+improves+T+cell+vaccination.">In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination.</a> Journal of Experimental Medicine. 199 (6), 815-824 (2004).</li><li>Boscardin, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+targeting+to+dendritic+cells+elicits+long-lived+T+cell+help+for+antibody+responses.">Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses.</a> Journal of Experimental Medicine. 203 (3), 599-606 (2006).</li><li>Hossain, M. K., Wall, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Dendritic+Cell+Receptors+as+Targets+for+Enhancing+Anti-Cancer+Immune+Responses.">Use of Dendritic Cell Receptors as Targets for Enhancing Anti-Cancer Immune Responses.</a> Cancers. 11 (3), 11030418 (2019).</li><li>Johnson, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+melanoma+growth+by+targeting+of+antigen+to+dendritic+cells+via+an+anti-DEC-205+single-chain+fragment+variable+molecule.">Inhibition of melanoma growth by targeting of antigen to dendritic cells via an anti-DEC-205 single-chain fragment variable molecule.</a> Clinical Cancer Research. 14 (24), 8169-8177 (2008).</li><li>Trumpfheller, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbial+mimic+poly+IC+induces+durable+and+protective+CD4++T+cell+immunity+together+with+a+dendritic+cell+targeted+vaccine.">The microbial mimic poly IC induces durable and protective CD4+ T cell immunity together with a dendritic cell targeted vaccine.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (7), 2574-2579 (2008).</li><li>Idoyaga, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (6), 2384-2389 (2011).</li><li>Sancho, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+therapy+in+mice+via+antigen+targeting+to+a+novel,+DC-restricted+C-type+lectin.">Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin.</a> Journal of Clinical Investigation. 118 (6), 2098-2110 (2008).</li><li>Volckmar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+antigen+delivery+to+dendritic+cells+elicits+robust+antiviral+T+cell-mediated+immunity+in+the+liver.">Targeted antigen delivery to dendritic cells elicits robust antiviral T cell-mediated immunity in the liver.</a> Scientific Reports. 7, 43985 (2017).</li><li>Kraal, G., Breel, M., Janse, M., Bruin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans'+cells,+veiled+cells,+and+interdigitating+cells+in+the+mouse+recognized+by+a+monoclonal+antibody.">Langerhans' cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody.</a> Journal of Experimental Medicine. 163 (4), 981-997 (1986).</li><li>Volckmar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Vaccine. 37 (35), 4963-4974 (2019).</li></ol>

Chemical conjugation of an endocytosis receptor-specific antibody and a protein antigen provides an efficient and, importantly, also flexible approach for in vivo DC targeting in pre-clinical mouse models. With our protocol we provide an efficient approach for the successful conjugation of the model antigen OVA to a DEC-205-specific IgG antibody.
In our protocol, &#945;DEC-205 is purified from a hybridoma cellline and in the past, we have purified the antibody using protein G sepharos...

Dendritic cells (DC) are central players of the immune system. They are a diverse group of cells specialized in antigen-presentation and their major function is to bridge innate and adaptive immunity1,2. Importantly, DC not only play an important role in efficient and specific pathogen-directed responses but are also involved in many aspects of antitumor immunity1,3.
Due to their exclusive role in host immunity, DC came into focus as target cells for vaccination4. One approach is to target antigens to DC in vivo to induce antigen-specific immune responses and over the last years, a large number of studies have been dedicated to defining suitable receptors and targeting strategies1,4. One example is the C-type lectin receptor DEC-205, which can be targeted by DEC-205-specific antibodies to induce endocytosis. Importantly, DEC-205 targeting in the combination with suitable adjuvants has been shown to efficiently induce long-lived and protective CD4+ and CD8+ T cells, as well as antibody responses, also against tumor antigens3,5,6,7,8,9.
There are a number of studies showing conjugated antigens targeted to DC to be superior to free un-conjugated antigen3,5,10,11,12. This makes the conjugation of the antigen to the respective DC targeting moiety a central step in DC targeting approaches. In the case of DC targeting via antibodies or antibody fragments, antigens can be either chemically or genetically linked and either strategy provides its own (dis)advantages1. On the one hand, in genetically engineered antibody-antigen constructs there is a control over the antigen dose as well as the location providing superior comparability between lots1. At the same time however, chemical conjugation needs less preparation and provides more flexibility especially when attempting to test and compare different antigens and/or vaccination strategies in experimental and pre-clinical models.
Here, we present a protocol for the efficient and reliable chemical conjugation of ovalbumin (OVA) as a model protein antigen to a DEC-205-specific IgG2a antibody (&#945;DEC-205) suitable for in vivo DC targeting in mice. First, &#945;DEC-205 is purified from&#160;NLDC-145 hybridoma cells13. For chemical conjugation, the heterobifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC), which contains NHS (N-hydroxysuccinimide) ester and maleimide groups, is used, allowing covalent conjugation of amine- and sulfhydryl-containing molecules. Specifically, the primary amines of the antibody initially react with sulfo-SMCC and the resulting maleimide-activated &#945;DEC-205 then reacts with the sulfhydryl-containing OVA protein reduced through TCEP-HCl (Tris(2-carboxyethyl) phosphine hydrochloride). The final product is chemically conjugated &#945;DEC-205/OVA (Figure 1). Beyond chemical conjugation itself, our protocol describes removal of excess OVA from the conjugate&#160;as well as the verification of successful conjugation through western blot analysis and a specific enzyme-linked immunosorbent assay. We have successfully employed this approach in the past to chemically conjugate OVA and other proteins or immunogenic peptides to &#945;DEC-205. We demonstrate efficient binding to CD11c+ cells in vitro as well as the efficient induction of cellular and humoral immunity in vivo.
Certainly, there are drawbacks to this method such as in lot-to-lot comparability and in the exact dosing of the antigen within the final conjugate. Nevertheless, chemical conjugation provides experimental flexibility in the choice of the antibody and the protein antigen as compared to genetically engineered constructs. Therefore, we believe this approach is especially valuable in evaluating different antigens for their efficiency in DC targeting in pre-clinical mouse models, importantly also in the context of specific antitumor immune responses.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>antibody buffer 2 %</td>
			<td></td>
			<td></td>
			<td>2 % (w/v) Slim-Fast Chocolate powder in TBS-T</td>
		</tr>
		<tr>
			<td>antibody buffer 5 %</td>
			<td></td>
			<td></td>
			<td>5 % milk powder (w/v) in TBS-T</td>
		</tr>
		<tr>
			<td>blocking buffer (ELISA)</td>
			<td></td>
			<td></td>
			<td>10 % FBS in PBS</td>
		</tr>
		<tr>
			<td>blocking buffer 4 %</td>
			<td></td>
			<td></td>
			<td>4 % (w/v) Slim-Fast Chocolate powder in TBS-T</td>
		</tr>
		<tr>
			<td>blocking buffer 10 %</td>
			<td></td>
			<td></td>
			<td>10 % milk powder (w/v) in TBS-T</td>
		</tr>
		<tr>
			<td>cell culture flask T25</td>
			<td>Greiner Bio-One</td>
			<td>690175</td>
			<td>we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (690195 )</td>
		</tr>
		<tr>
			<td>cell culture flask T75</td>
			<td>Greiner Bio-One</td>
			<td>658175</td>
			<td>we use standard CELLSTAR&#160; filter cap cell culture flasks; alternatively use suspension culture flask (658195)&#160;</td>
		</tr>
		<tr>
			<td>cell culture flask T175</td>
			<td>Greiner Bio-One</td>
			<td>661175</td>
			<td>we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (661195)</td>
		</tr>
		<tr>
			<td>centrifugal concentrator MWCO 10 kDa</td>
			<td>Sartorius</td>
			<td>VS2001</td>
			<td>Vivaspin 20 centrifugal concentrator</td>
		</tr>
		<tr>
			<td>centrifugal protein concentrator MWCO 100 kDa, 5 - 20 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>88532</td>
			<td>Pierce Protein Concentrator, PES 5 -20 ml; we use the Pierce Concentrator 150K MWCO 20mL (catalog number 89921), which is however no longer available&#160;</td>
		</tr>
		<tr>
			<td>centrifuge bottles</td>
			<td>Nalgene</td>
			<td>525-2314</td>
			<td>PPCO (polypropylene copolymer) with PP (polypropylene) screw closure, 500 ml; obtained from VWR, Germany</td>
		</tr>
		<tr>
			<td>coating buffer (ELISA)</td>
			<td></td>
			<td></td>
			<td>0.1 M sodium bicarbonate (NaHCO3) in H2O (pH 9.6)</td>
		</tr>
		<tr>
			<td>desalting columns MWCO 7 kDa</td>
			<td>Thermo Fisher Scientific</td>
			<td>89891</td>
			<td>Thermo Scientific Zeba Spin Desalting Columns, 7K MWCO, 5 mL</td>
		</tr>
		<tr>
			<td>detection reagent ELISA (HRPO substrate)</td>
			<td>Sigma-Aldrich/Merck</td>
			<td>T8665-100ML</td>
			<td>3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB) liquid substrate system</td>
		</tr>
		<tr>
			<td>detection reagent western blot (HRPO substrate)</td>
			<td>Roche/Merck</td>
			<td>12 015 200 01</td>
			<td>Lumi-Light Western Blotting Substrate (Roche)</td>
		</tr>
		<tr>
			<td>dialysis tubing MWCO 12 - 14 kDa</td>
			<td>SERVA Electrophoresis</td>
			<td>44110</td>
			<td>Visking dialysis tubing, 16 mm diameter</td>
		</tr>
		<tr>
			<td>ELISA 96-well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>442404</td>
			<td>MaxiSorp Nunc-Immuno Plate</td>
		</tr>
		<tr>
			<td>fetal calf serum</td>
			<td>PAN-BIOtech</td>
			<td>P40-47500</td>
			<td>FBS Good forte</td>
		</tr>
		<tr>
			<td>ISF-1 medium</td>
			<td>Biochrom/bioswisstec</td>
			<td>F 9061-01</td>
			<td></td>
		</tr>
		<tr>
			<td>milk powder</td>
			<td>Carl Roth</td>
			<td>T145.2</td>
			<td>powdered milk, blotting grade, low in fat; alternatively we have also used conventional skimmed milk powder from the supermarket</td>
		</tr>
		<tr>
			<td>NLDC-145 hybridoma</td>
			<td>ATCC</td>
			<td>HB-290</td>
			<td>if not already at hand, the hybridoma cells can be acquired from ATCC</td>
		</tr>
		<tr>
			<td>non-reducing SDS sample buffer 4 x&#160;</td>
			<td></td>
			<td></td>
			<td>for 12 ml: 4 ml of 10 % SDS, 600 &#181;l 0.5 M Tris-HCl (ph 6.8), 3.3 ml sterile H2O, 4 ml glycerine, 100 &#181;l of 5 % Bromphenol Blue</td>
		</tr>
		<tr>
			<td>ovalbumin</td>
			<td>Hyglos (via BioVendor)</td>
			<td>321000</td>
			<td>EndoGrade OVA ultrapure with &#60;0.1 EU/mg</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Gibco Penicillin/Streptomycin 10.000 U/ml; alternatively Gibco Penicillin/Streptomycin 5.000 U/ml (15070-063) can be used</td>
		</tr>
		<tr>
			<td>PETG polyethylene terephthalate glycol cell culture roller bottles</td>
			<td>Nunc In Vitro</td>
			<td>734-2394</td>
			<td>standard PDL-coated, vented (1.2X), 1050 cm&#178;, 100 - 500 ml volume; obtained from VWR, Germany&#160;&#160;</td>
		</tr>
		<tr>
			<td>pH indicator strips</td>
			<td>Merck</td>
			<td>109535</td>
			<td>pH indicator strips 0-14</td>
		</tr>
		<tr>
			<td>polyclonal goat &#945;rat-IgG(H+L)-HRPO (western blot)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>112-035-062</td>
			<td>obtained from Dianova, Germany; used at 1:5000 for western blot</td>
		</tr>
		<tr>
			<td>polyclonal goat &#945;rat-IgG+IgM-HRPO antibody&#160; (ELISA)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>112-035-068</td>
			<td>obtained from Dianova, Germany; used at 1:2000 for ELISA</td>
		</tr>
		<tr>
			<td>polyclonal goat &#945;rabbit-IgG-HRPO (western blot)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>111-035-045&#160;</td>
			<td>obtained from Dianova, Germany; used at 1:2000 for western blot</td>
		</tr>
		<tr>
			<td>polyclonal rabbit &#945;OVA (ELISA)</td>
			<td>Abcam</td>
			<td>ab181688</td>
			<td>used at 3 ng/&#181;l</td>
		</tr>
		<tr>
			<td>polyclonal rabbit &#945;OVA antibody (western blot)</td>
			<td>OriGene</td>
			<td>R1101</td>
			<td>used at 1:3,000 for western blot</td>
		</tr>
		<tr>
			<td>Protein G Sepharose column</td>
			<td>Merck/Millipore</td>
			<td>P3296</td>
			<td>5 ml Protein G Sepharose, Fast Flow are packed onto an empty column PD-10 (Merck, GE 17-0435-01)</td>
		</tr>
		<tr>
			<td>protein standard</td>
			<td>Thermo Fisher Scientific</td>
			<td>26616</td>
			<td>PageRuler Prestained Protein ladder 10 - 180 kDa</td>
		</tr>
		<tr>
			<td>PVDF (polyvinylidene difluoride) membrane</td>
			<td>Merck/Millipore</td>
			<td>IPVH00010</td>
			<td>immobilon-P PVDF (polyvinylidene difluoride) membrane</td>
		</tr>
		<tr>
			<td>rubber plug</td>
			<td>Omnilab</td>
			<td>5230217</td>
			<td>DEUTSCH &#38; NEUMANN rubber stoppers (lower &#934; 17 mm; upper &#934; 22 mm)</td>
		</tr>
		<tr>
			<td>silicone tube</td>
			<td>Omnilab</td>
			<td>5430925</td>
			<td>DEUTSCH &#38; NEUMANN (inside &#934; 1 mm; outer &#934; 3 mm)</td>
		</tr>
		<tr>
			<td>Slim-Fast</td>
			<td></td>
			<td></td>
			<td>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</td>
		</tr>
		<tr>
			<td>stopping solution (ELISA)</td>
			<td></td>
			<td></td>
			<td>1M H2SO4</td>
		</tr>
		<tr>
			<td>sulfo-SMCC</td>
			<td>Thermo Fisher Scientific</td>
			<td>22322</td>
			<td>Pierce Sulfo-SMCC Cross-Linker; alternatively use catalog number A39268 (10 x 2 mg)</td>
		</tr>
		<tr>
			<td>syringe filter unit 0.22 &#181;m&#160;</td>
			<td>Merck/Millipore</td>
			<td>SLGV033RS</td>
			<td>Millex-GV Syringe Filter Unit, 0.22 &#181;m, PVDF, 33 mm, gamma sterilized&#160;</td>
		</tr>
		<tr>
			<td>syringe 10 ml</td>
			<td>Omnilab</td>
			<td></td>
			<td>Disposable syringes Injekt&#174; Solo B.Braun</td>
		</tr>
		<tr>
			<td>Sterican&#174; cannulas</td>
			<td>B. Braun</td>
			<td></td>
			<td>Sterican&#174; G 20 x 1 1/2&#34;&#34;; 0.90 x 40 mm; yellow</td>
		</tr>
		<tr>
			<td>TBS-T</td>
			<td></td>
			<td></td>
			<td>Tris-buffered saline containing 0.1 % (v/v) Tween 20</td>
		</tr>
		<tr>
			<td>TCEP-HCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>A35349</td>
			<td></td>
		</tr>
		<tr>
			<td>tubing connector</td>
			<td>Omnilab</td>
			<td></td>
			<td>Kleinfeld miniature tubing connectors for silicone tube</td>
		</tr>
	</tbody>
</table>

chemical conjugation of a purified dec-205-directed antibody with full-length protein for targeting mouse dendritic cells in vitro and in vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical conjugation of &#945;DEC-205 to OVA protein using this protocol will typically allow efficient generation of &#945;DEC-205/OVA for in vivo DC targeting approaches. There are different strategies to verify the technique itself and to test the functionality of the yielded conjugate. Western blot analysis and ELISA are used to verify successful conjugation and at the same time detect potentially left free OVA (Figure 2). In vitro bindi...

Watch this Scientific Journal Video about Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo at JoVE.com

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

This protocol for which all of the experimental condition have been carefully optimized can be used to facilitate a successful and efficient chemical conjugation of the DEC-205 antibody to OVA antigen. The main advantages of this technique are that it permits a flexible selection of the protein antigen and antibody, and that it does not rely on genetic fusion of these components. We have also used this protocol to generate conjugates of hepatitis C virus proteins and anti-DEC-205 for in vivo DC targeting.This could likewise be valuable for anti-tumor vaccination approaches. To start anti-DEC-205 production, resuspend a one milliliter volume of one to five million thawed cryo-preserved NLDC 145 cells in nine milliliters of 37 degrees Celsius ISF-1 medium supplemented with 1%penicillin and streptomycin, and seed the cells into a 25 square centimeter cell culture flask for incubation at 37 degrees Celsius and 5%carbon dioxide for 24 to 48 hours. When the cells reach 70%confluency, transfer the entire cell suspension into a 15 milliliter conical tube and collect the cells by centrifugation.Resuspend the pellet in 12 milliliters of fresh complete 37 degree ISF-1 medium and re-plate the cells in a 75 square centimeter flask. After 48 to 72 hours of culture, split the 70%confluent culture between each of two new 75 square centimeter flasks and add six milliliters of fresh complete ISF-1 medium to each flask. When the cultures reach 70%confluency, use a 10 milliliter pipette to flush the cell culture flask bottoms and culture surfaces with the cell suspension to collect all of the cells and transfer 10 milliliters of each expanded NLDC 145 cell suspension into individual PETG roller bottles.Then add 140 milliliters of complete ISF-1 medium to each roller bottle culture and culture the roller bottles at 37 degrees Celsius, 5%carbon dioxide and 25 rounds per minute for three days. For antibody purification from the NLDC 145 supernatant, place the end of a silicon tube into the supernatant-filled beaker and allow 800 milliliters of supernatant to run dropwise through a Protein G Sepharose column. For elution, add 100 microliters of 1.5 molar Tris-HCL into each of twenty 1.5 milliliter tubes and remove the rubber plug from the column.Transfer one milliliter of 0.1 molar glycine to the upper chamber of the column and collect the antibody containing eluent into one of the prepared 1.5 milliliter tubes and repeat for each tube. When all of the antibody has been collected and the antibody-containing elutions have been pooled, close the bottom of a 20 centimeter length dialysis tubing with an appropriate dialysis tubing closure, and carefully pipette the antibody elution into the tubing. Close the top of the dialysis tubing with a second clamp and fix the upper clamp of the dialysis tubing to a floating stand.Add a magnetic stir bar to a beaker of PBS and place the beaker onto a magnetic stirrer. After overnight dialysis at four degrees Celsius, open one clamp to allow loading of the complete dialysate to a centrifugal concentrator with a 10 kiloDalton molecular weight cutoff, and centrifuge the concentrator for 30 minutes at 693 times G and four degrees Celsius. At the end of the spin, load the centrifugal concentrator with 10 milliliters of PBS for a second centrifugation until a final one to 1.5 milliliter volume of antibody solution is obtained.To conjugate OVA to the anti-DEC-205 antibody, add 200 microliters of PBS supplemented with 500 micrograms of OVA protein, 240 microliters of a freshly prepared TCEP-HCL solution, and 560 microliters of sterile ultrapure water to a 1.5 milliliter tube for a 1.5 hour incubation at room temperature. At the same time, add 2.5 milligrams of anti-DEC-205 in 900 microliters of PBS and 100 microliters of freshly prepared sulfo-SMCC to a second 1.5 milliliter tube for a 30-minute incubation at 37 degrees Celsius at 550 revolutions per minute in a heating block. At the end of the incubations, place desalting columns with loosened caps and twisted off bottoms into individual 15 milliliter conical tubes, and centrifuge the columns to remove the liquid.After centrifugation, place the columns in new tubes with the caps removed, and slowly load the antibody sulfo-SMCC and OVA TCEP-HCL solutions to the center of the compact resin bed of one column per solution. After centrifuging, discard the columns and immediately mix the antibody and OVA solutions with gentle pipetting. Load the anti-DEC-205/OVA solution onto a centrifugal protein concentrator and fill the concentrator with PBS to a 15 milliliter volume.Centrifuge the concentrator for five minutes at 2, 000 times G and room temperature and fill the concentrator with an additional 10 milliliters of PBS. After centrifuging the concentration for at least eight minutes under the same centrifuge conditions, gently collect the concentrated sample from the upper chamber. To verify the success of the conjugation by Western blot analysis, load an aliquot out of each sample on a protein standard onto an SDS gel and run the gel according to standard SDS-PAGE analysis protocols.After gel blotting, stain the membranes with horseradish peroxidase conjugated antibodies to detect anti-DEC-205 or OVA according to standard protocols. The membranes can then be analyzed for the presence of the respective protein in each sample. To verify the success of the conjugation by ELISA, coat an appropriate 96-well ELISA plate with 100 microliters of the anti-OVA antibody per well and serially dilute anti-DEC-205/OVA at a one to two ratio and blocking buffer to obtain dilutions from six micrograms per milliliter to 93.8 nanograms per milliliter.Add 100 microliters of each dilution to the appropriate wells of the antibody-coated plate for a one-hour incubation at room temperature. At the end of the incubation, add 100 microliters of horseradish peroxidase conjugated antibody against the anti-DEC-205/OVA conjugate to the plate for a one-hour incubation at room temperature, then add 50 microliters of horseradish peroxidase substrate to each well to allow analysis of the color reaction by spectrophotometry. Parallel Western blot analysis can be used to detect both conjugated OVA as well as conjugated anti-DEC-205.Staining for OVA would allow the detection of excess free OVA if present, and staining for anti-DEC-205 verifies the success of the conjugation through an increase in the molecular weight between naked anti-DEC-205 and the conjugate. ELISA can also be used to assess the success of the conjugation with a positive association between the detected signal and the analyzed amount of protein indicating the successful generation of anti-DEC-205/OVA conjugant. Flow cytometry and immunofluorescence analysis clearly show that the anti-DEC-205 core efficiently binds bone marrow-derived CD11c-positive cells.Vaccination with anti-DEC-205/OVA in conjunction with adjuvant induces an increase in OVA-specific IgG titers in mouse recipients compared to vaccination with OVA and adjuvant alone. Furthermore, anti-DEC-205/OVA efficiently induces over-specific CD4 and CD8-positive T-cell responses with the anti-DEC-205/OVA-induced CD8-positive T-cell responses significantly exceeding that induced by OVA alone. For a successful conjugation of OVA antigen to anti-DEC-205 antibody, it is important to prepare the materials as demonstrated and to perform the conjugation steps under consistent conditions.Following successful conjugation of the antigen and the antibody, numerous subsequent approaches are possible, including binding analysis and the induction of pathogen or tumor-related immunity in vivo.

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