Truncated Antibody Fab Fragments for Site-Specific Effector Attachment
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Solution Overview
Problem
Current methods for attaching effector molecules to antibody fragments are inefficient due to the need for interchain disulphide bond maintenance, which limits the controlled and specific attachment of effector molecules, leading to reduced production efficiency and potential loss of activity or affinity.
Innovation Solution
Development of antibody Fab fragments where the heavy chain constant region terminates at the interchain cysteine of CH1, allowing for site-specific attachment of effector molecules without inter-chain covalent linkages, enabling higher efficiency and stability comparable to wild-type fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If mild reducing conditions are used to remove thiol capping agents while maintaining interchain disulphide bonds, then the stability of antibody fragments is preserved, but the efficiency of effector molecule attachment is reduced
Solution Approach 1:
The invention extracts the interchain disulphide bond from the constant region by truncating the heavy chain at the CH1 interchain cysteine, separating the stabilizing function from the region where effector attachment occurs. This allows free thiols to be generated without compromising overall fragment stability, as the variable region disulphide bonds remain intact.
Solution Approach 2:
The antibody fragment is segmented into regions with distinct functions: the variable region maintains antigen binding stability through intact disulphide bonds, while the truncated constant region provides accessible free thiols for efficient effector attachment. This segmentation resolves the contradiction between stability and reactivity.
2Manufacturing precision
If site specific attachment of effector molecules is achieved through cysteine residues in hinge regions, then control over attachment location is improved, but the complexity of the modification process increases
Solution Approach 1:
The invention extracts the cysteine residues from the hinge region context and relocates them to the C-terminus of the constant region. This provides site-specific attachment capability without requiring hinge region engineering, simplifying the overall process while maintaining precision.
Solution Approach 2:
Instead of modifying hinge regions to create attachment sites, the invention inverts the approach by using the natural C-terminal cysteines of the constant region. This reverses the conventional strategy and achieves site-specific attachment with less complexity.
3Ease of manufacture
If random attachment of effector molecules is performed through available amino acid side chains, then the ease of modification is improved, but the precision of attachment location is reduced
Solution Approach 1:
The invention creates local quality by concentrating reactive cysteine residues at the C-terminal region of the constant chain, making this specific location highly reactive while leaving other regions unchanged. This enables site-specific attachment without requiring complex engineering throughout the entire antibody fragment.
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 allows for efficient attachment of effector molecules with maintained antigen affinity and stability, improving production efficiency and reducing the need for engineered hinge regions, thus providing a cost-effective alternative for therapeutic applications.
Implementation Method 1
Site specific attachment of effector molecules is most commonly achieved by attachment to cysteine residues
Implementation Method 2
This is usually achieved by using thiol based reductants such as β-mercaptoethanol (β-ME), β-mercaptoethylamine (β-MA) and dithiothreitol (DTT)
Data Source
AI summary
The present invention provides antibody Fab fragments in which the heavy chain constant region terminates at the interchain cysteine of CH1. Also provided are antibody Fab fragments in which the heavy chain constant region terminates at the interchain cysteine of CH1 to which one or more effector molecules are attached.