Discovery and Functional Characterization of hPT3, a Humanized Anti-Phospho Tau Selective Monoclonal Antibody

Abstract

Background: As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer’s disease. Objective: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. Methods: By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. Results and Conclusion: Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer’s disease.

The structure of the unstructured: mosaic of tau protein linear motifs obtained by high-resolution techniques and molecular simulation

“…Phospho-specific antibody PT3 that binds tau phosphorylated at threonine residues 212 and 217 has been generated and was reported to bind AD PHF and phosphorylated recombinant tau with picomolar affinity (Van Kolen et al 2020). The whole tau peptide 210 SRpTPSLPpTPPTRE 222 used for co-crystallization (with acetylated N-terminus) could be modelled in the complex structure.…”

Plasma p217+tau vs NAV4694 amyloid and MK6240 tau PET across the Alzheimer continuum

“…More recently a high sensitivity Simoa assay has been developed using a capture antibody (pT3) that was raised against tau in paired helical filaments (PHF) of AD brain [12,13]. The core requirement for this antibody binding is phosphorylation at aa217 (p217) with enhanced binding when other nearby phosphorylated sites are present, predominantly at aa212 [12].…”

Development and Validation of a High Sensitivity Assay for Measuring p217 + tau in Cerebrospinal Fluid

“…Since both assays (p217 + tau short and p217 + tau long) reveal nearly identical performance and diagnostic potential, the core reagent PT3 appears to be the key element to the assay power. The PT3 mAb actually probes an epitope that contains not just pT212 and pT217 but additional nearby phospho-residues [ 18 ]. Hence the assays are described as recognizing tau with p217 “plus” (epitope containing at least p212 and p217).…”

“…The PT3 mAb (epitope = aa210-220 of human tau, requiring phosphorylation at aa212 & 217, "p217 + tau") was evaluated as capture reagent on Simoa HD-1 platform, pairing it with either HT43 (epitope = aa7-20 of human tau), PT82 (epitope = aa119-126 of human tau), Janssen A (epitope = aa151-158 of human tau), Janssen B (epitope = aa166-182 of human tau), BT2 (epitope = aa193-198 of human tau), or Quanterix anti-tau kit detection reagent (epitope = aa16-24 of human tau). The focused nature of the screen was based on a) preference to rely on PT3 for p217 + tau specificity [18] and b) pairing with a reagent targeting the N-terminus or mid-region of tau since extracellular tau is C-terminally truncated [15,17,19].…”

“…Here we describe a novel method for measuring tau phosphorylated at amino acid T212, T217, and surrounding residues (“p217 + tau”), that shows potential as an improved CSF-based p-tau assay, which may be useful for staging. The method relies on the high affinity and specificity of the p217 + tau targeted monoclonal antibody (mAb) PT3, described in Van Kolen et al, 2020 [ 18 ], paired with either HT43 (anti-N-terminal tau) or PT82 (anti-mid-region tau) mAbs on the Simoa HD-1 platform. These assays show greater differentiation of HC versus AD than traditional tau assays, modest correlation with cognition, and can be combined with a denaturing technique to use as p217 + tau immunotherapy target engagement biomarkers.…”