Dual-Host Expression and Secretion for Fab-to-IgG Discovery
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Solution Overview
Problem
The laborious process of subcloning and reformatting Fab fragments for phage display and subsequent IgG expression in phage-based antibody discovery is inefficient, with a significant failure rate in reformatted Fabs, increasing the number of clones required and reducing success probability.
Innovation Solution
An expression and secretion system using mammalian signal sequences, such as mBiP, to drive expression of Fab-phage fusion in E. coli and full-length IgG in mammalian cells, bypassing the need for subcloning by employing mRNA splicing to insert a synthetic intron in the human IgG1 hinge region.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If subcloning and reformatting Fab fragments into full-length IgG is performed, then the Fab fragments can be expressed in mammalian cells, but the process becomes laborious and time-consuming
Solution Approach 1:
The patent incorporates a mammalian signal sequence and intron structure directly into the phage display vector before Fab fragment selection. This preliminary preparation of eukaryotic expression elements allows that after Fab selection, the same vector can be directly transfected into mammalian cells without requiring subsequent subcloning operations, thereby eliminating the time-consuming subcloning step while maintaining IgG expression capability
Solution Approach 2:
The patent creates a universal vector system that serves dual purposes: it functions as a phage display vector for Fab fragment selection in prokaryotes, and simultaneously as a mammalian expression vector for IgG production after splicing. By integrating both prokaryotic and eukaryotic expression capabilities into a single vector platform, the system eliminates the need for separate subcloning operations into different vector types, thus reducing time loss
2Reliability
If multiple clones are carried through subcloning and screening process, then the probability of finding functional Fabs increases, but the labor and time required increase proportionally
Solution Approach 1:
By pre-installing the mammalian signal sequence and splicing machinery components into the phage display vector, the system enables direct expression of functional IgG in mammalian cells after Fab selection. This eliminates the bottleneck of subcloning dozens or hundreds of selected clones, allowing high-throughput screening while maintaining the reliability of finding functional Fabs through extensive library screening
Solution Approach 2:
The patent replaces the mechanical subcloning process with a molecular splicing-based expression system. Instead of physically isolating and re-cloning Fab fragments into separate IgG expression vectors, the system uses RNA splicing to convert the Fab-phage fusion protein into full-length IgG in mammalian cells, thereby maintaining high screening throughput while eliminating labor-intensive subcloning operations
3Reliability
If Fab fragments are reformatted into full-length IgG, then they can be evaluated in functional assays, but a substantial percentage fail to perform satisfactorily
Solution Approach 1:
The patent incorporates a mammalian signal sequence and properly structured intron elements directly into the phage display vector before Fab fragment selection. This preliminary setup ensures that when Fabs are selected and the vector is transfected into mammalian cells, the splicing machinery correctly processes the mRNA to produce properly folded and functional full-length IgG, thereby improving the correctness of the reformatting process and reducing the failure rate of functional assays
4Device complexity
If a single nucleic acid sequence is used for both phage display and IgG expression, then subcloning is eliminated, but the system must function in both prokaryotic and eukaryotic cells
Solution Approach 1:
The patent creates a universal vector system that contains both prokaryotic and eukaryotic expression elements, allowing a single nucleic acid sequence to function in both E. coli for phage display and mammalian cells for IgG expression. The vector includes a mammalian signal sequence, intron structure, and promoter elements that enable dual-host compatibility, thereby reducing device complexity while achieving broad adaptability
Solution Approach 2:
The patent employs local quality by having different functional regions within the same vector sequence perform different functions in different host cells. The prokaryotic promoter and ribosome binding sites enable expression in E. coli, while the mammalian signal sequence and intron structure enable proper processing and secretion in mammalian cells. This localized functional differentiation allows a single vector to adapt to different host cell types without increasing overall complexity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Facilitates the selection and purification of Fab fragments with antigen binding capability in both prokaryotic and eukaryotic cells without subcloning, enhancing the efficiency and success rate of antibody discovery.
Implementation Method 1
Using mammalian mRNA splicing to remove a synthetic intron containing a phage fusion peptide inserted within the hinge region of the human IgG1 HC, we are able to generate two distinct proteins in a host cell-dependent fashion
Implementation Method 2
Using mammalian mRNA splicing to remove a synthetic intron containing a phage fusion peptide inserted within the hinge region of the human IgG1 HC
Data Source
AI summary
The invention provides an expression and secretion system, and methods of using the same, for the expression and secretion of one fusion protein in prokaryotic cells and a second fusion protein in eukaryotic cells. Also provided herein are nucleic acid molecules, vectors and host cells comprising such vectors and nucleic acid molecules.