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Abstract

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Protocol

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Acknowledgements

Materials

References

Cancer Research

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

Published: February 5th, 2021

DOI:

10.3791/62018

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
* These authors contributed equally

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.

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.

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....

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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 a.......

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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.......

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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.......

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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 ''Immunotherapy of Cancers" (HCC_WP2b).

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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

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