Aß Immunotherapy:Aß Immunotherapy:Lessons From Mice, Monkeys Lessons From Mice, Monkeys

and Menand Men

Cynthia A. Lemere

Center for Neurologic Diseases

Brigham and Women’s Hospital

Harvard Medical School

Boston, MA USA

Silver Stained Plaques and TanglesSilver Stained Plaques and Tangles

Alzheimer’s DiseaseAlzheimer’s Disease

A syndrome arising from a chronic imbalance between Aß production and Aß clearance that leads to gradual cerebral accumulation of Aß42.

The imbalance can be caused by numerous distinct genetic (or environmental) alterations (e.g., mutations in APP, PS1 or PS2 lead to increased Aß production and early onset FAD).

Aß aggregates: toxic in vitro and cause impaired memory and learning, gliosis and neuritic dystrophy in vivo (APP, PSAPP tg mice)

• inhibit Aß-generating proteases (ß- or inhibit Aß-generating proteases (ß- or -secretase)-secretase)

• prevent the aggregation of Aß into oligomers and fibrilsprevent the aggregation of Aß into oligomers and fibrils

• prevent the deposition of Aß into plaquesprevent the deposition of Aß into plaques

• enhance Aß clearanceenhance Aß clearance

• interfere with the toxic response of neurons to amyloidinterfere with the toxic response of neurons to amyloid

• inhibit the inflammatory process around amyloid plaquesinhibit the inflammatory process around amyloid plaques

Several Therapeutic Approaches BasedSeveral Therapeutic Approaches BasedUpon the Importance of ß-Amyloid in ADUpon the Importance of ß-Amyloid in AD

Aß Vaccination: Potential Therapy Aß Vaccination: Potential Therapy For Alzheimer’s DiseaseFor Alzheimer’s Disease

Mouse StudiesMouse Studies

ActiveActive Aß immunization in APP and APP/PS1 Tg mice: Aß immunization in APP and APP/PS1 Tg mice:

• Generates anti-Aß titersGenerates anti-Aß titers

– Lowers cerebral Aß levels (if early enough)Lowers cerebral Aß levels (if early enough) (Schenk et al., (Schenk et al., NatureNature 1999; Weiner, Lemere et al., 1999; Weiner, Lemere et al., Ann NeurolAnn Neurol 2000; and many other 2000; and many other publications)publications)

– B cell epitopes within Aß amino-terminus, Aß1-15B cell epitopes within Aß amino-terminus, Aß1-15

– T cell epitopes within Aß16-42T cell epitopes within Aß16-42

• Increases peripheral levels of Aß in bloodIncreases peripheral levels of Aß in blood (Lemere et al, (Lemere et al, Neurobiol DisNeurobiol Dis 2003) 2003)

• Improves behavior/cognition on certain tasksImproves behavior/cognition on certain tasks (Janus et al., (Janus et al., NatureNature 2000; Morgan et al., 2000; Morgan et al., NatureNature 2000; Dodart et al., 2000; Dodart et al., Nature Nature

NSNS 2002, etc.) 2002, etc.)

• Results: - direct application of Aß antibodies to brain surface Results: - direct application of Aß antibodies to brain surface clears local plaques clears local plaques (Bacskai et al., (Bacskai et al., Nat MedNat Med 2001) 2001)

- i.p. injections decreased brain Aß - i.p. injections decreased brain Aß (Bard et al., (Bard et al., Nat MedNat Med 2000) 2000)- increased Aß in blood - increased Aß in blood (DeMattos et al., (DeMattos et al., PNASPNAS 2001) 2001)

- improved behavior - improved behavior (Dodart et al, (Dodart et al, Nat NeurosciNat Neurosci 2002; Kotilinek et al., 2002; Kotilinek et al., J NeurosciJ Neurosci 2002; Wilcock et al., 2002; Wilcock et al., J J NeuroinflammationNeuroinflammation 2004) 2004)• HC injections of 2 Aß Mabs in 3xAD-Tg mice cleared HC injections of 2 Aß Mabs in 3xAD-Tg mice cleared

extracellular and intracellular Aß as well as early tau extracellular and intracellular Aß as well as early tau aggregates but not hyperphosphorylated tau aggregates but not hyperphosphorylated tau (Oddo et al., (Oddo et al., NeuronNeuron 2004) 2004)

• Intracerebral ventricular injections of Aß antibodies Intracerebral ventricular injections of Aß antibodies protected protected APP tg mice from synaptic loss and gliosis APP tg mice from synaptic loss and gliosis (Chuahan (Chuahan et al., et al., J J Neurosci Res,Neurosci Res, 2002) 2002)

• Passive transfer protected neurons from induced seizures in Passive transfer protected neurons from induced seizures in APP tg mice APP tg mice (Mohajeri et al., (Mohajeri et al., J Biol ChemJ Biol Chem 2002) 2002)

• Microhemorrhage observed in very old APP tg mice with CAAMicrohemorrhage observed in very old APP tg mice with CAA(Pfiefer et al., (Pfiefer et al., ScienceScience 2002; Wilcock et al., 2002; Wilcock et al., J NeuroinflammJ Neuroinflamm 2004; Racke et 2004; Racke et al., al., J NeurosciJ Neurosci 2005 ) 2005 )

Passive Transfer of Aß Antibodies in MicePassive Transfer of Aß Antibodies in Mice

Passive immunization in PDAPP Tg micePassive immunization in PDAPP Tg mice

Bard et al., Bard et al., Nature MedicineNature Medicine, 2000, 2000

Aß Pab by i.p. weekly for 6 months: 93% reductionAß Pab by i.p. weekly for 6 months: 93% reduction

Aß Immunization of Aß Immunization of Caribbean Vervet Monkeys Caribbean Vervet Monkeys

(Chlorocebus atheiops)(Chlorocebus atheiops)

-- 8 s.c. injections 1 mg Aß40/42 + CFA/IFA over 10 months -- 5 vervets immunized, 5 controls (all between 15 and 30 yr)

-- B cell epitope in Aß N-terminus, reduced cerebral Aß load and gliosis, increased plasma Aß, no T or B cells in brain

(Lemere et al., Am J Pathol 2004)

• African Green Monkeys

• Imported to St. Kitts ~1670

• Collaboration with BSF

Aßx-42 in Brain Homogenates

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Soluble Aßx-42 Insoluble Aßx-42

Aßx-42 (µg/ml)

+ SEM

controlimmunized

**

Plaque Burden in Frontal Cortex

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edAß Immunization Reduced Cerebral AßAß Immunization Reduced Cerebral Aß

• Aß antibodies prevent Aß aggregation and/or dissolve Aß

aggregates in vitro (Solomon et al., PNAS 1996, 1997)

• Fc-mediated microglial phagocytosis of Aß in brain (Bard et al., Nature Med 2000 )

-- Fc-mediated response not required (Fab; FcR-/- mice)

(Bacskai et al., J Neurosci 2002; Das et al., J Neurosci 2003)

• Peripheral Sink Hypothesis: shift in gradient across the BBB such that there is an increase in efflux of Aß from brain to blood (DeMattos et al., PNAS 2001; Lemere et al., Neurobiol Dis, 2003)

Proposed MechanismsProposed Mechanisms

ELAN/AHP/Wyeth Human Clinical TrialsELAN/AHP/Wyeth Human Clinical Trials• Trial halted (January 2002): ~ 6 % of Aß-immunizedTrial halted (January 2002): ~ 6 % of Aß-immunized patients

(18/300) developed meningoencephalitis; anti-Aß antibodies detected in serum in subset (Schenk, Nature Rev 2002; Orgogozo et al, Neurology 2003; Gilman et al., Neurology 2005)

• Anti-Aß antibodies recognize AD plaques and CAA but not soluble Aß42 nor APP (Hock et al., Nature Med 2002); epitopes against free N-terminus of Aß (Lee et al., Ann Neurol 2005)

• Some slowing of cognitive decline (Hock et al., Neuron 2003; Gilman et al.,

Neurology 2005)

• Reduced Aß deposition in brain (Case Studies: Nicoll et al., Nature Med 2003; Ferrer et al., Brain Pathol 2004; Masliah et al., Neurology 2005)

• Reduced tau levels in CSF (Gilman et al., Neurology 2005)

• Cortical shrinkage by MRI in patients who generated Aß antibodies even though some clinical improvement observed (Fox et al., Neurology 2005)

Reduced Plaque Burden in Immunized PtReduced Plaque Burden in Immunized Pt

From Nicoll et al., Nature Medicine 2003

• The immunogen, Aß1-42, may have been recognized as self-antigen by some, leading to an autoimmune, toxic T cell response in the CNS

• The adjuvant, QS-21, purified saponin, is known to have

prodominantly Th1/CD4+ cellular immune effects and can induce CTL against antigens (Kenney et al., Vaccine 2002; Cribbs et al., Int Immunol 2003)

• Increased T cell response to Aß in aged humans (Monsonego et al., JCI 2003)

• Addition of polysorbate 80 to vaccine formulation (Nicholl et al., Nature Med 2003)

• Thus, development of an active Aß vaccine that would produce a strong humoral response but avoid an Aß-specific T cell response may be beneficial.

Possible explanations for the adverse effects?Possible explanations for the adverse effects?

Novel Immunogens to Short Aß Peptides:Novel Immunogens to Short Aß Peptides:Publications in PreparationPublications in Preparation

(cannot show immunogens yet)(cannot show immunogens yet)

Intranasal Aß Immunization:Intranasal Aß Immunization:Our previous workOur previous work

• Intranasal (i.n.) Aß immunization without adjuvant resulted in a modest humoral immune response that lowered cerebral A in PDAPP tg mice (Weiner et al., Ann Neurol, 2000; Lemere et al., NY Acad Sci, 2000).

• Adjuvant LT(R192G), a mutant form of E. coli heat labile toxin that is less toxic than its native form, enhanced the generation of Aß antibodies when administered intranasally with Aß peptides (Lemere et al., Neurobiol Aging, 2002).

Immunization of J20 APP tg mice withImmunization of J20 APP tg mice with 2 novel Aß immunogens 2 novel Aß immunogens

• J20 APP-tg mice (hAPPSw, Ind)/PDGF-promoter on a mixed C57BL/6 x

DBA background (L. Mucke, UCSF)

• 6 month-old mice received intranasal immunization weekly

(n = 7 mice/ group)

Treatment Groups:

100g Aß Immunogen 1 + 5g LT(R192G)

50 µg Aß Immunogen 2 + 5 µg LT(R192G)

5 µg LT(R192G)

H2O

• Treated for 24 weeks; at end, 5 mice per group except vehicle (6).

New Aß Immunogens: SummaryNew Aß Immunogens: Summary

• Intranasal immunization using our novel short Aß immunogens led to a robust humoral response in both WT and J20 APP tg mice.

• Ig isotypes were mainly of the Th2 type; B cell epitopes were located within the amino-terminus of Aß.

• Splenocytes proliferated against the immunizing peptides but not full-length Aß.

• Immunization resulted in lowering of plaque burden and plaque-associated pathology in APP tg mice.

• No toxicity or untoward affects were observed. B and T cells were absent from brain.

Aß Immunotherapy: ConclusionsAß Immunotherapy: Conclusions

• Active Aß vaccination prevents or reduces AD-like pathology and improves cognition but needs to be made safer.

• Generating a humoral immune response while avoiding an Aß-specific cellular response may improve safety.

• Passive transfer of Aß antibodies is also effective at lowering cerebral Aß and improving cognition but has been associated with microhemorrhage in the presence of abundant CAA in old APP tg mice. Human trials are underway.

• Ultimately, an active vaccine would be less costly and would allow coverage of a much larger population compared to passive transfer.

• Our novel immunogens appear to be safe. Further testing is required but first an animal model is needed.

““All truth passes through three All truth passes through three stages. First, it is ridiculed. stages. First, it is ridiculed.

Second, it is violently opposed. Second, it is violently opposed. Third, it is accepted as being self-Third, it is accepted as being self-

evident.”evident.”

Arthur Schopenhauer (1788-1860)Arthur Schopenhauer (1788-1860)German PhilosopherGerman Philosopher

AcknowledgementsAcknowledgementsCenter for Neurologic Diseases

Lemere Lab OthersTim Seabrook Gal Bitan Marcel Maier Weiming XiaLiying Jiang Jackie Sears Katelyn Thomas Dominic WalshYing Peng Noel LazoAmy Huynh

Former Lab MembersEdward Spooner Jeanne BloomDiana Li

Dr. Lennart Mucke (J20 APP tg mice) -- Gladstone Institute, SF, CA

Dr. John Clements [LT(R192G)] -- Tulane U. Medical School, LA

Elan Pharmaceuticals (ELISA Mabs) -- So. San Francisco, CA

Drs. Todd Golde and Pritam Das (ELISA) -- Mayo Clinic, Jacksonville, FA

Funding: Alzheimer’s Association, NIH (AG20159) to CAL, and a special thanks to the Brunozzi Family.