Multispecific Fab Antibody Linkers for Stable Heterodimer Assembly
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current bispecific and multispecific antibody forms face challenges such as poor stability, excessive renal clearance, and short in vivo half-life due to issues like light chain mismatching and the absence of the Fc region, which affects heterodimer formation and stability.
Innovation Solution
A novel bispecific antibody design featuring Fab fragments with specific peptide linkers allowing only one disulfide bond formation between heavy and light chains, enhancing stability and preventing homodimer formation, thereby increasing molecular weight and reducing renal clearance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of moving object
If full-length bispecific antibody with Fc region is used, then long half-life and Fc effector function are achieved, but heterodimer formation stability is poor due to light chain mismatching and knobs-into-holes requirements
Solution Approach 1:
The patent extracts and removes the Fc region from the antibody molecule, retaining only the Fab fragments (variable regions and constant regions up to CH1). This eliminates the heterodimerization problems associated with Fc region while maintaining the bispecific binding capability through the Fab fragments alone.
Solution Approach 2:
The antibody is segmented into separate heavy chain and light chain Fab fragments that are connected by flexible peptide linkers. This segmentation allows independent optimization of each fragment's binding specificity while avoiding the heterodimerization issues of full-length antibodies.
2Device complexity
If single-chain bisspecific antibody form (BITE, TandAb, DART) is used, then Fc interfering effect is avoided and smaller size is achieved, but in vivo half-life is very short due to renal clearance
Solution Approach 1:
The patent changes the molecular weight parameter by using Fab fragments with constant regions (approximately 50-60 kDa per fragment) rather than small single-chain formats. This increased size reduces renal clearance while maintaining the simplicity of the Fab-only structure without Fc region.
3Ease of manufacture
If Fab fragments are directly fused via common linker, then bispecificity is achieved, but random association of light chains results in inactive by-products
Solution Approach 1:
The patent introduces asymmetric design elements including different peptide linker sequences for heavy chain and light chain connections, and uses specific cysteine residue positioning to control disulfide bond formation. This asymmetry prevents random light chain association and ensures correct heterodimer formation.
Solution Approach 2:
The patent uses peptide linkers as intermediary elements that specifically connect heavy chain and light chain Fab fragments. These linkers contain controlled cysteine residues that mediate disulfide bond formation, ensuring proper pairing and preventing random association by-products.
4Strength
If knobs-into-holes technology is employed to improve heterodimerization, then Fc region stability is improved, but light chain mismatching still results in poor stability and non-target products
Solution Approach 1:
The patent removes the Fc region entirely, eliminating the need for knobs-into-holes technology. The heterodimerization stability is achieved through the Fab fragment design with controlled disulfide bonds and peptide linkers, avoiding the limitations of Fc-based approaches.
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 new design results in higher stability, reduced aggregation, and extended in vivo half-life, facilitating efficient production and targeted antigen binding without the need for screening common light or heavy chains.
Implementation Method 1
only one disulfide bond can be formed between the first and the second peptide linkers
Data Source
AI summary
The present application provides a multispecific antibody and preparation method and use thereof. The multispecific antibody comprises:a) a Fab fragment specifically binding to a first antigen, wherein the Fab fragment consists of a light chain and CH1 and a variable region of a heavy chain;b) a first peptide linker with the N-terminal end fused to the heavy chain;c) a second peptide linker with the N-terminal end fused to the light chain,wherein only one disulfide bond can be formed between the first and the second peptide linkers, and each of the first and the second peptide linkers is independently selected from the group consisting of a peptide linker comprising any of the sequences as set forth in SEQ ID NO.1-2, wherein X represents any amino acid other than Cys, or is absent.