catalytic antibodies and their use as therapeutic agents
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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
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
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
Yang, G.; Chun, J.; Arakawa-Uramoto, H.; Wang, X.; Gawinowicz, M.; Zhao, K.; Landry, D. J. Am. Chem. Soc. 1996, 118, 5881.
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
Yang, G.; Chun, J.; Arakawa-Uramoto, H.; Wang, X.; Gawinowicz, M.; Zhao, K.; Landry, D. J. Am. Chem. Soc. 1996, 118, 5881.
Other Synthesized Transition-State Analogues
NH3C O
O
OP
HN
ONH
O
Transition State Analogue 1
Transition State Analogue 3
Transition State Analogue 2
NH3C O
OCH3
OP
O
OH
NH
O
O
NH
N O
OCH3
OP
O
OH
HN
OHN
O
O
OH
Yang, G.; Chun, J.; Arakawa-Uramoto, H.; Wang, X.; Gawinowicz, M.; Zhao, K.; Landry, D. J. Am. Chem. Soc. 1996, 118, 5881.
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
Deng, S.; Prada, P.; Landry, D. J. Immunol. Meth. 2002, 269, 299.
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
Deng, S.; Prada, P.; Landry, D. J. Immunol. Meth. 2002, 269, 299.
Conclusions for Cocaine Degrading Antibody 15A10
Efficacy Km = 220 µMKcat = 2.3 min-1
ImprovementsLinker lengthButyrylcholinesterase studiesSite-directed mutagenesis
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
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
Isomura, S.; Wirsching, P.; Janda, K. J. Org. Chem. 2001, 66, 4115.
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%
Isomura, S.; Wirsching, P.; Janda, K. J. Org. Chem. 2001, 66, 4115.
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
Isomura, S.; Wirsching, P.; Janda, K. J. Org. Chem. 2001, 66, 4115.
Screening of Catalytic AntibodiesThe elicited monoclonal antibodies were screened against nicotine and irradiated with UV light in the presence of riboflavin
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
Preliminary ResultsNo catalysis was observedConclusion
Antibodies might be binding too strongly to substrateDesign hapten that binds weaker
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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
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
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
Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R.; Rader, C.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 2001, 98, 7528.
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
Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R.; Rader, C.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 2001, 98, 7528.
Prodrug 6 Activation ResultsComparison in growth inhibition activity of prodrug 6 in the absence and presence of 38C2
Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R.; Rader, C.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 2001, 98, 7528.
Prodrug 6 Activation Results The effect of 38C2-mediated prodrug 6 activation
Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R.; Rader, C.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 2001, 98, 7528.
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
Sinha, S.; Li, L.; Miller, G.; Dutta, S.; Rader, C.; Lerner, R. Proc. Natl. Acad. Sci. 2004, 101, 3095.
Results Using 38C2 to Activate Dynemicin
N
O
OO
HO
H
O
N
O
O
H
O
3a
4a
Sinha, S.; Li, L.; Miller, G.; Dutta, S.; Rader, C.; Lerner, R. Proc. Natl. Acad. Sci. 2004, 101, 3095.
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
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