Naturally occurring antibodies isolated from PD patients inhibit synuclein seeding in vitro and recognize Lewy pathology.

Deposition of α-synuclein into Lewy bodies and Lewy neurites is the hallmark of Parkinson's disease (PD). It is hypothesized that α-synuclein pathology spreads by a "prion-like" mechanism (i.e., by seeded aggregation or templated misfolding). Therefore, various extracellular α-synuclein conformers and/or posttranslational modifications may serve as biomarkers of disease or potential targets for novel interventions. To explore whether the antibody repertoires of PD patients contain anti-α-synuclein antibodies that can potentially be used as markers or immunotherapy, we interrogated peripheral IgG+ memory B cells from PD patients for reactivity to α-synuclein. In total, ten somatically mutated antibodies were recovered, suggesting the presence of an ongoing antigen-driven immune response. The three antibodies that had the highest affinity to recombinant full-length α-synuclein, aSyn-323.1, aSyn-336.1 and aSyn-338.1, were characterized further and shown to recognize epitopes in the C terminus of α-synuclein with binding affinities between 0.3 and 2.8 µM. Furthermore, all three antibodies were able to neutralize the "seeding" of intracellular synuclein aggregates in an in vitro α-synuclein seeding assay. Finally, differential reactivities were observed for all three human anti-α-synuclein antibodies across tissue treatment conditions by immunohistochemistry. Our results suggest that the memory B-cell repertoire of PD patients might represent a potential source of biomarkers and therapies.

Structural Basis for Recognition of a Unique Epitope by a Human Anti-tau Antibody.

Aggregation of the hyperphosphorylated protein tau into neurofibrillary tangles and neuropil threads is a hallmark of Alzheimer disease (AD). Identification and characterization of the epitopes recognized by anti-tau antibodies might shed light on the molecular mechanisms of AD pathogenesis. Here we report on the biochemical and structural characterization of a tau-specific monoclonal antibody CBTAU-24.1, which was isolated from the human memory B cell repertoire. Immunohistochemical staining with CBTAU-24.1 specifically detects pathological tau structures in AD brain samples. The crystal structure of CBTAU-24.1 Fab with a phosphorylated tau peptide revealed recognition of a unique epitope (Ser235-Leu243) in the tau proline-rich domain. Interestingly, the antibody can bind tau regardless of phosphorylation state of its epitope region and also recognizes both monomeric and paired helical filament tau irrespective of phosphorylation status. This human anti-tau antibody and its unique epitope may aid in development of diagnostics and/or therapeutic AD strategies.

Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau.

Aggregation of tau protein and spreading of tau aggregates are pivotal pathological processes in a range of neurological disorders. Accumulating evidence suggests that immunotherapy targeting tau may be a viable therapeutic strategy. We have previously described the isolation of antibody CBTAU-22.1 from the memory B-cell repertoire of healthy human donors. CBTAU-22.1 was shown to specifically bind a disease-associated phosphorylated epitope in the C-terminus of tau (Ser422) and to be able to inhibit the spreading of pathological tau aggregates from P301S spinal cord lysates in vitro, albeit with limited potency. Using a combination of rational design and random mutagenesis we have derived a variant antibody with improved affinity while maintaining the specificity of the parental antibody. This affinity improved antibody showed greatly enhanced potency in a cell-based immunodepletion assay using paired helical filaments (PHFs) derived from human Alzheimer's disease (AD) brain tissue. Moreover, the affinity improved antibody limits the in vitro aggregation propensity of full length tau species specifically phosphorylated at position 422 produced by employing a native chemical ligation approach. Together, these results indicate that in addition to being able to inhibit the spreading of pathological tau aggregates, the matured antibody can potentially also interfere with the nucleation of tau which is believed to be the first step of the pathogenic process. Finally, the functionality in a P301L transgenic mice co-injection model highlights the therapeutic potential of human antibody dmCBTAU-22.1.

A common antigenic motif recognized by naturally occurring human VH5-51/VL4-1 anti-tau antibodies with distinct functionalities.

Misfolding and aggregation of tau protein are closely associated with the onset and progression of Alzheimer's Disease (AD). By interrogating IgG+ memory B cells from asymptomatic donors with tau peptides, we have identified two somatically mutated VH5-51/VL4-1 antibodies. One of these, CBTAU-27.1, binds to the aggregation motif in the R3 repeat domain and blocks the aggregation of tau into paired helical filaments (PHFs) by sequestering monomeric tau. The other, CBTAU-28.1, binds to the N-terminal insert region and inhibits the spreading of tau seeds and mediates the uptake of tau aggregates into microglia by binding PHFs. Crystal structures revealed that the combination of VH5-51 and VL4-1 recognizes a common Pro-Xn-Lys motif driven by germline-encoded hotspot interactions while the specificity and thereby functionality of the antibodies are defined by the CDR3 regions. Affinity improvement led to improvement in functionality, identifying their epitopes as new targets for therapy and prevention of AD.

Immunological memory to hyperphosphorylated tau in asymptomatic individuals.

Several reports have described the presence of antibodies against Alzheimer's disease-associated hyperphosphorylated forms of tau in serum of healthy individuals. To characterize the specificities that can be found, we interrogated peripheral IgG+ memory B cells from asymptomatic blood donors for reactivity to a panel of phosphorylated tau peptides using a single-cell screening assay. Antibody sequences were recovered, cloned, and expressed as full-length IgGs. In total, 52 somatically mutated tau-binding antibodies were identified, corresponding to 35 unique clonal families. Forty-one of these antibodies recognize epitopes in the proline-rich and C-terminal domains, and binding of 26 of these antibodies is strictly phosphorylation dependent. Thirteen antibodies showed inhibitory activity in a P301S lysate seeded in vitro tau aggregation assay. Two such antibodies, CBTAU-7.1 and CBTAU-22.1, which bind to the proline-rich and C-terminal regions of tau, respectively, were characterized in more detail. CBTAU-7.1 recognizes an epitope that is similar to that of murine anti-PHF antibody AT8, but has different phospho requirements. Both CBTAU-7.1 and CBTAU-22.1 detect pathological tau deposits in post-mortem brain tissue. CBTAU-7.1 reveals a similar IHC distribution pattern as AT8, immunostaining (pre)tangles, threads, and neuritic plaques. CBTAU-22.1 shows selective detection of neurofibrillary changes by IHC. Taken together, these results suggest the presence of an ongoing antigen-driven immune response against tau in healthy individuals. The wide range of specificities to tau suggests that the human immune repertoire may contain antibodies that can serve as biomarkers or be exploited for therapy.

Immune responses against domain I of ß(2)-glycoprotein I are driven by conformational changes: domain I of ß(2)-glycoprotein I harbors a cryptic immunogenic epitope.

OBJECTIVE: The presence of autoantibodies against a cryptic epitope in domain I of ß(2)-glycoprotein I (ß(2)GPI) is strongly associated with thrombotic events in patients with the antiphospholipid syndrome. We hypothesized that a conformational change could be a trigger for the formation of antibodies against domain I of ß(2)GPI. Therefore, we investigated whether immune responses against ß(2)GPI are related to its conformation. METHODS: Conformational changes in ß(2)GPI were studied using various techniques, either upon binding to cardiolipin or after disruption of the internal disulfide bonds. The immunogenicity of ß(2)GPI in different conformations as well as the individual domains of ß(2)GPI were studied in vivo by monitoring the generation of antibodies after intravenous administration of ß(2)GPI to mice. Furthermore, plasma samples from these mice were assessed for lupus anticoagulant activity and thrombin-antithrombin complex levels. RESULTS: We observed that the interaction of ß(2)GPI with cardiolipin induced a conformational change in ß(2)GPI: electron microscopy revealed that ß(2)GPI assembled into polymeric meshworks. We next investigated the immunogenicity of both human and murine ß(2)GPI in mice. Both human and murine ß(2)GPI combined with cardiolipin and misfolded ß(2)GPI triggered antibody formation against the native protein as well as against domain I of ß(2)GPI, while native ß(2)GPI was not immunogenic. In addition, we observed that anti-domain I antibodies developed in mice injected with domain I of ß(2)GPI, and that antibodies did not develop in mice injected with domains II-V. The induced anti-domain I antibodies prolonged the dilute Russell's viper venom plasma clotting time. The plasma of mice with anti-domain I antibodies had increased levels of circulating thrombin-antithrombin complexes. CONCLUSION: The results of our studies indicate that the exposure of cryptic epitopes due to conformational changes in ß(2)GPI can induce autoantibody formation.