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Methods of identifying and validating affinity reagents

A specific, cell-based technology, applied in the field of affinity reagents, can solve the problems of high cost and low throughput of display technology

Inactive Publication Date: 2018-03-27
AXIOMX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current display techniques are costly and low-throughput

Method used

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  • Methods of identifying and validating affinity reagents
  • Methods of identifying and validating affinity reagents
  • Methods of identifying and validating affinity reagents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1. Materials and methods

[0108] Bacterial Strains and Vectors

[0109] Escherichia coli ( E. coli ) strain TG1[F'( tra D36, pro AB+ lac I q , lac ZΔM15), sup E, thi -1, Δ( lac-pro AB), Δ( mcr B- hsd SM) 5, (rK - mK - )] were purchased from Lucigen (Middleton, WI) and used to encode Eco The pAX1492 plasmid of the 29kI RM operon was transformed into TG1 to develop E. coli ( E. coli ) strain AXE688. Escherichia coli ( E. coli ) strain CJ236 (FΔ (Hin DIII )::cat( Tra + , Pil + ,Cam R ) / ung-1, relA1, dut-1, thi- 1, spoT1, mcrA ) were purchased from New England BioLabs (NEB; Waverly, MA). Electrocompetent and chemically competent strains will be prepared according to the NEB protocol. The template plasmid used for AXM mutagenesis was a derivative of the phagemid pCH103, and the same scFv template was genetically fused to the coat protein gp3 of phage M13. Each of the six CDRs of this template scFv will be modified to include...

Embodiment 2

[0124] Example 2. Overview of antibody framework and library design

[0125] We have identified Ab libraries based on constant framework scFvs that encode the NNK codon (see e.g. Benhar, Expert Opin Biol Ther. 7(5):763-79, 2007; Chan et al., Int Immunol. 26(12): 649-657, 2014; Miersch et al., Methods.57(4):486-98, 2012; Mondon et al., Front Biosci.13:1117-29, 2008; Tohidkia et al., J Drug Target.20(3) :195-208, 2012), with coding positions at 18 different positions within the six complementarity-determining regions (CDRs) (Mandrup et al., PLoS One. 8(10):e76834, 2013). The B2A framework of the library identified and used in these experiments was based on its ability to function in phage display (ɸD) (when fused to the M13 gp3 protein) and yeast two-hybrid (Y2H) (when fused to the activation domain) Make a selection. It is worth noting that although the platform of the present invention for generating rAbs is currently used for single chain variable fragments (scFv), this pla...

Embodiment 3

[0131] Example 3.pCH103 cloning vector system can eliminate the pair used in ɸD, yeast two-hybrid, and Escherichia coli (E. coli) The need for subcloned proteins tested in protein expression

[0132] Techniques for Y2H and ɸD can be adapted to develop selective and scalable systems for obtaining, characterizing and affinity-matured antibodies in a very high-throughput and cost-effective manner. A dual-function vector system (pCH103) has been constructed, which has both Y2H and Escherichia coli ( E. coli ) the ability to function in the ɸD approach ( figure 2 ). in Escherichia coli ( E. coli ), protein expression depends on E. coli ( E. coli )genotype. The main features of the pCH103 vector system and its beneficial effects are described below.

[0133] pCH103 vector design

[0134] like figure 2 As shown in A, an amber stop codon was inserted between the protein of interest (scFv in this case) and gp3. Therefore, in non-inhibitory E. coli ( E. coli ) middle , ...

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Abstract

The invention features methods of identifying and validating affinity reagents, such as antibodies. The methods of the invention generally involve screening an antibody library by, for example, phagedisplay on bacteria (e.g., E. coli) to identify particular antibody clones capable of binding a desired target polypeptide. Clones identified in this way can then be validated using yeast 2-hybrid. Insome instances, antibodies identified by their capacity to binding a partial antigen can be validated by their capacity to bind to the full-length antigen. Validated clones can be further screened byadditional rounds of phage display and / or yeast 2-hybrid. Between each round, additional variants of particular antibody clones can be generated and screened to identify variants that demonstrate higher binding affinity to the target of interest.

Description

[0001] sequence listing [0002] This patent application contains a Sequence Listing, which has been filed electronically in ASCII format, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on February 12, 2016, is named 50881_011WO2_Sequence_Listing_2_12_16_ST25.txt and is 5150 bytes in size. technical field [0003] The present invention relates to identifying and validating affinity reagents capable of binding a target polypeptide. Background of the invention [0004] The most common method used to produce antibodies is by immunization of animals. However, this approach is generally low-throughput, expensive, time-consuming, and the antibodies produced are not always reproducible. Furthermore, there are no intermediate validation steps to ensure that such antibodies isolated against haptens will effectively bind the full-length antigen. An alternative approach is the production of recombinant antibodies (rAbs), which is currently accompli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C07K16/00G01N33/68
CPCC12N15/1086G01N33/6854C07K16/00C07K2317/567C07K2317/622C12N15/1037C12N15/1055
Inventor M.维纳V.巴西吉纳
Owner AXIOMX
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