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

Summary

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.

Abstract

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

Introduction

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. Importantly, DC not only play an important role in efficient and specific pathogen-directed responses but are also 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 vaccination⁴. One approach is to target antigens to....

Protocol

All of the described animal experiments were approved by the local government agency (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit; file number 33.12-42502-04-10/0108) and were performed according to the national and institutional guidelines.

1. Production of αDEC-205 from the hybridoma cell line NLDC-145

1. For antibody production, thaw cryopreserved NLDC-145 cells producing αDEC-205 at 37 °C in a water bath. Expand the cells at 37 °C and 5% CO₂. Two 75 cm² bottles will be needed to proceed to antibody production. Cell culture procedures should be ....

Results

Chemical conjugation of αDEC-205 to OVA protein using this protocol will typically allow efficient generation of αDEC-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.......

Discussion

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, αDEC-205 is purified from a hybridoma cellline and in the past, we have purified the antibody using protein G sepharos.......

Materials

Name	Company	Catalog Number	Comments
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