Site-Specific Antibody-Drug Conjugates via Enzymatic Cyclase
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Solution Overview
Problem
Current antibody-drug conjugates (ADCs) face challenges in achieving site-specific conjugation, stability, and immunogenicity, particularly in generating homogeneous ADCs with defined drug-to-antibody ratios (DARs) for targeted therapies beyond oncology applications.
Innovation Solution
Development of bifunctional antibody-CD38 fusion proteins coupled with designer dinucleotide-based covalent inhibitors, such as 2′-modified araNAD+ analogues, for rapid generation of site-specific ADCs with defined DARs of 2 or 4, targeting HER2 and CLL-1, using a single-step conjugation process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If nonspecific conjugation to surface cysteine or lysine residues is used, then ADC production is simplified, but the resulting ADCs are heterogeneous with varied drug-to-antibody ratios and distinct pharmacological properties
Solution Approach 1:
The patent introduces an engineered enzyme (horseradish peroxidase or TdT) as an intermediary that catalyzes the formation of a unique reactive intermediate (formylated tyrosine or terminal deoxynucleotidyl transferase-mediated addition) at a specific site on the antibody. This intermediary enables site-specific conjugation without requiring complex multi-step chemistry, resolving the contradiction between manufacturing simplicity and precision by providing a single-step enzymatic route to homogeneous ADCs with defined DARs.
2Manufacturing precision
If engineered amino acids, carbohydrates, or unnatural amino acids are used for site-specific conjugation, then homogeneous ADCs with defined DARs are generated, but the conjugation processes require multiple steps or long reaction times
Solution Approach 1:
The patent replaces complex multi-step chemical conjugation mechanisms with a single-step enzymatic catalysis mechanism. The engineered enzyme (peroxidase or TdT) catalyzes the conjugation reaction directly at the desired site, eliminating the need for multiple protection/deprotection steps, purification steps, and long reaction times required by traditional chemical methods. This substitution of chemical mechanics with enzymatic catalysis achieves both high precision and high productivity.
3Productivity
If genetic fusions of peptide motifs or engineered enzymes are used for site-specific ADC production, then more efficient production is achieved, but the introduced mutations or non-human derived sequences may raise considerable immunogenicity concerns
Solution Approach 1:
The patent modifies the enzyme's substrate specificity or catalytic properties through targeted mutations rather than introducing foreign protein sequences. For example, the peroxidase enzyme is engineered to recognize and catalyze formylation at specific tyrosine residues, or TdT is engineered to add nucleotides at specific sites. These parameter changes in enzyme behavior allow site-specific conjugation using human-compatible enzymes, reducing immunogenicity while maintaining high production efficiency.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The resulting ARC-ADCs demonstrate enhanced stability and potency against HER2-positive breast cancer and CLL-1 positive acute myeloid leukemia both in vitro and in vivo, offering a new strategy for producing ADCs with improved properties for various diseases.
Implementation Method 1
ADP-ribosyl cyclases...a catalytic subunit of an ADP-ribosyl cyclase...cyclic ADP-ribose hydrolase
Data Source
AI summary
Antibody-drug conjugates, compositions thereof, and methods use. The antibody-drug conjugates include a fusion protein comprising an antibody covalently linked to an ADP-ribosyl cyclase protein via a peptide linker moiety at one or more of a C-terminus or N-terminus of a heavy or light chain of the antibody, a NAD or NMN analogue, and a payload such that the NAD or NMN analogue is conjugated to both the payload and the ADP-ribosyl cyclase protein.