Multi-arm linker constructs for treating pathological blood clots

• US 9,623,118 B2
• Filed: 07/18/2016
• Issued: 04/18/2017
• Est. Priority Date: 09/01/2015
• Status: Active Grant

• Current Assignee: Immunwork Inc.
• Original Assignee: Immunwork Inc.
• Inventors: Chang, Tse-Wen, Chu, Hsing-Mao, Lin, Chun-Yu
• Primary Examiner(s): Alstrum Acevedo, James H
• Assistant Examiner(s): Coffa, Sergio
• Application Number:
  US15/212,298
• Publication Number:
  US 20170056517A1
• Time in Patent Office: 274 Days
• Field of Search: None
• 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 linker unit comprising a center core, a plurality of linking arms, a plurality of first elements, and optionally a coupling arm, wherein

the center core comprises, (1) a first polypeptide comprising a plurality of lysine (K) residues, wherein each K residue and its next K residue are separated by a filler sequence comprising glycine (G) and serine (S) residues, and the number of K residues ranges from 2 to 15;

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

the plurality of linking arms are respectively linked to the K residues of the center core;

the plurality of first elements are respectively linked to the plurality of linking arms via forming an amide bond therebetween, or via thiol-maleimide reaction, copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, strained-promoted azide-alkyne click chemistry (SPAAC) reaction, or inverse electron demand Diels-Alder (iEDDA) reaction, wherein each of the first elements is an single-chain variable fragment (scFv) specific for fibrin; and

the amino acid residue at the N- or C-terminus of the center core has the azide or the alkyne group;

or the amino acid residue at the N- or C-terminus of the center core is a cysteine residue, and the thiol group of the cysteine residue is linked with the coupling arm having an azide, an alkyne, a tetrazine, a cyclooctene, or a cyclooctyne group at the free terminus of the coupling arm, wherein,

when the plurality of first elements are respectively linked to the plurality of linking arms via CuAAC or SPAAC reaction, then the amino acid residue at the N- or C-terminus of the center core is a cysteine residue, and the free terminus of the coupling arm is the tetrazine or the cyclooctene group;

or

when the plurality of first elements are respectively linked to the plurality of linking arms via iEDDA reaction, then the amino acid residue at the N- or C-terminus of the center core has the azide or the alkyne group, or the amino acid residue at the N- or C-terminus of the center core is a cysteine residue, and the free terminus of the coupling arm is the azide, the alkyne, or the cyclooctyne group.

2. The linker unit of claim 1, wherein the filler sequence has one of the sequences selected from the group consisting of GS, GGS, GSG, or one of SEQ ID NOs:

1-16.

3. The linker unit of claim 1, wherein the first polypeptide comprises 2-15 units of the sequence of G_1-5SK.

4. The linker unit of claim 3, wherein the first polypeptide comprises the sequence of (GSK)_2-15.

5. The linker unit of claim 1, wherein each of the linking arms is a PEG chain having 2-20 repeats of EG units.

6. The linker unit of claim 1, wherein the coupling arm is a PEG chain having 2-12 repeats of EG units.

7. The linker unit of claim 1, 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.

8. The linker unit of claim 1, wherein the amino acid residue having the alkyne group is L-homopropargylglycine (L-HPG), D-homopropargylglycine (D-HPG), or beta-homopropargylglycine (β

-HPG).

9. The linker unit of claim 1, wherein the cyclooctene group is trans-cyclooctene (TCO);

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

10. The linker unit of claim 1, wherein the tetrazine group is 1,2,3,4-tetrazine, 1,2,3,5-tetrazine or 1,2,4,5-tetrazine, or derivatives thereof.

11. The linker unit of claim 1, further comprising a second element that is linked to the center core via any of the following reactions,

CuAAC reaction occurred between the azide or alkyne group and the second element;

SPAAC reaction occurred between the azide or cyclooctyne group and the second element; and

iEDDA reaction occurred between the cyclooctene group or tetrazine group and the second element.

12. The linker unit of claim 11, wherein the second element is a tissue plasminogen activator or an inhibitor of Factor Xa or thrombin.

13. The linker unit of claim 12, wherein the tissue plasminogen activator is alteplase, reteplase, tenecteplase, or lanoteplase.

14. The linker unit of claim 12, wherein the inhibitor of Factor Xa is apixaban, edoxaban, or rivaroxaban;

and the inhibitor of thrombin is argatroban or melagatran.

15. The linker unit of claim 11, wherein the second element is linked to the azide or alkyne group of the N- or C-terminal amino acid residues of the center core via CuAAC reaction.

16. The linker unit of claim 15, further comprising a third element that is linked to the coupling arm via iEDDA reaction.

17. The linker unit of claim 16, wherein the third element is a long PEG chain having a molecular weight of about 20,000 to 50,000 Daltons.

18. The linker unit of claim 11, wherein the second element is linked to the azide group of the N- or C-terminal amino acid residues of the center core via SPAAC reaction.

19. The linker unit of claim 18, further comprising a third element that is linked to the coupling arm via iEDDA reaction.

20. The linker unit of claim 19, wherein the third element is a long PEG chain having a molecular weight of about 20,000 to 50,000 Daltons.

21. A method for preventing the formation of blood clot in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the linker unit of claim 12.

22. The method of claim 21, wherein the second element is the inhibitor of Factor Xa or thrombin.

23. The method of claim 22, wherein,

the inhibitor of Factor Xa is apixaban, edoxaban, or rivaroxaban;

and

the inhibitor of thrombin is argatroban or melagatran.

24. A method for treating thrombosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the linker unit of claim 12.

25. The method of claim 24, wherein the second element is the tissue plasminogen activator.

26. The method claim 24, wherein the tissue plasminogen activator is alteplase, reteplase, tenecteplase, or lanoteplase.