Humanized Anti-CD20 Antibody Reducing Antigenicity
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Solution Overview
Problem
Current anti-CD20 monoclonal antibodies, such as rituximab, have limitations including antigenicity, short blood half-life, and variable response rates in treating B cell-mediated tumors and diseases, with challenges in maintaining the natural three-dimensional structure of the CD20 protein and inducing effective biological responses.
Innovation Solution
Development of murine anti-CD20 monoclonal antibodies that bind specifically to human CD20 antigen, with direct cell growth inhibitory activities and apoptosis induction, followed by chimerization and humanization to enhance complement- and antibody-dependent cell-mediated cytotoxicity, using GST-fused CD20 protein as an immunogen and expressing human CD20 on mammalian cells for immunization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If chimeric anti-CD20 monoclonal antibodies are used as therapeutic agents, then they can effectively target and eliminate B cells, but they induce antigenicity and have short blood half-life
Solution Approach 1:
The patent applies parameter changes by modifying the antibody structure from chimeric to humanized format, changing the amino acid sequence composition to increase human-like characteristics. This transformation extends the blood half-life from 3-4 days to potentially longer durations while maintaining therapeutic effectiveness against B cell-mediated diseases.
Solution Approach 2:
The patent creates composite antibody structures by combining human antibody frameworks with selected CDR regions from murine antibodies. This composite approach (humanized antibodies) reduces antigenicity while preserving the ability to bind CD20 and eliminate B cells, thereby extending circulation time in the bloodstream.
2Reliability
If chimeric anti-CD20 monoclonal antibodies are used as therapeutic agents, then they can effectively target and eliminate B cells, but they induce antigenicity
Solution Approach 1:
The patent changes the compositional parameters of the antibody by replacing murine protein sequences with human sequences in the constant regions and framework regions. This parameter change reduces the immunogenic difference between the therapeutic antibody and human proteins, thereby minimizing antigenicity and the formation of neutralizing antibodies.
Solution Approach 2:
The patent constructs composite antibodies with human-derived protein frameworks and selected functional CDR regions. This composite structure reduces overall antigenicity while preserving the specific binding capability to CD20, eliminating the harmful antigenic response that occurs with fully chimeric antibodies.
3Quantity of substance
If CD20 protein is solubilized using surfactant or strong alkali, then it can be obtained from cell lysate, but the natural three-dimensional structure cannot be maintained
Solution Approach 1:
The patent introduces mild detergents as intermediary substances that enable solubilization of the hydrophobic CD20 protein from cell membranes without causing denaturation. These intermediary agents facilitate protein extraction while preserving the native three-dimensional structure required for proper antibody binding and biological function.
Solution Approach 2:
The patent changes the solubilization parameters from extreme conditions (strong alkali) to mild conditions (controlled detergent concentration, physiological pH). This parameter optimization allows extraction of sufficient CD20 protein while maintaining its folded three-dimensional structure, enabling effective immunogen preparation.
4Shape
If CD20 positive B cell strain is used as immunogen, then the natural structure is preserved, but immunostimulating property is weak and it is not easy to obtain mature antibody-producing clones
Solution Approach 1:
The patent creates composite immunogen preparations by combining intact CD20 positive B cells with solubilized CD20 protein or GST-CD20 fusion protein. This composite approach provides both structural integrity and enhanced immunogenicity, stimulating stronger immune responses and facilitating the generation of mature antibody-producing hybridoma clones.
Solution Approach 2:
The patent merges two immunogen approaches: using whole CD20 positive B cells to preserve natural structure and using solubilized CD20 protein to enhance immunostimulating properties. This combination strategy overcomes the limitations of each individual approach, achieving both structural preservation and high clone maturation efficiency.
Data Source
AI summary
A murine anti-CD20 monoclonal antibody having cell growth inhibitory activities is disclosed. Cell growth inhibitory activities include apoptosis against human CD20 antigen expressing cells in culture of the CD20 antigen expressing cells without effector cells. The anti-CD20 monoclonal antibody is incorporated into chimeric anti-CD20 monoclonal antibodies in which the amino acid sequences of the variable regions of the anti-CD20 monoclonal antibody and the amino acid sequences of the constant regions of human immunoglobulin are fused. Also a humanized anti-CD20 monoclonal antibody is described which includes all of the variable region CDRs of the H chain of the anti-CD20 monoclonal antibody and all of the variable region CDRs of the L chain of the anti-CD20 monoclonal antibody and an amino acid sequence of human immunoglobulin. A nucleotide sequence encoding the amino acid sequence of the chimeric or humanized anti-CD20 monoclonal antibody can be expressed in mammalian cells.