Thermostable Protein Scaffold for Mono-Specific B-Cell Epitope Presentation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods lack a systematic approach to identify and improve mono-specific B-cell epitopes for eliciting protective antibody responses, as existing techniques fail to accurately synthesize and couple B-cell epitopes onto carrier proteins, leading to suboptimal antibody responses due to antigen competition and downregulation, and are ineffective in neutralizing native proteins or cancer cells.
Innovation Solution
A thermostable protein scaffold, such as GFP, is used to constrain and evolve mono-specific B-cell epitopes, allowing for high-affinity interactions with antibodies through ribosome display and error-prone PCR, ensuring the epitopes maintain native conformation and immunogenicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If linear peptides are synthesized and coupled onto carrier protein for immunization, then antibodies are induced that react with linear peptide or denatured protein, but the antibodies are not cross-reactive with native protein and cannot neutralize toxins or cancer cells
Solution Approach 1:
The patent creates a conformational peptide library that copies the three-dimensional structure of native protein epitopes. Instead of using linear sequences, the invention synthesizes peptides that fold into native-like conformations, allowing antibodies to recognize both the peptide library and native proteins effectively.
Solution Approach 2:
The invention changes the structural parameters of peptide presentation by using a scaffold protein to constrain peptides into specific three-dimensional conformations. This transforms linear, flexible sequences into structured, native-like epitopes that maintain immunogenicity while enabling cross-reactivity with native proteins.
2Measurement precision
If overlapped synthetic peptide sequences are coupled to carrier protein, then mono-specific antibody response can be raised, but antigen competition and downregulation occur due to presence of multiple B-cell epitopes
Solution Approach 1:
The patent extracts the essential B-cell epitope sequences from complex native proteins and presents them in isolation on a scaffold protein. This extraction eliminates competing epitopes that would otherwise cause antigen competition and downregulation, allowing focused antibody responses against specific target epitopes.
Solution Approach 2:
The scaffold protein serves multiple functions: it presents multiple different B-cell epitopes simultaneously, maintains their native-like conformations, and prevents aggregation. This universal platform can be applied to various target proteins by simply changing the inserted peptide sequences.
3Shape
If foreign B-cell antigenic loops are grafted in CDR regions of immunoglobulin scaffold, then constrained antigenic conformations similar to native loop are achieved, but solubility is affected and conformational stability is reduced
Solution Approach 1:
The patent uses a disposable, highly stable scaffold protein (such as GFP or other thermostable proteins) that can tolerate the insertion of foreign antigenic sequences without losing its structural integrity. The scaffold acts as a sacrificial stable framework that protects the inserted epitopes while maintaining overall protein stability and solubility.
Data Source
AI summary
A method is disclosed wherein an antigenic B-cell epitope is discovered by in vitro transcription and translation of pre-determined sequence on thermostable protein scaffolds detected by antibodies to a native protein. Immune reactivities of IgE B-cell epitopes from pre-determined IgE sequences from the constant region, engaged in binding to high affinity IgE Fc receptors, placed in a loop of green fluorescent protein (GFP) were shown immuno-reactive with anti-IgE. Moreover, the antigenic B-cell epitope can be further optimized by molecular evolution, selected by conformer antibody and receptor on solid phase via ribosome display. Alternatively, a random aptameric antigenic sequence inserted into a loop of the protein scaffold, mimicking a pre-determined sequence of a native protein, can be selected by solid phase conformer antibody and receptor via ribosome display. High affinity binding of B-cell epitopes selected from the random aptameric library with antibodies to native pre-determined B-cell epitope of a native protein can be optimized by molecular evolution with error-prone RT-PCR by ribosome display.