Humanized CD3 Binding Domain Stability
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Solution Overview
Problem
Existing CD3 binding antigen binding proteins face challenges with stability, expression, and biophysical properties, particularly when humanized from murine VL frameworks, leading to poor recognition and cytotoxic potency.
Innovation Solution
The use of a human VL framework of the κ chain (Vκ1_39) instead of the murine λ chain, combined with specific mutations at positions VH111 and VL49, enhances CD3 binding affinity, stability, and cytotoxic potency in bispecific tandem diabodies, while maintaining binding properties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If murine VL framework (λ chain) is used for humanization, then CD3 binding affinity is maintained, but stability and expression are poor
Solution Approach 1:
The patent applies parameter changes by switching the VL framework type from murine λ chain to human κ chain (Vκ1_39), and by introducing specific amino acid mutations at positions 49 and 50. This fundamental parameter change in the framework structure resolves the contradiction by providing both stable expression and maintained CD3 binding affinity, as the human κ framework is more compatible with human cellular machinery while the specific mutations preserve the binding interface.
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions at specific positions (49 and 50) within the VL framework, rather than changing the entire framework. These localized changes at the CDR3 region preserve the essential CD3 binding interactions while improving overall protein stability and expression characteristics, demonstrating how local modifications can resolve global stability issues.
2Stability of the object's composition
If human VL framework (Vκ1_39) is used, then stability and expression are improved, but initial binding affinity is reduced
Solution Approach 1:
The patent uses parameter changes by introducing specific amino acid substitutions at positions 49 and 50 of the Vκ1_39 framework. These parameter modifications restore and optimize the CD3 binding affinity that was initially reduced by the framework switch, while maintaining the stability and expression benefits of the human κ framework.
Solution Approach 2:
The patent applies preliminary action by pre-optimizing the Vκ1_39 framework with specific mutations at positions 49 and 50 before final antibody construction. This preliminary optimization ensures that the framework is pre-configured to maintain both stability and binding affinity, preventing the need for later corrective modifications and ensuring consistent performance in the final therapeutic antibody.
3Stability of the object's composition
If specific mutations (VH111 to Y or H, VL49 from G to A) are introduced, then stability is improved, but binding mechanism is altered
Solution Approach 1:
The patent applies local quality by introducing mutations at specific positions (VH111 and VL49) that directly contact each other at the CD3 binding interface. These localized changes improve stability through enhanced inter-chain interactions while maintaining the essential binding mechanism, as the mutations are positioned to optimize structural stability without disrupting the overall binding geometry and affinity.
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 3A~3B
AI summary
The disclosure relates to a humanized CD3 binding site, which comprises (a) a variable heavy chain domain (VH) as depicted in SEQ ID NO:8 and a variable light chain domain (VL) as depicted in SEQ ID NO:3; or (b) a variable heavy chain domain (VH) as depicted in SEQ ID NO:9 and a variable light chain domain (VL) as depicted in SEQ ID NO:4. The CD3 binding sites have an increased stability, while the binding affinity has been retained due to mutations at positions VH111 and VL49.