Non-covalent Yeast Antibody Display and Secretion

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Solution Overview

Problem

Current methods for antibody production and screening, such as phage display, face limitations in producing high-affinity eukaryotic antibodies and post-translational modifications like glycosylation, and existing yeast surface display technologies have practical disadvantages including covalent binding and complex isolation processes.

Innovation Solution

A non-covalent surface display method using yeast S. cerevisiae cells with an Fc binding domain from Staphylococcus aureus protein A, allowing for the display and secretion of antibodies and fragments, enabling high-throughput screening and biochemical characterization without the need for subcloning or reformatting steps.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If phage display is used for antibody production and screening, then antibody libraries can be generated and screened, but the method cannot produce high-affinity eukaryotic antibodies with post-translational modifications like glycosylation

Engineering Contradiction:

Improveantibody affinity and functionalityVSAvoidpost-translational modification capability

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The patent transitions from a prokaryotic expression system (phage display in E. coli) to a eukaryotic expression system (yeast surface display in S. cerevisiae). This parameter change in the host organism enables post-translational modifications such as glycosylation while maintaining high-affinity antibody production and screening capabilities.

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If yeast surface display is used for antibody display, then eukaryotic proteins with post-translational modifications can be produced, but covalent binding and complex isolation processes are required

Engineering Contradiction:

Improvepost-translational modification capabilityVSAvoidisolation process complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent extracts the antibody from the complex covalent binding system by using a non-covalent interaction system instead. The antibody is displayed on yeast surface through non-covalent binding to the ZZ domain, and can be easily released into the supernatant by simple centrifugation, eliminating complex isolation processes.

Inventive Principle:

Principle #2Taking out (Extraction)

3Stability of the object's composition

If stable genotype-phenotype coupling is achieved by covalent fusion of antibody with cell wall protein, then surface display is stable, but the antibody cannot be easily secreted for biochemical characterization

Engineering Contradiction:

Improvesurface display stabilityVSAvoidsecretion ease

Core Design Contradiction:

Stability of the object's compositionVSEase of manufacture

Solution Approach 1:

The patent introduces a dynamic, switchable system where the antibody can transition between surface-displayed and secreted states. By controlling the expression of the ZZ domain anchor protein, the system allows stable surface display when ZZ is expressed and easy secretion when ZZ expression is repressed, providing flexibility for both stability and biochemical characterization needs.

Inventive Principle:

Principle #15Dynamics

4Productivity

If high-throughput screening is performed to select specific variants, then desired antibodies can be identified, but additional subcloning and reformatting steps are traditionally required for production

Engineering Contradiction:

Improvescreening efficiencyVSAvoidsubcloning and reformatting time

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The patent merges the screening function with the production function in a single yeast host system. The same yeast cells used for high-throughput screening via surface display can directly produce and secrete the selected antibodies into the supernatant, eliminating the need for separate subcloning and reformatting steps required in traditional phage display workflows.

Inventive Principle:

Principle #5Merging (Combining)

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

This method simplifies the production and selection of antibodies by ensuring correct folding, secretion, and stability, allowing for the direct use of selected clones for protein production and enabling the display of diverse IgG molecules and fragments with desired properties.

Implementation Method 1

The non-covalent manner of the surface display renders possible the selection of specific variants

Methodology Applied

Scientific EffectNon-covalent binding: Absorption (physical)

Implementation Method 2

expressing the fusion proteins with simultaneous co-expression of the diversity of antibodies, antibody fragments or antibody domains in the yeast cells in the presence of polyethylene glycol (PEG) having a molecular weight of >5,000 in the cultivation medium, wherein said IgG molecules and also the fusion protein are secreted from the yeast cell in soluble form

Methodology Applied

Scientific EffectPolyethylene glycol effect:

Data Source

PatentUS10138477B2Method of producing secretable antibodies by expression in saccharomyces cerevisiae

Publication Date: 2018.11.27 MERCK PATENT GMBH

AI summary

The invention relates to a method for the production and non-covalent surface display of antibodies and derived fragments as well as molecule libraries based thereon on the surface of S. cerevisiae cells. The non-covalent manner of the surface display renders possible the selection of specific variants by means of high throughput screening and the subsequent switchable secretion of the selected binding molecule into the culture supernatant for biochemical characterization.