Study of Novel IgG-like and Fragment-based Bispecific Antibodies

Introduction

Bispecific antibodies (bsAbs) represent a promising class of therapeutic molecules capable of simultaneously binding to multiple antigens, distinguishing them from conventional monoclonal antibodies (mAbs) with single antigen-binding paratopes. In scenarios involving tumors with multiple antigen epitopes, combination therapies using different mAbs can be both cost-prohibitive and potentially induce autoimmune responses. To address these challenges, bsAbs have emerged as a solution by enabling the simultaneous targeting of multiple antigens. This is especially valuable since many tumors rely on multiple survival and growth pathways.

Some bsAbs can also redirect effector T-cells or natural killer cells to target tumor cells, bypassing the multi histocompatibility complex (MHC)-related immune responses. Notable examples of bsAbs approved for medicinal purposes include Catumaxomab and Blinatumomab.

BsAbs come in two primary forms: full-length IgG-like asymmetric antibodies and single-chain variable fragment-based antibodies (scFv). Given their high variability, the quality, quantity, and stability of these novel antibodies are critical factors during production.

Objective

The present study focuses on utilizing biophysical techniques to analyze the stability, aggregation, conformation, and overall functionality of bsAbs, with the aim of improving their purification efficiency and therapeutic potency.

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Materials and Methods

The study encompasses various types of bsAbs, each characterized by distinct structures:

All these bsAb types possess unique biophysical properties that warrant further investigation to enhance their functionality and production efficiency.

Experimental Methods

1. Surface Plasmon Resonance (SPR) Experiments: SPR experiments were conducted to determine the forward and backward reaction rates for the binding between the CD3 polypeptide and bsAbs. SPR is advantageous due to its minimal reactant requirement and high accuracy. However, it demands costly equipment and sensors, which may lead to immobilization traces and mass transfer limitations.
2. Isothermal Titration Calorimetry (ITC) Experiments: ITC experiments were employed to compare the binding affinity of bsAbs to the CD3 polypeptide complex (the ligand). ITC allows the calculation of numerous thermodynamic parameters without requiring ligand immobilization. However, it necessitates a larger sample volume for analysis.
3. Small Angle X-ray Scattering (SAXS): SAXS is a technique utilized to measure protein structures in solution. Its primary advantage lies in its ease of implementation and the ability to cover the total protein surface area. Challenges include numerical integrations and optimization.
4. Differential Scanning Calorimetry (DSC): DSC was used to assess the stability of bsAbs by analyzing transition or melting temperatures. Its strengths include high resolution and sensitivity, but it may exhibit some accuracy and precision limitations, as well as protein loss due to unfolding during analysis.
5. Sedimentation Velocity - Analytical Ultracentrifugation (SV - AUC): SV - AUC experiments provide a rapid means of analyzing protein geometrical properties such as shape, size, and structure. This technique is applicable across a wide range of protein density distributions and buffer conditions.
6. Size Exclusion Chromatography (SEC): SEC involves the elution of protein samples based on size without altering their biological properties. However, its resolution may be unsatisfactory when differences in molecular mass between components are minimal.

Experiments

Binding Affinity of bsAbs to CD3 Polypeptide

ITC experiments were conducted to assess the binding affinity of various bsAbs with the CD3 polypeptide complex. Figure 3 presents the Dissociation constant (Kd) values for each interaction:

All ITC results were cross-verified with SPR experiments, demonstrating good agreement between the two methods (Figure 3).

Aggregation and Oligomerization Studies

Sedimentation velocity experiments (SV-AUC) were employed to investigate the size and aggregation tendencies of bsAbs (Figure 5). The sedimentation coefficients and observed oligomers are summarized below:

Smaller-sized peaks in the SV-AUC profiles correspond to oligomers in bsAb solutions. Notably, mAb, DVD-Ig, and DART exhibit minimal aggregation, while BiTE, tandAb, and nanobodies show a propensity for aggregation. Additionally, SEC results corroborate the presence of dimers in BiTE, tandAb, and nanobody solutions, consistent with SV-AUC findings.

Stability of bsAbs

Differential Scanning Calorimetry (DSC) was employed to assess the stability of bsAb folded structures (Figure 6). The melting temperatures (Tm) for different bsAbs are summarized below:

Analysis of Kratky plots (Figure 4) revealed that DVD-Ig, tandAb, and DART exhibit bell-shaped curves, indicating a higher degree of structural compactness and reduced flexibility. In contrast, nanobodies and BiTEs display more flexible structures. This aligns with the general consensus that DARTs possess greater stability compared to BiTEs.

Conclusion

CD3 receptors play a pivotal role in activating cytotoxic CD4+ and helper CD8+ T cells to eliminate tumor cells. Anti-CD3 monoclonal antibodies (mAbs) bind to these receptors via their variable regions (Vl and Vh). In contrast, bispecific antibodies (bsAbs) represent an innovative class of antibodies capable of not only binding to tumor cells but also stimulating T cell functions by targeting CD3 receptors. The strength of binding is a crucial factor when designing new drugs, as higher binding affinity results in prolonged half-life and reduced dosing frequency for patients.

In this study, we employed both Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) experiments to assess the binding affinities between bsAbs and the CD3 polypeptide. TandAb exhibited the highest affinity, attributed to its compact structure and dual binding sites, while crossmAb, IgG mAb, and DVD-Ig showed similar affinities due to their structural similarities.

Additionally, we investigated the stability of bsAbs using Small Angle X-ray Scattering (SAXS) and Differential Scanning Calorimetry (DSC). TandAb, mAb, and crossmAb displayed globular, compact structures, while DVD-Ig, BiTE, and nanobodies exhibited flexible, less compact folding. Melting temperature analysis indicated that most bsAbs had melting temperatures in the range of 68-70°C, except for BiTE with a lower Tm of approximately 57°C.

Furthermore, we considered the size of bsAbs, recognizing its importance for tissue and cell penetration, ultimately affecting dosage requirements. Analytical Ultracentrifugation-Sedimentation Velocity (AUC-SV) experiments revealed that DVD-Ig had the largest size, while nanobodies were the smallest. TandAb and BiTE showed evidence of oligomerization in solution, with tandAb having a 5% dimerization rate and BiTE exhibiting 17% dimerization.

In conclusion, this research has provided insights into the biophysical characteristics of bsAbs, which are crucial for their purification processes and their interactions with T-cell receptors. Understanding these properties contributes to the development of more effective and efficient therapeutic antibodies.

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