Immunotherapy targeting the C-terminal domain of TDP-43 decreases neuropathology and confers neuroprotection in mouse models of ALS/FTD.

Effective therapies are urgently needed to safely target TDP-43 pathology as it is closely associated with the onset and development of devastating diseases such as frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). In addition, TDP-43 pathology is present as a co-pathology in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Our approach is to develop a TDP-43-specific immunotherapy that exploits Fc gamma-mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP-43 function. Thus, using both in vitro mechanistic studies in conjunction with the rNLS8 and CamKIIa inoculation mouse models of TDP-43 proteinopathy, we identified the key targeting domain in TDP-43 to accomplish these therapeutic objectives. Targeting the C-terminal domain of TDP-43 but not the RNA recognition motifs (RRM) reduces TDP-43 pathology and avoids neuronal loss in vivo. We demonstrate that this rescue is dependent on Fc receptor-mediated immune complex uptake by microglia. Furthermore, monoclonal antibody (mAb) treatment enhances phagocytic capacity of ALS patient-derived microglia, providing a mechanism to restore the compromised phagocytic function in ALS and FTD patients. Importantly, these beneficial effects are achieved while preserving physiological TDP-43 activity. Our findings demonstrate that a mAb targeting the C-terminal domain of TDP-43 limits pathology and neurotoxicity, enabling clearance of misfolded TDP-43 through microglia engagement, and supporting the clinical strategy to target TDP-43 by immunotherapy. SIGNIFICANCE STATEMENT: TDP-43 pathology is associated with various devastating neurodegenerative disorders with high unmet medical needs such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Thus, safely and effectively targeting pathological TDP-43 represents a key paradigm for biotechnical research as currently there is little in clinical development. After years of research, we have determined that targeting the C-terminal domain of TDP-43 rescues multiple patho-mechanisms involved in disease progression in two animal models of FTD/ALS. In parallel, importantly, our studies establish that this approach does not alter the physiological functions of this ubiquitously expressed and indispensable protein. Together, our findings substantially contribute to the understanding of TDP-43 pathobiology and support the prioritization for clinical testing of immunotherapy approaches targeting TDP-43.

TLR4- and TRIF-dependent stimulation of B lymphocytes by peptide liposomes enables T cell-independent isotype switch in mice.

Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell-dependent and vaccination in T cell-deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptide-specific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cell-independent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domain-containing adapter inducing interferon-ß (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are assembled and appropriately presented directly to B lymphocytes. A TI vaccine could enable efficient prophylactic and therapeutic vaccination of patients with T-cell deficiencies and find application in diseases where induction of T-cell responses contraindicates vaccination, for example, in Alzheimer disease.

Targeting the TDP-43 low complexity domain blocks spreading of pathology in a mouse model of ALS/FTD.

Abnormal cytoplasmic localization and accumulation of pathological transactive response DNA binding protein of 43 kDa (TDP-43) underlies several devastating diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP). A key element is the correlation between disease progression and spatio-temporal propagation of TDP-43-mediated pathology in the central nervous system. Several lines of evidence support the concept of templated aggregation and cell to cell spreading of pathological TDP-43. To further investigate this mechanism in vivo, we explored the efficacy of capturing and masking the seeding-competent region of extracellular TDP-43 species. For this, we generated a novel monoclonal antibody (mAb), ACI-6677, that targets the pathogenic protease-resistant amyloid core of TDP-43. ACI-6677 has a picomolar binding affinity for TDP-43 and is capable of binding to all C-terminal TDP-43 fragments. In vitro, ACI-6677 inhibited TDP-43 aggregation and boosted removal of pathological TDP-43 aggregates by phagocytosis. When injecting FTLD-TDP brain extracts unilaterally in the CamKIIa-hTDP-43NLSm mouse model, ACI-6677 significantly limited the induction of phosphorylated TDP-43 (pTDP-43) inclusions. Strikingly, on the contralateral side, the mAb significantly prevented pTDP-43 inclusion appearance exemplifying blocking of the spreading process. Taken together, these data demonstrate for the first time that an immunotherapy targeting the protease-resistant amyloid core of TDP-43 has the potential to restrict spreading, substantially slowing or stopping progression of disease.

An effector-reduced anti-ß-amyloid (Aß) antibody with unique aß binding properties promotes neuroprotection and glial engulfment of Aß.

Passive immunization against ß-amyloid (Aß) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aß monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aß, protected against Aß1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aß oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aß, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aß antibody that takes advantage of a unique Aß binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

Targeting amyotrophic lateral sclerosis by neutralizing seeding-competent TDP-43 in CSF.

In amyotrophic lateral sclerosis, a disease driven by abnormal transactive response DNA-binding protein of 43 kDa aggregation, CSF may contain pathological species of transactive response DNA-binding protein of 43 kDa contributing to the propagation of pathology and neuronal toxicity. These species, released in part by degenerating neurons, would act as a template for the aggregation of physiological protein contributing to the spread of pathology in the brain and spinal cord. In this study, a robust seed amplification assay was established to assess the presence of seeding-competent transactive response DNA-binding protein of 43 kDa species in CSF of apparently sporadic amyotrophic lateral sclerosis patients. These samples resulted in a significant acceleration of substrate aggregation differentiating the kinetics from healthy controls. In parallel, a second assay was developed to determine the level of target engagement that would be necessary to neutralize such species in human CSF by a therapeutic monoclonal antibody targeting transactive response DNA-binding protein of 43 kDa. For this, evaluation of the pharmacokinetic/pharmacodynamic effect for the monoclonal antibody, ACI-5891.9, in vivo and in vitro confirmed that a CSF concentration of ≍1100 ng/mL would be sufficient for sustained target saturation. Using this concentration in the seed amplification assay, ACI-5891.9 was able to neutralize the transactive response DNA-binding protein of 43 kDa pathogenic seeds derived from amyotrophic lateral sclerosis patient CSF. This translational work adds to the evidence of transmission of transactive response DNA-binding protein of 43 kDa pathology via CSF that could contribute to the non-contiguous pattern of clinical manifestations observed in amyotrophic lateral sclerosis and demonstrates the ability of a therapeutic monoclonal antibody to neutralize the toxic, extracellular seeding-competent transactive response DNA-binding protein of 43 kDa species in the CSF of apparently sporadic amyotrophic lateral sclerosis patients.

Novel Phospho-Tau Monoclonal Antibody Generated Using a Liposomal Vaccine, with Enhanced Recognition of a Conformational Tauopathy Epitope.

The microtubule-associated protein Tau is an intrinsically unfolded, very soluble neuronal protein. Under still unknown circumstances, Tau protein forms soluble oligomers and insoluble aggregates that are closely linked to the cause and progression of various brain pathologies, including Alzheimer's disease. Previously we reported the development of liposome-based vaccines and their efficacy and safety in preclinical mouse models for tauopathy. Here we report the use of a liposomal vaccine for the generation of a monoclonal antibody with particular characteristics that makes it a valuable tool for fundamental studies as well as a candidate antibody for diagnostic and therapeutic applications. The specificity and affinity of antibody ACI-5400 were characterized by a panel of methods: (i) measuring the selectivity for a specific phospho-Tau epitope known to be associated with tauopathy, (ii) performing a combination of peptide and protein binding assays, (iii) staining of brain sections from mouse preclinical tauopathy models and from human subjects representing six different tauopathies, and (iv) evaluating the selective binding to pathological epitopes on extracts from tauopathy brains in non-denaturing sandwich assays. We conclude that the ACI-5400 antibody binds to protein Tau phosphorylated at S396 and favors a conformation that is typically present in the brain of tauopathy patients, including Alzheimer's disease.

Development of a small D-enantiomeric Alzheimer's amyloid-ß binding peptide ligand for future in vivo imaging applications.

Alzheimer's disease (AD) is a devastating disease affecting predominantly the aging population. One of the characteristic pathological hallmarks of AD are neuritic plaques, consisting of amyloid-ß peptide (Aß). While there has been some advancement in diagnostic classification of AD patients according to their clinical severity, no fully reliable method for pre-symptomatic diagnosis of AD is available. To enable such early diagnosis, which will allow the initiation of treatments early in the disease progress, neuroimaging tools are under development, making use of Aß-binding ligands that can visualize amyloid plaques in the living brain. Here we investigate the properties of a newly designed series of D-enantiomeric peptides which are derivatives of ACI-80, formerly called D1, which was developed to specifically bind aggregated Aß1-42. We describe ACI-80 derivatives with increased stability and Aß binding properties, which were characterized using surface plasmon resonance and enzyme-linked immunosorbent assays. The specific interactions of the lead compounds with amyloid plaques were validated by ex vivo immunochemistry in transgenic mouse models of AD. The novel compounds showed increased binding affinity and are promising candidates for further development into in vivo imaging compounds.