Monoclonal Antibodies as Probes to Protein Structure and Function : Insights into human Chorionic gonadotropin

Abstract

Thanks to Köhler and Milstein, monoclonal antibodies (mAbs) can serve as excellent probes for protein structure and function. However, the method used to study the interaction between an antigen and its cognate mAb has a significant bearing on the results obtained and their interpretation. For example, indiscriminate use of ELISA procedures has sometimes produced ambiguous results. Among contemporary methods to study protein interactions, BIAcore is the most popular. Similarly, isothermal titration calorimetry (ITC), used to study the thermodynamic aspects of ligand-ligate interactions, is well accepted in the scientific community. However, both methods suffer from limitations that often go unnoticed.

In this thesis, we have developed methods to study protein-protein interactions and used them to obtain valuable information about protein structure and function, using human Chorionic Gonadotropin (hCG) and its monoclonal antibodies (mAbs) as a model system.

General Introduction The introduction reviews the literature and highlights important aspects of protein-protein interaction, with special reference to the antigen-antibody system. It describes the nature and types of forces driving complex formation and stabilization. Since hCG is the antigen studied here, its salient structural features are outlined. The concluding section illustrates how monoclonal antibodies have been used in protein structure-function analysis. Although mAbs are versatile probes, their use has been limited by the lack of quantitative methods to assess antigen-antibody binding in vitro.

Chapter Summaries Chapter 1 - Development of a method to study real-time kinetics of ligand-ligate interaction.

Traditionally studied by fluorescence or SPR.

This chapter reports analysis of dissociation data of the hCG-mAb system using nitrocellulose as a solid support and radio-iodinated hCG (I-hCG) as a probe.

Data fit well into a two-step model, with a fraction of bound antigen being non-dissociable (TBP).

Rate constants obtained were comparable to those from liquid-phase reactions.

Chapter 2 - Thermodynamic analysis of hCG-mAb interaction.

Dissociation profiles conformed to a two-step model.

van’t Hoff enthalpies were calculated from equilibrium constants at different temperatures.

Free energy and entropy changes were derived.

For mAb VM7, enthalpy ( H) was favorable above 30 °C, with entropy ( S) favorable at all temperatures.

For mAb VM4a, binding was driven by large favorable entropic contributions, highlighting hydrophobic interactions.

Chapter 3 - Use of BIAcore to study real-time kinetics of hCG-mAb interaction.

Most studies assume simple 1:1 Langmuir binding and complete reversibility.

In high-affinity systems, dissociation is negligible, limiting sensogram analysis.

A method was developed using a two-minute sensogram to measure association rate constants and functional capacity of immobilized hCG.

At higher mAb concentrations, binding conformed to a two-step model, possibly due to surface heterogeneity.

Chapter 4 - Structural insights into CTP- (residues 88-92) of hCG.

This region is important for hormone bioactivity but not resolved in crystal structures.

Monoclonal antibody probes, modified hCG derivatives, epitope mapping, molecular modeling, and dynamics simulations revealed consistent topographies.

Demonstrates how mAbs can probe fine structural features of proteins.

Chapter 5 - Identification of a peptide antagonist of the LH/CG receptor using phage display libraries.

Glycoprotein hormones are heterodimers with disulfide-constrained epitopes.

Inhibitory mAbs recognizing discontinuous epitopes were used to screen peptide libraries.

A 15-mer cyclic peptide emerged as the most potent antagonist, blocking hCG binding and hormone-induced steroidogenesis in vitro.

This strategy has therapeutic potential for designing antagonists of other glycoprotein hormones.

Chapter 6 - Role of electrostatic forces in antigen-antibody interaction.

Rate constants from kinetic analysis and epitope mapping were used to identify electrostatic contributions.

Dependence of forward and backward rate constants on ionic strength indicated long-range repulsive forces in the hCG-mAb system.

High ionic strength overcame these forces, and charged residues in hCG were identified as contributors.

Highlights the importance of non-covalent forces in protein-protein interactions.