Humanized Anti-DR5 Antibody CDR Grafting Reduces Immunogenicity
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Solution Overview
Problem
Murine monoclonal antibodies used in cancer therapy induce a human anti-mouse antibody response, reducing their therapeutic efficacy and causing immunogenicity, necessitating the development of a humanized antibody with reduced immunogenicity while maintaining affinity and specificity.
Innovation Solution
A humanized monoclonal antibody against the extracellular domain of human death receptor 5 (DR5) is created by grafting complementarity-determining regions (CDRs) from a murine antibody onto a human antibody framework, combined with a recombinant eukaryotic expression vector for efficient production, using a process that includes transfecting HEK293 cells and purifying the antibody via affinity chromatography.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If murine monoclonal antibody is used for cancer therapy, then therapeutic efficacy is achieved, but immunogenicity increases causing HAMA response
Solution Approach 1:
The patent applies CDR grafting technology to replace only the antigen-binding CDR regions of the murine antibody with human CDR regions, while maintaining the murine framework regions that provide structural stability. This local substitution reduces immunogenicity while preserving therapeutic efficacy against DR5-expressing cancer cells
Solution Approach 2:
The patent creates a chimeric antibody structure combining human constant regions with murine variable regions, forming a composite molecular architecture that reduces immunogenicity while maintaining antigen-binding capability. The humanized antibody consists of human IgG1 or IgG4 constant regions paired with engineered variable regions containing both human and murine sequences
2Object-affected harmful factors
If CDR grafting is used to humanize antibody, then immunogenicity is reduced, but affinity and specificity may be compromised
Solution Approach 1:
The patent systematically optimizes the amino acid sequences in both CDR and framework regions through rational design and back-mutation. Key murine-specific residues are retained in framework regions to maintain structural integrity, while human sequences are introduced to reduce immunogenicity. The balancing of human and murine sequences achieves both reduced HAMA response and preserved high-affinity binding to DR5
Solution Approach 2:
The patent uses computational modeling and structural analysis as intermediaries to guide the CDR grafting process. By analyzing the three-dimensional structure of the antigen-antibody complex, the patent identifies critical residues for maintaining binding affinity while selecting appropriate human sequences for substitution, ensuring that affinity and specificity are preserved during humanization
3Object-affected harmful factors
If chimeric antibody structure is created, then HAMA response is reduced, but spatial separation of variable and constant regions occurs
Solution Approach 1:
The patent divides the antibody molecule into distinct functional segments: human constant regions (Fc) that mediate effector functions and reduce immunogenicity, and engineered variable regions (Fab) that contain both human and murine sequences to maintain antigen-binding capability. This segmentation allows each region to optimize its function while working together as an integrated molecule
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 humanized antibody effectively induces apoptosis in cancer cells, inhibits tumor growth in vivo, and combines with TRAIL or other agents for enhanced therapeutic efficacy without significant immunogenicity or toxicity to normal cells, demonstrating improved pharmacokinetic properties and safety.
Implementation Method 1
purifying the antibody via affinity chromatography
Data Source
AI summary
The present invention provides a humanized monoclonal antibody against extracellular domain of human death receptor 5, comprising a light chain variable region, whose amino acid sequence has at least 90% identity with the amino acid sequence shown as SEQ ID NO: 1, a heavy chain variable region, whose amino acid sequence has at least 90% identity with the amino acid sequence shown as SEQ ID NO: 2, and constant region derived from human antibody. The present invention also provides nucleotide sequence encoding said humanized monoclonal antibody, a recombinant eukaryotic expression vector, a process for preparing the humanized monoclonal antibody, and the composition and use therefore. Said humanized monoclonal antibody of the present invention shows specific apoptosis-inducing activity against various cancer cells both in vivo and in vitro, and thus it can be used alone or in combination with natural ligand of DR5, apoptosis-inducing ligand associated with tumor nerosis factor or other medicaments for the treatment of a variety of cancers as well as other diseases associated with high DR5 expression.