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Systemic vaccination with anti-oligomeric monoclonal antibodies improves cognitive function by reducing beta-amyloid deposition and tau pathology in 3xTg-AD mice
P706
P4-201
Poster Presentations: P4
BENEFICIAL CATALYTIC AUTOIMMUNITY TO BETA-AMYLOID PEPTIDE
Yasuhiro Nishiyama1, Stephanie Planque1, Mariko Hara1, Kenji Watanabe1, Xiaoyun Xu2, Hiroaki Taguchi2, Einar M. Sigurdsson3, Brian O’Nuallain4, Ian Murray5, Robert P. Friedland6, Ken-ichiro Fukuchi7, Richard Massey8, Sudhir Paul1, 1University of Texas Health Science Center-Medical School at Houston, Houston, Texas, United States; 2 University of Texas Health Science Center-Medical School at Houston, Houston, Texas, United States; 3New York University School of Medicine, New York, New York, United States; 4Harvard Medical School-Brigham & Womens Hospital, Boston, Massachusetts, United States; 5Texas A&M Health Science Center, Bryan, Texas, United States; 6University of Louisville, Louisville, Kentucky, United States; 7University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States; 8Covalent Bioscience, Inc., Houston, Texas, United States. Background: We previously reported human catalytic autoantibodies to amyloid b peptide (Ab). We hypothesize that recognition of electrophilic amyloid epitopes by nucleophilic autoantibodies is an innate immune function that is recruited for catalytic clearance of amyloid deposits associated with aging and Alzheimer’s disease (AD). Methods: Ab cleavage was measured by HPLC, acid precipitation, mass spectroscopy or electrophoresis. Electrophilic Ab (E-Ab) was prepared by carbonylation with the lipid peroxidation end products 4-hydroxynonenal (HNE)/malonaldehyde (MDA) or phosphonate diester insertion. Covalent immune complexes were analyzed by SDS-electrophoresis. Ab1-42 aggregates were identified by antibody or Thioflavin-T staining. Results: IgM from healthy human sera, the first antibody class produced during B cell differentiation, catalyzed Ab cleavage at rates superior to IgGs. Preferential Ab cleavage by IgMs was also observed for antibodies from the sera and cerebrospinal fluid from patients with AD. Two Ab cleaving antibody fragments were isolated from a phage library, a heterodimeric V L -V L construct (2E6) and a single domain V L construct (5D3). Treatment with antibody 2E6 induced disappearance of oligomeric and fibrillar Ab. Intracranial antibody injection in Ab-overexpressing transgenic mice cleared the Ab plaques. Traditional antibodies bind antigens at complementarity determining regions (CDRs). The Ab cleaving antibodies contained CDRs with no or minimum mutations acquired by antigen-driven diversification. Deleting the CDRs did not attenuate Ab cleavage by antibody 2E6, but the catalytic activity was lost by replacing the framework regions (FRs) with corresponding FRs from a non-catalytic antibody. The FRs are evolutionarily conserved segments important for innate recognition of B cell superantigens without requirement for adaptive immune processes. From protease inhibitor and epitope mapping studies, the catalytic mechanism entails noncovalent binding at the Ab C terminus followed by nucleophilic peptide bond cleavage. Antibody 2E6 reacted covalently with an electrophilic phosphonate-containing Ab analog and the naturally-occurring Ab-HNE/Ab-MDA analogs (E-Ab). Monoclonal murine antibodies (MAbs) that cleaved Ab at low substrate concentrations were identified by immunization with non-electrophilic Ab. A subset of MAbs induced by immunization with E-Ab cleaved Ab robustly. Conclusions: Amplification of the innate noncovalent recognition and catalytic functions of antibodies driven by age/ disease-associated Ab accumulation can remove toxic amyloid deposits. P4-202
SYSTEMIC VACCINATION WITH ANTIOLIGOMERIC MONOCLONAL ANTIBODIES IMPROVES COGNITIVE FUNCTION BY REDUCING BETA-AMYLOID DEPOSITION AND TAU PATHOLOGY IN 3XTG-AD MICE
Suhail Rasool, Hilda Martinez Coria, Charles Glabe, UC Irvine, Irvine, California, United States. Background: Alzheimer’s disease (AD) is a devastating disorder that is clinically characterized by a comprehensive cognitive decline. Accumulation of the amyloid-beta (Ab) peptide plays a pivotal role in the
pathogenesis of AD. In AD, the conversion of Ab from a physiological water-soluble monomeric form into the neurotoxic oligomeric and beta (b)-sheet rich fibrillar conformation is an important event. The most toxic form of Ab are oligomers. In transgenic AD animal models, both active and passive anti-Ab immunotherapies improve cognitive function and clear the parenchymal accumulation of amyloid plaques in the brain. Methods: Vaccination Behavioral Studies Immunohistochemistry ELISA. Results: In this report we studied immunotherapy of sequence-independent anti-oligomeric monoclonal antibodies on the cognitive function and amyloid load in 3xTg -AD mice. Anti-oligomeric monoclonal antibodies significantly reduce the amyloid load and improve the cognition. The clearance of amyloid load was significantly correlated with reduction in tau hyperphosphorylation and improvement in cognition. In summary, we demonstrate that prolonged systemic immunotherapyusing oligomer-specific monoclonal antibodies effectively attenuates behavioral and pathological impairments in the 3xTg -AD mice. Conclusions: Our findings demonstarate that conformation-dependent antibodies efficiently clear soluble and insoluble Ab deposits and highlight that an early initiation of immunotherapy with oligomer-specific antibodies, perhaps even when patients are asymptomatic, might be necessary to inhibit both soluble and insoluble Ab-mediated pathology in Alzheimer’s disease.
P4-203
EXPRESSION AND CHARACTERIZATION OF A NOVEL Ab OLIGOMER AND FIBRIL CONFORMER-SPECIFIC SINGLE-CHAIN VARIABLE FRAGMENT (SCFV) THAT WAS SELECTED USING Ab1-16 LINEAR SEQUENCE
Susanne Funke1, Silke Dornieden1, Andreas M€uller-Schiffmann2, Heinrich Sticht3, Carsten Korth2, Dieter Willbold1, 1Forschungszentrum J€ulich, J€ulich, Germany; 2Heinrich-Heine-Universit€at D€usseldorf, D€usseldorf, Germany; 3Friedrich-Alexander-Universit€at Erlangen-N€urnberg, Erlangen, Germany. Background: Alzheimer’s disease is a progressive neurodegenerative disorder with devastating effects. Currently, only palliative therapies are available. In recent years, Ab immunotherapy has become a promising tool for the treatment of AD. Both, entire antibodies as well as antibody fragments, have been shown to reduce amyloid- b (Ab) plaque burden and ameliorate cognitive deficits in AD mouse models. However, clinical trials with active immunization using Ab encountered severe complications, for example meningoencephalitis. Alternatively, single-chain variable fragments (scFvs) can be used in therapeutic approaches. These were shown to alter Ab aggregation and toxicity. Methods: We selected an IgG using the N-terminal region of A b in the selection procedure. The corresponding single-chain variable fragment (scFv) was expressed in E. coli, purified and characterized regarding its interaction with different A b species and its influence on A b fibril formation using biophysical and in silico methods. Results: We were able to show that the identified scFv strongly influenced the aggregation behavior of A b. Moreover, it also bound to A b oligomers and fibrils with higher affinity than to Ab monomers, although it was selected using Ab1-16. Docking experiments suggest that the scFv binds to His14 und Lys16 of the N-terminal pile of Ab fibrils. Conclusions: Because the identified scFv specifically targets higher oligomeric species of Ab and prevents Ab fibril formation, it would be a valuable candidate for therapeutic approaches.
P4-204
THE WEAK COMBINED MAGNETIC FIELDS INDUCE THE REDUCTION OF BRAIN Ab LEVELS IN TWO ANIMAL MODELS OF ALZHEIMER’S DISEASE
Vadim Novikov, Cell Biophysics Institute, Russian Academy of Sciences, Puschino, Russia. Background: During the twenty years’ long large-scale research carried out in our laboratory, various effects of the weak low frequency magnetic
P4-201
Poster Presentations: P4
BENEFICIAL CATALYTIC AUTOIMMUNITY TO BETA-AMYLOID PEPTIDE
Yasuhiro Nishiyama1, Stephanie Planque1, Mariko Hara1, Kenji Watanabe1, Xiaoyun Xu2, Hiroaki Taguchi2, Einar M. Sigurdsson3, Brian O’Nuallain4, Ian Murray5, Robert P. Friedland6, Ken-ichiro Fukuchi7, Richard Massey8, Sudhir Paul1, 1University of Texas Health Science Center-Medical School at Houston, Houston, Texas, United States; 2 University of Texas Health Science Center-Medical School at Houston, Houston, Texas, United States; 3New York University School of Medicine, New York, New York, United States; 4Harvard Medical School-Brigham & Womens Hospital, Boston, Massachusetts, United States; 5Texas A&M Health Science Center, Bryan, Texas, United States; 6University of Louisville, Louisville, Kentucky, United States; 7University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States; 8Covalent Bioscience, Inc., Houston, Texas, United States. Background: We previously reported human catalytic autoantibodies to amyloid b peptide (Ab). We hypothesize that recognition of electrophilic amyloid epitopes by nucleophilic autoantibodies is an innate immune function that is recruited for catalytic clearance of amyloid deposits associated with aging and Alzheimer’s disease (AD). Methods: Ab cleavage was measured by HPLC, acid precipitation, mass spectroscopy or electrophoresis. Electrophilic Ab (E-Ab) was prepared by carbonylation with the lipid peroxidation end products 4-hydroxynonenal (HNE)/malonaldehyde (MDA) or phosphonate diester insertion. Covalent immune complexes were analyzed by SDS-electrophoresis. Ab1-42 aggregates were identified by antibody or Thioflavin-T staining. Results: IgM from healthy human sera, the first antibody class produced during B cell differentiation, catalyzed Ab cleavage at rates superior to IgGs. Preferential Ab cleavage by IgMs was also observed for antibodies from the sera and cerebrospinal fluid from patients with AD. Two Ab cleaving antibody fragments were isolated from a phage library, a heterodimeric V L -V L construct (2E6) and a single domain V L construct (5D3). Treatment with antibody 2E6 induced disappearance of oligomeric and fibrillar Ab. Intracranial antibody injection in Ab-overexpressing transgenic mice cleared the Ab plaques. Traditional antibodies bind antigens at complementarity determining regions (CDRs). The Ab cleaving antibodies contained CDRs with no or minimum mutations acquired by antigen-driven diversification. Deleting the CDRs did not attenuate Ab cleavage by antibody 2E6, but the catalytic activity was lost by replacing the framework regions (FRs) with corresponding FRs from a non-catalytic antibody. The FRs are evolutionarily conserved segments important for innate recognition of B cell superantigens without requirement for adaptive immune processes. From protease inhibitor and epitope mapping studies, the catalytic mechanism entails noncovalent binding at the Ab C terminus followed by nucleophilic peptide bond cleavage. Antibody 2E6 reacted covalently with an electrophilic phosphonate-containing Ab analog and the naturally-occurring Ab-HNE/Ab-MDA analogs (E-Ab). Monoclonal murine antibodies (MAbs) that cleaved Ab at low substrate concentrations were identified by immunization with non-electrophilic Ab. A subset of MAbs induced by immunization with E-Ab cleaved Ab robustly. Conclusions: Amplification of the innate noncovalent recognition and catalytic functions of antibodies driven by age/ disease-associated Ab accumulation can remove toxic amyloid deposits. P4-202
SYSTEMIC VACCINATION WITH ANTIOLIGOMERIC MONOCLONAL ANTIBODIES IMPROVES COGNITIVE FUNCTION BY REDUCING BETA-AMYLOID DEPOSITION AND TAU PATHOLOGY IN 3XTG-AD MICE
Suhail Rasool, Hilda Martinez Coria, Charles Glabe, UC Irvine, Irvine, California, United States. Background: Alzheimer’s disease (AD) is a devastating disorder that is clinically characterized by a comprehensive cognitive decline. Accumulation of the amyloid-beta (Ab) peptide plays a pivotal role in the
pathogenesis of AD. In AD, the conversion of Ab from a physiological water-soluble monomeric form into the neurotoxic oligomeric and beta (b)-sheet rich fibrillar conformation is an important event. The most toxic form of Ab are oligomers. In transgenic AD animal models, both active and passive anti-Ab immunotherapies improve cognitive function and clear the parenchymal accumulation of amyloid plaques in the brain. Methods: Vaccination Behavioral Studies Immunohistochemistry ELISA. Results: In this report we studied immunotherapy of sequence-independent anti-oligomeric monoclonal antibodies on the cognitive function and amyloid load in 3xTg -AD mice. Anti-oligomeric monoclonal antibodies significantly reduce the amyloid load and improve the cognition. The clearance of amyloid load was significantly correlated with reduction in tau hyperphosphorylation and improvement in cognition. In summary, we demonstrate that prolonged systemic immunotherapyusing oligomer-specific monoclonal antibodies effectively attenuates behavioral and pathological impairments in the 3xTg -AD mice. Conclusions: Our findings demonstarate that conformation-dependent antibodies efficiently clear soluble and insoluble Ab deposits and highlight that an early initiation of immunotherapy with oligomer-specific antibodies, perhaps even when patients are asymptomatic, might be necessary to inhibit both soluble and insoluble Ab-mediated pathology in Alzheimer’s disease.
P4-203
EXPRESSION AND CHARACTERIZATION OF A NOVEL Ab OLIGOMER AND FIBRIL CONFORMER-SPECIFIC SINGLE-CHAIN VARIABLE FRAGMENT (SCFV) THAT WAS SELECTED USING Ab1-16 LINEAR SEQUENCE
Susanne Funke1, Silke Dornieden1, Andreas M€uller-Schiffmann2, Heinrich Sticht3, Carsten Korth2, Dieter Willbold1, 1Forschungszentrum J€ulich, J€ulich, Germany; 2Heinrich-Heine-Universit€at D€usseldorf, D€usseldorf, Germany; 3Friedrich-Alexander-Universit€at Erlangen-N€urnberg, Erlangen, Germany. Background: Alzheimer’s disease is a progressive neurodegenerative disorder with devastating effects. Currently, only palliative therapies are available. In recent years, Ab immunotherapy has become a promising tool for the treatment of AD. Both, entire antibodies as well as antibody fragments, have been shown to reduce amyloid- b (Ab) plaque burden and ameliorate cognitive deficits in AD mouse models. However, clinical trials with active immunization using Ab encountered severe complications, for example meningoencephalitis. Alternatively, single-chain variable fragments (scFvs) can be used in therapeutic approaches. These were shown to alter Ab aggregation and toxicity. Methods: We selected an IgG using the N-terminal region of A b in the selection procedure. The corresponding single-chain variable fragment (scFv) was expressed in E. coli, purified and characterized regarding its interaction with different A b species and its influence on A b fibril formation using biophysical and in silico methods. Results: We were able to show that the identified scFv strongly influenced the aggregation behavior of A b. Moreover, it also bound to A b oligomers and fibrils with higher affinity than to Ab monomers, although it was selected using Ab1-16. Docking experiments suggest that the scFv binds to His14 und Lys16 of the N-terminal pile of Ab fibrils. Conclusions: Because the identified scFv specifically targets higher oligomeric species of Ab and prevents Ab fibril formation, it would be a valuable candidate for therapeutic approaches.
P4-204
THE WEAK COMBINED MAGNETIC FIELDS INDUCE THE REDUCTION OF BRAIN Ab LEVELS IN TWO ANIMAL MODELS OF ALZHEIMER’S DISEASE
Vadim Novikov, Cell Biophysics Institute, Russian Academy of Sciences, Puschino, Russia. Background: During the twenty years’ long large-scale research carried out in our laboratory, various effects of the weak low frequency magnetic