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