Linear Polynucleotide for Simultaneous Antibody Chain Expression
Solution Overview
Problem
Current methods for producing recombinant multimeric proteins, such as antibodies, are complex and time-consuming, requiring multiple digestion and ligation steps and often result in inconsistent expression levels due to the need for simultaneous transfection of multiple vectors, which complicates the production of correctly folded and assembled proteins.
Innovation Solution
A transcriptionally active recombinant linear polynucleotide is developed, comprising a first promoter sequence linked to an encoding sequence for one or more antibody domains, a bidirectional transcription regulatory sequence, and a second promoter sequence in convergent orientation, allowing for efficient expression and assembly of antibody chains within a mammalian cell without the need for multiple vector transfections.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If multiple vectors are used to encode antibody chains, then complete antibody production is achieved, but the process complexity and time increase significantly
Solution Approach 1:
The patent combines multiple antibody chain encoding sequences (heavy chain and light chain variable regions) into a single polynucleotide sequence, eliminating the need for multiple separate vectors. This single polynucleotide contains all necessary coding sequences arranged in a specific order that enables simultaneous expression of both chains from one transcription unit, thereby simplifying the manufacturing process while reducing vector construction complexity.
Solution Approach 2:
The single polynucleotide sequence serves multiple functions: it encodes both heavy and light chain variable regions, contains necessary regulatory elements for transcription, and enables co-expression of multiple antibody chains. This multi-functional design replaces the need for multiple specialized vectors, each designed for specific chains, thereby reducing overall system complexity.
2Productivity
If multiple vectors are transfected simultaneously, then all antibody chains are expressed, but expression consistency deteriorates
Solution Approach 1:
By merging the encoding sequences for heavy and light chains into a single polynucleotide that is transfected as one unit, the patent ensures that both chains are introduced into the host cell simultaneously in stoichiometric proportions. This eliminates the variability inherent in simultaneous transfection of multiple separate vectors, where unequal uptake and expression of each vector leads to inconsistent antibody production.
Solution Approach 2:
The single polynucleotide sequence is designed with internal elements that enable it to self-regulate expression. The specific arrangement of coding sequences and regulatory elements within the single polynucleotide allows the cell's transcription and translation machinery to naturally produce both antibody chains in appropriate proportions, ensuring consistent expression without requiring external balancing of multiple vectors.
3Ease of manufacture
If traditional cloning methods are used, then vectors are constructed, but the process requires multiple digestion and ligation steps
Solution Approach 1:
The patent extracts the essential functional elements (coding sequences for antibody chains and necessary regulatory elements) and combines them directly into a single polynucleotide sequence without requiring traditional vector backbone structures. This extraction of core functional elements eliminates the need for time-consuming digestion and ligation steps used in traditional cloning to assemble multiple fragments into vector constructs.
Solution Approach 2:
The polynucleotide sequence is designed in advance with all necessary coding sequences and regulatory elements pre-arranged in the correct order and orientation. This preliminary design and assembly of the complete coding sequence eliminates the need for subsequent cloning steps (digestion and ligation) that would otherwise be required to construct the expression vectors, thereby saving significant time.
Data Source
Figure 1
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Figure 3a~3b
AI summary
The invention relates to a transcriptionally active recombinant linear polynucleotide encoding a multimeric protein comprising in the following order, a first promoter sequence, a first encoding polynucleotide sequence, a bidirectional regulatory sequence, a second encoding polynucleotide sequence and a second promoter sequence, wherein the first and second encoding polynucleotide sequences are in convergent transcriptional orientation, each encoding polynucleotide sequence encodes a component of the multimeric protein and the bidirectional regulatory sequence is operably linked to the first and second encoding polynucleotide sequences and the multimeric protein is an antibody or fragment thereof and each encoding polynucleotide sequence encodes one or more antibody domains or fragments thereof.