Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Summary

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

Abstract

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long α-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

Introduction

Antibodies play important roles in humoral immune response for the neutralization of pathogenic bacteria and viruses¹. However, some antibodies exhibit harmful impacts to the organisms, such as cross-reactive antibodies in the ADE effect during DENV infection and over-reactive antibodies in myasthenia gravis, which is an autoimmune diseases^2,3. ADE effect is mediated by the cross-reactive antibodies that make the bridge to connect DENV and Fc receptor presenting cells^4,5, while myasthenia gravis is caused by the excessive antibo....

Protocol

1. Chemical synthesis of the hydrazine derivative of a Fc-III peptide

1. Converting 2-Cl-(Trt)-Cl resin to 2-Cl-(Trt)-NHNH₂ resin
   1. Weigh 625 mg of 2-Cl-(Trt)-Cl resin (0.25 mmol) into a 25 mL peptide synthesis vessel.
   2. Add 5 mL of N,N-dimethylformamide (DMF) to the resin from the top of the vessel, put the cap back, gently shake the vessel for 15 s and then drain it. Repeat DMF washing for two more times.
   3. Add 5 mL of dichloromethane (DCM) to the vessel, put the cap back, gently shake the vessel for 15 s and then drain it. Repeat DCM washing for two more times.
   4. Repeat step 1.1.2 three times....

Results

The flowchart for the synthesis route by native chemical ligation in this article is shown in Figure 1. Figures 2-6 show the chromatograms (A) and mass spectra (B) of chemical synthesized hydrazine derivative of a Fc-III peptide, recombinant expressed linker and antigen part, the purified product from NCL reaction, the purified product from desulfurization reaction and the purified final product DCAF1, respectively. The chromatograms show th.......

Discussion

The protocol here describes the semi-synthesis and detection of DCAF1 by using NCL approach, which is shown in Figure 1. Briefly, the two fragments of DCAF1 are chemical synthesized and recombinantly expressed, respectively; then, the full length DCAF1 molecule is assembled, modified and purified. For the hydrazine derived Fc-III fragment synthesis, using low-capacity 2-Cl resin is quite important, because high-capacity has a negative effect for hydrazine generation and leads to low yield of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Chlorotrityl resin	Tianjin Nankai HECHENG S&T
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium hexafluorophosphate 3-oxide	GL Biochem	00703
2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate	GL Biochem	00706
2,2′-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride	J&K Scientific	503236
4G2 antibody	Thermo	MA5-24387
4-mercaptophenylacetic acid	Alfa Aesar	H27658
96-well microtiter plates	NEST	701001
Acetonitrile	Thermo-Fisher	A955	MS Grade
AgOAc	Sinopharm Chemical Reagent	30164324
anti-GST antibody	Abclonal	AE001
Anti-mouse IgG, HRP-linked Antibody	Cell Signaling Technology	7076P2
BSA	Beijing DINGGUO CHANGSHENG BOITECHNOL
CD spectrometer	Applied Photophysics Ltd
dialysis bag	Sbjbio	SBJ132636
Dichloromethane	Sinopharm Chemical Reagent	80047360
diethyl ether	Sinopharm Chemical Reagent	10009318
DNA Gel Extraction Kit	Beyotime	D0056
Fusion Lumos mass spectrometer	Thermo
GSH Sepharose	GE Lifesciences
Guanidine hydrochloride	Sinopharm Chemical Reagent	30095516
Hydrazine hydrate	Sinopharm Chemical Reagent	80070418
Hydrochloric acid	Sinopharm Chemical Reagent	10011018
imidazole	SIGMA	12399-100G
Isopropyl β-D-Thiogalactoside	SIGMA	5502-5G
kanamycin	Beyotime	ST101
Methanol	Thermo-Fisher	A456	MS Grade
N, N-Diisopropylethylamine	GL Biochem	90600
N, N-Dimethylformamide	Sinopharm Chemical Reagent	8100771933
NcoI	Thermo	ER0571
PBS buffer	Solarbio	P1022
Peptide BEH C18 Column	Waters	186003625
piperidine	Sinopharm Chemical Reagent	80104216
Plasmid Extraction Kit	Sangon Biotech	B611253-0002
QIAexpress Kit	QIAGEN	32149
Rapid DNA Ligation Kit	Beyotime	D7002
Sodium dihydrogen phosphate dihydrate	Sinopharm Chemical Reagent	20040718
Sodium hydroxide	Sinopharm Chemical Reagent	10019762
Sodium nitrite	Sinopharm Chemical Reagent	10020018
sodium chloride	Sinopharm Chemical Reagent	10019318
Standard Fmoc-protected amino acids	GL Biochem
sterilizing pot	Tomy	SX-700
SUMO Protease	Thermo Fisher	12588018
stop solution	Biolegend	423001
the whole gene sequence that can express SUMO-linker-antigen	Taihe Biotechnology Compay
TMB reagent	Biolegend	421101
Trifluoroacetic acid	SIGMA	T6508
Triisopropylsilane	GL Biochem	91100
Tris(2-carboxyethyl)phosphine hydrochloride	Aladdin	T107252-5g
tryptone	OXOID	LP0042
Tween 20	Solarbio	T8220
Ultimate 3000 HPLC	Thermo
vacuum pump	YUHUA	SHZ-95B
XhoI	Thermo	IVGN0086
yeast extract	OXOID	LP0021