WO2023143463A1

WO2023143463A1 - Fusion protein and application thereof

Info

Publication number: WO2023143463A1
Application number: PCT/CN2023/073419
Authority: WO
Inventors: Yin JI; Dongping LI; Jiahui ZHAO
Original assignee: Nanjing Reju Therapeutics , Inc.
Priority date: 2022-01-30
Filing date: 2023-01-20
Publication date: 2023-08-03
Also published as: CN117186239A

Abstract

The present disclosure relates to a fusion protein including Annexin A5 and one or more cell-penetrating peptides. The fusion protein can be used for treating hemorrhage, vascular injury, blood-brain barrier damage and inflammatory-related diseases of the central or peripheral system.

Description

FUSION PROTEIN AND APPLICATION THEREOF

Field of the Invention

The present disclosure relates to the biomedical field, and more particularly, to fusion proteins and application thereof.

Background of the Invention

Annexins are a family of soluble Ca²⁺-dependent phospholipid-binding proteins that widely distribute in various tissues and cells of animals and plants. There are 12 annexins in total, namely Annexin A1-A11 and Annexin 13, in vertebrates. Annexins can bind to biological membranes containing negatively charged phospholipids (mainly phosphatidylserine (PS) ) in a Ca²⁺-dependent manner. Annexin A5 is the smallest member of the annexins, with a molecular weight of about 35～36kDa. Annexin A5 has unique membrane-binding and self-assembling properties. It is capable of binding to PS with high affinity, and forming an ordered two-dimensional protein network on membrane surface and finally internalizing into cells. It has been proved that Annexin A5 is widely involved in biological functions both inside and outside cells, such as anticoagulant, signal transduction, anti-inflammation, membrane transport and ion channels. Annexin A5 not only exerts functions by binding to cell membrane PS, but also has intracellular biological functions. For example, both intracellular and extracellular Annexin A5 can be, in membrane repair mechanism, recruited to damaged sites of the cell membrane to block the damaged sites and promote the membrane repairmen. Annexin A5 can also act as a signaling protein for a vascular endothelial growth factor receptor-2 (VEGFR-2) and promote the proliferation of vascular endothelial cells through direct interaction with the intracellular domain of VEGFR-2, playing a role in vascular neogenesis and protection.

Annexin A5 is used for the treatment of, for example, post-operative syndrome (EP2694538B1) , arterial stenosis (AU2009216543B2) , peripheral arterial disease (US9649355B2) , plaque rupture (EP1755642B1) , wound healing (chronic) (US8377881B2) , cholestasis (CN112618696A) , diabetes (JP2019043924A) , and the like. There are also some prior arts relating to the modification of Annexin A5, for example, Dianexin (Annexin A5 dimer) for the treatment of diseases associated ischemia-reperfusion injury (WO2011069090A1) ; fusion proteins formed by Annexin A5 and HPV tumor antigen for the treatment of tumors (WO2020028794A1) ; and fusion proteins formed by Annexin A5 and TNF-α for the treatment of breast cancer (WO2016179430A1) . However, there is no relevant report targeting a fusion protein composed of a cell-penetrating peptide and Annexin A5.

Due to the existence of a blood-brain barrier (BBB) , the central nervous system prevents most of the neurotherapeutic drugs from entering the brain to exert their effects. It poses a significant barrier to the development of drugs for the central nervous system due to the lack of effective means for delivering drugs and genes to the brain. Brain-penetrating molecular transport vectors, such as brain permeable peptides or BBB shuttle peptides, have shown promise in overcoming BBB and transporting drug molecules to the brain. At present, brain-targeting peptides or BBB shuttle peptides that achieve the transportation via receptors are more commonly used as the brain-penetrating molecular transport vectors, to increase the transportation through the BBB by recognizing BBB-specifically expressed receptors. The molecular strategies aim to hijack endogenous BBB transport by chemically linking drugs to BBB-targeting ligands or BBB shuttle peptides, in which the latter binds to their receptors to trigger cellular transport to deliver the drugs into the brain. The common receptors for the brain-targeting peptides which trigger the receptor-mediated transport comprise insulin receptors, transferrin receptors, low density lipoprotein (LDL) receptors, LDL-associated proteins, leptin receptors, nicotinic acetylcholine receptors, glutathione transporters and the like.

Cell-penetrating peptides (CPPs) are investigated for the development of ideal drug delivery systems because they can be transported into cells in vitro and in vivo, either alone or in combination with drugs. An important advantage of CPPs lies in that they can penetrate most types of cell membranes, thereby improving drug absorption. In addition, CPPs also has the advantages of low cytotoxicity, the ability of penetrating a variety of cell types, dose dependence, being not limited by the sizes or types of drugs delivered, high biocompatibility, ease of synthesis, small size, and the like.

To date, there is no report related to the construction of fusion proteins of specific brain-targeting peptides and non-specific cell-penetrating peptides with Annexin A5.

Summary of the Invention

The present disclosure relates to a fusion protein including Annexin A5 and one or more cell-penetrating peptides. The fusion protein can be used for treating hemorrhage, vascular injury, BBB damage and inflammation-related diseases of the central or peripheral system, with increasing the targeting ability of Annexin A5 and enhancing the efficacy of Annexin A5 in certain diseases.

In a first aspect, the present disclosure provides a fusion protein which includes Annexin A5 and one or more cell-penetrating peptides.

In some embodiments, the fusion protein may include a first domain which includes one or more Annexin A5, active fragment (s) or variant (s) thereof having activity.

In some embodiments, a plurality of Annexin A5, the active fragments or variants thereof having activity may be connected in series or spaced apart from each other.

In some embodiments, the fusion protein may further include a second domain which includes one or more non-specific cell-penetrating peptide (s) , active fragment (s) or variant (s) thereof having activity, wherein a plurality of non-specific cell-penetrating peptides, the active fragments or variants thereof having activity may be the same or different from each other.

In some embodiments, the plurality of non-specific cell-penetrating peptides, the active fragments or variants thereof having activity may be connected in series or spaced apart from each other.

In some embodiments, the fusion protein may further include a third domain which include one or more brain-targeting peptide (s) , active fragment (s) or variant (s) thereof having activity, wherein a plurality of brain-targeting peptides, the active fragments or variants thereof having activity may be the same or different from each other.

In some embodiments, the plurality of brain-targeting peptides, the active fragments or variants thereof having activity may be connected in series or spaced apart from each other.

In some embodiment, the first domain, the second domain and/or the third domain may be connected to each other directly or by a linker.

In some embodiments, the one or more Annexin A5, the active fragment (s) or variant (s) thereof having activity; the one or more non-specific cell-penetrating peptide (s) , the active fragment (s) or variant (s) thereof having activity; and/or the one or more brain-targeting peptide (s) , the active fragment (s) or variant (s) thereof having activity may be connected to each other directly or by a linker.

In some embodiments, the linker may have an amino acid sequence selected from a group consisting of:

(GS) _n, wherein n is 1-5;

(GGGGS) _n, wherein n is 1-5;

(GGGGS) _n (GS) _m, wherein n is 1-5 and m is 1-5;

(EAAAK) _n, wherein n is 1-5;

A (EAAAK) _nA, wherein n is 1-5;

(GGGGS) _n (EAAAK) _m (GGGGS) _p, wherein n is 1-5, m is 1-5, and p is 0-5;

(EAAAK) _m (GGGGS) _n (GGGGS) _p, wherein m is 1-5, n is 1-5, and p is 0-5;

(GGGGS) _m (PPPPP) _n (GGGGS) _p, wherein m is 1-5, n is 1-5, and p is 0-5;

EPKSSDKTHTPPPPP (SEQ ID NO: 51) ;

DKTHTCPPCP (SEQ ID NO: 52) ;

GGGGGDKTHTCPPCP (SEQ ID NO: 53) ;

EPKSSDKTHTPPPPPRT (SEQ ID NO: 54) ;

LGGGGSGGGGSGGGGSRT (SEQ ID NO: 55) ;

LEPKSSDKTHTPPPPPRT (SEQ ID NO: 56) ;

KLEPKSSDKTHTPPPPPRT (SEQ ID NO: 57) ;

RTGGGGSKL (SEQ ID NO: 58) ;

RTGGGGSGGGGSGGGGSKL (SEQ ID NO: 59) ;

RTGGGGSGGGGSGGGGSGGGGSGGGGSKL (SEQ ID NO: 60) ;

EPKSCDKTHTCPPCP (SEQ ID NO: 61) ;

EPKSSDKTHTCPPCP (SEQ ID NO: 62) ;

ERKCCVECPPCP (SEQ ID NO: 63) ;

ESKYGPPCPSCP (SEQ ID NO: 64) ; or

ESKYGPPCPPCP (SEQ ID NO: 65) .

In some embodiments, the fusion protein may have a structure selected from a group consisting of:

A_n1-R_n2-B_n3,

R_n1-A_n2-B_n3,

B_n3-R_n1-A_n2,

B_n3-A_n1-R_n2, or

A_n1/R_n1-B_n3-A_n2/R_n2,

wherein A is a brain-targeting peptide, an active fragment or variant thereof which is capable of specifically crossing a blood-brain barrier; R is a non-specific cell-penetrating peptide, an active fragment or variant thereof having activity; and B is Annexin A5, an active fragment or variant thereof having activity, 0≤n1≤10, 0≤n2≤10, and 0≤n3≤10. Preferably, 0≤n1≤5, 0≤n2≤5, and 0≤n3≤5. More preferably, 0≤n1≤3, 0≤n2≤3, and 0≤n3≤3. More preferably, n3=1.

In some embodiments, the brain-targeting peptide may be a ligand of any one receptor which is selected from, but not limited to, an insulin receptor, a transferrin receptor, a low density lipoprotein (LDL) receptor, an LDL receptor-associated protein, a leptin receptor, a nicotinic acetylcholine receptor and a glutathione transporter.

In some embodiments, the brain-targeting peptide may be a ligand selected from, but not limited to, Angiopep-2, L57, ApoE (159-167) 2, ApoB (3371-3409) , AEP, LRPep2, THR, CRT, HAI, RVG29, D-CDX, GSH, Leptin30 or g21 (12-32) .

In some embodiments, the brain-targeting peptide may be any one selected from, but not limited to, Angiopep-2 set forth in SEQ ID NO: 2; L57 set forth in SEQ ID NO: 3; ApoE (159-167) 2 set forth in SEQ ID NO: 4; ApoB (3371-3409) set forth in SEQ ID NO: 5; AEP set forth in SEQ ID NO: 6; LRPep2 set forth in SEQ ID NO: 7; THR set forth in SEQ ID NO: 8; CRT set forth in SEQ ID NO: 9; HAI set forth in SEQ ID NO: 10; RVG29 set forth in SEQ ID NO: 11; D-CDX set forth in SEQ ID NO: 12; GSH; Leptin30 set forth in SEQ ID NO: 13; or g21 (12-32) set forth in SEQ ID NO: 14.

In some embodiments, the non-specific cell-penetrating peptide may be any one selected from, but not limited to, TAT, R9, Penetratin, MAP, ARF (1-22) , P28, CADY, R5, R6, R7, R8, R10, R11, BPrPr (1-28) , VT5, Bac7, pVEC, Pep-1, Trasportan, Trasportan 10, VP22, gH625, INF, MPG, C105Y, BIP, Pep-7, Antp, pTAT (48-60) , Buforin II, hClock (35-47) , K-FGF, Mouse PrP. sup. c (1-28) , SynBl, HN-1 and pISL.

In some embodiments, the non-specific cell-penetrating peptide may be any one selected from, but not limited to, TAT set forth in SEQ ID NO: 15, R9 set forth in SEQ ID NO: 16, Penetratin set forth in SEQ ID NO: 17, MAP set forth in SEQ ID NO: 18, ARF (1-22) set forth in SEQ ID NO: 19, P28 set forth in SEQ ID NO: 20, CADY set forth in SEQ ID NO: 21, R5 set forth in SEQ ID NO: 22, R6 set forth in SEQ ID NO: 23, R7 set forth in SEQ ID NO: 24, R8 set forth in SEQ ID NO: 25, R10 set forth in SEQ ID NO: 26, R11 set forth in SEQ ID NO: 27, BPrPr (1-28) set forth in SEQ ID NO: 28, VT5 set forth in SEQ ID NO: 29, Bac7 set forth in SEQ ID NO: 30, pVEC set forth in SEQ ID NO: 31, Pep-1 set forth in SEQ ID NO: 32, Trasportan set forth in SEQ ID NO: 33, Trasportan 10 set forth in SEQ ID NO: 34, VP22 set forth in SEQ ID NO: 35, gH625 set forth in SEQ ID NO: 36, INF set forth in SEQ ID NO: 37, MPG set forth in SEQ ID NO: 38, C105Y set forth in SEQ ID NO: 39, BIP set forth in SEQ ID NO: 40, Pep-7 set forth in SEQ ID NO: 41, Antp set forth in SEQ ID NO: 42, pTAT (48-60) set forth in SEQ ID NO: 43, Buforin II set forth in SEQ ID NO: 44, hClock (35-47) set forth in SEQ ID NO: 45, K-FGF set forth in SEQ ID NO: 46, Mouse PrP. sup. c (1-28) set forth in SEQ ID NO: 47, SynBl set forth in SEQ ID NO: 48, HN-1 set forth in SEQ ID NO: 49, and pISL set forth in SEQ ID NO: 50.

In some embodiments, the Annexin A5 may be human Annexin A5 or a mammalian ortholog thereof, an allelic variant or a genetic variant or a functional analog thereof, or a variant that has at least 70%, at least 80%, at least 90%, or even at least 95%identity to human Annexin A5 and has activity as Annexin A5.

In some embodiments, the Annexin A5 may have an amino acid sequence set forth in SEQ ID NO: 1, or a variant that has at least 70%, at least 80%, at least 90%, or even at least 95%identity to the amino acid sequence set forth in SEQ ID NO: 1 and has activity as Annexin A5.

In some embodiments, the fusion protein may further include a fourth domain which includes an Fc domain derived from an immunoglobulin.

Preferably, the immunoglobulin may be selected from IgA, IgG, IgM, IgD and IgE, preferably from IgG, more preferably from a group consisting of IgG1, IgG2, IgG3 or IgG4.

In some embodiments, the Fc domain may be Fc domain derived from human IgG1, preferably, the Fc includes mutation (s) L234A, L235A, and/or P329G.

In some embodiments, the Fc domain may be Fc domain derived from human IgG4, and preferably, the Fc includes mutations L234A and L235A.

In some embodiments, the Fc domain may have structural Knob-in-Hole mutations.

In some embodiments, the fusion protein may be a homodimer or a heterodimer.

In some embodiments, the fusion protein may include a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain constitute a homodimer or a heterodimer.

In some embodiments, the first polypeptide chain may include a first Fc domain, the second polypeptide chain may include a second Fc domain so as to constitute a dimer Fc region, and the first polypeptide chain and/or the second polypeptide chain may include Annexin A5, an active fragment or variant thereof having activity.

In some embodiments, the first polypeptide chain may further include the first domain, the second domain, and/or the third domain.

In some embodiments, the second polypeptide chain may further include the first domain, the second domain, and/or the third domain.

In some embodiments, the first domain, the second domain, and/or the third domain in the first polypeptide chain may be connected to each other directly or by a linker.

In some embodiments, in the first polypeptide chain, the one or more Annexin A5, the active fragment (s) or variant (s) thereof having activity; the one or more non-specific cell-penetrating peptide (s) , the active fragment (s) or variant (s) thereof having activity; and/or the one or more brain-targeting peptide (s) , the active fragment (s) or variant (s) thereof having activity may be connected to each other directly or by a linker.

In some embodiments, the first domain, the second domain, and/or the third domain in the second polypeptide chain may be connected to each other directly or by a linker.

In some embodiments, in the second polypeptide chain, the one or more Annexin A5, the active fragment (s) or variant (s) thereof having activity; the one or more non-specific cell-penetrating peptide (s) , the active fragment (s) or variant (s) thereof having activity; and/or the one or more brain-targeting peptide (s) , the active fragment (s) or variant (s) thereof having activity may be connected to each other directly or by a linker.

In some embodiments, in the first polypeptide chain, a C-terminus or a N-terminus of the first domain, a C-terminus or a N-terminus of the second domain, and/or a C-terminus or a N-terminus of the third domain may be connected to a N-terminus or a C-terminus of the fourth domain directly or by a linker.

In some embodiments, in the second polypeptide chain, a C-terminus or a N-terminus of the first domain, a C-terminus or a N-terminus of the second domain, and/or a C-terminus or a N-terminus of the third domain may be connected to a N-terminus or a C-terminus of the fourth domain directly or by a linker.

In some embodiments, the first polypeptide chain may include:

(a) the first Fc domain;

(b) the first Fc domain and Annexin A5;

(c) the first Fc domain and a brain-targeting peptide;

(d) the first Fc domain and a non-specific cell-penetrating peptide;

(e) the first Fc domain, Annexin A5 and a non-specific cell-penetrating peptide;

(f) the first Fc domain, Annexin A5 and a brain-targeting peptide;

(g) the first Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide; or

(h) the first Fc domain, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, wherein

the domains are connected directly or by a linker.

In some embodiments, the second polypeptide chain may include:

(a) the second Fc domain;

(b) the second Fc domain and Annexin A5;

(c) the second Fc domain and a brain-targeting peptide;

(d) the second Fc domain and a non-specific cell-penetrating peptide;

(e) the second Fc domain, Annexin A5 and a non-specific cell-penetrating peptide;

(f) the second Fc domain, Annexin A5 and a brain-targeting peptide;

(g) the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide; or

(h) the second Fc domain, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, wherein

the domains are connected directly or by a linker.

In some embodiments, the first polypeptide chain may include:

(a) the first Fc domain;

(b) sequentially from a C-terminus to a N-terminus, the first Fc domain and Annexin A5, and vice versa;

(c) sequentially from a C-terminus to a N-terminus, the first Fc domain and a brain-targeting peptide, and vice versa;

(d) sequentially from a C-terminus to a N-terminus, the first Fc domain and a non-specific cell-penetrating peptide, and vice versa;

(e) sequentially from a C-terminus to a N-terminus, the first Fc domain, Annexin A5 and a non-specific cell-penetrating peptide; or the first Fc domain, a non-specific cell-penetrating peptide and Annexin A5; or a non-specific cell-penetrating peptide, the first Fc domain and Annexin A5; and vice versa;

(f) sequentially from a C-terminus to a N-terminus, the first Fc domain, Annexin A5 and a brain-targeting peptide; or the first Fc domain, a brain-targeting peptide and Annexin A5; or a brain-targeting peptide, the first Fc domain and Annexin A5; and vice versa;

(g) sequentially from a C-terminus to a N-terminus, the first Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide; or the first Fc domain, a brain-targeting peptide and a non-specific cell-penetrating peptide; or a non-specific cell-penetrating peptide, the first Fc domain and a brain-targeting peptide; and vice versa;

(h) sequentially from a C-terminus to a N-terminus, the first Fc domain, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide; or the first Fc domain, a non-specific cell-penetrating peptide, Annexin A5, and a brain-targeting peptide; or the first Fc domain, a brain-targeting peptide, Annexin A5, and a non-specific cell-penetrating peptide; or the first Fc domain, Annexin A5, a brain-targeting peptide, and a non-specific cell-penetrating peptide; or the first Fc domain, a non-specific cell-penetrating peptide, a brain-targeting peptide, and Annexin A5; or the first Fc domain, a brain-targeting peptide, a non-specific cell-penetrating peptide, and Annexin A5; or a non-specific cell-penetrating peptide, a brain-targeting peptide, the first Fc domain, and Annexin A5; or a non-specific cell-penetrating peptide, Annexin A5, the first Fc domain, and a brain-targeting peptide; or a non-specific cell-penetrating peptide, the first Fc domain, Annexin A5, and a brain-targeting peptide; or a non-specific cell-penetrating peptide, the first Fc domain, a brain-targeting peptide, and Annexin A5; or Annexin A5, the first Fc domain, a non-specific cell-penetrating peptide, and a brain-targeting peptide; or Annexin A5, a non-specific cell-penetrating peptide, the first Fc domain, and a brain-targeting peptide; and vice versa, wherein

the domains are connected directly or by a linker.

In some embodiments, the second polypeptide chain may include:

(a) the second Fc domain;

(b) sequentially from a C-terminus to a N-terminus, the second Fc domain and Annexin A5, and vice versa;

(c) sequentially from a C-terminus to a N-terminus, the second Fc domain and a brain-targeting peptide, and vice versa;

(d) sequentially from a C-terminus to a N-terminus, the second Fc domain and a non-specific cell-penetrating peptide, and vice versa;

(e) sequentially from a C-terminus to a N-terminus, the second Fc domain, Annexin A5 and a non-specific cell-penetrating peptide; or the second Fc domain, a non-specific cell-penetrating peptide and Annexin A5; or a non-specific cell-penetrating peptide, the second Fc domain and Annexin A5; and vice versa;

(f) sequentially from a C-terminus to a N-terminus, the second Fc domain, Annexin A5 and a brain-targeting peptide; or the second Fc domain, a brain-targeting peptide and Annexin A5; or a brain-targeting peptide, the second Fc domain and Annexin A5; and vice versa;

(g) sequentially from a C-terminus to a N-terminus, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide; or the second Fc domain, a brain-targeting peptide and a non-specific cell-penetrating peptide; or a non-specific cell-penetrating peptide, the second Fc domain and a brain-targeting peptide; and vice versa;

(h) sequentially from a C-terminus to a N-terminus, the second Fc domain, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide; or the second Fc domain, a non-specific cell-penetrating peptide, Annexin A5 and a brain-targeting peptide; or the second Fc domain, a brain-targeting peptide, Annexin A5 and a non-specific cell-penetrating peptide; or the second Fc domain, Annexin A5, a brain-targeting peptide and a non-specific cell-penetrating peptide; or the second Fc domain, a non-specific cell-penetrating peptide, a brain-targeting peptide and Annexin A5; or the second Fc domain, a brain-targeting peptide, a non-specific cell-penetrating peptide and Annexin A5; or a non-specific cell-penetrating peptide, a brain-targeting peptide, the second Fc domain and Annexin A5; or a non-specific cell-penetrating peptide, Annexin A5, the second Fc domain and a brain-targeting peptide; or a non-specific cell-penetrating peptide, the second Fc domain, Annexin A5 and a brain-targeting peptide; or a non-specific cell-penetrating peptide, the second Fc domain, a brain-targeting peptide and Annexin A5; or Annexin A5, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide; or Annexin A5, a non-specific cell-penetrating peptide, the second Fc domain and a brain-targeting peptide; and vice versa, wherein

the domains are connected directly or by a linker.

In some embodiments, the fusion protein may have a structure selected from the following group :

(a) the first polypeptide chain includes the first Fc domain, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, the second Fc domain, a linker, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(b) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, the first Fc domain, a linker, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, and the second polypeptide chain includes the second Fc domain, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(c) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5, a linker and the first Fc domain, and the second polypeptide chain includes the second Fc domain, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(d) the first polypeptide chain includes the first Fc domain, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, a linker and the second Fc domain, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(e) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide and the first Fc domain, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, Annexin A5, a linker and the second Fc domain, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(f) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, Annexin A5, a linker and the first Fc domain, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide and the second Fc domain, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(g) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, the first Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, the second Fc domain, a linker and Annexin A5, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(h) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, the first Fc domain, a linker and Annexin A5, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(i) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide and the first Fc domain, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, the second Fc domain, a linker and Annexin A5, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(j) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, the first Fc domain, a linker and Annexin A5, and the second polypeptide chain includes the second Fc domain, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(k) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, the first Fc domain, a linker and Annexin A5, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, the second Fc domain, a linker and Annexin A5, wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer;

(l) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, the first Fc domain, a linker and Annexin A5, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, Annexin A5, a linker, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(m) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, Annexin A5, a linker, the first Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, Annexin A5, a linker, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide, wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer;

(n) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, the first Fc domain, a linker, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, the second Fc domain, a linker, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer;

(o) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, the first Fc domain, a linker, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5, a linker and the second Fc domain, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(p) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5, a linker and the first Fc domain, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5, a linker and the second Fc domain, wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer;

(q) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, Annexin A5 and the first Fc domain, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide and the second Fc domain, wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(r) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, Annexin A5, the first Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, Annexin A5, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide, wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer; and

(s) the first polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5 and the first Fc domain, and the second polypeptide chain sequentially includes, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5 and the second Fc domain, wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer.

In a second aspect, the present disclosure provides an isolated nucleic acid molecule, which encodes the fusion protein according to the first aspect.

In a third aspect, the present disclosure provides an expression vector, which includes the isolated nucleic acid molecule according to the second aspect.

The expression vector may be a eukaryotic expression vector or a prokaryotic expression vector, preferably a plasmid. The expression vector may also be a viral vector, such as an adenovirus vector or a lentivirus vector.

In a fourth aspect, the present disclosure provides a host cell, which includes the isolated nucleic acid molecule according to the second aspect, or the expression vector according to the third aspect.

In a fifth aspect, the present disclosure provides a method for preparing the fusion protein according to the first aspect, wherein the method includes the step of preparing the fusion protein according to the first aspect by using the isolated nucleic acid molecule according to the second aspect, the expression vector according to the third aspect or the host cell according to the fourth aspect.

In a sixth aspect, the present disclosure provides a pharmaceutical composition, including the fusion protein according to the first aspect, the isolated nucleic acid molecule according to the second aspect or the expression vector according to the third aspect, and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition may further include a second therapeutic agent. For example, the second therapeutic agent is a tissue plasminogen activator (tPA) .

Preferably, the pharmaceutical composition may be an aqueous solution, a non-aqueous solution or a suspension. The pharmaceutically acceptable carrier may be a conventional carrier in the art, and may be any suitable physiologically or pharmaceutically acceptable pharmaceutical accessories. The pharmaceutical accessories may be a conventional pharmaceutical accessories in the art and preferably includes a pharmaceutically acceptable excipient, a filler or a diluent and the like.

The pharmaceutical composition may be administered parenterally, by injection or orally. The pharmaceutical composition may be prepared into a form suitable for administration, such as a solid, semi-solid or liquid form. The pharmaceutical composition may be in the form of aqueous solution, non-aqueous solution, suspension, powder, tablets, capsules, granules, injection or infusion. The pharmaceutical composition may be administered intravascularly, subcutaneously, intraperitoneally, intramuscularly, inhalationally, intranasally, by airway instillation or intrathoracic instillation. The pharmaceutical composition may also be administered in an aerosol or spray form, e.g. intranasally; or, intrathecally, intramedullarily, intraventricularly, transdermally, percutaneously, topically, enterally, intravaginally, sublingually or rectally. The pharmaceutical composition may be prepared into various dosage forms as desired, and may be administered at a dose beneficial to a patient determined by a physician according to factors such as the type, age, body weight and general condition of the patient, mode of administration, and the like.

In a seventh aspect, the present disclosure provides use of the fusion protein according to the first aspect, the isolated nucleic acid molecule according to the second aspect, the expression vector according to the third aspect, the host cell according to the fourth aspect, or the pharmaceutical composition according to the sixth aspect for the prevention, treatment and/or diagnosis of hemorrhage, vascular injury, BBB damage or inflammatory-related diseases of central or peripheral system.

The hemorrhage may be caused by vascular rupture.

The hemorrhage and vascular injury may be caused by trauma or other causes such as pathology, or may be caused by a drug which includes, but is not limited to, a thrombolytic drug (such as tPA) , an antiplatelet drug (such as aspirin) , a fibrinogen-decreasing drug, an antipyretic analgesics and the like.

In some embodiments, the hemorrhage, vascular injury, BBB damage and inflammatory-related central nervous system disease may include, but be not limited to, a number of diseases or conditions caused by excessive activation of immune cells in brain, neurological injury, neurobehavioral deficit, a neurodegenerative disease, excitotoxicity in nervous system, pathological elevation of calcium ions, excessive NOS activation, or the like; or clinically identified central nervous diseases, such as stroke caused by hemorrhage or ischemia, hemorrhage caused by tPA treatment for ischemic stroke; epilepsy, encephaledema caused by BBB damage, micro-circulation disorder, cerebral hemorrhage, ischemia and the like; brain injury, brain-and spinal cord-trauma; cerebral vascular sclerosis, encephalitis, meningitis, encephalomyelitis, viral or autoimmune encephalitis, multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, amyotrophic lateral sclerosis, AIDS-related neurodegeneration, Alzheimer’s disease, Parkinson’s disease, limb weakness and limpness caused by neurobehavioral deficits; cognitive neurological impairment caused by nerve injury, such as visual, gustatory, olfactory, auditory, and facial nerve impairments; psychiatric or behavioral disorders caused by nerve injury and central inflammation, such as mania, affective disorders, depression, anxiety, schizophrenia, various types of phobias, and other psychiatric disorders.

In some embodiments, the hemorrhage, vascular injury, BBB damage and inflammatory-related peripheral system disease may include, but be not limited to, vasculitis, dermatitis caused by various causes, herpes-like dermatitis, psoriasis, atopic dermatitis, osteoarthritis, rheumatoid arthritis, inflammatory myopathy, glomerulonephritis, uveitis, endophthalmitis, gingivitis, periodontitis, allergic and non-allergic rhinitis, inflammatory bowel disease, lupus nephritis, thyroiditis, alcoholic and non-alcoholic fatty liver, viral and non-viral hepatitis, autoimmune hepatitis, chronic relapsing hepatitis, liver cirrhosis, conjunctivitis, autoimmune uveitis, autoimmune hemolytic anemia, temporal arteritis, Crohn’s disease, colitis, ulcerative colitis, lupus erythematosus, ankylosing spondylitis, immune complex vasculitis, myocarditis, ischemic heart disease, hypercholesterolemia, atherosclerosis, preeclampsia, various inflammatory diseases in diabetes, various inflammation diseases caused by autoimmune disorders, sepsis and lung injury.

Brief Description of the Drawings

Fig. 1 shows a schematic diagram of a fusion protein.

Fig. 2 shows a schematic diagram of the construction of the vector for the fusion protein.

Fig. 3 shows a schematic diagram of the structure of fusion proteins R6, R7 and R8.

Fig. 4 shows a schematic diagram of the construction of vector for the fusion protein.

Fig. 5 shows the results of the membrane permeation experiments of the FITC-labeled fusion protein with hCMEC/D3 as an example.

Fig. 6 shows the crystal violet staining experiments.

Fig. 7 shows the protective effects of Annexin A5 derivatives on inflammatory factors produced by LPS-stimulated BV2 cells detected by ELISA assay.

Fig. 8 shows the results of the protein membrane permeation experiments of the FITC-labeled fusion protein with RAW264.7 as an example.

Fig. 9 shows the effect of Annexin A5 derivatives on macrophage detected by qPCR.

Fig. 10 shows the HE staining results of the liver from the NASH model.

Fig. 11 shows that Annexin A5 derivatives can effectively alleviate imiquimod-induced psoriasis symptoms.

Fig. 12 shows the changes in body weight of the mice treated with Annexin A5 derivatives VS. A5.

Detailed Description of the Embodiments

The present disclosure provides a fusion protein that specifically permeates brain, so as to treat central nervous system (CNS) -related diseases caused by hemorrhage, BBB damage or inflammation. The present disclosure solves the problem that large molecules are difficult to enter brain. The present disclosure further provides cell membrane-penetrating fusion proteins, which increase intracellular drug levels and enhance the drug efficacy in certain disease states.

In some specific embodiments, in the fusion protein, the C terminus of the one or more brain-targeting peptide (s) may be connected to the N-terminus of the one or more cell-penetrating peptides, and the C terminus of the one or more cell-penetrating peptide (s) may be connected to the N-terminus of Annexin A5.

In some specific embodiments, in the fusion protein, the C terminus of the one or more brain-targeting peptide (s) may be connected to the N-terminus of Annexin A5.

In some specific embodiments, in the fusion protein, the C terminus of the one or more cell-penetrating peptide (s) may be connected to the N-terminus of the one or more brain-targeting peptides, and the C-terminus of the one or more brain-targeting peptide (s) may be connected to the N-terminus of Annexin A5.

In some specific embodiments, in the fusion protein, the C-terminus of one or more cell-penetrating peptide (s) may be connected to the N-terminus of Annexin A5.

In some specific embodiments, in the fusion protein, the C-terminus of Annexin A5 may be connected to the N-terminus of the one or more cell-penetrating peptide (s) , and the C-terminus of the one or more cell-penetrating peptide (s) may be connected to the N-terminus of the one or more brain-targeting peptide (s) .

In some specific embodiments, in the fusion protein, the C-terminus of Annexin A5 may be connected to the N-terminus of the one or more brain-targeting peptide (s) , and the C-terminus of the one or more brain-targeting peptide (s) may be connected to the N-terminus of one or more cell-penetrating peptide (s) .

In some specific embodiments, in the fusion protein, the C-terminus of Annexin A5 may be connected to the N-terminus of the one or more brain-targeting peptide (s) .

In some specific embodiments, in the fusion protein, the C-terminus of Annexin A5 may be connected to the N-terminus of the one or more cell-penetrating peptide (s) .

In some specific embodiments, in the fusion protein, the C-terminus of the one or more brain-targeting peptide (s) or the one or more cell-penetrating peptide (s) may be connected to the N-terminus of Annexin A5, and the C-terminus of Annexin A5 may be connected to the N-terminus of the one or more cell-penetrating peptide (s) or the one or more brain-targeting peptide (s) .

In some embodiments, the fusion protein may include: an immunoglobulin Fc domain having a hinge region, a CH2 domain, and a CH3 domain; Annexin A5 and two types of cell-penetrating peptides, wherein Annexin A5 and the two types of cell-penetrating peptides are provided at two ends of the immunoglobulin Fc domain, respectively, or at the same end of the Fc domain; or the two types of cell-penetrating peptides are connected to the N-terminus of Annexin A5, and the C-terminus of Annexin A5 is connected to the hinge region or the CH3 domain of the immunoglobulin Fc domain; and one Fc domain is connected to one or two Annexin A5 to form a dimer; the cell-penetrating peptides include either brain-targeting peptide (s) or non-specific cell-penetrating peptide (s) or include both of them, and one or more cell-penetrating peptides is included in the fusion protein.

Fig. 1 shows exemplary fusion proteins of the present disclosure.

As used herein, the “N-terminus” means an amino terminus of a peptide or protein, and for the purposes of the present disclosure, refers to a site capable of binding to another peptide or protein. For example, the “N-terminus” may include one or more amino acid residues located at the N-terminus, as well as the amino acid residue at the N-terminal of the peptide or protein. In particular, the “N-terminus” may include the ten amino acid residues from the N-terminal, but is not limited thereto.

the “C-terminus” means a carboxyl terminus of a peptide or protein, and for the purposes of the present disclosure, refers to a site capable of binding to another peptide or protein. For example, the “C-terminus” may include one or more amino acid residues located at the C-terminus, as well as the amino acid residue at the C-terminal of the peptide or protein. In particular, the “C-terminus” may include the amino acid residues from the C-terminal, but is not limited thereto.

As used herein, the “brain-targeting peptide” refers to a peptide that crosses the BBB via a receptor-mediated transport pathway. In particular, the brain-targeting peptide may cross the BBB via a receptor-mediated transport pathway which may be any one selected from, but not limited to, insulin receptors, transferrin receptors, low density lipoprotein receptors, low density lipoprotein receptor-associated protein, leptin receptors, nicotinic acetylcholine receptors, glutathione transporters, and the like. In the present disclosure, the brain-targeting peptide may be used interchangeably with a brain-penetrating peptide.

In some specific embodiments, the brain-targeting peptide may include one or more selected from the followings, but be not limited to:

(1) Angiopep-2: TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 2) ;

(2) L57: TWPKHFDKHTFYSILKLGKH (SEQ ID NO: 3) ;

(3) ApoE (159-167) 2: (LRKLRKRLL) 2 (SEQ ID NO: 4) ;

(4) ApoB (3371-3409) : SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO: 5) ;

(5) AEP: LRKLRKRLLR (SEQ ID NO: 6) ;

(6) LRPep2: HPWCCGLRLDLR (SEQ ID NO: 7) ;

(7) THR: THRPPMWSPVWP (SEQ ID NO: 8) ;

(8) CRT: c [CRTIGPSVC] c (SEQ ID NO: 9) ;

(9) HAI: HAIYPRH (SEQ ID NO: 10) ;

(10) RVG29: YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 11) ;

(11) D-CDX: D- [GREIRTGRAERWSEKF] (SEQ ID NO: 12) ;

(12) GSH: γ-ECG;

(13) Leptin30: YQQILTSMPSRNVIQISNDLENLRDLLHVL (SEQ ID NO: 13) ; and

(14) g21 (12-32) : TLIKTIVTRINDISHTQSVSA (SEQ ID NO: 14) .

As used herein, the “non-specific cell-penetrating peptide” can pass through the membrane of most types of cells alone or in combination with a drug, In some specific embodiments, the cell-penetrating peptide may include one or more selected from the followings, but be not limited to: TAT set forth in SEQ ID NO: 15, R9 set forth in SEQ ID NO: 16, Penetratin set forth in SEQ ID NO: 17, MAP set forth in SEQ ID NO: 18, ARF (1-22) set forth in SEQ ID NO: 19, P28 set forth in SEQ ID NO: 20, CADY set forth in SEQ ID NO: 21, R5 set forth in SEQ ID NO: 22, R6 set forth in SEQ ID NO: 23, R7 set forth in SEQ ID NO: 24, R8 set forth in SEQ ID NO: 25, R10 set forth in SEQ ID NO: 26, R11 set forth in SEQ ID NO: 27, BPrPr (1-28) set forth in SEQ ID NO: 28, VT5 set forth in SEQ ID NO: 29, Bac7 set forth in SEQ ID NO: 30, pVEC set forth in SEQ ID NO: 31, Pep-1 set forth in SEQ ID NO: 32, Trasportan set forth in SEQ ID NO: 33, Trasportan 10 set forth in SEQ ID NO: 34, VP22 set forth in SEQ ID NO: 35, gH625 set forth in SEQ ID NO: 36, INF set forth in SEQ ID NO: 37, MPG set forth in SEQ ID NO: 38, C105Y set forth in SEQ ID NO: 39, BIP set forth in SEQ ID NO: 40, Pep-7 set forth in SEQ ID NO: 41, Antp set forth in SEQ ID NO: 42, pTAT (48-60) set forth in SEQ ID NO: 43, Buforin II set forth in SEQ ID NO: 44, hClock (35-47) set forth in SEQ ID NO: 45, K-FGF set forth in SEQ ID NO: 46, Mouse PrP. sup. c (1-28) set forth in SEQ ID NO: 47, SynBl set forth in SEQ ID NO: 48, HN-1 set forth in SEQ ID NO: 49, and pISL set forth in SEQ ID NO: 50.

In some embodiments of the present disclosure, the term “Fc fusion protein of Annexin A5” can be used interchangeably with the term “Fc fusion protein” or “fusion protein” .

The fusion protein of the present disclosure can be used for treating hemorrhage, vascular injury, BBB damage and inflammatory-related central system nervous diseases.

The hemorrhage may be caused by vascular rupture.

The hemorrhage and vascular injury may be caused by trauma or other causes such as pathology, or may be caused by a drug which may include, but be not limited to, the followings: thrombolytic drugs (such as tPA) , antiplatelet drugs (such as aspirin) , fibrinogen-lowering drugs, antipyretic analgesics, and the like.

In some embodiments, the hemorrhage, vascular injury, BBB damage and inflammatory-related central system disease may include, but be not limited to, a number of diseases or conditions caused by excessive activation of immune cells in brain, neurological injury, neurobehavioral deficit, a neurodegenerative disease, excitotoxicity in nervous system, pathological elevation of calcium ions, excessive NOS activation, or the like; or clinically identified central nervous diseases, such as stroke caused by hemorrhage or ischemia, hemorrhage caused by tPA treatment for ischemic stroke; epilepsy; encephaledema caused by BBB damage, micro-circulation disorder, cerebral hemorrhage, ischemia and the like; brain injury; brain-and spinal cord-trauma; cerebral vascular sclerosis; encephalitis; meningitis; encephalomyelitis; viral or autoimmune encephalitis; multiple sclerosis; chronic inflammatory demyelinating polyneuropathy; amyotrophic lateral sclerosis; AIDS-related neurodegeneration; Alzheimer’s disease; Parkinson’s disease; limb weakness and limpness caused by neurobehavioral deficits; cognitive neurological impairment caused by nerve injury, such as visual, gustatory, olfactory, auditory, and facial nerve impairments; psychiatric or behavioral disorders caused by nerve injury and central inflammation, such as mania; affective disorders; depression; anxiety; schizophrenia; various types of phobias; and other psychiatric disorders.

In some embodiment, the hemorrhage, vascular injury, BBB damage and inflammatory-related peripheral system disease may include, but be not limited to, vasculitis, dermatitis caused by various causes, herpes-like dermatitis, psoriasis, atopic dermatitis, osteoarthritis, rheumatoid arthritis, inflammatory myopathy, glomerulonephritis, uveitis, endophthalmitis, gingivitis, periodontitis, allergic and non-allergic rhinitis, inflammatory bowel disease, lupus nephritis, thyroiditis, alcoholic and non-alcoholic fatty liver, viral and non-viral hepatitis, autoimmune hepatitis, chronic relapsing hepatitis, liver cirrhosis, conjunctivitis, autoimmune uveitis, autoimmune hemolytic anemia, temporal arteritis, Crohn’s disease, colitis, ulcerative colitis, lupus erythematosus, ankylosing spondylitis, immune complex vasculitis, myocarditis, ischemic heart disease, hypercholesterolemia, atherosclerosis, preeclampsia, various inflammatory diseases in diabetes, various inflammation diseases caused by autoimmune disorders, sepsis and lung injury.

In some embodiments of the present disclosure, hCMEC/D3 (an immortalized human brain microvascular endothelial cell line) cells were selected as an in vitro model in the present disclosure, in order to in vitro verify the protective effect of Annexin A5 and the fusion protein thereof on the BBB. The hCMEC/D3 cell line was derived from human temporal lobe microvessels isolated from tissues excised during surgery for epilepsy., The isolated tissues were rich in cerebral endothelial cells (CEC) . The cells were immortalized by lentivirus vector transduction with the catalytic subunit of human telomerase (hTERT) and SV40 large T antigen, following which CECs extensively characterized for brain endothelial phenotype characteristics were screened by cloning. The hCMEC/D3 cell line expresses a variety of protein markers on surface that are closely associated with the BBB, such as VE cadherin, claudin-3, claudin-5, and the like. The hCMEC/D3 cell line represents one such in vitro model of human BBB that is stable, and can be easily grown and stably maintain normal BBB phenotypes, and is commonly used for studying BBB characteristics and drug transportion into the BBB.

In the present disclosure, specific brain-targeting peptides and non-specific cell-penetrating peptides are combined with Annexin A5 to form fusion proteins. The fusion proteins provided by the present disclosure can be used to treat hemorrhage, vascular injury, BBB damage and inflammatory-related central or peripheral system diseases. Further, the fusion proteins provided by the present disclosure can be used in combination with drugs which are prone to cause hemorrhage, so as to reduce the risk of hemorrhage and improve the therapeutic efficacy. For example, the fusion proteins can be used in combination with a tissue plasminogen activator (tPA) to reduce hemorrhage caused by tPA treatment for ischemic stroke, and improve the therapeutic window and safety of tPA. tPA is a standard drug for clinical treatment for acute ischemic stroke. However, tPA may also increase the risk of hemorrhagic transformation. Especially, when tPA was administered beyond 4.5 h after ischemia onset, it would lead to poor clinical prognosis for stroke patients. There is evidence that the hemorrhagic transformation resulting from stroke treatment with tPA is associated with BBB disruption, which may occur in the early stage after stroke. It largely limits the clinical application of tPA for thrombolysis in stroke patients. Therefore, the fusion proteins comprising the cell-penetrating peptide and Annexin A5 can be used to treat hemorrhagic transformation due to tPA. The advantages lies in that the cell-penetrating peptide (s) can increase the efficiency of drug permeation, cross the BBB and increase the drug delivery to focal sites. In addition, because Annexin A5 has a protective effect on vascular endothelium both inside and outside the cells, the fusion proteins can have increased intracellular concentration of Annexin A5 and enhanced efficacy of Annexin A5 by the fusion of the cell-penetrating peptide and Annexin A5.

Therefore, the present disclosure has the advantages that the specific brain-targeting peptide (s) and the non-specific cell-penetrating peptide (s) are combined with Annexin A5 to form new fusion proteins, which can increase the drug permeation efficiency, crossing ability of physiological barriers such as BBB and the like, and drug delivery at focal sites, and have better therapeutic effect than Annexin A5 for certain diseases. The fusion proteins including the Fc domain can also prolong the in vivo half life of drugs, prolong the interval time of administration and improve the compliance of patients, and be particularly suitable for the treatment and prevention of chronic diseases and other diseases requiring long-term administration.

In the specific examples hereinafter, the embodiments of the present disclosure will be described in more detail. However, the following examples are exemplary only. That is, the present disclosure is not limited by the following examples.

Example 1. Preparation of the fusion proteins

In this example, TAT-A5, R9-A5, agiopep2-A5, A5-agiopep2, agiopep2-TAT-A5, HAI-A5 and HAI-TAT-A5 were prepared as exemplary fusion proteins. TAT-A5 was commissioned to be prepared by AtaGenix, and the rest were commissioned to be prepared by GenScript. Escherichia coli pET30a was used as the construction and culture system. The fusion proteins were constructed in a vector which included: an initiation codon, a HIS tag, a TEV digestion site, a cell-penetrating peptide and Annexin A5. The specific constructs are shown in Fig. 2.The gene and protein sequences of the fusion proteins are shown in Table 1

Table 1. Preparation information of the fusion proteins.

Example 2. Preparation of the fusion proteins

In this example, R6, R7, and R8 were prepared as exemplary Fc fusion proteins of Annexin A5. R6, R7, and R8 were commissioned to be prepared by GenScript. The structures of the Fc fusion proteins of Annexin A5 are shown in Fig. 3. HD CHO cells were used as the construction and culture system. The Fc fusion proteins were constructed in a vector which included: EcoRI--Kozak sequence--Artificial signal peptide--target protein--Stop codon-HindIII. The specific constructs are shown in Fig. 4. The gene and protein sequences of the fusion proteins are shown in Table 2 below.

Table 2: Sequence information of the Fc fusion proteins of Annexin A5.

Example 3. In vitro membrane permeation effects of the fusion proteins of Annexin A5 (the central system)

The main purpose of this example was to use immortalized human brain microvascular endothelial cells to determine the in vitro membrane permeability and brain permeability of the fusion proteins. Further, the function of the cell-penetrating peptides was evaluated by comparing the intracellular amounts of the Annexin A5 fusion proteins of the present disclosure with Annexin A5 (A5) . The specific method and results were as follows.

Method: HCMEC/D3 cells were plated at a cell density of 3×10⁴ cells/well. When the cells substantially cover all over the wells, 1μM of A5, TAT-A5, R9-A5, agiopep2-TAT-A5 and HAI-TAT-A5 were added respectively. After 24h, the nuclei of living cells were stained with hoechst 33342 (Beyotime) and the fluorescence intensity in the cells was observed under a fluorescence microscope. Wherein, all of A5, TAT-A5, R9-A5, agiopep2-TAT-A5 and HAI-TAT-A5 were coupled to FITC, and none of A5, TAT-A5, R9-A5, agiopep2-TAT-A5 and HAI-TAT-A5 had the Fc domain.

Results: as shown in Fig. 5, the intracellular FITC fluorescence intensities of TAT-A5, R9-A5, agiopep2-TAT-A5 and HAI-TAT-A5 were significantly higher than that of A5. This indicated that the cell-penetrating peptide had certain membrane permeation effect, such that the amounts of the fusion proteins in the cells were increased.

Example 4. Protective effect of Annexin A5 fusion proteins on the BBB disrupted by tPA

The main purpose of this example was to compare the protective effects of the fusion proteins of Annexin A5 of the present disclosure and Annexin A5 (A5) on the BBB. The result shows that the protective effect of the fusion proteins of Annexin A5 on the BBB are better than that of Annexin A5. The specific method and results were as follows.

Method: HCMEC/D3 cells were used to plate at a cell density of 3×10⁴ cells/well. When the cells substantially cover all over the wells, tPA (at a concentration of 10 μg) and plgn (at a concentration of 50 nM) were added. In the meantime, A5, TAT-A5, R9-A5, agiopep2-TAT-A5, HAI-TAT-A5, R6 and R7 were administered, respectively, at a concentration of 200 nM. After 24h, the cells were stained with crystal violet and then photographed for cell morphology comparison.

Results: as can be seen from Fig. 6, TAT-A5, R9-A5, agiopep2-TAT-A5, HAI-TAT-A5, R6 and R7 have better protective effect on HCMEC/D3 cells as compared to A5. This indicated that the protective effect of the fusion proteins of Annexin A5 on the BBB were better than that of Annexin A5. Wherein, none of A5, TAT-A5, R9-A5, agiopep2-TAT-A5 and HAI-TAT-A5 had the Fc domain.

Example 5. Protective effect of the fusion proteins of Annexin A5 from inflammatory factors produced by BV2 cells stimulated by LPS

The main purpose of this example was to investigate the anti-inflammatory effect of the fusion proteins of Annexin A5. The specific method and results were as follows.

Method: BV2 cells were used to plate at a density of 1.5×10⁵ cells/well. In each group, A5, TAT-A5, R9-A5 and HAI-TAT-A5 were added respectively. For LPS treatment, 100 ng/ml of LPS was added. After adding 100 ng/ml of LPS, A5, TAT-A5, R9-A5 and HAI-TAT-A5 were added at a concentration of 200 nM, respectively. After 24h, the cell supernatants were collected for the detection of inflammatory factors TNF-α and IL-6 by ELISA. The ELISA kit was purchased from Dakewe Biotech.

Results: as shown in Tables 3 and 4, as well as Fig. 7, the fusion proteins, per se, did not stimulate BV2 cells to produce inflammatory factors. Moreover, A5, TAT-A5, R9-A5 and HAI-TAT-A5 all decreased the expression of TNF-α and IL-6, and TAT-A5, R9-A5 and HAI-TAT-A5had stronger effects. Wherein, none of A5, TAT-A5, R9-A5 and HAI-TAT-A5 had the Fc domain.

Table 3: Effect of fusion proteins on secretion of inflammatory factors in BV2 cells.

Table 4: Anti-inflammatory effects of fusion proteins on LPS-stimulated BV2 cells.

Example 6. In vitro membrane permeation effects of the fusion proteins of Annexin A5 (the peripheral system)

Annexin A5 exerts peripheral anti-inflammation effects by entering the cells to regulate intracellular signaling pathways. The main purpose of this example was, using RAW264.7 (macrophages) , to compare the membrane permeation effect of the fusion proteins of Annexin A5 of the present disclosure with Annexin A5. Better membrane permeation effect would lead to better anti-inflammatory effects. The specific method and results were as follows.

Method: RAW264.7 cells were plated at a cell density of 3×10⁴ cells/well. When the cells substantially cover all over the wells, 1 μM of A5, TAT-A5 and R9-A5 were added respectively. After 24h, the nuclei of living cells were stained with hoechst 33342 (Beyotime) and the fluorescence intensity in the cells was observed under a fluorescence microscope.

Results: as shown in Fig. 8, the intracellular FITC fluorescence intensities of TAT-A5 and R9-A5 were significantly higher than that of A5. This indicated that the cell-penetrating peptide had certain membrane permeation effect. That is, the amounts of the fusion proteins in the cells were increased. Wherein, none of A5, TAT-A5 and R9-A5 had the Fc domain.

Example 7. Effect of the fusion proteins of Annexin A5 on the transformation of macrophages from M1-type (pro-inflammatory) to M2-type (anti-inflammatory)

The purpose of this example was to evaluate that anti-inflammatory effect of the fusion proteins of Annexin A5. The specific method and results were as follows.

Method: RAW264.7 cells ( “N” represents the quiescent state) were used. After differentiation into M1-type and M2-type under certain conditions, 200nM of TAT-A5, R9-A5 and R6 were added. After 24 h, the cells were collected. The relative expression levels of IL-1β, IL-6 and TNF-α in the M1-type cells, and the relative expression levels of CD206, Fizz1 and Ym1 in the M2-type cells were detected by qPCR. For the differentiation into M1-type, Raw 264.7 cells were stimulated with 10 ng/mL of LPS and 10 ng/mL of IFN-γ for 6 h. For the differentiation into M2-type, Raw 264.7 cells were stimulated with 20 ng/mL of IL-4 for 6 h. The primers used in the qPCR experiment were as follows.

IL-1β:

5’-CTTCAGGCAGGCAGTATCACTC-3’ (SEQ ID NO: 92) (forward) ;

5’-TGCAGTTGTCTAATGGGAACGT-3’ (SEQ ID NO: 93) (reverse) .

IL-6:

5’-ACAACCACGGCCTTCCCTAC-3’ (SEQ ID NO: 94) (forward) ;

5’-TCTCATTTCCACGATTTCCCAG-3’ (SEQ ID NO: 95) (reverse) .

TNF-α:

5’-CGAGTGACAAGCCTGTAGCCC-3’ (SEQ ID NO: 96) (forward) ;

5’-GTCTTTGAGATCCATGCCGTTG-3’ (SEQ ID NO: 97) (reverse) .

CD206:

5’-GCAGGTGGTTTATGGGATGT-3’ (SEQ ID NO: 98) (forward) ;

5’-GGGTTCAGGAGTGTTGTGG-3’ (SEQ ID NO: 99) (reverse) .

Fizz1:

5’-AGGAGCTGTCATTAGGGACATC-3’ (SEQ ID NO: 100) (forward)

5’-GGATGCCAACTTTGAATAGG-3’ (SEQ ID NO: 101) (reverse) .

Ym1:

5’-AGAAGGGAGTTTCAAACCTGGT-3’ (SEQ ID NO: 102) (forward)

5’-GTCTTGCTCATGTGTGTAAGTGA-3’ (SEQ ID NO: 103) (reverse) .

Results: as shown in Fig. 9, both TAT-A5, R9-A5 and R6 had strong anti-inflammatory effects and could promoted the transformation of M1-type to M2-type. Neither TAT-A5 nor R9-A5 had the Fc domain, and R6 had the Fc domain.

Example 8. In vivo efficacy of the fusion proteins of Annexin A5

The specific method and results were as follows.

Method: a nonalcoholic steatohepatitis model (NASH) was used for this example, in order to evaluate the effect of the fusion proteins of Annexin A5 on peripheral diseases. In particular, male C57BL/6 mice, aged 6-8 weeks, were selected and fed with a high-fat diet. The mice of the control group were fed with a normal diet. After 8 weeks, the mice were grouped, i.e., divided into a control group, a model group, an A5 group, a TAT-A5 group and a R9-A5 group. The mice in each group were administered with normal saline, A5, TAT-A5 and R9-A5 via tail vein injection, respectively, at a dosage of 30 μg/kg once a day, and continued with being fed with the high-fat diet (except for the control group) for 4 weeks. The liver tissues of the mice were taken for HE staining to observe the degree of liver lesions.

Results: as shown in Fig. 10, A5, TAT-A5 and R9-A5 can all alleviate the pathological changes of fatty liver. Further, TAT-A5 and R9-A5 had better effects than that of A5. Wherein, none of A5, TAT-A5 and R9-A5 had the Fc domain.

Example 9. Anti-inflammatory effect of the fusion proteins of Annexin A5 on autoimmune diseases

Number of mice: 25

Animal model: BALB/c mice, aged 7-8 weeks

Modeling method: shave off most of the hair on the backs of BALB/c mice, and further shave off fine hair with depilatory cream; wash with water and wipe dry; administer with normal saline, A5 or R9-A5 according to the following groupign; after 1h, apply about 62.5 mg of imiquimod cream evenly on the backs of the mice for psoriasis modeling, once a day, for 7 days. The mice were weighed, photographed, and scored with PASI from the day prior to the administration.

Specific grouping:

1. control group: five BALB/c mice, aged 7-8 weeks, administration of normal saline, once a day, subcutaneous injection;

2. model group: five BALB/c mice, aged 7-8 weeks, administration of normal saline, once a day, subcutaneous injection;

3. A5 group: five BALB/c mice, aged 7-8 weeks, administration of 5 mg/kg of A5, once a day, subcutaneous injection; and

4. R9-A5 group: five BALB/c mice, aged 7-8 weeks, administration of 5 mg/kg of R9-A5, once a day, subcutaneous injection; and

5. R6 group: five BALB/c mice, aged 7-8 weeks, administration of 5 mg/kg of R6, once a day, subcutaneous injection.

Referring to Fig. 11, it can be observed that R9-A5 and R6 can effectively alleviate imiquimod-induced psoriasis-like symptoms such as skin thickening, erythema and scaling in mice. Referring to Figs. 11 and 12, it can be seen that R9-A5 and R6 has significantly higher efficacy than that of A5. Especially in terms of body weight, R9-A5 was effective in alleviating the weight loss due to the disease. Wherein, neither A5 nor R9-A5 had the Fc domain, and R6 had the Fc domain.

Claims

A fusion protein, comprising Annexin A5 and one or more cell-penetrating peptides.
The fusion protein according to claim 1, comprising a first domain which comprises one or more Annexin A5, active fragment (s) or variant (s) thereof having activity,

preferably, a plurality of Annexin A5, active fragments or variants thereof having activity are connected in series or spaced apart from each other.
The fusion protein according to claim 1, further comprising a second domain comprising one or more non-specific cell-penetrating peptide (s) , active fragment (s) or variant (s) thereof having activity,

wherein a plurality of non-specific cell-penetrating peptides, active fragments or variants thereof having activity are the same or different from each other, and

wherein a plurality of non-specific cell-penetrating peptides, active fragments or variants thereof having activity are connected in series or spaced apart from each other.
The fusion protein according to claim 1, further comprising a third domain comprising one or more brain-targeting peptides, active fragment (s) or variant (s) thereof having activity,

wherein a plurality of brain-targeting peptides, active fragments or variants thereof having activity are the same or different from each other, and

wherein a plurality of brain-targeting peptides, active fragments or variants thereof having activity are connected in series or spaced apart from each other.
The fusion protein according to any one of claims 2 to 4, wherein the first domain, the second domain and/or the third domain are connected to each other directly or by a linker, or

one or more Annexin A5, active fragment (s) or variant (s) thereof having activity; one or more non-specific cell-penetrating peptide (s) , active fragment (s) or variant (s) thereof having activity; and/or one or more brain-targeting peptide (s) , active fragment (s) or variant (s) thereof having activity are connected to each other directly or by a linker,

preferably, the linker has an amino acid sequence selected from a group consisting of:

(GS) _n, wherein n is 1-5;

(GGGGS) _n, wherein n is 1-5;

(GGGGS) _n (GS) _m, wherein n is 1-5 and m is 1-5;

(EAAAK) _n, wherein n is 1-5;

A(EAAAK) _nA, wherein n is 1-5;

(GGGGS) _n (EAAAK) _m (GGGGS) _p, wherein n is 1-5, m is 1-5, and p is 0-5;

(EAAAK) _m (GGGGS) _n (GGGGS) _p, wherein m is 1-5, n is 1-5, and p is 0-5;

(GGGGS) _m (PPPPP) _n (GGGGS) _p, wherein m is 1-5, n is 1-5, and p is 0-5;

EPKSSDKTHTPPPPP (SEQ ID NO: 51) ;

DKTHTCPPCP (SEQ ID NO: 52) ;

GGGGGDKTHTCPPCP (SEQ ID NO: 53) ;

EPKSSDKTHTPPPPPRT (SEQ ID NO: 54) ;

LGGGGSGGGGSGGGGSRT (SEQ ID NO: 55) ;

LEPKSSDKTHTPPPPPRT (SEQ ID NO: 56) ;

KLEPKSSDKTHTPPPPPRT (SEQ ID NO: 57) ;

RTGGGGSKL (SEQ ID NO: 58) ;

RTGGGGSGGGGSGGGGSKL (SEQ ID NO: 59) ;

RTGGGGSGGGGSGGGGSGGGGSGGGGSKL (SEQ ID NO: 60) ;

EPKSCDKTHTCPPCP (SEQ ID NO: 61) ;

EPKSSDKTHTCPPCP (SEQ ID NO: 62) ;

ERKCCVECPPCP (SEQ ID NO: 63) ;

ESKYGPPCPSCP (SEQ ID NO: 64) ; or

ESKYGPPCPPCP (SEQ ID NO: 65) .
The fusion protein according to claim 1, wherein the fusion protein has a structure selected from a group consisting of:

A_n1-R_n2-B_n3,

R_n1-A_n2-B_n3,

B_n3-R_n1-A_n2,

B_n3-A_n1-R_n2, or

A_n1/R_n1-B_n3-A_n2/R_n2,

wherein A is a brain-targeting peptide, an active fragment or variant thereof which is capable of specifically crossing a blood-brain barrier; R is a non-specific cell-penetrating peptide, an active fragment or variant thereof having activity; and B is Annexin A5, an active fragment or variant thereof having activity,

wherein 0≤n1≤10, 0≤n2≤10, and 0≤n3≤10,

preferably, 0≤n1≤5, 0≤n2≤5, and 0≤n3≤5;

more preferably, 0≤n1≤3, 0≤n2≤3, and 0≤n3≤3, and

more preferably, n3=1.
The fusion protein according to any one of claims 2 to 4 or claim 6, wherein the brain-targeting peptide is a ligand of any one receptor selected from a group consisting of an insulin receptor, a transferrin receptor, a low density lipoprotein (LDL) receptor, an LDL receptor-associated protein, a leptin receptor, a nicotinic acetylcholine receptor and a glutathione transporter;

preferably, the brain-targeting peptide is any one ligand selected from Angiopep-2, L57, ApoE (159-167) 2, ApoB (3371-3409) , AEP, LRPep2, THR, CRT, HAI, RVG29, D-CDX, GSH, Leptin30 or g21 (12-32) ;

the cell-penetrating peptide is any one selected from a group consisting of TAT, R9, Penetratin, MAP, ARF (1-22) , P28, CADY, R5, R6, R7, R8, R10, R11, BPrPr (1-28) , VT5, Bac7, pVEC, Pep-1, Trasportan, Trasportan 10, VP22, gH625, INF, MPG, C105Y, BIP, Pep-7, Antp, pTAT (48-60) , Buforin II, hClock (35-47) , K-FGF, Mouse PrP. sup. c (1-28) , SynBl, HN-1 and pISL;

the Annexin A5 is human Annexin A5 or a mammalian ortholog thereof, an allelic variant, a genetic variant or a functional analog thereof, or a variant that has at least 70%, at least 80%, at least 90%, or at least 95%identity to human Annexin A5 and has activity as human Annexin A5;

preferably, the Annexin A5 has an amino acid sequence set forth in SEQ ID NO: 1, or a variant having an amino acid sequence that has at least 70%, at least 80%, at least 90%, or at least 95%identity to the amino acid sequence set forth in SEQ ID NO: 1 and has activity as Annexin A5.
The fusion protein according to claim 1, further comprising a fourth domain which comprises an Fc domain derived from an immunoglobulin,

preferably, the immunoglobulin is selected from a group consisting of IgA, IgG, IgM, IgD and IgE, preferably from IgG, more preferably from a group consisting of IgG1, IgG2, IgG3 or IgG4,

preferably, the Fc domain is derived from human IgG1, or the Fc domain comprises mutation (s) L234A, L235A, and/or P329G; or

the Fc domain is derived from human IgG4, or the Fc domain comprises mutations L234A and L235A.
The fusion protein according to claim 1, wherein the fusion protein is a homodimer or a heterodimer,

the fusion protein comprises a first polypeptide chain and a second polypeptide chain, which constitute a homodimer or a heterodimer,

wherein, the first polypeptide chain comprises a first Fc domain and the second polypeptide chain comprises a second Fc domain so as to constitute a dimer Fc region, and the first polypeptide chain and/or the second polypeptide chain comprise Annexin A5, an active fragment or variant thereof having activity.
The fusion protein according to claim 9, wherein the first polypeptide chain further comprises a first domain, a second domain, and/or a third domain, which preferably are connected to each other directly or by a linker; the second polypeptide chain further comprises a first domain, a second domain, and/or a third domain, which preferably are connected to each other directly or by a linker, or

wherein the first polypeptide chain further comprises one or more Annexin A5, active fragments or variants thereof having activity, one or more non-specific cell-penetrating peptides, active fragments or variants thereof having activity, and/or one or more brain-targeting peptides, active fragments or variants thereof having activity, which are connected to each other directly or by a linker;

the second polypeptide chain further comprises one or more Annexin A5, active fragments or variants thereof having activity, one or more non-specific cell-penetrating peptides, active fragments or variants thereof having activity, and/or one or more brain-targeting peptides, active fragments or variants thereof having activity, which are connected to each other directly or by a linker;

preferably, in the first polypeptide chain, a C-terminus or a N-terminus of the first domain, a C-terminus or a N-terminus of the second domain, and/or a C-terminus or a N-terminus of the third domain is connected to a N-terminus or a C-terminus of the fourth domain directly or by a linker;

preferably, in the second polypeptide chain, a C-terminus or a N-terminus of the first domain, a C-terminus or a N-terminus of the second domain, and/or a C-terminus or a N-terminus of the third domain are connected to a N-terminus or a C-terminus of a fourth domain directly or by a linker.
The fusion protein according to claim 9, wherein the first polypeptide chain comprises:

(a) the first Fc domain;

(b) the first Fc domain and Annexin A5;

(c) the first Fc domain and a brain-targeting peptide;

(d) the first Fc domain and a non-specific cell-penetrating peptide;

(e) the first Fc domain, Annexin A5 and a non-specific cell-penetrating peptide;

(f) the first Fc domain, Annexin A5 and a brain-targeting peptide;

(g) the first Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide; or

(h) the first Fc domain, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, wherein

the domains are connected directly or by a linker;

the second polypeptide chain comprises:

(a) the second Fc domain;

(b) the second Fc domain and Annexin A5;

(c) the second Fc domain and a brain-targeting peptide;

(d) the second Fc domain and a non-specific cell-penetrating peptide;

(e) the second Fc domain, Annexin A5 and a non-specific cell-penetrating peptide;

(f) the second Fc domain, Annexin A5 and a brain-targeting peptide;

(g) the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide; or

(h) the second Fc domain, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, wherein

the domains are connected directly or by a linker.
The fusion protein according to claim 9, wherein the first polypeptide chain comprises:

(a) the first Fc domain;

(b) sequentially from a C-terminus to a N-terminus, the first Fc domain and Annexin A5, and vice versa;

(c) sequentially from a C-terminus to a N-terminus, the first Fc domain and a brain-targeting peptide, and vice versa;

(d) sequentially from a C-terminus to a N-terminus, the first Fc domain and a non-specific cell-penetrating peptide, and vice versa;

(e) sequentially from a C-terminus to a N-terminus, the first Fc domain, Annexin A5 and a non-specific cell-penetrating peptide; or the first Fc domain, a non-specific cell-penetrating peptide and Annexin A5; or a non-specific cell-penetrating peptide, the first Fc domain and Annexin A5; and vice versa;

(f) sequentially from a C-terminus to a N-terminus, the first Fc domain, Annexin A5 and a brain-targeting peptide; or the first Fc domain, a brain-targeting peptide and Annexin A5; or a brain-targeting peptide, the first Fc domain and Annexin A5; and vice versa;

(g) sequentially from a C-terminus to a N-terminus, the first Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide; or the first Fc domain, a brain-targeting peptide and a non-specific cell-penetrating peptide; or a non-specific cell-penetrating peptide, the first Fc domain and a brain-targeting peptide; and vice versa;

(h) sequentially from a C-terminus to a N-terminus, the first Fc domain, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide; or the first Fc domain, a non-specific cell-penetrating peptide, Annexin A5, and a brain-targeting peptide; or the first Fc domain, a brain-targeting peptide, Annexin A5, and a non-specific cell-penetrating peptide; or the first Fc domain, Annexin A5, a brain-targeting peptide, and a non-specific cell-penetrating peptide; or the first Fc domain, a non-specific cell-penetrating peptide, a brain-targeting peptide, and Annexin A5; or the first Fc domain, a brain-targeting peptide, a non-specific cell-penetrating peptide, and Annexin A5; or a non-specific cell-penetrating peptide, a brain-targeting peptide, the first Fc domain, and Annexin A5; or a non-specific cell-penetrating peptide, Annexin A5, the first Fc domain, and a brain-targeting peptide; or a non-specific cell-penetrating peptide, the first Fc domain, Annexin A5, and a brain-targeting peptide; or a non-specific cell-penetrating peptide, the first Fc domain, a brain-targeting peptide, and Annexin A5; or Annexin A5, the first Fc domain, a non-specific cell-penetrating peptide, and a brain-targeting peptide; or Annexin A5, a non-specific cell-penetrating peptide, the first Fc domain, and a brain-targeting peptide; and vice versa, wherein

the domains are connected directly or by a linker;

the second polypeptide chain comprises:

(a) the second Fc domain;

(b) sequentially from a C-terminus to a N-terminus, the second Fc domain and Annexin A5, and vice versa;

(c) sequentially from a C-terminus to a N-terminus, the second Fc domain and a brain-targeting peptide, and vice versa;

(d) sequentially from a C-terminus to a N-terminus, the second Fc domain and a non-specific cell-penetrating peptide, and vice versa;

(e) sequentially from a C-terminus to a N-terminus, the second Fc domain, Annexin A5 and a non-specific cell-penetrating peptide; or the second Fc domain, anon-specific cell-penetrating peptide and Annexin A5; or a non-specific cell-penetrating peptide, the second Fc domain and Annexin A5; and vice versa;

(f) sequentially from a C-terminus to a N-terminus, the second Fc domain, Annexin A5 and a brain-targeting peptide; or the second Fc domain, a brain-targeting peptide and Annexin A5; or a brain-targeting peptide, the second Fc domain and Annexin A5; and vice versa;

(g) sequentially from a C-terminus to a N-terminus, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide; or the second Fc domain, a brain-targeting peptide and a non-specific cell-penetrating peptide; or a non-specific cell-penetrating peptide, the second Fc domain and a brain-targeting peptide; and vice versa;

(h) sequentially from a C-terminus to a N-terminus, the second Fc domain, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide; or the second Fc domain, a non-specific cell-penetrating peptide, Annexin A5 and a brain-targeting peptide; or the second Fc domain, a brain-targeting peptide, Annexin A5 and a non-specific cell-penetrating peptide; or the second Fc domain, Annexin A5, a brain-targeting peptide and a non-specific cell-penetrating peptide; or the second Fc domain, a non-specific cell-penetrating peptide, a brain-targeting peptide and Annexin A5; or the second Fc domain, a brain-targeting peptide, a non-specific cell-penetrating peptide and Annexin A5; or a non-specific cell-penetrating peptide, a brain-targeting peptide, the second Fc domain and Annexin A5; or a non-specific cell-penetrating peptide, Annexin A5, the second Fc domain and a brain-targeting peptide; or a non-specific cell-penetrating peptide, the second Fc domain, Annexin A5 and a brain-targeting peptide; or a non-specific cell-penetrating peptide, the second Fc domain, a brain-targeting peptide and Annexin A5; or Annexin A5, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide; or Annexin A5, a non-specific cell-penetrating peptide, the second Fc domain and a brain-targeting peptide; and vice versa, wherein

the domains are connected directly or by a linker.
The fusion protein according to claim 9, wherein,

(a) the first polypeptide chain comprises the first Fc domain, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, the second Fc domain, a linker, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(b) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, the first Fc domain, a linker, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, and

the second polypeptide chain comprises the second Fc domain,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(c) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5, a linker and the first Fc domain, and

the second polypeptide chain comprises the second Fc domain,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(d) the first polypeptide chain comprises the first Fc domain, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, a linker and the second Fc domain,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(e) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide and the first Fc domain, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, Annexin A5, a linker and the second Fc domain,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(f) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, Annexin A5, a linker and the first Fc domain, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide and the second Fc domain,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(g) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, the first Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, the second Fc domain, a linker and Annexin A5,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(h) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, the first Fc domain, a linker and Annexin A5, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(i) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide and the first Fc domain, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, the second Fc domain, a linker and Annexin A5,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(j) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, the first Fc domain, a linker and Annexin A5, and

the second polypeptide chain comprises the second Fc domain,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(k) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, the first Fc domain, a linker and Annexin A5, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, the second Fc domain, a linker and Annexin A5,

wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer;

(l) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, the first Fc domain, a linker and Annexin A5, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, Annexin A5, a linker, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(m) the first polypeptide chain sequentially comprising, from a C-terminus to a N-terminus, Annexin A5, a linker, the first Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, Annexin A5, a linker, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide,

wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer;

(n) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, the first Fc domain, a linker, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, the second Fc domain, a linker, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide,

wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer;

(o) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, the first Fc domain, a linker, Annexin A5, a non-specific cell-penetrating peptide and a brain-targeting peptide, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5, a linker and the second Fc domain,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(p) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5, a linker and the first Fc domain, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5, a linker and the second Fc domain,

wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer;

(q) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, Annexin A5 and the first Fc domain, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide and the second Fc domain,

wherein the first polypeptide chain and the second polypeptide chain constitutes a heterodimer;

(r) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, Annexin A5, the first Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, Annexin A5, the second Fc domain, a non-specific cell-penetrating peptide and a brain-targeting peptide,

wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer; and

(s) the first polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5 and the first Fc domain, and

the second polypeptide chain sequentially comprises, from a C-terminus to a N-terminus, a brain-targeting peptide, a non-specific cell-penetrating peptide, Annexin A5 and the second Fc domain,

wherein the first polypeptide chain and the second polypeptide chain constitutes a homodimer.
An isolated nucleic acid molecule, encoding the fusion protein according to any one of claims 1-13.
An expression vector, comprising the isolated nucleic acid molecule according to claim 14, wherein

the expression vector is a eukaryotic expression vector or a prokaryotic expression vector, preferably a plasmid, an adenovirus vector or a lentivirus vector.
A host cell, comprising the isolated nucleic acid molecule according to claim 14, or the expression vector according to claim 15.
A method for preparing the fusion protein according to any one of claims 1-13, comprising the step of preparing the fusion protein using the isolated nucleic acid molecule according to claim 14, the expression vector according to claim 15, or the host cell according to claim 16.
A pharmaceutical composition, comprising the fusion protein according to any one of claims 1-13, the isolated nucleic acid molecule according to claim 14 or the expression vector according to claim 15, and a pharmaceutically acceptable carrier.
The pharmaceutical composition according to claim 18, further comprising a second therapeutic agent,

preferably, the second therapeutic agent is a tissue plasminogen activator.
Use of the fusion protein according to any one of claims 1-13, the isolated nucleic acid molecule according to claim 14, the expression vector according to claim 15, the host cell according to claim 16, or the pharmaceutical composition according to claim 18 for the prevention, treatment and/or diagnosis of hemorrhage, vascular injury, blood-brain barrier damage or inflammatory-related central or peripheral system disease,

preferably, the hemorrhage is caused by vascular rupture,

preferably, the hemorrhage or vascular injury is due to trauma or pathological causes, or to a thrombolytic drug, an antiplatelet drug, a fibrinogen-decreasing drug or an antipyretic analgesic,

preferably, the hemorrhage, vascular injury, blood-brain barrier damage or inflammatory-related central system disease is selected from a group consisting of: a disease or condition caused by excessive activation of immune cells in brain, neurological injury, neurobehavioral deficit, a neurodegenerative disease, excitotoxicity in nervous system, pathological elevation of calcium ions or excessive NOS activation; central nervous diseases, stroke caused by hemorrhage or ischemia; hemorrhage caused by tPA treatment for ischemic stroke; epilepsy; encephaledema caused by blood-brain barrier damage, micro-circulation disorder, cerebral hemorrhage and ischemia; brain injury; brain-and spinal cord-trauma; cerebral vascular sclerosis; encephalitis; meningitis; encephalomyelitis; viral or autoimmune encephalitis; multiple sclerosis; chronic inflammatory demyelinating polyneuropathy; amyotrophic lateral sclerosis; AIDS-related neurodegeneration; Alzheimer’s disease; Parkinson’s disease; limb weakness and limpness caused by neurobehavioral deficits; cognitive neurological impairment caused by neurological injury; mania caused by neurological injury and central inflammation; affective disorder, depression, anxiety, schizophrenia, and phobia,

preferably, the hemorrhage, vascular injury, blood-brain barrier damage and inflammatory-related peripheral system disease is selected from a group consisting of: vasculitis, dermatitis, herpes-like dermatitis, psoriasis, atopic dermatitis, osteoarthritis, rheumatoid arthritis, inflammatory myopathy, glomerulonephritis, uveitis, endophthalmitis, gingivitis, periodontitis, allergic and non-allergic rhinitis, inflammatory bowel disease, lupus nephritis, thyroiditis, alcoholic and non-alcoholic fatty liver, viral and non-viral hepatitis, autoimmune hepatitis, chronic relapsing hepatitis, liver cirrhosis, conjunctivitis, autoimmune uveitis, autoimmune hemolytic anemia, temporal arteritis, Crohn’s disease, colitis, ulcerative colitis, lupus erythematosus, ankylosing spondylitis, immune complex vasculitis, myocarditis, ischemic heart disease, hypercholesterolemia, atherosclerosis, preeclampsia, inflammatory diseases in diabetes, inflammation diseases in autoimmune disorders, sepsis and lung injury.