Каталитические аутоантитела в медецине и биотехнологии Габибов А.Г.

Каталитические аутоантитела в медецине и биотехнологии Габибов А.Г. Лекции 7-8

Ферменты – биокатализаторы жизни – были отобраны естественной эволюцией для обеспечения развития живых клеток и целых организмов в их естественной среде. Поэтому, природные ферменты не всегда обладают адекватными свойствами, специфичностью, стабильностью и каталитической активностью для применения их в биотехнологии. Jacques Fastrez

AntiBodies Каталитические антитела ENzymes Abzymes (Абзимы)

Каталитические антитела.

Catalysis Binding Drug discovery Artificial Enzymes Directed Enzyme evolution Searching for new substrates Recombinant antibodies The main characteristic of binding process is Kd=k1/k-1 Km=(k-1 +kcat)/k1 The main characteristic of catalysis is Km/kcat

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

The Claisen rearrangement of chorismate to prephenate catalyzed by antibody 1F7 chorismate prephenate Transision state of Claisen rearrengment Transision state analog Jackson D. et al. (1988) J. Am. Chem. Soc., 110: p. 4941-42.

Abzyme catalyzed Diels-Alder reaction Hilvert D. et al. (1989) J. Am. Chem. Soc. 111, 9261-9262. Braisted A. C. et al. (1990) J. Am. Chem. Soc., 112, 7430-1.

Основные пути получения каталитических антител Иммунизация аналогом переходного состояния реакции Получение анти-идиотипических антител Реакционная иммунизация Индукция аутоиммунного заболевания Скрининг фаг-дисплейных библиотек

The same amount of target may be inactivated by smaller amount of catalytic antibody Binding antibody Catalytic antibody

• Долгое время жизни в кровотоке • Высокая специфичность • Одно и то же количество антигена может быть инактивированно меньшим количеством каталитического антитела + Богатый мировой опыт в антительной терапии + Широкие возможности по созданию и производству мАт Почему терапия каталитическими антителами может быть эффективна? + Мышиный репертуар может быть заменен на человеческий Низкая скорость

Каталитические антитела: • Способны акцептировать или каталитически уничтожать: фосфорорганические вещества, химическое оружие, пестициды (Smirnov et al. PNAS 2011) • Способны каталитически уничтожать: белки-патогены белки оболочки вирусов и бактерий высокий уровень цитокинов при инфекциях • Способны гидролизовать препараты в кровотоке кокаин (Mets B. et al PNAS (1998),95,10176-81) • Способны превращать пролекарства в их активную форму раковая химеотерапия (Wentworth P. et al PNAS (1996),93,799-83)

Presumed tetrahedral intermediate of benzoyl ester hydrolysis and the corresponding phosphonate monoester analog Transition State Benzoyl Esterolisis (Approximation) Transition-State Analog (TSA) Free TSA: R=H TSA-I: R=(CH2)3NHCO(CH2)2CONH-BSA

Hydrolysis of cocaine at the Benzoyl ester and methyl ester Cocaine Ecgonine Methyl Ester Benzoyl Ecgonine

Dose-response relationship for mab 15A10 on rat’s survival after infusion an LD90 (16 mg/kg) cocaine

Rat Self-Administration * * * * An asterisk indicates a significant difference from cocaine

Schematic representation of antibody-directed enzyme prodrug therapy Tumour Inactive prodrug Cytotoxic agent Tumour Associated Antigenic determinants

Lineweaver-Burk analysis of EA11-D7-catalized hydrolysis of prodrug 1/S * 104, M 1/V * 109 M-1.s

The ß-lactamase model (amidase activity) The enzyme is ß-lactamase I (E.C. 3.5.2.6) from Bacillus cereus. - low molecular weight enzyme (29 kDa) - the 3-D structure is known. - the catalytic mechanism is well known - high kcat value (>2000 sec-1 with penicillin) - numerous substrates (penicillins, cephalosporins)  - the ß-lactamase activity is absent in mammals.

Inhibition of Bacillus cereus ß-lactamase by mAb 7AF9. 7AF9/ß-lactamase : ki = 10-5M. B. Avalle, D. Thomas & A. Friboulet, FASEB J. (1998) 12, 1055.

Mice immunization by Ab1 7AF9. Friboulet et al. (1999) Immunol. Today 20, 474-475

Catalytic properties of 9G4H9 Avalle, D. Thomas & A. Friboulet, FASEB J. (1998) 12, 1055 Conditions: 0.1 M phosphate buffer , pH 7.4 PADAC Ampicillin

The ß-lactamase model (amidase activity) The enzyme is ß-lactamase I (E.C. 3.5.2.6) from Bacillus cereus. - low molecular weight enzyme (29 kDa) - the 3-D structure is known. - high kcat value (>2000 sec-1 with penicillin)  - the ß-lactamase activity is absent in the serum of mammals. Avalle et al. (1998) FASEB J., 12, 1055-1060.

ß-lactamase 9G4H9 Comparison : ß-lactamase/9G4H9 Glu 166 S er130 S er70 Ly s 73 Glu 98 Gln 95 S er26 S er28 Ly s 27 Stabilization of the active site or general base Deacylation Binding and nucleophilic attack of ß-lactams Tight binding of substrate Padiolleau-Lefèvre et al. (2003) Biocat. Biotransf., 21, 79-85

Exo-amylolytic And A-glucosidase Activities Of Human Abzymes Exo-amylase action: a-Glucosidase action:

Human autoantibodies with amylolytic activity *Unit definition: one unit of activity was defined as the quantity of abzymes that released 1 μmole of reducing sugar from PNPG6 (5 mM) per minute at 30ºC, pH 7.5.

Catalysis by Anti-Idiotypic Antibodies. “The Lock and Key Approach” Enzyme Substrate Antibodies repertoire First antibody Anti-Idiotypic Antibodies formation Abzyme reaction Anti-Idiotypic Antibodies Enzyme reaction Abzyme Substrate

Antiidiotypic Origin of Catalytic Antibodies

Антиидиотипическое антитело 9A8 с ацетилхолинэстеразной активностью Суперпозиция активных сайтов эстеролитических антител 9A8 (зеленый) и 17E8 (голубой). Показана диада Ser99 - His35. Водородные связи отмечены пунктирными линиями. PNAS 2000 Dec 5;97(25):13526-31.

Acetylcholine Esterase-like Antiidiotypic 9A8 Antibody Fv fragment of 9А8 antibody (blue) Predicted catalytic diade in green 9A8 may covalently accept and delete acetylcholine esterase poisons from blood stream

BIACORE Analysis of Affinity Binding of Idiotype AE2 and Aniidiotype 9A8 (SPR) Concentrations of 9A8 are indicated on sensograms. K =2,26 ·10-9 М-1

Phosphonates Inhibitors of Enzymes and Abzymes Compound I: Coumarinyl ethyl p-trifluoroacetamidophenylmetyl phosphonate Compound (II): N-Nitrophenyl methyl-p-biotinamidophenylmethyl phosphonate

Recombinant 9A8 Fab Interacts with Idiotypic AB AE2. ELISA Interaction of idiotypic AE2 AB with recombinant heavy (HC) and light (LC) chains and Fab fragment. Purified extract from bacteria without expression plasmid served as negative control (K).

Catalytic Activity of Recombinant 9А8 A. Acetylcholine hydrolysis was measured according to Elman`s method. Recombinant heavy (HC) and light (LC) chains and recombinant Fab fragment were taken in concentration of 850 nM. Inhibition of Fab - mediated catalysis was performed by addition of corresponding idiotypic antibody AE in molar ratio Fab:AE2 1:1 and 1:2. Uncatalyzed acetylcholine hydrolysis served as negative control (K). B. Initial rate of acetylcholine hydrolysis dependence from concentration of Fab fragment.

Fluorescence Kinetic Analysis of the Interaction of Abzymes and Enzymes with Phosphonate (I) Aminco SPF-1000CS. excitation 1=365 nm, emission 2= 450 nm. 1 -butyrylcholinesterase 2 - AB 9А8 3 -acetylcholinesterase 4 - AB 2Н11 kBuChE=0,062 min-1 k9A8=0,015 min-1

Western Analysis of Proteins by Biotinilated Inhibitors 1 – Molecular mass markers 2 – Trypsin labeled by biotinil-AEBSF; 3 – Antibody 9А8 labeled by biotinil-AEBSF; 4 – Antibody 2H11 labeled by biotinil -AEBSF; 5 –Antibody 9А8 labeled by phosphonate (II); 6 – Trypsin labeled by phosphonate (II); 7 – Antibody 2H11 labeled by phosphonate (II); 1 2 3 4 5 6 7 70 kDa 24 kDa

MALDI MS analysis of tryptic digestion of Fab fragment of 9A8 The peptides of the variable domain of the heavy chain are indicated.  - Peptide labeled by Compound (I) 99-SYYGPDYWGQGTTLTVSSESQSFPLVSCESPLSDK-137

PNAS 2000 Dec 5;97(25):13526-31. mAb 9A8 Биокатализатор kкат/kнекат x10-8 81 0.60 n.i HE AcChoE 8000 0.13 FBS AcChoE 5800 0.11 kкат с-1 Ki мМ 22 18 1.35 615 527 4.15 410 297 Рекомбинантное 9А8 <10 8 Кинетические константы гидролиза ацетилхолина ферментами и абзимами Км мкМ kкат/Км х10-5 М-1с-1 - - - Антитело 9A8 может ковалентно акцептировать и выводить из кровотока нервнопаралитические яды

Antibody therapy. 3D-modeling of active site of 9A8 antibody. Rendered images of the 3D models of 9A8 wild type. Catalytic dyad Ser99-His35 is shown.

Soman r9A8 interact with soman-MCA RFU Time, h

Scheme of anti-idiopytic antibodies Subtilisin Substrate Products Subtilisin Anti subtilisin active center antibody (5H3H4) Ab2 Antiidiotypic antibodies (6B8E12) Ab2 Antiidiotypic antibodies activity screening Model substrates AAPFpNa и GGLpNa

Proteolytic activity of 6B8-E12 anti-idiotypic mAb The anti-idiotypic antibody 6B8-E12 (100 and 200 ng, lanes 2 and 3, respectively) was separated by 10% SDS-PAGE with FBSA impregnated in the gel during polymerization (see Materials and Methods for details). After electrophoresis, proteins were in-gel renatured by Triton X-100 washes. Proteolytic degradation was visualized by the increase in fluorescence intensity, seen as bright bands at the dark background. Mouse anti-c-myc antibody 9E10 was used as the negative control (lane 1). Bovine trypsin (100 pg and 1 ng, lanes 4 and 5, respectively) was used as the positive control.

Time course of unquenching of the FBSA fluorescence by the anti-idiotypic abzyme. Concentrations of antibody and F(ab) fragment were 0.5 μM, substrate was 0.16 μM. Measurements of fluorescence (!exc: 495 nm; !em: 525 nm) were done directly on microtiter plates at different times. F(ab) fragment of 6B8-E12 (gray); Ab2 6B8-E12 (black); IgG 4H7-H3 (white) used as negative control (29). Degree of total trypsin digestion of FBSA was 235 fluorescence a.u. (not shown). Fab 6B8E12 4H7-H3 Proteolytic activity of 6B8-E12 anti-idiotypic mAb

Comparison Of Kinetic Parameters For Amidase And Esterase Activities Of Ab2 6B8-E12 And Subtilisin Carlsberg Amidase activity of subtilisin Carlsberg and of 6B8-E12 (1 mM final concentration) was assayed by monitoring cleavage of succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide (AAPFpNa) and glutaryl-Gly-Gly-Leu-p-Nitroanilide (GGLpNa) at 410 nm in 0.1 M Tris-HCl pH 8.6. Esterase activity was estimated by monitoring hydrolysis of p-Nitrophenyl acetate by subtilisin Carlsberg and 6B8-E12 (0.46 !M final concentration) at 410 nm in 0.1 M phosphate buffer pH 7.6.

Hydrolysis Of Bioactive Peptides By 6B8-E12 Monoclonal Antibody Hydrolysis of natural bioactive peptides by antiidiotypic antibody 6B8-E12 as studied by mass spectrometry. Scissile bonds are marked by arrows; aromatic amino-acids residues (squares); aliphatic amino-acids residues (circles).

Hydrolysis of BSA-FITC by 6B8E12. Time, h BSA-FITC (0.16 mM) was incubated at 37oC with different concentration of 6B8E12 abzyme in the presence of NaN3

Idiotype-antiidiotype interaction 5H3H4/6B8E12 BSA-FITC cleavage inhibition by idiotype 5H3H4 Ab1+Ab2 ka kd Ab1/Ab2 Inhibition of FBSA cleavage reaction by the Ab1. The abzyme concentration was kept constant at 1 mM, the Ab1 concentration increased from 0 to 3 mM. Vi, cleavage rate in the presence of 5-H4; V0, cleavage rate with the abzyme alone.

6B8E12 activity inhibition BSA-FITC (0.16 mM) was incubated with 0.5 mM of the abzyme in the absence or presence of 10 mM PMSF or 1 mM aprotinin. 0,5µM of 6B8E12 0,5µM of 6B8E12+1µM aprotinin 0,5µM of 6B8E12+10µM PMSF

PCR with specific primers NcoI-SalI PCR PCR Cloning into pUC19 Fv regions of heavy chains into pUC19/SmaI FvL FvL Vkf Vkr FvH and FvL were cloned into expression vector pET32b FvL NcoI-BglII/BamHI Fv region of light chain into pUC19/SmaI Cloning of FvН и FvL regions of MAb

Catalytic activity of recombinant ScFv6B8E12

Rendered images of the 3-D models of Ab1 5-H4 and Ab2 6B8-E12 scFvs A shows a side view and a top view of the modeled structure of 5-H4 scFv with the characterictic protruding finger-like structure formed by VL CDR1 and CDR3. B is a rendered top view of 5-H4 scFv. C and D show side and top views respectively for the modeled structure of 6B8-E12 scFv with the typical cleft between VH and VL CDRs. Colors indicate VH CDR1 (yellow), CDR2 (green), CDR3 (red) and VL CDR1 (orange), CDR2 (cyan), CDR3 (pink).

SCIENCE VOL 298, pp 2195-2199 Antibody-Catalyzed Ozone Formation E. coli O112a,c cell after exposure to antigen-specific murine monoclonal IgG (15404, 20 M), HPIX in PBS and visible light for 1 hour at 4°C (5% viable). To visualize the sites of antibodyattachment gold-labeled goat anti-mouse antibodies were added after completion of the bactericidal assay (6). The arrow points to a puncture in the cell and plasma membrane.

PNAS (2002) 9, 3376–81 The reaction of 1O2 with H2O to form H2O3. The top pathway (TS0 at 61.1) is uncatalyzed. The middle pathway is H2O-catalyzed (TS1 at 28.9), whereas the lowest pathway (TS2 at 16.4) is (H2O)2-catalyzed. Either reaction (1) or (2) can be part of Pathways I and II. Antibody-Catalyzed Ozone Formation

Methods for generation and screening of combinatorial chemical libraries Tagged Methodologies: Phage Technology Peptides on Plasmids Peptide coded Libraries Electrophoric Polyhalobenzene Coded Libraries Encoded Combinatorial Libraries Based on a physical link between a protein and its encoded gene – the best known system is phage display. Filamentous phage The product of gene 8 (g8p) – is a small protein that forms the cylinder of the capsid; its number of copies (2700 for the wild-type phage). The other coat proteins (g3p, g6p, g7p and g9p) close the extremities of the cylinder. The product of gene 3 – present in three to five copies – is responsible for phage infectivity. Proteins of interest are usually fused by their C-terminal to g3p or to g8p.

Three critical aspects in any combinatorial library: The chemical units that go to the library The technique for generating the library Identification of the library members that interact with the biological target of interest

Improvement of thermodynamic stability and folding folded unfolded binding Affinity matrix 1. Conventional Phage Display 2. Selectively Infective Phage folded ligands binding and infection E.coli F pilus 3. Resistance Against Proteolysis folded folded folded unfolded unfolded unfolded digestion and infection E.coli F pilus unfolded

Enzyme Phage Display binding Affinity matrix TSA/SI Enzyme-substrate Phage Display N2 N1 N2 N1 Catalysis and product binding Interacting domain/peptide N2 N1 N2 N1 Affinity matrix

Substrate Phage-display N2 N1 N2 N1 substrate Catalysis and product binding Affinity matrix product Affinity matrix product Release by catalysis

DG= uncat DGS DG= cat DGP DGTS Substrate Enzyme *Substrate Catalyzed Transition State Uncatalyzed Transition State Enzyme *Product Product DG= uncat-DG= cat = DGTS-DGS or kcat/kuncat = KM/Ki Transition State Theory Of Biocatalysis

Philippe Vayron et al. Proc. Natl. Acad. Sci. USA, Vol. 97, Issue 13, 7058-7063, June 20, 2000 Immunization with ground state analog Antibody-catalyzed hydrolysis of organophosphorus poisons

Antibody toward pentacoordinate organophosphorus compound may destroy phosphonates and other tetracoordinate organophosphorus compound Hapten – transition state analog

Hyper Variability Of Antibody Molecule Provides The Best Template To Generate Artificial Binder (Scavenger)/Biocatalysts

Antibody fragments suitable for functional expression in E.coli

Химическая селекция фаг- дисплейных библиотек Ферменты Миллионы лет эволюции Искусственные биокатализаторы, полученные комбинаторными способами Несколько месяцев или недель эволюции Отбор из 108÷109 индивидуальных клонов Классы ферментов могут быть заменены суперсемейством иммуноглобулинов

+ Poisoning of OPC leads to collapse nervous system covalent coupling of toxin with acetylcholine esterase leads to increase of acetylcholine in synapse and collapse of nervous system acetylcholine

Question of OPC-poising therapy The major target of organophosphorous toxins are cholinesterase-like enzymes; Extremely low LD50 value OPC-associated mortality is 200000 people per year; There are real threats of acts of terrorism, for example sarin attack in Tokyo underground at 20 March of 1995

Risk group Soldier Farmers Users Stuff of OP poisons utilization plant

Antibody therapy Antidotes based on antibody OPC Antibody approach using internal image of enzyme active centre Fishing of antibody against OPC from phage library Antibodies has several advantages: Low immunogenic High stability in bloodstream They may used as «catalytic» vaccines

Antibody therapy. Antibody from phage library Phosphonate molecule has the high reactivity against esterase and amidase In contrast of «real» OPC phosphonates are less toxicity and stability in water, which enough for screening experiments. Tramontano et al., Applied Biochemistry and Biotechnology (2000)

-Молекула стрептавидина -Молекула BSA -Биотиновая группа -Фосфонатная группа Контрольная лунка Экспериментальная лунка Пул фагов после реакции с Bt-X Элюция трипсином Амплификация фагов Последующий раунд селекции Bt-X Реакционная (ковалентная) селекция Journal of the American Chemical Society 2007

Alignment of covalent and non-covalent binding clones The conserved amino acid residues of the heavy chain are highlighted in yellow and those of light chain in green. CDR regions are enclosed in blue boxes. Reactive clones are shown in bold font. All selected clones are highly conservative, but those clones which could bind phosphonate non-covalently deviates more significant. .

Stoichiometry of phosphorylation reactions Active center titration of a typical reaction of A.17 (18 M) with 100 M of reactive posphonate 18 мкМ А.17 А.17 А.17+Bt-X SELDI MS analysis of A.17 labeled with Bt-X phosphonate and unmodified A.17. The mass difference between labeled and unlabeled scFv corresponded to one phosphonate residue.

scFv A17 and A5 active site mapping Phosphonate labeled Control A17 VTISCSGSSSNIGNNYVSWYQQLPGTAPK A5 VTISCSGSSSNIGSNYVDWYQQLPGTAPK CDR1L FR1L FR2L MS/MS analysis of modified tryptic peptide A17 VTISCSGSSSNIGNNYVSWYQQLPGTAPK A5 VTISCSGSSSNIGSNYVDWYQQLPGTAPK Tyr36L Tyr32L MS analysis revealed that Tyr36L and Tyr32L are modified by reactive phosphonate in case of A17 and A5 respectively.

Position Of Nucleopholic Tyrosine Residues

A5 WT - control A5 Y36F Active site - mutagenesis A5 Y32F A17 WT - control A17 Y32F A17 Y36F A17 Y32,36F A17 Y36FS strept-HRP staining c-myc-HRP staining A17 A5 A5 Y32F and A17 Y36F; A17 Y32,36F; A17 Y36S were fully inactive in reaction with phosphonate. A5 Y36F and A17 Y32F retained its activity.

A5 active site modeling D93L Y33L N32L D93L F33L-mut N32L Y37L N105H F37L-mut N105H A B C D A:The model shows that Tyr33L, which is located on the surface of the protein, in CDR1 of the light chain, forms H-bonds (pink lines) with Asn32L (CDR1) and Asp93L (CDR3 of the light chain). B: When Tyr33L is mutated in Phe33L no H-bonds were found. C: Tyr37L does not form any H-bond with Asn105H (CDR3 of the heavy chain). The distance between the Tyr and Asn residues is 3.28 angstroms. D: When Tyr37L is mutated into Phe37L, the distance between the Tyr and Phe residues is found to increase to 4.84 angstroms The model shows that active Tyr33L forms H-bonds (pink lines) with Asn32L (CDR1) and Asp93L (CDR3 of the light chain)

A17 active site modeling Y37L N105H F37L-mut N105H D93L Y33L N32L A B C A: The model shows that Tyr33L does not form any H-bond with Asn32L (CDR1) and Asp93L (CDR3 of the light chain). The distances between the Tyr residue and the Asp and Asn residues are found to be 3.33 and 3.80 angstroms respectively (to be compared with the 2.18 and 2.04 found for scFv#5). B: At the contrary to scFv#5, in scFv#17, Tyr37L forms an H-bond with Asn105H. The distance between the two residues is found to be 1.79 angstroms. C: When Tyr37L is mutated to Phe37L there is no longer any H-bond, the distance between the two residues is found to be 2.93 angstroms The model shows that active Tyr37L forms H-bond (pink line) with Asn105H

Journal of the American Chemical Society 2007 Клоны, полученные после III раунда Реакционные клоны имеют несколько консервативных АК в CDR3H и состоят из комбинации из V1 и VH4 семейств (кроме A.21)

Кинетика модификации Китц-Вилсон Journal of the American Chemical Society 2007 Клон А17 проявлял наибольшую скорость модификации

Scavenger Receptors

Гидролиз Модификация Субстратная специфичность scFv A.17 Journal of the American Chemical Society 2007 В результате реакционной селекции были получены клоны с амидазной активностью

A.17 Catalytic Antibody 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 catalyzes paraoxon hydrolysis via covalent intermediate 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

The pre-existing primitive active site of the A.17 antibody stereo-selectively interacts with P(R)-isomer of the phosphonate molecule

Antigen binding center of unmodified Fab A17 hydrophobic aromatic aminoacids H chain Tyr33, Tyr53, Tyr59 L chain Tyr33, Tyr50, Trp92 aliphatic H chain Ser102, Ser103 L chain Leu96 two charged hydrophylic aminoacids L Lchain Asp51 and Lys54 the main chain of L chain Gly90-Thr91 the side chains of L chainSer35, Trp92, Pro98 and Phe100 Rings form sigma-like pocket opened opposite to the reactive Tyr. This may explain SN2 mechanism. The upper part stabilizes phosphonate molecule during first step of the non-covalent complex formation.

Active center of unmodified Fab A17 The light chain is in grey, the heavy chain in blue, the VH-loop is in magenta. Key residues are represented as sticks with nitrogen atoms in deep blue and oxygen atoms in red. Hydrogen bonds are represented by gray dashes. Electron density (2fo-fc) is represented by a blue mesh, contoured at 1.0 sigma.

Active site of FabA17 inhibited by OP molecule. The light chain is in grey, the heavy chain in blue, the VH-loop is in magenta and OP molecule in green. Key residues are represented as sticks with nitrogen atoms in deep blue, phosphorus in orange, and oxygen atoms in red. Hydrogen bonds are represented by gray dashes. Electron density (2fo-fc) is represented by a blue mesh, contoured at 1.0 sigma.

H chain CDR3 of non-modified (A) and phosphonate-modified (B) A17 superimposed on crystallographic electron density maps. The B-factors (e.i. protein crystallographic parameter reflecting the fluctuation of atoms about their average positions) for the CDR3 of both native and modified Fab are indicated. Structural changes at HCDR3 loop of A.17 upon phosphonylation. A B

The phenyl ring is confined in a pocket formed by the main chain of Gly90/Thr91 and the side chains of Ser35/Trp92/Pro98/Phe100, notably forming T-stacking interactions with Trp92 and Phe100

Superimposition of active sites of native (green) and OP-inhibited (blue) FabA17. This figure highlights the conformational changes of Trp92, Phe100, Tyr37 and residues of the VH-loop (Trp109, Ala107, Asp105).

Water molecules The phosphonyl oxygen is strongly H-bonded to water molecule W614 (2.55 Å). This suggests polarization of the P-O bond and subsequent increase in the electrophilicity of the phosphorus atom. There is a blob of electron density just above the face opposite to the OP-Tyr37 bond modeled as a cluster of mobile water molecules. These water molecules are at less than 5 Å from the phosphorus atom and are good candidate for in-line attack of the tyrosinyl-adduct. Tyr34 stabilizes this cluster by participating in the H-bond network. Tyr34 may also play a leading role during the phosphonylation step by aromatic-aromatic stacking interaction with the para-nitrophenyl leaving group.

Minimization of the energy: breathing movements FabA17 fragment movements and variable fragments movement alone resulting from the OP molecule entry. Yellow = light chain ; Blue = heavy chain ; Red = Heavy-CDR3; PO= grey (indicates the active site) View of the profile View of the profile The CDR-3 loop of the heavy chain is moving to reduce the entry of the cavity

Minimization of the energy: an amplitude of the loop movement Yellow = light chain and Orange = CDR-3 L ch ; Blue = heavy chain and Red= CDR-3 Hch; PO= grey (indicates the active site) The movements of the CDR-3’s towards the centre of the cavity cavity

H bonds interactions are in blue Chain H - Tyr34, Tyr53, His104 and Asn105 Chain L - Ser35, Asp51, Tyr37 Asn105 His104 Tyr34 Tyr53 Asp51 Ser35 Tyr37 Position OPX in the crystall

OPX which enters the pocket assembly of the residues wich are involved in the mimic of the enter of the PO molecule in the cavity Start Position : -3,77 kcal/mol Final Position : -6,33 kcal/mol

A.17 wt mutagenesis A17 L Y37F A17H H104A A17H N105A

Thermodynamic parameters of A17 modification

A.17 catalyzes paraoxon hydrolysis via covalent intermediate I. Smirnov, I. Kurkova et al, PNAS 2011

Titration of active sites A17 and Mutants with X phosphonate (Kitz-Wilson)

Molecular Mimicry (viral or bacterlial infection) Dendritic Cell/ B Cell Release of sequestered antigen Breakdown of T-cell supression Abnormalily in the thymic selection process Macrophage Activated TH cell Target tissue epithelium Antiself antibodies Help Inflammation and local DTH TISSUE DAMAGE

Hypotheses ON THE INCREASED ABZYME OCCURRENCE IN AUTOIMMUNITY Expanded repertoire of underevolved antibodies may favor the emergence of abzyme activity by following possible routes: 1.Spontaneous evolution of the existing enzyme plethora. 2.Expanded repertoire can result in the increased production of anti-idiotypes. Biological mechanisms of this phenomenon are still unknown.

Catalytic activity of Bence Jones Proteins 1845 W.MacIntire recognized the first case of Multiple myeloma Henry Bence Jones discussed the peculiarities of urinary proteins Gulstonian Lectures on chemical pathology to the Royal College of Physicians in London 1962 Edelman and Gally found that light chain of plasma immunoglobulin from patients with myeloma is identical with Bence Jones urine protein from the same patient. 1974 Erhan and Greller pointed out the statistically significant amino acid identity between immunoglobulin light chain and active cite of serine proteinase 1994 Matsuura et al, 1995 Paul et al. demonstrated catalytic activity of Bence Jones protein Catalytic Activity of Bence Jones Proteins is a possible Cause of Renal Failure Autoimmunity and Antibody Mediated Tissue Destructions

Role of B cells in the pathogenesis of rheumatoid arthritis The rheumatoid factors form large antigen/antibody complexes which may enhance the process of joint destruction. The poor prognosis of rheumatoid factor-positive patients is indicative of the critical role of immunoglobulin complexes in the continuous stimulation of the immune system and thus of the inflammatory processes. Gause A, Berek C. BioDrugs 2001;15(2):73-9 Sjogren's syndrome. i) defective apoptosis could lead to lymphoid cell accumulation and chronic inflammation in exocrine glands; ii) increased apoptosis of epithelial cells might explain the loss of secreting epithelium; and iii) orderly destruction of cellular components might induce autoantibody production. Bolstad AI, Jonsson R. Antineutrophil cytoplasmic antibodies induce human monocytes to produce oxygen radicals in vitro. Antineutrophil cytoplasmic antibodies (ANCA) are believed to play a pathogenetic role in necrotizing small-vessel vasculitis. Normal nonspecific IgG or their corresponding F(ab')2 fragments induced no release or very little release of oxygen radicals. Preincubation of monocytes with the Fcy receptor type II-blocking monoclonal antibody IV.3 before addition of ANCA greatly reduced formation of oxygen radicals Weidner S, et al. Arthritis Rheum 2001 Jul;44(7):1698-706 Immunopathogenic and clinical relevance of antibodies against myelin oligodendrocyte glycoprotein (MOG) in Multiple Sclerosis Immunopathogenic role of antibodies against myelin oligodendrocyte glycoprotein (MOG) and other myelin/nonmyelin targets in multiple sclerosis, as well as possible clinical implications for prognosis and therapy in the future. Berger T, Reindl M. J Neural Transm Suppl 2000;(60):351-60

VIP Тиреоглобулин неизвестно Фактор VIII ОБМ Сердечный миозин Grp94 Аутоантигены ДНК Белки астма тиреоидит Хасимото Множественная миелома (Бенс Джонса) Гемофилия A Рассеянный склероз Аутоиммунный миокардит Диабет СКВ Ревматоидный артрит Склеродермия Аутоиммунная патология Моноклональные антитела BV04-01 MRL-4 Аутоиммунные болезни Аутоантигены

Рассеянный склероз

Гидолиз MBP коррелирует с EDSS и стадией заболевания Immunol Lett. 2006;103(1):45-50

Inhibition of MBP-cleaving activity Can we stop the disease by inhibitors?

Гибридизация каталитических антител на срезах головного мозга крысы Human autoantibodies and EAE SJL antibodies recognize myelin basic protein in rat brain. A - Wright's stain of rat brain section. B,C,D - double-label immunofluorescence of EAE SJL and human ab. B - SJL antibodies (red), C - Human Antibody (green), D - colocalization (yellow) of both antibodies detected by fluorecsent microscopy. E,F - double-label immunofluorescence of MAB 382 and human ab. E - MAB 382 (red), F - human ab (green). G - Western blotting of MBP (M) and rat brain tissue homogenate (B) Каталитические антитела колокализуются с мАт к MBP PNAS 2006 Jan 10,103(2):281–6

Сайт-специфический гидролиз MBP Analysis of major MBP cleavage products. Reverse-phase HPLC-MS analysis of major MBP cleavage products. Column eluate fractions, corresponding to dominant chromatography peaks (A), were collected, freeze dried, redissolved, and applied to SELDI H4 chip and tricine-SDSPAGE (B). Gel was stained by Sypro Orange. Peptides, unambiguously identified by SELDI and clearly seen in corresponding gel lanes, are indicated. (C) Schematic description of the preferential antibody cleavage sites in the MBP sequence. Sequence fragments identical to the immunodominant MBP-derived peptides (12–31, 82–98, 110–128, and 144–169) are shown in yellow rectangles, and the encephalitogenic peptide region (86–98) is marked by the red box. (D) 32P-MBP degradation by autoantibodies. Autoradiography of 32P-phosphorylated MBP hydrolysis by proteolytic mouse (EAE SJL) and human (MS) IgG. Line M, molecular mass markers (range 2.5–16.9 kDa, Amersham Pharmacia). Сайты протеолиза расположены внутри иммунодоминантных фрагментов PNAS 2006 Jan 10,103(2):281–6

Конструкция эпитопной библиотеки Корректная презентация фрагментов MBP возможна лишь в составе слитного белка с тиоредоксином PNAS 2006 Jan 10,103(2):281–6

Деградация эпитопной библиотеки модельными ферментами и абзимами Journal of Immunology 2007 (in press)

Метод измерения специфической абзиматической активности на основе FRET-пары Journal of Immunology 2008

EPeFRET подвергается гидролизу абзимами и модельными протеазами Инновационный биомаркер рассеянного склероза Journal of Immunology 2008

Абзиматический гидролиз EPeFRET эффективно ингибируется энцефалитогенным пептидом и Copaxone

Кинетические характеристики протеолиза EPeFRET абзимами и модельными протеазами

Паттерн связывания аутоантител при PC и других нейродегенеративных заболеваниях с эпитопной библиотекой MBP Новые подходы к дифференциальной диагностике РС

Паттерн связывания аутоантител с эпитопной библиотекой MBP при РС и различных его животных моделях

Каталитические антитела к gp120 в качестве терапии ВИЧ Протеолитическое разрушение молекулы gp120 приводит к ее поверхностной реорганизации. Это может помочь иммунной системе бороться с ВИЧ. Разрушение gp120 абзимами, пассивная вакцинация

Ponomarenko et al. Biochemistry 2006 Durova et al. Molecular Immunology, 2009

Индукция специфичных каталитических антител на фоне аутоиммунного заболевания. Принцип Epitope spreading. Иммунизация (короткий курс) 6-8 недельных SJL/J (SPF) мышей. 1 Контрольная группа 2 MBP85-97 peptide (170 μg ) 3 gp120I-III-CHmbp85-101 (150 mg) 4 gp120I-III-CHmbp85-101 (300 mg) 5 gp120I-III (150 mg) 6 gp120I-III (300 mg) in CFA, Pertussis toxin. День 10-14 Предклиническая стадия День 17 Иммунизация аналогичным антигеном в IFA День 21 Отбор спленоцитов, слияние с миеломой Мышь SJL/J Иммунизация

SJL mice immunized with MBP-gp120 develop autoimmune disease. Immunological status revealed the disease progression

Паттерн абзиматической деградации различных форм белка gp120I-III Proteolytic degradation pattern of non-glycosilated (A) and glycosilated baculovirus-expressed (B) forms of gp120I-III protein. Antibodies heavy chains, light chains, gp120I-III and it`s degradation products are marked as Hc, Lc, gp and arrow sign, respectively. Biochemistry 2006, 45:324-30 Медленная скорость: <2·10-2 нг/мг Абзима/мин

Сайт-специфический гидролиз gp120 был показан с помощью SELDI MS -P484-LGVAPTKAKLDPHHHHHHGSGEQKLISEEDLNSSSVDKLAAALEHHHHHH Приблизительная масса 5528 Да Biochemistry 2006, 45:324-30 Абзим

Immunization Usual Antibody binds the hapten due to Hydrogen bonds Salt bridges Hydrophobic interaction Reactive Antibody binds the hapten due to Covalent bonds

Phosphonates form covalent bond with active site of serine proteases

Accessible Sites of Proteolysis onto The Surface of Gp120

Reactive Immunization of Autoimmune Mice by Peptidylphosphonate Biot-LAE-V-PO(OPh)2 Biot-V- PO(OPh)2 1 2 3 4 5 6 1 2 3 4 5 6 Phoresis and immunoblotting of polyclonal antibodies (1mg), BSA (10mg) and trypsin (1mg) modified with peptidylphosphonate. Line 1 – BSA; 2 – trypsin; 3 – polyclonal antibodies from non-immunized control mouse BALB/c; lines 4 – 6 immunized SJL/J, MRL and NZB/NZW mouse correspondingly. Phoresis Streptavidin-HRP The antigen specific catalytic antibodies have been obtained by reactive immunization.

Proteolitic activity of monoclonal antibodies raised against gp120I-III-CH-MBP fusion protein in SJL mice on the background of experimental autoimmune encephalomyelitis affinity purified antibodies were incubated with FITC-overlabeled BSA at 37oC for 24 hours. Percent of proteolysis was calculated as ratio of the obtained values of resulting fluorescence to that of complete FITC-BSA trypsinolysis conducted in the same experimental conditions. Affinity purified antibodies were incubated with RNase A in presence of NaN3 and Triton X-100 at 37oC for 14 h. Value of RNase A hydrolysis was calculated as a ratio of resulting ribonucleolitic activity and a ribonucleolitic activity in a control probe. Data are averaged from three independent experiments. Error bars represents standard deviation. degree of RNase hydrolysis, % hybridoma subclones hybridoma subclones degree of subsrate conversion, %

Biotinylated factor VIII (385 nM) was incubated alone (Control) or in the presence of IgG antibody (167 nM) for 24 hours at 37°C. Immune globulin (IVIg) was used as a source of normal IgG and as a negative control. Samples were separated by 10 percent sodium dodecyl sulfate–polyacrylamide-gel electrophoresis. BU denotes Bethesda units, and ND not determined. †Hydrolysis of factor VIII was quantified by scanning of immunoblots.Spontaneous hydrolysis that occurred on incubation of factor VIII in the presence of buffer alone was considered to represent the background level and was subtracted from each analysis. Plus-minus values are means ±SD of two or three experiments. The mean coefficient of variation was 0.33 (range, 0.01 to 1.41). P<0.05 for the comparison with immune globulin Hydrolysis of Factor VIII by IgG Purified from Plasma of Patients with Severe Hemophilia A Inhibitory activity against factor VIII of IGG from 24 patients with severe hemophilia A and rates of hydrolysis of factor VIII

1. antibodies with DNA-hydrolyzing activity biotinylated anti-/ chain antibodies (Sigma) 2. streptavidin coated magnetic beads (Dynal) DNA-hydrolyzing antibody - biotinylated anti-/ chain antibody complexes antibodies binding streptavidin-biotin interaction 1h incubation 5h incubation Affinity Capture Assay For Catalytic Activity Of Antibodies

4. streptavidin wiith 100 mM Gly-HCl pH2.6 control for DNA-hydr.activity in solution ACTIVITY RESTORED magnetic interaction between the beads and separation stand, three times washing with sterile buffer 3. control for DNA-hydr.activity in the solution NONE DETECTED

Possible Ways Of Dna Cleavage P P 3’ 3’ nucleotide nucleotide 5’ 5’ DNA-hydrolyzing Abs, Nuclease S1, Phosphodiesterase Micrococcal nuclease

DNA hydrolyzing activity of IgG from plasma of SLE patients dsDNA + + - + - + - + + - - - - + ENA - - - - - - - - + + - - - - Cardiolipin - - - - - - - - + - - - - - 1 2 3 4 5 6 7 8 9 10 11 D1 D2 D3 K relaxed supercoiled

Control Balb/c NZB/W MRL SJL * offscale Cleavage of scDNA (pBlueScript) and autoantigens binding by antibodies from autoimmune mice

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

Two hypotheses of DNA-abzyme mediated cell-death. Myosin I Annexin V SnRNP A. Belogurov Jr. et al, BioEssays 2009

Anti-DNA Autoantibodies Frequency In Several Diseases +++ - more than in 30% cases analyzed ++ - 15-30% + - 5-15% +/- - 0-5% - - never detected

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 PNAS, 1995; Mol Immunol. 1997 Oct;34(15):1083-93.

L32Tyr/L27dHis (WT) -9 Catalytic activity of mutants of DNA-abzyme Site-directed mutagenesis shows that DNA-abzyme participates in the chemical steps of catalysis.

Перспективы: создание медленных биокатализаторов in vivo IL6 TNF-a gp120 ??? AbP

Der Mensch als Industriepalast (Man as Industrial Palace) Stuttgart, 1926. Chromolithograph. National Library of Medicine. Fritz Kahn (1888-1968) Kahn’s modernist visualization of the digestive and respiratory system as "industrial palace," really a chemical plant

Институт биоорганической химии РАН им. Академиков М.М. Шемякина и Ю.А. Овчинникова Авторы: Александр Габибов, Алексей Белогуров, Иван Смирнов, Андрей Решетняк, Инна Куркова, Мария Захарова, Иван Воробьев, Оксана Дурова, Наталья Пономаренко.