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Effects of mutations on MUC1-C/ED protein stability and antibody Binding: Structural insight
Dwipanjan Sanyal1, Danny Muzata2, Vladimir N Uversky3
1XYone Therapeutics, Canton, MA, USA.
Abstract:
Antibody-drug conjugates (ADCs) represent a transformative approach in cancer therapy, combining the specificity of monoclonal antibodies with targeted delivery of potent cytotoxic drugs to tumors. Discovery of novel antigens has been the hallmark for development of ADC therapeutics and MUC1 is one such oncoprotein, which has garnered renewed interest recently. The 3D1 antibody, engineered to bind specifically to the alpha 3 (α3) helix of the MUC1-C/extracellular domain, is being actively developed towards clinical translation. The study examined the conformational coupling in the residues spanning the MUC1 sea urchin sperm protein, enterokinase, and agrin domain (SEA), which incorporates MUC1-C. 3D1 has been shown to be sensitive to three specific point mutations-D85E, V86A, and T88A- at the interaction surface between MUC1-C and the 3D1 antibody. Our findings reveal that D85E and T88A mutations cause significant conformational shifts and reduced binding affinity due to altered electrostatic interactions and hydrogen bonding. The V86A mutation, while maintaining the overall conformation, disrupts local hydrophobic interactions, leading to decreased binding efficiency. Further, residue 85 in the wild-type (WT) MUC1 SEA domain interacts with other residues, contributing to local frustration and destabilization. Mutants D85E, V86A, and T88A exhibited similar frustration patterns but with increased local energy, indicating reduced stability. Frustration calculations highlighted the crucial role of the α3 helix in protein stability and antibody interaction. Collectively, these data highlight the importance of specific residues contributing to the local frustration in maintaining effective antibody binding and suggest that mutations impacting these residues can significantly alter antigen binding dynamics.
Insights
Understanding MUC1 antigen mutations is key for antibody-drug conjugate (ADC) cancer therapy. Specific MUC1 mutations impact antibody binding, affecting ADC efficacy and stability.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Antibody-drug conjugates (ADCs) are a promising cancer therapy, utilizing monoclonal antibodies for targeted drug delivery.
- MUC1 is an oncoprotein and a target antigen for ADC development, with the 3D1 antibody specifically binding its MUC1-C extracellular domain.
- The MUC1 SEA domain, including MUC1-C, is crucial for antibody interaction.
Purpose of the Study:
- To investigate the impact of specific point mutations (D85E, V86A, T88A) on the MUC1 SEA domain on 3D1 antibody binding affinity and antigen stability.
- To elucidate the molecular mechanisms underlying altered binding dynamics caused by these MUC1 mutations.
Main Methods:
- Analysis of conformational changes and binding affinity using computational methods.
- Examination of electrostatic, hydrogen bonding, and hydrophobic interactions at the MUC1-C and 3D1 antibody interface.
- Calculation of local frustration and energy to assess protein stability.
Main Results:
- Mutations D85E and T88A induced significant conformational shifts and reduced binding affinity by altering electrostatic and hydrogen bonding interactions.
- Mutation V86A disrupted local hydrophobic interactions, decreasing binding efficiency while maintaining overall conformation.
- All three mutations (D85E, V86A, T88A) increased local energy and frustration, indicating reduced MUC1 SEA domain stability.
- The α3 helix of MUC1 was identified as critical for both protein stability and antibody interaction.
Conclusions:
- Specific residues within the MUC1 SEA domain are vital for maintaining effective 3D1 antibody binding through contributions to local frustration and stability.
- Mutations at these critical residues can significantly alter antigen binding dynamics, impacting ADC therapeutic potential.
- Understanding these residue-specific interactions is essential for designing and optimizing MUC1-targeted ADCs.
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