Circular Trifab Antibody Design for Multispecific Binding
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Solution Overview
Problem
Current monoclonal antibodies developed for human therapy often cause unwanted side effects due to anti-rodent antibodies and lack specificity, leading to the need for more compact and multispecific antibody formats that can effectively target multiple antigens with reduced molecular weight and improved efficacy.
Innovation Solution
A trivalent, bi- or tri-specific, bi-circular polypeptide design featuring three variable domain-constant domain (V-C) dimers, where each dimer forms part of an antigen binding site, allowing for covalent or non-covalent association to create compact multispecific antibodies with enhanced binding capabilities, including the use of disulfide bonds for stabilization and heterodimerization of constant heavy chain domains.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional monoclonal antibodies are used for human therapy, then they can bind to targets, but they cause unwanted side effects due to anti-rodent antibodies and lack specificity
Solution Approach 1:
The antibody molecule is segmented into distinct functional modules: three separate antigen binding sites (first, second, and third binding sites) that can be independently configured. Each binding site is formed by specific variable domain combinations (VH-VL pairs), allowing the molecule to be divided into functional units that can be optimized for different targets, thereby achieving multispecificity and reduced cross-reactivity with rodent antibodies.
Solution Approach 2:
The antibody molecule is designed with universal multivalency, incorporating three antigen binding sites that can recognize different antigens. This multi-functional design allows a single molecule to perform multiple binding functions simultaneously, improving specificity for multiple targets while reducing dependence on rodent antibody responses.
2Volume of moving object
If traditional antibody formats are used, then they provide binding capability, but they have high molecular weight and are not sufficiently compact
Solution Approach 1:
The invention extracts and eliminates unnecessary components from the traditional antibody structure. Specifically, the molecule lacks the conventional Fc region and hinge region, retaining only the essential variable domains and minimal constant domains required for binding. This extraction of non-essential elements significantly reduces molecular weight while maintaining binding capability through the three integrated antigen binding sites.
Solution Approach 2:
The invention merges the antigen binding sites directly into the polypeptide chain structure. The first, second, and third binding sites are integrated within a single continuous polypeptide sequence, eliminating the need for separate Fc regions and hinge regions. This merging of binding functions into a compact single-chain structure reduces overall molecular size and weight while maintaining functional integrity.
3Reliability
If multispecific antibody formats are developed, then binding affinity and specificity are improved, but the structural complexity increases
Solution Approach 1:
The antibody molecule incorporates dynamic flexibility through its circular polypeptide structure with V-C dimers that can rotate and adjust their orientations. The first and second polypeptides are connected through flexible linkages, allowing the three antigen binding sites to dynamically adjust their spatial arrangements to optimize binding affinity for different antigens while maintaining a relatively simple overall structure.
Solution Approach 2:
The invention employs a nested structural arrangement where the first and second polypeptides are circularized through intrachain associations, with the third binding site formed by the association of these two polypeptides. This nesting of functional elements within a compact polypeptide framework achieves multispecificity without proportionally increasing structural complexity.
4Stability of the object's composition
If covalent association is used to circularize polypeptides, then stability is improved, but the complexity of formation increases
Solution Approach 1:
The invention uses disulfide bonds as intermediary linkages to circularize the polypeptide chains. These disulfide bonds act as stable covalent bridges that form between cysteine residues at specific positions (e.g., between the C-terminus of the first variable domain and the N-terminus of the first constant domain). This intermediary bonding mechanism provides high stability while maintaining relative simplicity in formation, as disulfide bonds can form spontaneously during protein folding under oxidative conditions.
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 multispecific antibodies are highly compact, exhibit improved binding affinity and specificity, and can perform effector functions such as ADCC and CDC, with increased stability and flexibility compared to conventional antibodies, making them suitable for therapeutic applications.
Implementation Method 1
Each of the associations can be independently of each other either non-covalently or covalently. In case the association is covalently it is not by a peptide bond, but, e.g. by a disulfide bond
Data Source
AI summary
Herein is reported a multispecific antibody comprising two circular fusion polypeptides each of them comprising a VH/VL-pair and thereby a first and a second binding site, whereby a third VH/VL-pair and thereby a third binding site is formed by the associated of the two circular fusion polypeptides.