Can an antibody-drug conjugate be successfully engineered to achieve the targeted degradation of extracellular tau?

By Arjun Gupta, USA

Abstract:

416 million people worldwide are affected by Alzheimer’s disease, whether it is in its prodromal, preclinical, or dementia stage (Gustavsson et al.). AD and other tauopathies are characterized by the accumulation of neurotoxic tau protein aggregates in the brain (Iqbal et al.). The mechanism this proposal outlines is an antibody-drug conjugate inspired by cancer treatments. This conjugate is composed of the MC1 antibody, shown to efficiently target extracellular tau, and Epigallocatechin gallate, a natural compound known to disrupt tau fibrils. These molecules are linked via Sulfo-SMCC, a ADC linker that is structurally compatible for these compounds.

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Introduction:

Neurodegenerative diseases marked by the pathological aggregation of tau protein, collectively known as tauopathies, represent a significant and growing global health burden. Among these, Alzheimer's disease (AD) is the most prevalent (Iqbal et al.). In AD and other tauopathies, tau transitions from a soluble state to insoluble filamentous aggregates. These aggregates are widely recognized as one of the key drivers of neuronal dysfunction (Lee et al.). Current AD therapies fail to effectively target extracellular tau effectively, and this proposal addresses this critical gap. The treatment described in this proposal is a combination of Epigallocatechin gallate (ECGC), which disaggregates extracellular tau (Gonçalves et al.), and an MC1 monoclonal antibody, known for targeting this tau (Söderberg et al.). The linker to join the two compounds is Sulfo-SMCC, a linker chosen to promote stability. This combination, known as an antibody-drug conjugate, is designed for intrathecal administration to avoid issues with the Blood Brain Barrier. The concept of this ADC class is purely theoretical at present, representing an exciting frontier in the field.

Literature Review:

Antibody-Drug Conjugates (ADCs): ADCs have been revolutionary in cancer therapy and combine a tumor-targeting antibody with a smaller molecule to break the tumor down. This technology has revolutionized cancer treatment by minimizing systemic toxicity; however, adapting ADCs for neurodegenerative diseases remains a novel strategy (Punyakoti et al.). Targeting extracellular tau via an ADC to hinder, and even reverse, the progression of AD has never been tested (Punyakoti et al.).

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Monoclonal Antibodies: Monoclonal antibodies have emerged as a prominent therapeutic strategy in Alzheimer's disease (AD), targeting key pathological hallmarks like amyloid-beta (Aβ) plaques and tau tangles (Yoshiyuki Soeda et al.). Early efforts focused on clearing Aβ with these antibodies such as Aducanumab, Lecanemab, and Gantenerumab, demonstrated modest clinical benefits in slowing cognitive decline by reducing amyloid burden in the brain (Söderberg et al.). However, the MC1 antibody exhibits high selectivity for tau oligomers (extracellular tau) by identifying folds in the tangles (Yoshiyuki Soeda et al.). Clinical testing of this molecule was stopped due to its inability to untangle tau independently (Carroll).

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Epigallocatechin gallate (EGCG): EGCG, a green tea catechin, has shown promise in inhibiting tau aggregation by interacting with a variety of proteins linked to protein misfolding (Gonçalves et al.). Its clinical application for targeting the increasingly recognized extracellular tau pathology is significantly limited by its inability to effectively reach these extracellular targets (Nag et al.).

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Linker: Sulfo-SMCC is a heterobifunctional cross-linking reagent designed for usage in ADCs (Thermo Fisher Product Description). Its heterobifunctional nature allows for efficient and stable covalent attachment of a molecule to a large antibody (Thermo Fisher Scientific). The amine-reactive sulfo-NHS ester readily binds to residues on the MC1 antibody, while the maleimide group specifically reacts with introduced thiols on EGCG, ensuring a sturdy linkage.

Methodology:​ 

Limited literature on this ADC class means conjugate formation protocols are scarce. Therefore, this methodology was developed de novo, based on the molecule’s chemistry. As with any new procedure, minor adjustments based on observations in a lab will likely be necessary for optimization.

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Section 1 - Preparing MC1 Antibody: A 1-10 mg/mL concentration of an MC1 antibody solution will be centrifuged to remove aggregates. Next, TCEP, a reducing agent, will be added to partially reduce the antibody's disulfide bonds for controlled linker attachment (Hampton Research). Excess TCEP will be immediately removed using a desalting column to prevent the newly formed thiol groups from reoxidizing, ensuring their availability for conjugation.

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Section 2 - Preparation of Amine-Functionalized EGCG: EGCG lacks readily available functional groups for a direct reaction with Sulfo-SMCC (Nag et al.). This section introduces a primary amine group onto the EGCG molecule through a reaction with ethylenediamine (EDA) to allow EGCG to bind. EGCG will be dissolved in anhydrous dimethyl sulfoxide (DMSO) and reacted with EDA at a molar ratio of 5:1 for 2 hours at room temperature. Excess EDA will be removed by precipitation with cold diethyl ether, followed by centrifugation. The resulting EGCG solution will be resuspended in a suitable reaction buffer (PBS pH 7.0).

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Section 3 - Activation of EGCG with Sulfo-SMCC: The Sulfo-SMCC linker does not require modification because it has an NHS ester that reacts with primary amines (Thermo Fisher Scientific); therefore, the EGCG solution and Sulfo-SMCC solution can be gently stirred at room temperature to gain an optimal conjugation.

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Section 4 - Conjugation of EGCG-Mal to Reduced MC1 Antibody: The reactive MC1 antibody from section 1 will be mixed with the EGCG solution from section 3 at varying molar ratios (e.g., 1:5 to 1:10 antibody to EGCG-linker) to optimize the drug-to-antibody ratio (DAR) for testing later. The conjugation reaction will proceed for 2-4 hours at room temperature under gentle agitation and an inert atmosphere. This is the final conjugation step, where a maleimide group on the EGCG-linker reacts with the free thiol groups that were generated on the MC1 antibody.

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Section 5 - Testing: The conjugate's specific binding to extracellular tau oligomers will be quantified using Enzyme-Linked Immunosorbent Assays and visualized via immunocytochemistry on cell cultures expressing extracellular tau (Alhajj et al.). The effectiveness of the conjugate will be measured using Thioflavin S fluorescence assays and confirmed by Transmission Electron Microscopy imaging (Hudson et al.). The toxicity of the final conjugate to neuronal cell lines will be evaluated using cell viability assays such as MTT release assays (ScienceDirect). Different DAR ratios from section 4 will be tested for safety and optimization.

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Conclusion:

The proposed MC1-Sulfo-SMCC-EGCG antibody-drug conjugate seeks to overcome the limitations of systemic EGCG administration and the prior inability of the MC1 antibody alone to resolve tau tangles. This targeted approach to extracellular tau degradation could represent a significant advancement in the development of disease-modifying therapies for neurodegenerative conditions like Alzheimer's disease.

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References :

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