Ligand

a technology of ligands and ligands, applied in the field of ligands, can solve the problems of inability to control the ratio of binding sites to each antigen or epitope in the assembled molecule, inability to control the ratio of binding sites to each antigen or epitope, and inability to combine variable domains. to achieve the effect of increasing the half-life of ligands

Inactive Publication Date: 2017-05-25
DORMANTIS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The dual-specific ligands exhibit extended persistence in the body, ensuring longer functional activity and overcoming the limitations of rapid clearance and instability in conventional bispecific antibodies.

Problems solved by technology

Each of these techniques presents its particular disadvantages; for instance in the case of hybrid hybridomas, inactive VH / VL pairs can greatly reduce the fraction of bispecific IgG.
It is therefore impossible to control the ratio of binding sites to each antigen or epitope in the assembled molecule and thus many of the assembled molecules will bind to one antigen or epitope but not the other.
However, the variable domains were not complementary.
However, the camel heavy chain single domains are unusual in that they are derived from natural camel antibodies which have no light chains, and indeed the heavy chain single domains are unable to associate with camel light chains to form complementary VH and VL pairs.
Furthermore, these single domains have been shown to have a very short in vivo half-life.
Therefore such domains are of limited therapeutic value.
The disadvantage with this approach is that isolated antibody variable domains may have a hydrophobic interface that normally makes interactions with the light chain and is exposed to solvent and may be “sticky” allowing the single domain to bind to hydrophobic surfaces.
Moreover, in this case the heavy chain variable domains would not be associated with complementary light chain variable domains and thus may be less stable and readily unfold (Worn & Pluckthun, 1998 Biochemistry 37, 13120-7).

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Selection of a Dual Specific scFv Antibody (K8) Directed Against Human Serum Albumin (HSA) and β-Galactosidase (β-Gal)

[0491]This example explains a method for making a dual specific antibody directed against β-gal and HSA in which a repertoire of Vκ variable domains linked to a germ line (dummy) VH domain is selected for binding to 3-gal and a repertoire of VH variable domains linked to a -germ line (dummy) Vκ domain is selected for binding to HSA. The selected variable VH HSA and Vκ β-gal domains are then combined and the antibodies selected for binding to β-gal and HSA. HSA is a half-life increasing protein found in human blood.

[0492]Four human phage antibody libraries were used in this experiment.

Library 1-Germ line Vκ / DVT VH8.46 × 107Library 2-Germ line Vκ / NNK VH9.64 × 107Library 3-Germ line VH / DVT Vκ1.47 × 108Library 4-Germ line VH / NNK Vκ1.45 × 108

[0493]All libraries are based on a single human framework for VH (V3-23 / DP47 and JH4b) and Vκ (O12 / O2 / DPK9 and Jκ1) with side chain ...

example 2

Characterisation of the Binding Properties of the K8 Antibody

[0499]Firstly, the binding properties of the K8 antibody were characterised by the monoclonal phage ELISA. A 96-well plate was coated with 100 μl of HSA and β-gal alongside with alkaline phosphatase (APS), bovine serum albumin (BSA), peanut agglutinin, lysozyme and cytochrome c (to check for cross-reactivity) at 10 μg / ml concentration in PBS overnight at 4° C. The phagemid from K8 clone was rescued with KM13 as described by Harrison et al., (1996) and the supernatant (50 μl) containing phage assayed directly. A standard ELISA protocol was followed (Hoogenboom et al., 1991) using detection of bound phage with anti-M13-HRP conjugate. The dual specific K8 antibody was found to bind to HSA and β-gal when displayed on the surface of the phage with absorbance signals greater than 1.0 (FIG. 4). Strong binding to BSA was also observed (FIG. 4). Since HSA and BSA are 76% homologous on the amino acid level, it is not surprising that...

example 4

Creation and Characterisation of the Dual Specific ScFv Antibodies (VH1 / VH2 Directed Against Antigens A and B and VK1 / VK2 Directed Against Antigens C and D)

[0506]This example demonstrates that dual specific ScFv antibodies (VH1 / VH2 directed against antigens A and B and VK1 / VK2 directed against antigens C and D) could be created by combining Vκ and VH single domains selected against respective antigens in a ScFv vector.

[0507]To create dual specific antibody VH1 / VH2, VH1 single domain is excised from variable domain vector 1 (FIG. 7) by NcoI / XhoI digestion and ligated into NcoI / XhoI digested variable domain vector 2 (FIG. 7) to create VH1 / variable domain vector 2. VH2 single domain is PCR amplified from variable domain vector 1 using primers to introduce SalI restriction site to the 5′ end and Nod restriction site to the 3′ end. The PCR product is then digested with SalI / NotI and ligated into SalI / NotI digested VH1 / variable domain vector 2 to create VH1 / VH2 / variable domain vector 2.

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Abstract

The invention provides a dual-specific ligand comprising a first immunoglobulin variable domain having a first binding specificity and a complementary or non-complementary immunoglobulin variable domain having a second binding specificity.

Description

RELATED APPLICATIONS[0001]This application is a divisional of U.S. application Ser. No. 11 / 704,832, filed Feb. 8, 2007, which is a continuation in part of U.S. application Ser. No. 11 / 023,959, filed Dec. 28, 2004, which is a continuation of International Application PCT / GB2003 / 002804, filed 30 Jun. 2003, which claims the priority of PCT / GB02 / 03014, filed 28 Jun. 2002 and Great Britain Application GB 0230202.4, filed 27 Dec. 2002, the contents of which are incorporated herein by reference. This application is also a continuation in part of WO2005118642, filed May 31, 2005, which claims the benefit of U.S. 60 / 576,271 filed Jun. 1, 2004, and U.S. 60 / 632,361 filed Dec. 2, 2004, the contents of which are incorporated herein by reference. The application also claims priority from U.S. Ser. No. 11 / 704,832, filed 8 Feb. 2007.[0002]The present invention relates to dual specific ligands. In particular, the invention provides a method for the preparation of dual-specific ligands comprising a f...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C07K16/18
CPCC07K16/18A61K47/48538C07K2317/569C07K2317/34C07K2317/567C07K2317/92C07K2317/565C07K2317/31A61K47/6843C07K16/241C07K16/2878C07K2317/35C07K2317/53C07K2317/55C07K2317/624C07K2317/76C07K2317/94C07K2318/20C07K2319/00C07K2319/01C07K2319/31C07K16/00C07K16/46
InventorTOMLINSON, IANJESPERS, LAURENTCLUBE, JASPERHOLT., LUCY J.SCHON, OLIVER
OwnerDORMANTIS LTD