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Antigen degradation machinery
Five ways to obtain catalytic antibodies
Immunization by transition state analog of reaction
Production of antiidiotypic antibody
Reactive immunization
Induction of autoimmune disease
Screening of phage- display libraries
A. Belogurov Jr. et al, BioEssays 2009
I. Smirnov, A. Belogurov Jr., A. Gabibov. Enzyme Catalysis in Organic Synthesis, Wiley-VCH
Combinatorial approach Rational design
High resolution 3D Effective screening
Directed evolution
A. Kolesnikov et al, PNAS 2000
• Superposition of the active
sites of esterolytic abzymes
9A8 (green) and 17E8
(blue).
• Ser99 - His35 diades are
indicated.
• Hydrogene bonds are
indicated by dashed lines.
9A8 may covalently accept and destroy anti-acetylcholine esterase poisons from blood stream
0
50
100
150
200
0 24 48
ButChE
rh9A8
control
Soman
r9A8 interact with soman-MCA
RFU
Time, h
S
P
“Reactibody” approach
Biotin-X-phosphonate
Griffin.1 scFv library
+
Screening for biotin-X binding
Conversion into the full-size human antibody
crystal
A. Reshetnyak et al, JACS 2007
A.17 antibody has unusual deep cavity with nucleophilic tyrosine at its base
I. Smirnov, I. Kurkova et al, PNAS 2011
A.17 antibody has unusual deep cavity with nucleophilic tyrosine at its base
I. Smirnov, I. Kurkova et al, PNAS 2011
Tyr59
Tyr53
Tyr33
Tyr34
Trp109 Tyr37
Trp92
Trp48
Phe100
OP compound
P
O
O
O
NO2
HN
Tyr37
OH
P(R)
P
O
OO
HN
Tyr37
P(S)
SN2
Tyr59 Tyr53
Tyr33
Tyr34
Trp109
Asn105
Ala107
Phe100
Trp92
Trp48
Cl-
2.5 Å 3.3 Å
3.2 Å
Ser51
2.8 Å
Tyr37
The pre-existing primitive active site of the A.17 antibody stereo-selectively interacts with P(R)-isomer of the phosphonate molecule
A.17 catalyzes paraoxon hydrolysis via covalent intermediate
I. Smirnov, I. Kurkova et al, PNAS 2011
CHO-based expression system employing genes encoding butyrylcholinestrase and
proline-rich peptide under control of EF promoter resulted in self-assembled active
enzyme multimers, which revealed enhanced pharmacodynamics being polysialylated.
Polysialylated bioscavenger revealed enhanced
protection of mice against 3.7 LD50 of nerve gas VR.
Perspectives: generation of artificial biocatalysts in vivo
Abzymes
IL6
TNFa
gp120
AbP
???
OPC
Prodrugs
Cocaine
Ponomarenko et al. Biochemistry 2006 Durova et al. Molecular Immunology, 2009
MESSAGE • Basically ALL Autoantigens may serve as substrates for autoantibodies. Shuster et al. Science, 1992
Kolesnikov et al. PNAS, 2000
Ponomarenko et al. Meth. Immunol., 2002
Kozyr et al. Imm.Lett., 2002
Ponomarenko et al. PNAS, 2006
Ponomarenko et al. Biochemistry 2006
Ponomarenko et al. Biochemistry, 2007
Belogurov et al. J.Immunology, 2008
Durova et al. Molecular Immunology, 2009
Belogurov et al. Autoimmunity, 2009
A. Belogurov Jr. et al, BioEssays 2009
Structural Similarity Between BV04-01 and MRL-4 anti-DNA Autoantibodies: DNA-binding and DNA-cleaving
Activities are Germline-Encoded CDRH3
DNA
Possible catalytic residues
Schuster et al, Science 1992 Gololobov et al, PNAS 1995 Gololobov et al, Mol. Immunol. 1997 Kozyr et al, Autoimmunity 2009
Two hypotheses of DNA-abzyme mediated cell-death.
Myosin I Annexin V SnRNP
A. Belogurov Jr. et al, BioEssays 2009
Do Myelin-Directed Antibodies Predict Multiple Sclerosis? N EJM, 2003
The B-Cell – Old Player, New Position on the Team NEJM, 2008
Multiple sclerosis
1. B-cells as one of the key players in the MS
2. Environmental hypothesis of MS induction. EBV virus involvement.
3. Is immunoproteasome is involved in the MS/EAE development?
Abzymatic Site-specific MBP hydrolysis
MBP
Musse et al, PNAS 2006
The cleavage sites are localized inside the encephalitogenic epitopes
N. Ponomarenko et al. PNAS 2006
Design of the MBP epitope library
A. Belogurov Jr. et al. J. Immunology 2008
EPeFRET is hydrolyzed by abzymes and model proteases
Control
Abzyme
Control
MMP-3
Trypsin
PS-CFP2 Phi-YFP MBP 82-101
Novel Biomarker for MS
A. Belogurov Jr. et al. J. Immunology 2008
MDS score versus days post disease induction (surrounding pictures). Peptides were applied at days 7-11 (120 μg/rat per day) after disease induction by nasal route. Maximum clinical score in each group of rats, median and 95% confidential interval (in the middle). NS - not significant
Administration of MBP peptides to DA rats with
EAE
A. Belogurov Jr. et al. Autoimmunity 2009
Pre-clinical trials of MBP46-62 formulation in DA rats
• SUV liposomes 50-90 nm mimicking virus particles
• Interaction with APC cells
• Dose-dependent effect
Design of specific B-cell killers
A. Stepanov, A. Belogurov Jr., et al. PLoS ONE 2011
Environmental hypothesis of MS induction. EBV virus involvement.
Antibodies selected from MS Phage-
display library are crossreactive towards
both, Myelin Basic Protein and EBV latent
membrane protein 1.
A. Gabibov, A. Belogurov Jr., et al. FASEB J. 2011
A. Gabibov, A. Belogurov Jr., et al. FASEB J. 2011
Environmental hypothesis of MS induction. EBV virus involvement.
P IP
Immunoproteasome generates increased amount of immunodominant MBP peptides
A. Belogurov Jr. et al. unpublished
A. Belogurov Jr. et al. unpublished
Immunoproteasome as a target for MS treatment
A. Belogurov Jr. et al. unpublished
COLLABORATION
RUSSIA
Prof. Vadim Govorun Institute of physicochemical medicine
Prof. Eugene Nikolaev Institute of Biochemical Physics RAS Prof. Alexey Boyko Moscow MS Center Prof. Natalia Sharova Koltsov Institute of Developmental Biology RAS Prof. Dmitry Knorre Prof. Olga Fedorova, Prof. Nina Tikunova, Dr. Nikita Kuznetsov Institute of chemical biology and Fundamental medicine
Dr. Dmitry Genkin ZAO Pharmsynthez
Dr. Dobroslav Melamed Melamed medicals
USA
Prof. Al Tramontano, UC Davis, Medical school National Institute of Allergy and Infectious
Diseases NIH
FRANCE
Prof. Daniel Thomas, Alain Friboulet The University of Technology, Compiegne
Prof. Patrick Masson, Drs Eugénie Carletti, Florian Nachon
Département de Toxicologie – CRSSA Institut de Recherche Biomédicale des Armées
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