catalytic antibodies and their use as therapeutic agents

Catalytic Antibodies and Their Use in Therapeutics

Samantha M. FrawleyOrganic Seminar

February 9th, 2005

Seminar OutlineBackground information

What are antibodies?What are catalytic antibodies?

Catalytic antibodies used in therapeuticsTransition-state analogue approach

Antibody catalyzed cocaine degradation

Hapten-substrate approachOxidative degradation of nicotine using catalytic antibodies

Reactive immunizationUsing catalytic antibodies to activate prodrug

Conclusion

Important TermsAntibody: An immunoglobulin protein produced by the immune system in response to the invasion of a foreign substanceHapten: A small molecule that reacts with a specific antibody but cannot induce the formation of antibodies unless it is bound to a carrier protein Antigen: Any substance that elicits an immune response when introduced into an animalIgG: The most common immunoglobulin, which is distributed between the blood and extravascular fluid. The IgG is used in the formation of catalytic antibodies

Voet, D.; Voet, J.; Pratt, C. Fundamentals of Biochemistry, Rev. Ed., John Wiley & Sons, New York, 1999.

Structure of Antibodies

Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. Molecular Biology of the Cell, 3rd Ed., Garland Publishing Co., 1994.

Antibodies vs. EnzymesHigh affinity and specific bindingStabilization of transition state vs. ground state

Tramontano, A.; Janda, K.D.; Lerner, R.A. Science 1986, 234, 1566.

http://bio.winona.edu/berg/308s04/Lec-note/chap6.htm

Catalytic AntibodiesCatalytic antibodies utilize

Antibody specificityEnzyme’s catalytic power

Methods of Forming Catalytic Antibodies







Conclusion

What is Cocaine?An alkaloid found in the leaves of Erythroxylon cocaBlocks removal of dopamine from a synapse in the reward pathway of the central nervous system

N O

OCH3

O O

(-)-Cocaine

Baird, T.; Deng, S.; Landry, D.; Winger, G.; Woods, J. J. Pharmacol. Exp. Ther. 2000, 295, 1127.

Previous Method to Treat Cocaine Addiction

This method depletes and inactivates available antibodies

Bunce, C.; Loudon, R.; Akers, C.; Dobson, J.; Wood, D. Curr. Opin. Mol. Ther. 2003, 5, 58.

Sherer, E.; Yang, G.; Turner, G.; Shields, G.; Landry, D. J. Phys. Chem. A 1997, 101, 8526.

Degradation of CocaineThe hydrolysis of cocaine’s benzoyl ester yields two biologically inactive productsThis transformation is an attractive approach to destroy cocaine prior to its absorption into the brain

N O

OCH3

O O

(-)-Cocaine

N

OH

O

OCH3

CO2H

EcgonineMethyl Ester

BenzoicAcid

+

Yang, G.; Chun, J.; Arakawa-Uramoto, H.; Wang, X.; Gawinowicz, M.; Zhao, K.; Landry, D. J. Am. Chem. Soc. 1996, 118, 5881.

Transition-State Analogue Approach to the Formation of Catalytic Antibodies

Transition-State Analogue ApproachPrevious work indicated transition-state analogue using the phosphonate monoester, yielded artificial esterases with the greatest activity

N O

OCH3

O OOH

N O

OCH3

O ON

OH

O

OCH3

CO2H

EcgonineMethyl Ester

BenzoicAcid(-)-Cocaine Transition State

+

NH3C O

OCH2R

OP

O

O

Phosphonate Mono-EsterTransition State Analog

R = tether carrier

Landry, D.; Zhao, K.; Yang, G.; Glickman, M.; Georgiadis, T. Science 1993, 259, 1899.

Importance of Using a LinkerAllows for exposure of the epitope to antibodyStimulate immune responseLinker variations

Tether siteLength

Formation of Transition State Analogue

N O

OH

(-)-Ecgonine

I(CH2)4N3

1) tetrazole PhP(O)Cl2

2) MeOH

1) P(CH3)3

2) 1,4-14C-succinic anhydride

3) DCC/DMAP Benzyl alcohol

4) H2, Pd/C

1) DCC N-OH phthalimide

2) TMSBr

N O

O(CH2)4N3

N

O

O

O(CH2)4N3

POCH3

O(CH3)4NOH

78% 89%

N

O

O

O

POCH3

O

HN

OOH

O

**

70%

N

O

O

O

PO

O

HN

OO

O

** N

O

O

~70%

OH OH


Coupling Transition-State Analogue to a Carrier Protein

Carrier protein BSA or ovalbumin

N

O

O

O

POCH3

O

HN

ONH

O

TSA1

* *N

O

O

O

POCH3

O

HN

OO

O

** N

O

O

Coupling ratio: 6:1 BSA15:1 ovalbumin

N OH

O

O


Other Synthesized Transition-State Analogues

NH3C O

O

OP

HN

ONH

O

Transition State Analogue 1



NH3C O

OCH3

OP

O

OH

NH

O

O

NH

N O

OCH3

OP

O

OH

HN

OHN

O

O

OH


How Catalytic Antibodies are Synthesized

http://www.scripps.edu/~yunfeng/personal/researchweb.html

Formation of Monoclonal AntibodiesThe half-life of regular IgGantibodies could not withstand testing-immortalize and culture

http://biology.kenyon.edu/courses/biol114/Chap08/Chapter_08b.html

Formation of Active Cocaine Degrading Antibodies

Deng, S.; Prada, P.; Landry, D. J. Immunol. Meth. 2002, 269, 299.

Results of Cocaine Degradation Using 15A10

LD90 (16 mg/kg) of cocaine was administered to rats previously injected with catalytic antibody 15A10


Results of Cocaine Degradation Using 15A10

0

20

40

60

80

100

120

0.000 0.015 0.030 0.060 0.125 0.250 0.500

Cocaine (mg/kg/injection)

Self-

Adm

inis

tere

d In

fusi

ons Saline

10 mg/kg Mab15A1030 mg/kg Mab15A10100 mg/kg Mab15A10


Conclusions for Cocaine Degrading Antibody 15A10

Efficacy Km = 220 µMKcat = 2.3 min-1

ImprovementsLinker lengthButyrylcholinesterase studiesSite-directed mutagenesis







Conclusion

Facts about NicotineNicotine is the most widely addictive drug in the world

Using nicotine gum and nicotine patches to quit smoking afforded less than 20% success rates

Nicotine binds to nicotinic cholinergic receptors in the mesolimbic dopamine system

N

N

Nicotine

H

Isomura, S.; Wirsching, P.; Janda, K. J. Org. Chem. 2001, 66, 4115.

Carrera, M.; Ashley, J.; Hoffman, T.; Isomura, S.; Wirsching, P.; Koob, G.; Janda, K. Bioorg. Med. Chem. 2004, 12, 563.

Previous Work on Nicotine AddictionNicotine gum/patchesNicotine vaccine

Bunce, C.; Loudon, R.; Akers, C.; Dobson, J.; Wood, D. Curr. Opin. Mol. Ther. 2003, 5, 58.

Oxidative Degradation of Nicotine

N

N

N

N

N

N

N

N

N

NH

N

N

N

N

OHO

OHHO CO2

-

O

O-

Nicotine

H

Six main metabolites of nicotine, many are formed from P-450 oxidation

Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.

Hapten-Substrate Approach

Hapten-Substrate Approach for Nicotine Degradation

Dickerson, T.; Yamamoto, N.; Janda, D. Bioorg. Med. Chem. 2004, 12, 4981.

Requirements for Nicotine Hapten(S)-configuration stereochemistry Similar structural properties Sufficient alkyl linker length, ~12 Å, to expose all structural features

NH

HN

O

OH

O

NIC hapten 3

N

NH

Nicotine


Synthesis of Hapten 3

N

Br CO2H

N

Br CO2Et

N

BrN

N

BrNH

H

EtOHH2SO4, reflux

1) 1-vinyl-2-pyrrolidinone, NaH, THF, reflux2) HCl(aq), reflux3) NaOH

NaBH4

1) MTPA salts

2) H2, Pd/C, Et3N, EtOH

DIEA, acetonitrileN

NH

H IO

HN

O

OBn

N

NH

O

HN

O

OBnN

NH

O

HN

O

OHH2, Pd/C,

MeOH

Hapten 3

94%72%

81%

54%48%

66%


Coupling Hapten 3 to a Carrier Protein

N

NH

O

HN

O

OH

Hapten 3

N

NH

O

HN

O

NHCarrier proteinKLH or BSA

Coupling ratio:19:1 BSA


Screening of Catalytic AntibodiesThe elicited monoclonal antibodies were screened against nicotine and irradiated with UV light in the presence of riboflavin


Preliminary ResultsNo catalysis was observedConclusion

Antibodies might be binding too strongly to substrateDesign hapten that binds weaker


Synthesis and Reasoning Behind Hapten 4

Substrate binding by antibodies to hapten 4 would not be as tight as hapten 3

N

N

O OH

O

Hapten 4

N

NH

O OO

DIEA72%

H H


Antibodies vs. OzonolysisThe two most proficient monoclonal antibodies, TD1-36H10 and TD1-10E8 were tested vs. ozonolysis

N

N

O3

N

N

N

N

N

NH

OO

N

N

N

N

NOH N

H

N

NH

OH

O O

O

1 2

or

TD1-36H10/TD1-10E8 andlight, riboflavin


Results of Nicotine DegradationAfter 6 hr of white light irradiation in the presence of either antibody, no detectable nicotine remained

0

10

20

30

40

50

60

0 45 88 180 270 375

Time (min)

Prod

uct 1

Con

cent

ratio

n ( µ

M)

TD1-36H10TD1-10E8w/o antibody

N

NOH

1


Further Results of Nicotine DegradationThere was a 10-fold rate enhancement vs. uncatalyzedreaction

0

10

20

30

40

50

60

70

0 45 88 180 270 375

Time (min)

Prod

uct 2

Con

cent

ratio

n ( µ

M)

TD1-36H10TD1-10E8w/o antibody

NH

N

OH

O O

O

2


Conclusions on Nicotine-Degradation Using Catalytic Antibodies

Antibodies with modest affinity may be more efficient Efficacy

Requires 20 µM antibody for 200 µM of nicotine

Practical applicationsNicotine mimic metabolitesBiologically active productsIn vivo systems







Conclusion

Prodrug ActivationAdministration of a drug in its non-toxic form (prodrug), which is enzymatically converted into its active form

X

OHO

XHEnzymatic cleavage

Inactive drug Active drug

drugdrug

Guo, X.; Lerner-Tung, M.; Chen, H.; Chang, C.N.; Zhu, J.; Chang, C.P.; Pizzorno, G.; Lin, T.; Cheng, Y. Biochem. Pharmacol. 1995, 49, 1111.

Catalytic Antibodies and Their Use in Prodrug Activation

Catalytic antibodies can access reactions that are not catalyzed by endogenous enzymes

Campbell, D.; Gong, B.; Kochersperger, L.; Yonkovich, S.; Gallop, M.; Schultz, P. J. Am. Chem. Soc. 1994, 116, 2165.

Reactive Immunization Approach

Selecting a Hapten for Aldolase AntibodyAppropriate reactivity Requires a mechanism-based “trap” in the active site of the antibody Ability to form the enamineProvide appropriate binding sites for intermolecular reaction

HN

OO

O

OH

O

Hapten 1

Wagner, J.; Lerner, R.; Barbas III, C. Science 1995, 270, 1797.

Formation of Hapten-Antibody Complex-Trapping Lysine

R

O O

Hapten 1

R

O

H2N Lys

BH

Ab

NH Lys Ab

HO

R

O N

H H

H Lys Ab

B R

O N

H

LysH Ab

R

O N

H

Lys AbH

Barbas III, C.; Heine, A.; Zhong, G.; Hoffmann, T.; Gramatikova, S.; Bjornestedt, R.; List, B.; Anderson, J.; Stura, E.; Wilson, I.; Lerner, R. Science 1997, 278, 2085.

Formation of Catalytic AldolaseAntibody

Antibodies are screened for the formation of the enaminoneintermediate

R

O N

H

LysH Ab

λ = 316 nm

Wagner, J.; Lerner, R.; Barbas III, C. Science 1995, 270, 1797

Mechanism Using Catalytic AldolaseAntibody

O HOHN N

H

N

R

O

H R

OH N

R

OOHH2N Lys Ab+

H2N Lys Ab -H2O

H2O

H Lys Ab H Lys Ab

H Lys Ab

Lys Ab

B

+N

H Lys Ab

Hoffmann, T.; Zhong, G.; List, B.; Shabat, D.; Anderson, J.; Gramatikova, S.; Lerner, R.; Barbas III, C. J. Am. Chem. Soc. 1998, 120, 2768.

Diversity of the 38C2 Catalytic AntibodyMore than 100 different aldoladditions or condensations have been formed by a single catalyst

Barbas III, C.; Heine, A.; Zhong, G.; Hoffmann, T.; Gramatikova, S.; Bjornestedt, R.; List, B.; Anderson, J.; Stura, E.; Wilson, I.; Lerner, R. Science 1997, 278, 2085.

Prodrug Activation by 38C2 via a Tandem Retro-Aldol-Retro-Michael Reaction

O

OHO

XH

Inactive drug

Active drug

drug

drug

O

O

38C2N

ON

O

X

H H

B

Odrug

N

ON

O

X

H H

O

drug XN

N

O

BH

CO2

NN

O

Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R.; Rader, C.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 2001, 98, 7528.

EtoposideEtoposide is an antitumor drug used in the treatment of the early stages of pediatric neuroblastomaCurrent survival rates of stage 4 neuroblastoma are quite poor

OO O

OHO OH

OO

OO

OCH3H3COOH

Etoposide


Activation of Prodrug 6 to Etoposide

OO O

OHO OH

OO

OO

OCH3H3CO

O

O

NN O

O OHO

O ONN

O

CO2

Ab38C2O

O OO

HO OH

OO

OO

OCH3H3COOH

Prodrug 6 Etoposide


Prodrug 6 Activation ResultsComparison in growth inhibition activity of prodrug 6 in the absence and presence of 38C2


Prodrug 6 Activation Results The effect of 38C2-mediated prodrug 6 activation


Activation of Doxorubicin and Camptothecin Prodrugs by 38C2

NN

O

O

OO

ON O

O OHO

O O

CO2

Camptothecin

Camptothecin-prodrug

38C2 NN

O

O

OOH

N

O

O

O

OH

OH

OH

OCH3O

OOH

O

HOHN O

O OHOO

38C2

O

O

O

OH

OH

OH

OCH3O

OOH

O

HO NH2

Doxorubicin

CO2

Doxorubicin-prodrug

Shabat, D.; Rader, C.; List, B.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 1999, 96, 6925.

A New Type of Prodrug Activation-Enediynes

Biologically active enediynes cleave DNA Benzenoid diradical abstracts hydrogen atoms from the DNA backbone

HN

OH

OOH

OH O

O

OMe

CO2H

Dynemicin A

OMe

Me

OH

O

O

O

O

O

O

OHO

OH

NH

O

Neocarzinostatin chromophore

Nicolaou, K.; Dai, W.; Tsay, S.; Estevez, V.; Wrasidlo, W. Science, 1992, 256, 1172.

Activation of Endiyne Prodrugs Under Basic Conditions

HN

X

HO

Nu

HN

X

HO

Nu

DNAcleaved DNA

O2

Bergmancyclization

N

O

O

X

OS

Ph

O O

HN

X

ON

X

PhS

O O

CO2

HN

X

HO

Nu

Nu-H

Base

HO

Sinha, S.; Li, L.; Miller, G.; Dutta, S.; Rader, C.; Lerner, R. Proc. Natl. Acad. Sci. 2004, 101, 3095.

Pathway of Activation of Prodrugs of Dynemicin Analogs

N

O

OO

HO

X

ON

O

O

X

O

N

O

O

X

OS

Ph

O O

HN

X

ON

X

PhS

O O

CO2

HN

X

HO

Nu

O

mAb 38C2

O

O

CO2

Nu-

HO HN

X

HO

Nu

HN

X

HO

Nu

DNAcleaved DNA

O2

Bergmancyclization


Results Using 38C2 to Activate Dynemicin

N

O

OO

HO

H

O

N

O

O

H

O

3a

4a


Other Approaches for Prodrug DeliveryADEPT-Antibody-directed enzyme prodrugtherapy

Johnston, N. Today’s Chemist at Work 2001, 10, 30.

Other Approaches to Activate ProdrugsADAPT: Antibody-directed abzymeprodrug therapy

Use of catalytic antibody instead of enzyme in drug activation

Transformations which cannot be performed by enzymesActivity of catalytic antibodies is lower

Conclusions on Aldolase ProdrugActivation

Catalyze transformations not available to enzymesEfficacy

Km = 54 µMKcat = 4.4 x 10-3 min-1

ConsequencesSubstrate selectivityLocalization

Final ThoughtsDifferent approaches can be employed to synthesize catalytic antibodies

Basic transformations-Transition-state analogue approachExternal energy source required-Hapten-substrate approachDiversity in substrates-Reactive immunization

Improvements need to be made before catalytic antibodies can be used in a clinical setting

Acknowledgements Dr. BorhanDr. TepeTepe group members

VasudhaTeriManasiAdamChrisJasonGwenJamesSamantha 2

catalytic antibodies and their use as therapeutic agents

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