FUSION PROTEIN FOR TREATING REJECTION REACTION IN TRANSPLANTATION

• US 20170056521A1
• Filed: 09/01/2016
• Published: 03/02/2017
• Est. Priority Date: 09/01/2015
• Status: Active Grant

• Current Assignee: Immunwork Inc.
• Original Assignee: Immunwork Inc.
• Inventors: DU, Li-Yun, CHANG, Tse-Wen, CHU, Hsing-Mao
• Granted Patent: US 10,293,018 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 31/137 Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
• CPC Class Codes: A61K 31/436 the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• CPC Class Codes: A61K 31/4545 containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
• CPC Class Codes: A61K 38/05 Dipeptides
• CPC Class Codes: A61K 39/44 Antibodies bound to carriers
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: A61K 47/68 the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
• CPC Class Codes: A61K 47/6811 the drug being a protein or peptide, e.g. transferrin or bleomycin
• CPC Class Codes: A61K 47/6843 the antibody targeting a material from animals or humans
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 14/70532 B7 molecules, e.g. CD80, CD86
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/36 against blood coagulation factors
• CPC Class Codes: C07K 2317/20 characterized by taxonomic origin
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/035 : containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 7/08 : having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12N 15/1037 : Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 9/6459 : t-plasminogen activator (3.4.21.68), i.e. tPA
• CPC Class Codes: C12Y 304/21068 : Tissue plasminogen activator (3.4.21.68), i.e. tPA

Abstract

The present disclosure provides various molecular constructs having a targeting element and an effector element. Methods for treating various diseases using such molecular constructs are also disclosed.

Claims

1. A molecular construct comprising,

a pair of CH2-CH3 segments of an IgG.Fc;

a pair of effector elements, wherein each effector element is an immune checkpoint protein, an antibody fragment specific for CD25, or a drug bundle comprising a plurality of immunosuppressant molecules; and

a pair of targeting elements, wherein each targeting element is an antibody fragment specific for a human leukocyte antigen (HLA) allotype present only on cells of a donor transplant, wherein,

when each effector element is the immune checkpoint protein, then the pair of effector elements is linked to the N-termini of the pair of CH2-CH3 segments, and the pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, or vice versa,

when each effector element is the antibody fragment specific for CD25, then (1) the pair of effector elements is linked to the N-termini of the pair of CH2-CH3 segments, and the pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, or vice versa, or (2) when the pair of effector elements and the pair of targeting elements are both in the form of single-chain variable fragments (scFvs), then the pair of targeting elements is linked to the N-termini of the pair of effector elements in a tandem or diabody configuration, thereby forming a pair of bispecific scFvs that are linked to the N-termini of the pair of CH2-CH3 segments, or

when each effector element is the drug bundle, then the pair of effector elements is linked to the C-termini of the pair of CH2-CH3 segments, and the pair of targeting elements is linked to the N-termini of the pair of CH2-CH3 segments.

2. The molecular construct of claim 1, wherein the HLA allotype is HLA-A, HLA-B, or HLA-C.

3. The molecular construct of claim 1, wherein the immunosuppressant is an inhibitor of mammalian target of rapamycin (mTOR) or inhibitor of calcineurin.

4. The molecular construct of claim 3, wherein the inhibitor of mTOR is sirolimus or everolimus.

5. The molecular construct of claim 3, wherein the inhibitor of calcineurin is tacrolimus.

6. The molecular construct of claim 1, wherein the immunosuppressant is fingolimod or derivatives thereof.

7. The molecular construct of claim 1, wherein the immune checkpoint protein is an extracellular domain of cytotoxic T lymphocyte associated protein 4 (CTLA-4) or an extracellular domain of programmed death-ligand 1 (PD-L1).

8. The molecular construct of claim 1, wherein the pair of CH2-CH3 segments is derived from human γ

1 or γ

4 immunoglobulin.

9. The molecular construct of claim 1, wherein the pair of targeting elements is in the form of an antigen-binding fragment (Fab) and is linked to the N-termini of the pair of CH2-CH3 segments, so that the molecular construct adopts an IgG configuration.

10. The molecular construct of claim 1, further comprising,

a peptide extension, having the sequence of (G_2-4S)_2-8C, and linked to the C-terminus of one of the pair of CH2-CH3 segments;

a coupling arm, linked to the C-terminus of the peptide extension via thiol-maleimide reaction occurred therebetween, wherein the drug bundle is linked to the coupling arm via inverse electron demand Diels-Alder (iEDDA) reaction, strain-promoted azide-alkyne click chemistry (SPAAC) reaction, or Copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction occurred therebetween.

11. The molecular construct of claim 10, wherein the drug bundle comprises,

a center core, which is (1) a first polypeptide comprising 2 to 15 K resides and one or more filler sequences separating each K residue and its next K residue, wherein the filler sequence comprises glycine (G) and serine (S) residues;

or (2) a second polypeptide comprising the sequence of (X_aa-K)_2-15, where X_aa is a PEGylated amino acid having 2 to 12 repeats of ethylene glycol (EG) unit; and

a plurality of linking arms, each having one terminus linked to the center core by reacting with one of the K residues of the center core, and carrying a maleimide group at the free terminus thereof, wherein each of the immunosuppressant molecules is linked to the center core via connecting through the linking arm by reacting with the maleimide group.

12. The molecular construct of claim 11, wherein the filler sequence has the sequence of GS, GGS, GSG, or SEQ ID NOs:

1-16.

13. The molecular construct of claim 12, wherein the first polypeptide comprises 2-15 units of the sequence of G_1-5SK.

14. The molecular construct of claim 13, wherein the first polypeptide comprises the sequence of (GSK)_2-15.

15. The molecular construct of claim 11, wherein each of the linking arms is a PEG chain having 2-20 repeats of EG units.

16. The molecular construct of claim 11, wherein the amino acid residue having the azide group is L-azidohomoalanine (AHA), 4-azido-L-phenylalanine, 4-azido-D-phenylalanine, 3-azido-L-alanine, 3-azido-D-alanine, 4-azido-L-homoalanine, 4-azido-D-homoalanine, 5-azido-L-ornithine, 5-azido-d-ornithine, 6-azido-L-lysine, or 6-azido-D-lysine.

17. The molecular construct of claim 11, wherein the amino acid residue having the alkyne group is L-homopropargylglycine (L-HPG), D-homopropargylglycine (D-HPG), or beta-homopropargylglycine (β

-HPG).

18. The molecular construct of claim 11, wherein the cyclooctene group is trans-cyclooctene (TCO);

and the cyclooctyne group is dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), or dibenzocyclooctyne (DICO).

19. The molecular construct of claim 11, wherein the tetrazine group is 1,2,3,4-tetrazine, 1,2,3,5-tetrazine or 1,2,4,5-tetrazine, or derivatives thereof.

20. A method for treating transplantation rejection in a subject receiving a donor transplant, comprising administering to the subject a therapeutically effective amount of the molecular construct of claim 1.