Fibronectin type iii domain-based multimeric scaffolds

a multimeric scaffold and fibronectin technology, applied in the field of antibody mimetics, can solve the problems of complex structure of heterotetrameric molecules, difficult to express most antibodies, and expensive mammalian cell expression systems

Inactive Publication Date: 2013-03-28
MEDIMMUNE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]In some embodiments, binding affinity for the target and protein stability are improved in the multimeric scaffold over those of a cognate FnIII monomer scaffold. In other embodiments, the binding avidity for the target and the protein stability of a multimeric scaffold are improved over those of a cognate FnIII monomer scaffold.
[0040]In some embodiments, the increased protein stability of at least one FnIII monomer scaffold is measured by differential scanning calorimetry (DSC), circular dichroism (CD), polyacrylamide gel electrophoresis (PAGE), protease resistance, isothermal calorimetry (ITC), nuclear magnetic resonance (NMR), urea denaturation, or guanidine denaturation.

Problems solved by technology

However, classical antibodies are structurally complex heterotetrameric molecules with are difficult to express in simple eukaryotic systems.
As a result, most antibodies are produced using complex and expensive mammalian cell expression systems.
However, this often causes aggregation when amino acid residues which would normally be buried in a hydrophobic environment such as an interface between variable and constant domain become exposed to the solvent.

Method used

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  • Fibronectin type iii domain-based multimeric scaffolds
  • Fibronectin type iii domain-based multimeric scaffolds
  • Fibronectin type iii domain-based multimeric scaffolds

Examples

Experimental program
Comparison scheme
Effect test

example 1

Design of Various Multivalent Tn3 Formats

[0671]Multivalent formats of the Tn3 scaffold have been designed. The multivalent formats contain one or more Tn3 modules fused to themselves, fused to other protein motifs that can oligomerize, or fused to themselves and to other protein motifs that can oligomerize are shown in FIG. 1. In each case, the resulting molecular entity contains at least 2 Tn3 modules. The polypeptide linkers connecting the Tn3 modules to each other or to other protein motifs can be structured or unstructured and with or without a function. Three exemplary classes of multivalent Tn3 scaffold proteins are specifically provided: (i) linear (L) multivalent proteins containing Tn3 modules fused to each other via a polypeptide linker; (ii) antibody-like (Ig) multivalent proteins containing one or more linearly fused Tn3 modules fused to the light and heavy chains of an antibody or antibody fragment and (iii) Fc-containing multivalent proteins containing one or more line...

example 2

Expression and Purification of Multivalent TRAIL R2-specific Tn3-containing Proteins

[0672]A series of eight multivalent Tn3-module containing scaffold proteins (also referred to as “Tn3 proteins” or “Tn3 scaffolds”) with binding specificity for human TRAIL R2 were prepared. Examples were prepared from each of the three multivalent formats described in Example 1, and all of these proteins presented 2 or more of the TRAIL R2-binding Tn3 module A1 (clone 1E11, G6 or 1C12). For several TRAIL R2-specific multivalent Tn3 protein, a corresponding control Tn3 protein (clone DE a Tn3 domain specific for the Synagis® antibody) that did not bind TRAIL R2 was also generated, differing only in the sequence and binding specificity of the component Tn3 modules. Tn3 clone D1 is a Tn3 protein wherein the BC, DE, and FG loops of a 1E11 clone are replaced with alternative loops with sequences corresponding to SEQ ID NO: 99, 38, and 107, respectively (see TABLE 4). Sequence identity numbers of the mult...

example 3

TRAIL R2Binding Affinity for Mono- and Polyvalent Tn3 Proteins

[0682]To measure the effect of Tn3 valency on binding affinity for a series of TRAIL R2-specific Tn3 proteins, a competition ELISA experiment was performed. A 96-well NUNC MaxiSorp plate (Thermo Fisher, Rochester, N.Y.), was coated with A9(1C12) (SEQ ID NO: 154+SEQ ID NO: 145) a TRAIL R2 specific scaffold in an antibody-like format, in PBS at 2 μg / ml overnight at 4° C. Plates were blocked with PBS 0.1% Tween 20+10 mg / ml BSA. Dilutions of A1 (1E11 monomer), and linear format A2 (1E11 bivalent) or A3 (1E11 tetravalent) multimeric scaffolds were incubated on the coated plate with 0.75 nM of biotinylated TRAIL R2-Fc for two hours at room temperature in PBS 0.1% Tween 20+1 mg / ml BSA, washed. Bound biotinylated TRAIL R2Fc was detected with streptavidin HRP, TMB, and neutralized with acid. Absorbance was read at 450 nm. Data is shown in FIG. 4. Binding affinities (IC50) are shown in TABLE 7 and were calculated as the concentrati...

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Abstract

The present invention provides fibronectin type III (Fn3)-based multimeric scaffolds that specifically bind to one or more specific target antigen. The invention further provides bispecific Fn3-derived binding molecules that bind to two or more target antigens simultaneously, fusions, conjugates, and methods to increase the stability of Fn3-based binding molecules. Furthermore, the present invention is related to a prophylactic, therapeutic or diagnostic agent, which contains Fn3-based multimeric scaffolds.

Description

REFERENCE TO THE SEQUENCE LISTING[0001]This application incorporates by reference a Sequence Listing submitted with this application via EFS-Web as text file entitled “2943.011PC01_sequence_listing.txt” created on Apr. 12, 2011 and having a size of 221 kilobytes.FIELD OF THE INVENTION[0002]The present invention relates in general to the field of antibody mimetics, specifically to multimeric scaffolds based on the fibronectin type III (Fn3) domain useful, for example, for the generation of products having novel binding characteristics.BACKGROUND[0003]Biomolecules capable of specific binding to a desired target epitope are of great importance as therapeutics, research, and medical diagnostic tools. A well known example of this class of molecules is the antibody. Antibodies can be selected that bind specifically and with affinity to almost any structural epitope. However, classical antibodies are structurally complex heterotetrameric molecules with are difficult to express in simple eu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K19/00
CPCA61K2039/505C07K14/001C07K16/1027C07K16/2878C07K16/468C07K2317/73C07K2317/90C07K2318/20C07K2319/00C07K2319/30C07K2319/33C07K2319/70C07K2319/74C07K19/00C07K14/78A61P29/00A61P31/00A61P35/00A61P37/06A61P43/00A61L27/56A61K39/395
Inventor BACA, MANUELTHISTED, THOMASSWERS, JEFFREY
Owner MEDIMMUNE LLC
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