A yeast phenotypic screening method for isolation of functional antibodies against g-protein coupled receptors

A functional antibody and antibody technology, applied in library screening, immunoglobulin, chemical instruments and methods, etc., can solve not-so-easy problems

Pending Publication Date: 2020-10-20
抗体私人有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But such mammalian phenotypic screening methods are unlikely to be applicable to GPCRs, because highly efficient screens like blocking cell death, while convenient for growth factor receptors, are unlikely, or at least not so easily, directly Used for most GPCR receptors in mammalian cell systems
[0007] There is currently no efficient method for isolating and identifying functional antibodies (especially activator antibodies) for GPCRs

Method used

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  • A yeast phenotypic screening method for isolation of functional antibodies against g-protein coupled receptors
  • A yeast phenotypic screening method for isolation of functional antibodies against g-protein coupled receptors
  • A yeast phenotypic screening method for isolation of functional antibodies against g-protein coupled receptors

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Experimental program
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example 1

[0127] Brief overview of the platform

[0128] figure 1 The platform shown is typical of autocrine signaling-based phenotypic screens. Antibody libraries are introduced into cells by efficient transformation and are secreted into the periplasmic space between the cell wall and membrane. Most antibodies are retained in the periplasmic space due to their size. When the activator antibody encounters its epitope on the receptor (Ste2p or human GPCR receptor), the antibody will bind to the receptor and activate the receptor, triggering a series of kinase reactions. Activates the Stel2p transcription factor at the end of a cascade of kinase reactions. Activated Stel2p binds to the promoter regions of a panel of α-factor-induced genes and activates their transcription. The promoters of two such genes, FUS2 and FIG1, were selected to drive the expression of the reporter gene CDC26 and antibiotic resistance genes (neomycin resistance gene neoR and bleomycin resistance gene zeoR), t...

example 2

[0130] Construction of Libraries, Vectors and Host Strains

[0131] The scFv library starts with >10 10 A diversity of , which is comparable or better than most phage-displayed antibody libraries, was produced in the universal shuttle vector pRS315. Sequence analysis of randomly selected library clones indicated that more than 80% of the library members contained antibody sequences correctly assembled into the vector. When performing the screening, the library was introduced into the screening cells by PCR of the scFv library and gap repair of the vector pKT103. This process ligated the scFv fragment to the FLO1 secretion signal and placed it under the control of the TEF1 promoter.

[0132] In order to construct strains suitable for this platform, certain modifications must be made to the Ste2p signaling pathway. First, as in other experiments using the yeast α-factor signaling pathway for heterologous GPCR studies, two genes, far1Δ and sst2Δ, were knocked out to preserve c...

example 3

[0135] Ste2p activating antibody

[0136] This platform was first used to find antibodies that target and activate the yeast GPCR receptor Ste2p. Use the scFv library to transform YKT099, spread the transformed cells on the SC-Leu plate (for selecting plasmids), and the SC-Leu plate also contains 20-40 μg / ml or 300-400 μg / ml of G418, respectively placed at 37 Cultivate at 30°C or 30°C. The "high temperature / low drug" selection condition was biased towards testing the expression of CDC26, while the "low temperature / high drug" condition was biased towards testing the expression of neoR. Colonies grown on the plates under each selection scheme were pooled and pooled, with each pool containing around 10 individual colonies. Plasmids were extracted from pooled cells and used again to transform YKT099. This time, transformants were screened using a reverse selection protocol. That is, if the first round of screening used high temperature / low drug selection, the second round of s...

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Abstract

The present invention provides a method for identifying a functional antibody or antigen-binding protein or a fragment thereof that is capable of binding to, and stimulating the activity of, a targettransmembrane protein comprising the steps of providing yeast cells transformed with yeast expression vectors encoding a library of antibodies or antigen-binding proteins or fragments thereof, whereinsaid yeast cells express said target transmembrane protein, expressing said library of antibodies or antigen-binding proteins or fragments thereof in the yeast cells, wherein said expressed antibodies or antigen-binding proteins or fragments thereof are secreted into the periplasmic space of said yeast cells, incubating the yeast cells in or on a selective medium or under a restrictive temperature, or a combination thereof, and detecting a predetermined phenotype in the yeast cells, wherein detection or manifestation of the predetermined phenotype is indicative of binding of the functional antibody or antigen-binding protein or a fragment thereof to said target transmembrane protein and stimulation of said target transmembrane protein by the functional antibody or antigen-binding proteinor a fragment thereof. Antibodies or antibody fragments that are agonists of G-protein coupled receptors identified by the method of the invention are also provided.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention belongs to the field of antibody isolation and identification through yeast phenotypic screening. Background technique [0002] G protein-coupled receptors (GPCRs), also known as seven transmembrane proteins, are a unique family of receptors with similar structures and diverse functions. Members of this large family exist in all eukaryotes from yeast to humans, and all have core structures unique to this family. The core structure consists of seven transmembrane α-helices connected by six protruding interhelical loops, three of which protrude into the extracellular space with the N-terminus, and three interhelical loops with the C-terminus exposed to the cytoplasm . The extracellular and intracellular domains of GPCRs respectively contain the action site of the ligand and the association site of the cell signal transduction system molecules, so that the receptor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B30/04C07K16/28
CPCC07K16/28C07K2317/75C07K2317/14C07K16/14C07K2317/21G01N33/6854G01N33/6845C12Q1/025C07K16/005C12N15/1037
Inventor 蔡名杰
Owner 抗体私人有限公司
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