Anti-addictive Drug Vaccine Platform Brian J. Kelly, Ph.D. Cornell Center for Technology Enterprise and Commercialization June 21, 2012
Winning the War
Strategy Use Gene Transfer Vectors to Develop Effective Antiaddiction Vaccines Gene transfer vaccine At risk for addiction Generate immunity against addictive drug
Why Use Viral Gene Transfer Vectors as Platforms? Effective adjuvants Delete genes to render replication incompetent Rapid induction of immunity Can deliver monoclonal antibodies Can generate active immunity against small molecules conjugated to surface proteins Enhance host defenses Viral gene transfer vector
Drug Addiction Market - world-wide problem for which there is no preventive treatment or effective therapy Strategy develop vaccines directed against preventing addictive drugs from reaching their receptors on brain cells Challenge all addictive drugs are small molecules not seen by the immune system Cocaine N O O O O
Platform Strategies for Vaccines for Addictive Drugs Active vaccine Passive vaccine Addictive drug Light chain Heavy chain Anti-addictive drug monoclonal antibody Anti-addictive drug AAV-based passive vaccine Anti-addictive drug adenovirus-based active vaccine
Platform Technology Couple Addictive Drugs to Adenovirus Capsid Proteins Adenoviruses are highly immunogenic By coupling a small addictive drug (or its analog) to the adenovirus capsid proteins, the immune system will think the addictive drug is immunogenic Antibodies generated against the addictive drug will prevent it from reaching the brain Addictive drug Adenovirus
Design of a Disrupted Adenovirus- based Anti-cocaine Vaccine to Treat Cocaine Addiction dad5gnc vaccine Conjugation of a cocaine analog to heat and detergent- disrupted adenovirus will present the hapten as an immunogen to evoke high titer anti-cocaine immunity N O Linker O O O GNC cocaine analog Activate with crosslinker Mix GNC with disrupted Ad5 E1 - E3 - Ad5 Disrupt with SDS at 56 C, 45 sec Fiber Hexon Penton base dad5gnc
dad5gnc-mediated Induction of High Titer Anti-GNC Antibodies in Mice dad5gnc dad5gnc 1 st boost 2 nd boost Balb/c n=20 Intramuscular Boost at 3 and 6 wk with dad5gnc Evaluate Serum anti-cocaine antibody titer Serum anti-gnc antibody titer -1 10 6 10 5 10 4 10 3 10 2 10 1 10 0 0 2 7 13 Time post-injection (wk)
Reduction in Ratio of Blood to Brain Cocaine Levels in Mice Challenged Intravenously with Cocaine Cocaine (ng/g brain, ) Naive dad5gnc n = 4 Intravenous 3 H-cocaine (2.5 mg) Sacrifice after 1 min 450 400 350 300 250 200 150 100 50 Ratio blood / brain 0.2 2.0 450 400 350 300 250 200 150 100 50 Cocaine (ng/ml serum, ) Evaluate Amount of cocaine in brain and blood 0 0 Brain Serum Brain Serum Naive dad5gnc100 dad5gnc vaccinated mice retain cocaine in the blood with a 10-fold difference in blood to brain ratio relative to naive mice
Cocaine-induced Locomotor Activity of Naive and dad5gnc-vaccinated Mice Naive + PBS Naive + cocaine (25 µg, IV) dad5gnc + cocaine (25 µg, IV) Naive + cocaine (50 µg, IV) dad5gnc + cocaine (50 µg, IV)
Cumulative ambulatory time (sec) dad5gnc Vaccine-mediated Reduction of Cocaine-induced Hyperactive Activity 200 Cocaine 50 µg IV Naive mice + cocaine 150 100 50 0 dad5gnc + cocaine dad5gnc + PBS Naive mice + PBS 0 1 2 3 4 5 6 7 8 9 10 Time post-injection (min)
Total distance traveled (cm x10 3 /10 min) Persistence of Vaccine-mediated Abrogation of Cocaine-induced Hyperactivity 5.0 Cocaine 50 µg 4.0 Naive + cocaine 3.0 2.0 1.0 0.0 dad5gnc + PBS Naive + PBS 0 1 2 Time of repeated challenge (wk) dad5gnc + cocaine
Anti-cocaine Antibody Titers in dad5gnevaccinated Non-human Primates dad5gne 100 µg, intramuscular 0-35 wk Evaluate Rhesus macaque n = 6 Serum anti-cocaine antibody titers (ELISA) Serum anti-cocaine antibody titer -1 10 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 dad5gne Limit of detection K d = 80 mm Controls, not vaccinated 0 5 10 15 20 25 30 35 Time post-injection (wk)
Change in blood pressure (mmhg) Ability of dad5gne Vaccination to Prevent the Cocaine-induced Cardiovascular Phenotype dad5gne 100 mg, intramuscular 30 20 A. Control Cocaine 10 Rhesus macaque 0 systolic diastolic 10 wk cocaine 1 mg/kg, IV 0 12 19 26 33 40 48 55 62 69 B. dad5gne vaccinated 30 Cocaine 20 Evaluate Blood pressure 10 0 0 12 19 26 33 40 48 55 62 69 Time (min)
Biology of Cocaine Action 1 Cocaine blocks the dopamine transporter Binding sites for dopamine and cocaine overlap in the transmembrane segments of the transporter Transmitting neuron Dopamine packaged in vesicles Result dopamine accumulation in synaptic clefts Enhanced, prolonged postsynaptic dopaminergic signaling of dopamine receptor in the receiving neurons Dopamine transporter functioning normally Dopamine receptors Receiving neuron Dopamine transporter blocked by cocaine Cocaine The cocaine high in humans requires >47% dopamine transporter occupancy by cocaine 1 1 Volkow et al, Nature 1997; 386: 827
Question Does the dad5gne anti-cocaine vaccine prevent cocaine from reaching its receptors in the brain?
Imaging Dopamine Transporter Occupancy with 11 C-PE2i 11 C-PE2i - cocaine analog which binds with high selectivity to the dopamine transporter Use 11 C-PE2i as a radiolabel for positron emission tomography imaging of the dopamine transporter Cocaine N O O O O CH 3 Cocaine displaces 11 C-PE2i in a dose-dependent manner, permitting quantitative assessment of cocaine occupancy of the dopamine transporter 11 C-PE2i I N O O O O 11 CH 3 CH 3
11 C-PE2i Imaging of the Dopamine Transporter in the Caudate and Putamen Non-human primate Rhesus macaque Caudate Putamen
Efficacy of dad5gne in Preventing Cocaine from Reaching CNS Dopamine Transporters dad5gne 100 mg, intramuscular No cocaine Pre-vaccination + Cocaine Rhesus macaque PET imaging of the dopamine transporter with 11 C-PE2i No cocaine 67% cocaine occupancy Post-vaccination + Cocaine Evaluate Caudate and putamen labeling with 11 C- PE2i before and after cocaine administration (1 mg/kg, intravenous) 23% cocaine occupancy
Conclusion Based on the knowledge that >47% of the CNS dopamine transporters have to be occupied by cocaine for humans to get a cocaine high, these studies in nonhuman primates suggest that the dad5gne vaccine should be effective as an anti-cocaine vaccine
How Many People Smoke Worldwide? Estimated 1.3 billion people smoke worldwide 5.4 million deaths/yr, estimated to increase to 10 million/yr in 2020 1 death every 6.5 sec 33-50% of all smokers die of a smoking related death Current strategies to help smokers quit are ineffective with an 80% recidivism rate Nicotine in the principal addictive component of cigarettes, mediating its effect through CNS nicotinic cholinergic receptors Immunotherapy against nicotine is a potential means to block nicotine from reaching the CNS
Anti-Nicotine Passive Immunization Hypothesis If high levels of anti-nicotine monoclonal antibody could be expressed via an adeno-associated virus (AAV) gene expression vector, it should be possible to induce persistent passive immunization that would prevent nicotine from reaching its receptors in the brain
Questions Can an AAV vector mediate persistent expression of high levels of a high affinity monoclonal anti-nicotine antibody? Will the anti-nicotine antibody expressed by the AAV vector sequester intravenous administered nicotine in the blood and shield the brain? Will anti-nicotine immunotherapy block nicotine-induced cardiovascular and behavioral responses upon challenge with nicotine?
Generation of an Adeno-associated Virus Gene Transfer Vector Coding for an Anti-nicotine Monoclonal Antibody Light Chain Igκ Heavy Chain IgG1 Anti-nicotine antibody (NIC9D9) Expression cassette ITR CAG Heavy chain AAVantiNic Furin 2A Light chain (A n ) ITR
Serum anti-nicotine antibody titer (µg/ml) Persistence of AAVantiNic-directed Expression of an anti-nicotine Monoclonal Antibody AAVantiNic (10 11 genome copies) 10 4 IV 10 3 AAVantiNic C57Bl/6 n=5 Evaluate Serum anti-nicotine antibody titers (ELISA) 10 2 10 1 0-18 wk K d = 43 ± 20 nmol/l 10 0 10-1 0 4 9 12 18 Time (wk) Limit of detection AAVcontrol
AAVantiNic Shields the Brain from Systemic Nicotine Nicotine (ng/g brain) Nicotine (ng/ml serum) AAVantiNic (10 11 genome copies) 100 80 A. Brain IV 60 4 months Nicotine (0.03 mg/kg, IV) 1 min C57Bl/6 n=5 40 20 0 600 500 400 300 B. Serum Assess Brain and blood 3 H- nicotine levels 200 100 0 Naive AAVantiNic
Vertical activity (sec) Total distance (m) AAVantiNic Immunization Blocks Nicotineinduced Hypo-locomotion AAVantiNic (10 11 genome copies) 7 wk Nicotine (0.5 mg/kg, subcutaneous) C57Bl/6 n=10 Assess Cumulative distance traveled (15 min) Vertical activity time (15 min) 8 7 6 5 4 3 2 1 0 250 200 150 100 A. Total distance 50 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time post-injection (min) B. Vertical activity AAVantiNic + nicotine Naive + PBS Naive + nicotine AAVantiNic + nicotine Naive + PBS Naive + nicotine 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time in chamber (min)
Mean arterial blood pressure (mmhg) Heart rate (bpm) AAVantiNic Prevents Cardiovascular Effects of Nicotine AAVantiNic (10 11 genome copies) A. Heart rate 700 600 500 Nicotine * 5 wk Nicotine (1.0 mg/kg, subcutaneous) C57Bl/6 n=4 Evaluate Heart rate and mean arterial pressure 400 300 200 B. Blood pressure Nicotine 130 120 110 100 90 80 70 60-20 0 20 40 60 80 100 120 140 160 180 * AAVantiNic + nicotine AAVantiNic + PBS AAVcontrol + nicotine AAVantiNic + nicotine AAVantiNic + PBS AAVcontrol + nicotine -20 0 20 40 60 80 100 120 140 160 180 Time (min)
Summary Passive immunization with AAVantiNic mediates persistent expression of high levels of a high affinity anti-nicotine antibody Upon challenge, AAVantiNic immunized mice retain nicotine in the blood and shield the brain from nicotine AAVantiNic blocks nicotine-mediated alterations in locomotor activity and cardiovascular effects AAV-based vaccines coding for a high affinity anti-nicotine monoclonal antibody may be a useful strategy to help nicotine-addicted subjects discontinue cigarette smoking
Anti-addictive Drug Vaccines Efficacious in experimental animal models Extensive clinical experience with adenoviruses Manufacturing capacity Versatile platform Pending patents Big market Clear path to clinical trials but need $$$! How can you help?
Vaccines to Shield the CNS from Addictive Drugs Addictive drug