Anti-αβTCR Antibody Light Chain Stabilization Against Fragmentation
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Solution Overview
Problem
Monoclonal antibodies suffer from fragmentation issues due to the disruption of native covalent bonds, particularly in the Asn-Pro motif, leading to reduced potency and potential immunogenicity during manufacture and storage.
Innovation Solution
Humanized anti-αβTCR binding polypeptides with specific amino acid substitutions, such as N93S in the light chain variable region, to enhance stability and reduce fragmentation, maintaining stability at pH greater than 5.0 and temperature above 4°C.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the Asn-Pro motif is present in the light chain variable region, then the antibody can bind to αβTCR, but the antibody undergoes fragmentation at the Asn clipping site
Solution Approach 1:
The patent removes the problematic Asn-Pro motif (specifically Asn at position 93) from the light chain variable region sequence. This extraction of the harmful element eliminates the fragmentation site while preserving the essential CDR sequences needed for αβTCR binding, thus resolving the contradiction between binding reliability and polypeptide stability.
Solution Approach 2:
The patent changes the amino acid sequence parameter at the critical position 93 from Asn to a different residue (such as Ser, Thr, or Pro). This parameter change in the polypeptide sequence eliminates the Asn-Pro cleavage motif while maintaining the structural and functional integrity of the variable region for antigen binding.
2Productivity
If the antibody is stored under accelerated conditions (elevated temperature and pH), then the fragmentation is more rapid, but the potency is lost
Solution Approach 1:
The patent introduces stabilizing mutations (such as N93S, N93D, N93H, or N93Y) in advance to cushion against the harmful effects of accelerated storage conditions. These pre-introduced mutations create structural rigidity that prevents fragmentation even when the antibody is exposed to elevated temperatures and pH levels during storage or stress testing.
3Stability of the object's composition
If the Asn-Pro motif is removed, then fragmentation is reduced, but the binding affinity may be affected
Solution Approach 1:
The patent applies local quality by making a targeted change only at the specific position 93 where fragmentation occurs, while leaving the rest of the variable region sequence (particularly the CDRs) unchanged. This localized modification eliminates fragmentation without affecting the overall binding affinity, as the critical antigen-contacting regions maintain their original sequences.
Data Source
AI summary
The present disclosure is related to improved compositions and methods for treating T-cell-mediated diseases and disorders (e.g., autoimmune disorders, graft-versus-host-disease, and graft rejection). Provided are anti-αβTCR binding polypeptides, including antibodies, which comprise at least one amino acid substitution or modification that increases the stability of the binding polypeptide by reducing fragmentation of the light chain variable region. The methods provided herein generally involve administering to a subject in need thereof an effective amount of a stabilized, humanized binding polypeptide that is specific to the alpha beta T-cell receptor (αβTCR).