PseudoFab Multispecific Proteins Mispairing
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Solution Overview
Problem
Current multispecific antibody technologies face challenges with mispairing of heavy and light chains, leading to undesired chain combinations and manufacturing issues, particularly for rare antibodies like broadly neutralizing anti-HIV antibodies, which cannot be adapted to common light-chain formats.
Innovation Solution
The introduction of a stabilized knockout domain, termed 'pseudoFab', which forms a pseudoFab moiety that incorporates a stabilized knockout VH domain paired with a VL domain, along with engineered interchain disulfide bonds, to facilitate preferential production and minimize mispairing in multispecific binding proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional multisspecific antibody technologies are used to create asymmetric binding arms, then multisspecific binding capability is achieved, but mispairing of heavy and light chains occurs leading to undesired chain combinations
Solution Approach 1:
The patent applies asymmetry by replacing the symmetric CH1-CL constant domain pair in one Fab arm with an asymmetric stabilized knockout domain (VHX-VLX). This asymmetric design creates distinct heterodimerization interfaces that enable correct chain pairing while preventing mispairing, thus resolving the contradiction between achieving multisspecific binding and maintaining manufacturing precision.
Solution Approach 2:
The patent changes the structural parameters of the antibody by substituting constant domains (CH1-CL) with variable domains (VHX-VLX) that contain engineered disulfide bonds. This parameter change transforms the pairing mechanism from relying on constant domain interactions to relying on engineered disulfide bond formation, thereby improving chain pairing accuracy while maintaining multisspecific binding capability.
2Adaptability or versatility
If common light chain format is used to circumvent light-chain problem, then binding flexibility to multiple antigens is enabled, but de novo antibody generation in transgenic mice is required
Solution Approach 1:
The patent extracts the light chain problem by removing the requirement for a common light chain across both Fab arms. Instead, each Fab arm retains its own light chain (CL or VLX), eliminating the need for transgenic mouse generation while maintaining the ability to bind multiple antigens through the stabilized knockout domain mechanism.
3Reliability
If stabilized knockout domain with engineered interchain disulfide bonds is introduced, then mispairing is minimized and thermal stability is enhanced, but structural complexity of the antibody increases
Solution Approach 1:
The patent segments the antibody structure by dividing it into distinct domains with specific functions: functional antigen binding sites (VH-VL-CH1-CL) and stabilized knockout domains (VHX-VLX with disulfide bonds). This segmentation allows each domain to perform its specialized function independently, improving chain pairing reliability while managing structural complexity through modular design.
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
This approach enhances the thermal stability and production efficiency of multispecific binding proteins by reducing mispairing and allowing for the creation of multispecific binding proteins that can bind multiple antigens effectively, including rare ones like anti-HIV antibodies.
Implementation Method 1
one or more engineered interchain disulfide bonds which confer enhanced thermal stability (Tm) of the pseudoFab relative to a reference Fab molecule
Data Source
AI summary
Binding proteins comprising a pseudoFab domain including a stabilised knockout domain and a second VH/VL that form a first functional antigen binding domain are provided. Multispecific binding proteins comprising at least one pseudoFab are also provided. Multispecific binding proteins, nucleic acids encoding binding proteins and multispecific binding proteins, expression vectors, host cells, pharmaceutical composition and methods of treatment administering the binding proteins or multispecific binding proteins described herein are also provided.