Antigen-binding chimeric proteins and methods and uses thereof

Patent metadata

Field	Value
Publication number	US-11873347-B2
Application number	US-201816760394-A
Country	US
Kind code	B2
Filing date	Oct 31, 2018
Priority date	Oct 31, 2017
Publication date	Jan 16, 2024
Grant date	Jan 16, 2024

Abstract

The present invention relates to the field of structural biology. More specifically, the present invention relates to novel antigen-binding chimeric proteins, their uses and methods in three-dimensional structural analysis of macromolecules, such as X-ray crystallography and high-resolution Cryo-EM, and their use as a therapeutic, diagnostic, or imaging tool. Even more specifically, the invention relates to a fusion of a scaffold protein and an antigen-binding domain wherein the scaffold protein of said fusion interrupts the Immunoglobulin domain topology to form a rigid chimer.

First claim

The invention claimed is:

1. An antigen-binding chimeric protein comprising an antigen-binding domain of an immunoglobulin fused with a scaffold protein, wherein the scaffold protein interrupts the primary topology of the antigen-binding domain at one or more exposed regions comprising a loop or β-turn of the antigen-binding domain, wherein said one or more exposed regions are not in complementarity-determining regions, wherein the antigen-binding domain retains the antigen-binding capacity, and wherein the scaffold protein is fused to the antigen-binding domain via at least two direct fusions or fusions made by a linker.

2. The antigen-binding chimeric protein of claim 1 , wherein the antigen-binding domain comprises at least 7 anti-parallel β-strands and at least three R turns, wherein the β-strands and βturns are defined according to IMGT nomenclature.

3. The antigen-binding chimeric protein of claim 1 , wherein the antigen-binding domain comprises an immunoglobulin (Ig) domain.

4. The antigen-binding domain of claim 1 , wherein the scaffold protein is inserted:
in the first β-turn that connects β-strand A and B of the antigen-binding domain; or
in the β-turn that connects β-strand C and C′ of the antigen-binding domain; or
in the β-turn that connects β-strand C″ and D of the antigen-binding domain; or
in the β-turn that connects β-strand D and E of the antigen-binding domain; or
in the β-turn that connects β-strand E and F of the antigen-binding domain,

and wherein the β-strands are defined according to IMGT nomenclature.

5. The antigen-binding chimeric protein of claim 1 , wherein the scaffold protein is a circularly permutated protein.

6. The antigen-binding chimeric protein of claim 1 , wherein the scaffold protein is a protein with symmetry, a multimeric scaffold having symmetry, or a protein forming virus-like particles.

7. The antigen-binding chimeric protein of claim 6 , wherein each of the monomers of the multimer is connected to said antigen-binding domain.

8. The antigen-binding chimeric protein of claim 1 , wherein the antigen-binding domain and the scaffold are connected via a disulphide bond.

9. The antigen-binding chimeric protein of claim 1 , wherein the scaffold protein has a total molecular mass of at least 30 kDa.

10. The antigen-binding chimeric protein of claim 1 , wherein the scaffold protein comprises an additional antigen-binding domain.

11. The antigen-binding chimeric protein of claim 1 , wherein the scaffold protein is a labelled protein.

12. The antigen-binding chimeric protein of claim 1 , wherein the antigen-binding chimeric protein is comprised in a cell.

13. The antigen-binding chimeric protein of claim 12 , wherein the cell further comprises the target antigen of the antigen-binding chimeric protein.

14. A complex comprising
(i) the antigen-binding chimeric protein of claim 1 , and
(ii) a target protein,
wherein the target protein is specifically bound to the antigen-binding chimeric protein.

15. A composition comprising the antigen-binding chimeric protein of claim 1 .

16. A composition comprising a first and a second antigen-binding chimeric protein, wherein each of the first and the second antigen-binding proteins are antigen binding proteins of claim 1 , wherein the antigen-binding domain of the second antigen-binding chimeric protein specifically binds the scaffold protein of the first antigen-binding chimeric protein.

17. The antigen-binding chimeric protein of claim 1 , wherein the antigen-binding chimeric protein is comprised in a virus-like particle.

18. A nucleic acid molecule encoding the antigen-binding chimeric protein of claim 1 .

19. The nucleic acid molecule of claim 18 , wherein the nucleic acid molecule is comprised in a vector.

20. The nucleic acid molecule of claim 19 , wherein the vector is a vector for surface display in yeast, phages, bacteria, or viruses.

21. A method of determining a 3-dimensional structure of a target protein, the method comprising:
Providing the complex of claim 14 ;
displaying the complex in suitable conditions for structural analysis, and
determining the 3D structure of the target protein;

wherein the 3D structure of the target protein is determined at high-resolution.

22. The method according to claim 21 , wherein determining the 3D structure of the target protein comprises single particle cryo-EM or crystallography.

Assignees

• Vib Vzw
• Univ Brussel Vrije
• Vrij Univ Brussel

Inventors

• Steyaert Jan
• Pardon Els
• Uchanski Tomasz
• Vranken Wim

Classifications

• C07K16/465 Primary

  with additional modified FR-residues · CPC title

• C07K14/005

  from viruses · CPC title

• C07K14/195

  from bacteria · CPC title

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title