Abstract
Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach invaccine 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 offull-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 (αDEC-205)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 αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-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.
Highlights
Dendritic cells (DC) are central players of the immune system
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 ( DEC-205) suitable for in vivo dendritic cells (DC) targeting in mice
Staining for OVA in the western blot analysis allows the detection of excess free OVA potentially still present next to the αDEC-205/OVA yielded in step 3.6., which is not the case for the blot shown
Summary
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 immunity[1,2]. DC play an important role in efficient and specific pathogendirected responses but are involved in many aspects of antitumor immunity[1,3]. Due to their exclusive role in host immunity, DC came into focus as target cells for vaccination[4]. DEC-205 targeting in the combination with suitable adjuvants has been shown to efficiently induce longlived and protective CD4+ and CD8+ T cells, as well as antibody responses, against tumor antigens[3,5,6,7,8,9]
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