Method to screen phage display libraries with different ligands

a technology of phages and libraries, applied in chemical libraries, animal/human proteins, combinational chemistry, etc., can solve the problems of antibody selection from these libraries may be poorly expressed, antibody folding cannot be correctly folded, and use consensus frameworks

Inactive Publication Date: 2004-02-26
DORMANTIS LTD
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Benefits of technology

[0015]In a third aspect, the invention provides a method for detecting, immobilising, purifying or immunoprecipitating one or more membe

Problems solved by technology

Although synthetic libraries help to overcome the inherent biases of the natural repertoire which can limit the effective size of phage libraries constructed from rearranged V genes, they require the use of long degenerate PCR primers which frequently introduce base-pair deletions into the assembled V genes.
This high degree of randomisation may also lead to the creation of antibodies which are unable to fold correctly and are also therefore non-functional.
Furthermore, antibodies selected from these libraries may be poorly expressed and, in many cases, will contain framework mutations that may effect the antibodies immunogenicity when used in human therapy.
Since it is desirable t

Method used

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  • Method to screen phage display libraries with different ligands
  • Method to screen phage display libraries with different ligands
  • Method to screen phage display libraries with different ligands

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example 2

[0143] [0144]Example 2 Library construction and selection with the generic ligandsThe "primary" and "somatic" libraries were assembled by PCR using the oligonucleotides listed in Table 1 and the germline V gene segments DPK9 (Cox et al. (1994) Eur. J. Immunol., 24: 827) and DP-47 (Tomlinson et al. (1992) J. Mol. Biol., 227: 7768). Briefly, first round of amplification was performed using pairs of 5' (back) primers in conjunction with NNK or DVT 3' (forward) primers together with the corresponding germline V gene segment as template (see Table 1). This produces eight separate DNA fragments for each of the NNK and DVT libraries. A second round of amplification was then performed using the 5' (back) primers and the 3' (forward) primers shown in Table 1 together with two of the purified fragments from the first round of amplification. This produces four separate fragments for each of the NNK and DVT libraries (a "primary" VH fragment, 5A; a "primary" Vκ fragment, 6A; a "somatic" VH frag...

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Abstract

Abstract of the Disclosure The present invention relates to methods for selecting repertoires of polypeptides using generic and target ligands. In particular, the invention relates to a library comprising a repertoire of polypeptides of the immunoglobulin superfamily, wherein the members of the repertoire have a known main chain conformation.

Description

Detailed Description of the InventionIntroduction[0001] The present invention relates to methods for selecting repertoires of polypeptides using generic and target ligands. In particular, the invention describes a method for selecting repertoires of antibody polypeptides with generic ligand to isolate functional subsets thereof.[0002] [0002]The antigen binding domain of an antibody comprises two separate regions: a heavy chain variable domain (VH) and a light chain variable domain (VL: which can be either Vκ or Vλ). The antigen binding site itself is formed by six polypeptide loops: three from VH domain (H1, H2 and H3) and three from VL domain (L1, L2 and L3). A diverse primary repertoire of V genes that encode the VH and VL domains is produced by the combinatorial rearrangement of gene segments. The VH gene is produced by the recombination of three gene segments, VH, D and JH. In humans, there are approximately 51 functional VH segments (Cook and Tomlinson (1995) Immunol Today, 16:...

Claims

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Application Information

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IPC IPC(8): G01N33/50C07K1/04C07K14/705C07K14/725C07K16/00C12N15/09C12N15/10C12P21/08C40B30/04C40B40/02G01N33/15G01N33/68
CPCC07K1/047C07K14/705C07K14/70503C07K14/7051C07K16/00C07K16/005G01N33/6854C07K2317/622C12N15/1037C40B30/04C40B40/02G01N33/6845C07K2317/21
Inventor TOMLINSON, IANWINTER, GREG
Owner DORMANTIS LTD
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