Binding polypeptides with restricted diversity sequences

Inactive Publication Date: 2005-05-19
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013] The present invention provides simplified and flexible methods of generating polypeptides comprising variant CDRs that comprise sequences with restricted diversity yet retain target antigen binding capability. Unlike conventional methods that are based on the proposition that adequate diversity of target binders can be generated only if a particular CDR(s), or all CDRs are diversified, and unlike conventional notions that adequate diversity is dependent upon the broadest range of amino acid substitutions (generally by substitution using all or most of the 20 amino acids), the invention provides methods capable of generating high quality target binders that are not necessarily dependent upon diversifying a particular CDR(s) or a particular number of CDRs of a reference polypeptide or source antibody. The invention is based, at least in part, on the surprising and unexpected finding that highly diverse libraries of high quality comprising functional polypept

Problems solved by technology

However, many of these libraries have limited diversity.
The size of the library is decreased by inefficiency of production due to improper folding of the antibody or antigen binding protein and the presence of stop codons.
However, these attempts have had varying success and have not been applied in a systematic and quantitative manner.
Creatin

Method used

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  • Binding polypeptides with restricted diversity sequences
  • Binding polypeptides with restricted diversity sequences
  • Binding polypeptides with restricted diversity sequences

Examples

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Effect test

example 1

Construction of Phage-Displayed Fab Libraries with CDR Residues Randomized as Only Tyr or Ser

[0484] Phage-displayed Fab libraries were constructed using a phagemid vector that resulted in the display of bivalent Fab moieties dimerized by a leucine zipper domain inserted between the Fab heavy chain and the C-terminal domain of the gene-3 minor coat protein (P3C). This vector comprises the sequence shown in FIG. 18 (SEQ ID NO:4). The vector (schematically illustrated in FIG. 19) comprises the humanized antibody 4D5 variable domains under the control of the IPTG-inducible Ptac promoter. The humanized antibody 4D5 is an antibody which has mostly human consensus sequence framework regions in the heavy and light chains, and CDR regions from a mouse monoclonal antibody specific for Her-2. The method of making the anti-Her-2 antibody and the identity of the variable domain sequences are provided in U.S. Pat. Nos. 5,821,337 and 6,054,297.

[0485] Two libraries were constructed. Library YS-A ...

example 2

Selection of Specific Antibodies from the Naïve Libraries YS-A and YS-B

[0489] Phage from library YS-A or YS-B (Example I) were cycled through rounds of binding selection to enrich for clones binding to targets of interest. Eight target proteins were analyzed separately with each library: human VEGF, murine VEGF, neutravidin, an apoptosis protein (AP), maltose binding protein, erbin-GST fusion, and Insulin. The binding selections were conducted using previously described methods (Sidhu et al., supra).

[0490] NUNC 96-well Maxisorp immunoplates were coated overnight at 4° C. with capture target (5 μg / mL) and blocked for 2 h with Superblock TBS (tris-buffered saline) (Pierce). After overnight growth at 37° C., phage were concentrated by precipitation with PEG / NaCl and resuspended in Superblock TBS, 0.05% Tween 20 (Sigma), as described previously (Sidhu et al., supra). Phage solutions (˜1012 phage / mL) were added to the coated immunoplates. Following a 2 h incubation to allow for phage b...

example 3

Construction of a Phage-Displayed Fab Library (F0505) with CDR Residues Randomized with Tetranomial Codons Encoding Four Amino Acids

[0496] Phage displayed libraries were constructed, as described in Example 1, with a previously described phagemid designed to display bivalent Fab moieties dimerized by a leucine zipper domain inserted between the Fab heavy chain and the C-terminal domain of the gene-3 minor coat protein (P3C) (as described in Example 1). CDR positions in the heavy chain were randomized, positions as shown in FIG. 1. Eleven separate mutagenesis reactions were performed with each mutagenesis reaction designed to randomize the CDR positions with a tetranomial codon that encoded for only four amino acids. In each mutagenesis reaction, the CDR positions were simultaneously replaced with only one type of tetranomial codon. The eleven tetranomial codons used for the eleven mutagenesis reactions and the amino acids they encode are shown in FIG. 6. For each mutagenesis, three...

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Abstract

The invention provides variant CDRs comprising highly restricted amino acid sequence diversity. These polypeptides provide a flexible and simple source of sequence diversity that can be used as a source for identifying novel antigen binding polypeptides. The invention also provides these polypeptides as fusion polypeptides to heterologous polypeptides such as at least a portion of phage or viral coat proteins, tags and linkers. Libraries comprising a plurality of these polypeptides are also provided. In addition, methods of and compositions for generating and using these polypeptides and libraries are provided.

Description

RELATED APPLICATIONS [0001] This application is a non-provisional application filed under 37 CFR 1.53(b)(1), claiming priority benefit under 35 USC 119(e) to provisional application No. 60 / 491,877 filed Aug. 1, 2003, the contents of which are incorporated herein in its entirety by reference.FIELD OF THE INVENTION [0002] The invention generally relates to variant CDRs diversified using highly limited amino acid repertoires, and libraries comprising a plurality of such sequences. The invention also relates to fusion polypeptides comprising these variant CDRs. The invention also relates to methods and compositions useful for identifying novel binding polypeptides that can be used therapeutically or as reagents. BACKGROUND [0003] Phage display technology has provided a powerful tool for generating and selecting novel proteins that bind to a ligand, such as an antigen. Using the techniques of phage display allows the generation of large libraries of protein variants that can be rapidly s...

Claims

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Application Information

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IPC IPC(8): C07H21/04C07K14/705C07K16/00C07K16/22C07K16/32C12N15/62C12P21/04
CPCA61K2039/505C07K16/005C07K16/22C07K16/32C07K2316/96G01N33/6845C07K2317/55C07K2317/56C07K2317/565C07K2317/73C07K2317/92C07K2317/31C07K2317/76A61P1/04A61P1/16A61P1/18A61P11/00A61P13/08A61P15/00A61P17/00A61P17/02A61P17/06A61P19/02A61P25/00A61P27/00A61P27/02A61P29/00A61P31/04A61P35/00A61P35/02A61P37/02A61P37/06A61P43/00A61P9/10A61K39/395C07K2317/24
Inventor FELLOUSE, FREDERICSIDHU, SACHDEV
Owner GENENTECH INC
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