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Dual specific single domain antibodies specific for a ligand and for the receptor of the ligand

A bispecific and specific technology, applied in the field of dual specific ligands, can solve problems such as poor stability, lack of light chain partners, and limited therapeutic value

Inactive Publication Date: 2006-03-08
DORMANTIS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, these individual functional domains have a short half-life in vivo, thus limiting their therapeutic value
[0009] It has been suggested that heavy chain variable domains of different specificities be joined together to produce bispecific antibody fragments (as described above), but this strategy has the disadvantage that the isolated antibody variable domains may have a region that normally interacts with the light chain. Hydrophobic interface of , which can be sticky when exposed to solvents, allowing individual domains to bind to hydrophobic surfaces
Furthermore, the combination of two or more different heavy chains lacking a light chain partner and possibly through a hydrophobic interface prevents them from binding one rather than two ligands that they would otherwise be able to bind
Moreover, in this case, the heavy chain variable region does not combine with the complementary light chain variable region, so the stability is poor and the folding is easy to open (Worn and Pluckthun, 1998, Biochemistry 37, 13120-7)

Method used

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  • Dual specific single domain antibodies specific for a ligand and for the receptor of the ligand
  • Dual specific single domain antibodies specific for a ligand and for the receptor of the ligand
  • Dual specific single domain antibodies specific for a ligand and for the receptor of the ligand

Examples

Experimental program
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Effect test

preparation example Construction

[0251] Preparation of immunoglobulin-based multispecific ligands

[0252] The dual-specific ligands of the present invention can be prepared according to the technologies previously established and applied to antibody engineering, regardless of whether they are in an open or closed conformation according to the expected configuration of the invention. Such as the preparation of scFv, phage antibodies and other engineered antibody molecules. Examples of techniques for preparing antibodies, particularly bispecific antibodies, are described in the following reviews and references: Winter & Milstein, (1991) Nature 349:293-299; Plueckthun (1992) Immunological Reviews 130:151-188; Wrightetal., (1992) Crti. Rev. Immunol. 12: 125-168; Holliger, P. & Winter, G. (1993) Curr. Op. Biotechn. 4, 446-449; Carter, et al. (1995) J. Hematother. 4, 463-470; Chester, KA & Hawkins, RE (1995) Trends Biotechn. 13, 294-300; Hoogenboom, HR (1997) Nature Biotechnol. 15, 125-126; Fearon, D. (1997) Nature Bi...

Embodiment 1

[0405] Example 1. Screening of bispecific scFv antibody (K8) specific for human serum albumin (HSA) and β-galactosidase (β-gal)

[0406] This example explains the method of preparing bispecific antibodies specific to β-gal and HSA. According to the ability to bind β-gal, it is selected as a dummy V H The Vκ variable region library connected to the Vκ region is selected based on the ability to bind HSA to select the V H Variable area library. Then combine the selected variable V H HSA and Vκβ-gal regions, and antibodies are selected based on their ability to bind β-gal and HAS. HSA is a half-life enhancing protein found in human blood.

[0407] Four human phage antibody libraries used in this experiment.

[0408] Antibody Library 1 Germline Vκ / DVT V H 8.46×10 7

[0409] Antibody Library 2 Germline Vκ / NNK V H 9.64×10 7

[0410] Antibody Library 3 Germline V H / DVTVκ 1.47×10 8

[0411] Antibody Library 4 Germline V H / NNKVκ 1.45×10 8

[0412] All antibody libraries are ba...

Embodiment 2K8

[0418] Example 2. Characteristics of K8 antibody binding performance

[0419] First, the monoclonal phage ELISA method was used to describe the binding characteristics of the K8 antibody. Use HSA, β-gal with alkaline phosphatase (APS), bovine serum albumin (BSA), peanut agglutinin (peanutagglufinin), lysozyme and cytochrome C at a concentration of 10μg / ml (to prevent cross-reaction ) 100 μl PBS 4°C overnight to coat a 96-well plate. The phagemid from the K8 clone was rescued with KM13, as described in Harrison et al. (1996), and the phage-containing supernatant (50 μl) was directly analyzed. Then a standard ELISA procedure (Hoogenboom et al., 1991) was carried out, using the detection method of bound phage with anti-M13-HRP conjugate. When the absorption signal displayed on the surface of the phage was greater than 1, the bispecific K8 antibody was found to bind to HAS and β-gal (Figure 4). Strong binding to BSA can also be observed (Figure 4). Since HSA and BSA are 76% homologous...

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Abstract

The invention provides a dual-specific ligand, comprising an immunoglobulin variable region having a first binding specificity for a target ligand and a complementary or immunoglobulin variable region having a second binding specificity for a receptor of the target ligand. Noncomplementary immunoglobulin variable regions.

Description

[0001] The present invention relates to dual specific ligands. Specifically, the present invention provides a method for preparing a dual-specific ligand, which includes a first immunoglobulin single variable domain that binds to a first antigen or epitope, and a second antigen or epitope that binds to a single variable domain. Two immunoglobulin single variable regions. More specifically, the present invention relates to a dual-specific ligand, which binds to at least one of the first and second antigens or epitopes, which can enhance the half-life of the ligand in vivo. The present invention describes open and closed conformation ligands containing more than one binding specificity. Background technique [0002] The antigen binding region in an antibody consists of two separate regions: the heavy chain variable region (V H ) And the light chain variable region (V L : V κ Or V λ ). The antigen binding site itself consists of 6 polypeptide loops: 3 from V H District (HI, H2 and H3)...

Claims

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Application Information

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IPC IPC(8): C07K16/24C07K16/28A61K39/395C12N15/63C12N15/62C12N15/13C07K16/00C07K16/18C07K16/40
CPCC07K16/2878C07K16/241C07K2317/31C07K16/18C07K2317/34C07K2317/622C07K2317/569C07K2317/92C07K2317/21C07K16/40C07K2317/55
Inventor 格雷格·温特伊恩·汤姆林森奥尔加·伊格纳托维奇本·伍尔文菲利普·琼斯
Owner DORMANTIS LTD
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