CN117209612A - Fusion proteins and uses thereof - Google Patents
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4721—Lipocortins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present application relates to fusion proteins comprising Annexin A5 and one or more transmembrane peptides. The fusion proteins are useful in the treatment of autoimmune diseases.
Description
Technical Field
The present application relates to the field of biological medicine, and more specifically, to fusion proteins and uses thereof.
Background
Annexins (Annexins) are a family of soluble calcium-dependent phospholipid-binding proteins that are widely distributed in various tissues and cells of animals and plants. There are 12 annexins in vertebrate animals, namely Annexin A1-A11 and Annexin 13, the main functions of which are toCa 2+ The dependent manner binds to biological membranes containing negatively charged phospholipids, mainly Phosphatidylserine (PS). Annexin A5 is the smallest member of Annexin family, has a molecular weight of about 35-36 kDa, has unique membrane binding and self-assembly characteristics, can be combined with PS with high affinity, and forms an ordered two-dimensional protein network on the surface of the membrane for final internalization into cells. Annexin A5 has been shown to be widely involved in intracellular and extracellular biological functions including anticoagulation, signaling, anti-inflammatory, membrane transport and ion channel activities. Annexin A5 not only functions by PS binding to cell membranes, but also has intracellular biological functions, for example, in plasma membrane injury repair mechanisms, intracellular Annexin A5 and extracellular Annexin A5 can be recruited to cell membrane injury sites simultaneously to block injury sites to promote cell membrane repair; annexin A5 can also be used as a signal protein of vascular endothelial growth factor receptor-2 (VEGFR-2), and can promote proliferation of vascular endothelial cells through direct interaction with an intracellular domain of VEGFR-2, thereby playing roles in angiogenesis and protection.
Annexin A5 is used to treat, for example, postoperative syndrome (EP 2694538B 1), arterial stenosis (AU 2009216543B 2), peripheral arterial disease (US 9649355B 2), plaque rupture (EP 1755642B 1), wound healing (chronic) (US 8377881B 2), cholestasis (CN 112618696A), diabetes (JP 2019043924A), and the like. Still other prior art is directed to modification of Annexin A5, for example: dianexin (Annexin A5 dimer) for the treatment of ischemia reperfusion injury related diseases (WO 2011069090 A1); annexin A5 forms a fusion protein with HPV tumor antigen for treating tumor (WO 2020028794A 1), and TNF-alpha forms a fusion protein for treating breast cancer (WO 2016179430A 1), etc., but no related report is made on fusion proteins composed of permeabilizing peptides and Annexin A5 at present.
Cell-penetrating peptides (CPPs) can be transported into cells both in vitro and in vivo, alone or in combination with drugs, and have therefore been investigated to develop ideal drug delivery systems. One important advantage of CPPs is the ability to cross most membranes, thereby enhancing drug absorption, and in addition, the advantages of CPPs include low cytotoxicity, ability to penetrate a range of cell types, dose dependence, freedom from size or type of drug delivered, high biocompatibility, ease of synthesis, small volume, etc.
Up to now, there have been no reports on the construction of a fusion protein of a permeant peptide with Annexin A5 and its use in the treatment of autoimmune diseases.
Disclosure of Invention
The present application relates to fusion proteins comprising Annexin A5 and one or more transmembrane peptides. The fusion protein can be used for treating autoimmune diseases, especially psoriasis, and the drug effect of Annexin A5 is increased.
In a first aspect, the application provides a fusion protein comprising Annexin A5 and one or more permeant peptides.
In some embodiments, the fusion protein comprises a first domain comprising one or more Annexin A5, an active fragment thereof, or a variant thereof that remains active.
In some embodiments, the plurality of Annexin A5, active fragments thereof, or variants thereof that remain active are linked in tandem, or are spaced apart from each other.
In some embodiments, the fusion protein further comprises a second domain comprising one or more permeant peptides, active fragments thereof, or variants thereof that remain active, which may be the same or different from each other.
In some embodiments, the plurality of permeant peptides, active fragments thereof, or variants thereof that remain active are linked in tandem, or are spaced apart from each other.
In some embodiments, the first domain and/or the second domain are linked to each other directly or via a linking peptide.
In some embodiments, the one or more Annexin A5, active fragments thereof, or variants thereof that remain active; and the one or more permeabilizing peptides, active fragments thereof, or variants thereof that remain active, are linked to each other either directly or through a linking peptide.
In some embodiments, the connecting peptide has an amino acid sequence selected from the group consisting of:
(GS) n n is 1-5;
(GGGGS) n n is 1-5;
(GGGGS) n (GS) m n is 1-5, and m is 1-5;
(EAAAK) n n is 1-5;
A(EAAAK) n a, n is 1-5;
(GGGGS) n (EAAAK) m (GGGGS) p n is 1-5, m is 1-5, and p is 0-5;
(EAAAK) m (GGGGS) n (GGGGS) p m is 1-5, n is 1-5, and p is 0-5;
(GGGGS) n (PPPPP) n (GGGGS) p m is 1-5, n is 1-5, and p is 0-5;
EPKSSDKTHTPPPPP(SEQ ID NO:2)；
DKTHTCPPCP(SEQ ID NO:3)；
GGGGGDKTHTCPPCP(SEQ ID NO:4)；
EPKSSDKTHTPPPPPRT(SEQ ID NO:5)；
LGGGGSGGGGSGGGGSRT(SEQ ID NO:6)；
LEPKSSDKTHTPPPPPRT(SEQ ID NO:7)；
KLEPKSSDKTHTPPPPPRT(SEQ ID NO:8)；
RTGGGGSKL(SEQ ID NO:9)；
RTGGGGSGGGGSGGGGSKL(SEQ ID NO:10)；
RTGGGGSGGGGSGGGGSGGGGSGGGGSKL(SEQ ID NO:11)；
EPKSCDKTHTCPPCP(SEQ ID NO:12)；
EPKSSDKTHTCPPCP(SEQ ID NO:13)；
ERKCCVECPPCP(SEQ ID NO:14)；
ESKYGPPCPSCP (SEQ ID NO: 15); or (b)
ESKYGPPCPPCP(SEQ ID NO:16)。
In some embodiments, the fusion protein has a structure selected from the group consisting of:
R n1 -B n2 ，
R n1 -B n2 -R n3 ，
B n2 -R n1 -B n3 ，
wherein R is a permeabilizing peptide, an active fragment thereof, or a variant thereof that retains activity; b is Annexin A5, an active fragment thereof, or a variant thereof which retains activity, 0.ltoreq.n1.ltoreq.10, 0.ltoreq.n2.ltoreq.10, and 0.ltoreq.n3.ltoreq.10. Preferably, 0.ltoreq.n1.ltoreq.5, 0.ltoreq.n2.ltoreq.5, and 0.ltoreq.n3.ltoreq.5. More preferably, 0.ltoreq.n1.ltoreq.3, 0.ltoreq.n2.ltoreq.3, and 0.ltoreq.n3.ltoreq.3.
In some embodiments, the permeant peptide is selected from, but is not limited to, any one of the following: TAT, R9, pennetrate, 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, hLock (35-47), K-FGF, mouse PrP.sup.c (1-28), synbl, HN-1, pSL.
In some embodiments, the permeant peptide is selected from, but is not limited to, any one of the following: SEQ ID NO:17, the TAT shown in SEQ ID NO:18, R9, SEQ ID NO:19, ARF (1-22) shown in SEQ ID NO. 20, P28 shown in SEQ ID NO. 21, CADY shown in SEQ ID NO. 22, R5 shown in SEQ ID NO. 24, R6 shown in SEQ ID NO. 25, R7 shown in SEQ ID NO. 26, R8 shown in SEQ ID NO. 27, R10 shown in SEQ ID NO. 28, R11 shown in SEQ ID NO. 29, BPrPr (1-28) shown in SEQ ID NO. 30, VT5 shown in SEQ ID NO. 31, bac7 shown in SEQ ID NO. 32, trasportan shown in SEQ ID NO. 33, pep-1 shown in SEQ ID NO. 34, trasportan 10 shown in SEQ ID NO. 36, VP22 shown in SEQ ID NO. 37, gH625 shown in SEQ ID NO. 38, INF shown in SEQ ID NO. 39, VP 5 shown in SEQ ID NO. 40, VP 7 shown in SEQ ID NO. 40, and clip NO. 48, P7 shown in SEQ ID NO. 48, P1-1 shown in SEQ ID NO. 33, pep-1, SEQ ID NO. 35 shown in SEQ ID NO. 48, hIV shown in SEQ ID NO. 48, P5, P1-48 shown in SEQ ID NO. 48, hC shown in SEQ ID NO. 48, hIV, 5, SEQ ID NO. 48, 5 shown in SEQ ID NO. 48, 5, SEQ ID NO. 35.
In some embodiments, the Annexin A5 is a human Annexin A5 or a mammalian ortholog, an allelic variant or genetic variant, or a functional analog of Annexin A5, or a variant having greater than 70%, e.g., greater than 80% or greater than 90%, even greater than 95% identity but retaining activity with human Annexin A5.
In some embodiments, the Annexin A5 is an Annexin A5 having the amino acid sequence shown in SEQ ID No. 1, or a variant having more than 70%, e.g., more than 80% or more than 90%, even more than 95% identity but retaining activity with an Annexin A5 having the amino acid sequence shown in SEQ ID No. 1.
In some embodiments, the fusion protein further comprises a third domain comprising an Fc region of an immunoglobulin.
Preferably, the immunoglobulin is selected from IgA, igG, igM, igD and IgE, preferably IgG, more preferably IgG1, igG2, igG3 or IgG4.
In some embodiments, the Fc is that of human IgG1, preferably the Fc comprises an L234A, L235A and/or P329G mutation.
In some embodiments, the Fc is that of human IgG4, preferably the Fc comprises L234A and L235A mutations.
In some embodiments, the Fc has a structural mutation of Knob-in-Hole.
In some embodiments, the fusion protein is a homodimer or a heterodimer.
In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain and the second polypeptide chain comprising a homodimer or a heterodimer.
In some embodiments, the first polypeptide chain comprises a first Fc domain, the second polypeptide chain comprises a second Fc domain, thereby constituting a dimeric Fc region, and the first polypeptide chain and/or the second polypeptide chain comprises Annexin A5, an active fragment thereof, or a variant thereof that retains activity.
In some embodiments, the first polypeptide chain further comprises the first domain, and/or the second domain.
In some embodiments, the second polypeptide chain further comprises the first domain, and/or the second domain.
In some embodiments, the first domain and the second domain in the first polypeptide chain are linked to each other directly or through a linking peptide.
In some embodiments, the one or more Annexin A5 in the first polypeptide chain, an active fragment thereof, or a variant thereof that remains active; and the one or more permeant peptides, active fragments thereof, or variants thereof that remain active, are linked to each other either directly or through a linking peptide.
In some embodiments, the first domain and the second domain in the second polypeptide chain are linked to each other directly or through a linking peptide.
In some embodiments, the one or more Annexin A5 in the second polypeptide chain, an active fragment thereof, or a variant thereof that remains active; and the one or more permeant peptides, active fragments thereof, or variants thereof that remain active, are linked to each other either directly or through a linking peptide.
In some embodiments, the first polypeptide chain, the C-terminus or N-terminus of the first domain, and/or the C-terminus or N-terminus of the second domain, is linked directly or via a linking peptide to the N-terminus or C-terminus of the third domain.
In some embodiments, the C-terminus or N-terminus of the first domain, and/or the C-terminus or N-terminus of the second domain, in the second polypeptide chain, is linked directly or through a linking peptide to the N-terminus or C-terminus of the third domain.
In some embodiments, the first polypeptide chain comprises a structure selected from the group consisting of:
(a) A first Fc domain;
(b) A first Fc domain and Annexin A5;
(c) A first Fc domain and a transmembrane peptide;
(d) A first Fc domain, annexin A5 and a permeabilizing peptide;
the individual moieties are linked directly or via a linker peptide.
In some embodiments, the second polypeptide chain comprises a structure selected from the group consisting of:
(a) A second Fc domain;
(b) A second Fc domain and Annexin A5;
(c) A second Fc domain and a transmembrane peptide;
(d) A second Fc domain, annexin A5 and a permeabilizing peptide;
the individual moieties are linked directly or via a linker peptide.
In some embodiments, the first polypeptide chain comprises a structure selected from the group consisting of:
(a) A first Fc domain;
(b) Comprising, in order from the C-terminus to the N-terminus, a first Fc domain and Annexin A5, and vice versa;
(c) Comprising, in order from the C-terminus to the N-terminus, a first Fc domain and a permeabilizing peptide, and vice versa;
(d) Comprising, in order from the C-terminus to the N-terminus, a first Fc domain, annexin A5 and a permeabilizing peptide; or a first Fc domain, a permeabilizing peptide and Annexin A5; or a permeabilizing peptide, a first Fc domain and Annexin A5; vice versa;
the individual moieties are linked directly or via a linker peptide.
In some embodiments, the second polypeptide chain comprises a structure selected from the group consisting of:
(a) A second Fc domain;
(b) Comprising, in order from the C-terminus to the N-terminus, a second Fc domain and Annexin A5, and vice versa;
(c) Comprising, in order from the C-terminus to the N-terminus, a second Fc domain and a permeabilizing peptide, and vice versa;
(d) Comprising, in order from the C-terminus to the N-terminus, a second Fc domain, annexin A5 and a permeabilizing peptide; or a second Fc domain, a permeabilizing peptide and Annexin A5; or a permeabilizing peptide, a second Fc domain and Annexin A5; vice versa;
the individual moieties are linked directly or via a linker peptide.
In some embodiments, the fusion protein has a structure selected from the group consisting of:
(a) The first polypeptide chain comprises a first Fc domain, and the second polypeptide chain comprises, in order from C-terminus to N-terminus, a second Fc domain, a connecting peptide, annexin A5, and a permeabilizing peptide, thereby forming a heterodimer;
(b) The first polypeptide chain comprises, in order from the C-terminus to the N-terminus, a first Fc domain, a connecting peptide, annexin A5, and a transmembrane peptide, and the second polypeptide chain comprises a second Fc domain, thereby forming a heterodimer;
(c) The first polypeptide chain comprises a transmembrane peptide, annexin A5, a connecting peptide and a first Fc domain in sequence from the C-terminal end to the N-terminal end, and the second polypeptide chain comprises a second Fc domain, thereby forming a heterodimer;
(d) The first polypeptide chain comprises a first Fc domain, and the second polypeptide chain comprises a permeabilizing peptide, a linker peptide and a second Fc domain in sequence from the C-terminus to the N-terminus, thereby forming a heterodimer;
(e) The first polypeptide chain comprises a transmembrane peptide and a first Fc domain in sequence from the C-terminal end to the N-terminal end, and the second polypeptide chain comprises Annexin A5, a connecting peptide and a second Fc domain in sequence from the C-terminal end to the N-terminal end, so that a heterodimer is formed;
(f) The first polypeptide chain comprises Annexin A5, a connecting peptide and a first Fc domain from the C end to the N end in sequence, and the second polypeptide chain comprises a permeabilizing peptide and a second Fc domain from the C end to the N end in sequence, so that a heterodimer is formed;
(g) The first polypeptide chain comprises a first Fc domain and a transmembrane peptide in sequence from the C-terminal end to the N-terminal end, and the second polypeptide chain comprises a second Fc domain, a connecting peptide and Annexin A5 in sequence from the C-terminal end to the N-terminal end, so that a heterodimer is formed;
(h) The first polypeptide chain comprises a first Fc structural domain, a connecting peptide and Annexin A5 from the C end to the N end in sequence, and the second polypeptide chain comprises a second Fc structural domain and a transmembrane peptide from the C end to the N end in sequence, so that a heterodimer is formed;
(i) The first polypeptide chain comprises a transmembrane peptide and a first Fc domain in sequence from the C-terminal end to the N-terminal end, and the second polypeptide chain comprises a second Fc domain, a connecting peptide and Annexin A5 in sequence from the C-terminal end to the N-terminal end, so that a heterodimer is formed;
(j) The first polypeptide chain comprises, in order from the C-terminus to the N-terminus, a transmembrane peptide, a first Fc domain, a linker peptide and Annexin A5, and the second polypeptide chain comprises a second Fc domain, thereby forming a heterodimer;
(k) The first polypeptide chain comprises a transmembrane peptide, a first Fc domain, a connecting peptide and Annexin A5 from the C end to the N end in sequence, and the second polypeptide chain comprises a transmembrane peptide, a second Fc domain, a connecting peptide and Annexin A5 from the C end to the N end in sequence, so that a homodimer is formed;
(l) The first polypeptide chain comprises a transmembrane peptide, a first Fc domain, a connecting peptide and Annexin A5 from the C end to the N end in sequence, and the second polypeptide chain comprises Annexin A5, a connecting peptide, a second Fc domain and a transmembrane peptide from the C end to the N end in sequence, so that a heterodimer is formed;
(m) a first polypeptide chain comprising Annexin A5, a linker peptide, a first Fc domain and a transmembrane peptide in that order from the C-terminus to the N-terminus, and a second polypeptide chain comprising Annexin A5, a linker peptide, a second Fc domain and a transmembrane peptide in that order from the C-terminus to the N-terminus, thereby forming a homodimer;
(N) the first polypeptide chain comprises, in order from the C-terminus to the N-terminus, a first Fc domain, a linker peptide, annexin A5 and a permeabilizing peptide, and the second polypeptide chain comprises, in order from the C-terminus to the N-terminus, a second Fc domain, a linker peptide, annexin A5 and a permeabilizing peptide, thereby forming a homodimer;
(o) the first polypeptide chain comprises, in order from the C-terminus to the N-terminus, a first Fc domain, a linker peptide, annexin A5 and a permeabilizing peptide, and the second polypeptide chain comprises, in order from the C-terminus to the N-terminus, a permeabilizing peptide, annexin A5, a linker peptide and a second Fc domain, and thereby comprises a heterodimer;
(p) a first polypeptide chain comprising, in order from the C-terminus to the N-terminus, a permeabilizing peptide, annexin A5, a linker peptide and a first Fc domain, and a second polypeptide chain comprising, in order from the C-terminus to the N-terminus, a permeabilizing peptide, annexin A5, a linker peptide and a second Fc domain, thereby forming a homodimer;
(q) a first polypeptide chain comprising Annexin A5 and a first Fc domain in sequence from C-terminus to N-terminus, and a second polypeptide chain comprising a permeabilizing peptide and a second Fc domain in sequence from C-terminus to N-terminus, thereby forming a heterodimer;
(r) a first polypeptide chain comprising Annexin A5, a first Fc domain and a permeabilizing peptide in order from C-terminus to N-terminus, and a second polypeptide chain comprising Annexin A5, a second Fc domain and a permeabilizing peptide in order from C-terminus to N-terminus, thereby forming a homodimer;
(s) the first polypeptide chain comprises a permeant peptide, annexin A5 and a first Fc domain in order from the C-terminus to the N-terminus, and the second polypeptide chain comprises a permeant peptide, annexin A5 and a second Fc domain in order from the C-terminus to the N-terminus, thereby forming a homodimer.
In a second aspect, the application provides an isolated nucleic acid encoding the fusion protein of the first aspect.
In a third aspect, the application provides an expression vector comprising the isolated nucleic acid of the second aspect.
The expression vector may be a eukaryotic or prokaryotic expression vector, preferably a plasmid. The expression vector may also be a viral vector, such as an adenovirus vector or a lentiviral vector.
In a fourth aspect, the application provides a host cell comprising the isolated nucleic acid of the second aspect, or the expression vector of the third aspect.
In a fifth aspect, the present application provides a method of preparing a fusion protein of the first aspect using the isolated nucleic acid of the second aspect, the expression vector of the third aspect or the host cell of the fourth aspect.
In a sixth aspect, the application provides a pharmaceutical composition comprising the fusion protein of the first aspect, the isolated nucleic acid of the second aspect or the expression vector of the third aspect, and a pharmaceutically acceptable carrier.
Preferably, the pharmaceutical composition is an aqueous solution, non-aqueous solution or suspension, and the pharmaceutically acceptable carrier may be any carrier conventional in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents and the like.
The route of administration of the pharmaceutical composition may be parenteral, injectable or oral. The pharmaceutical compositions may be prepared in a form suitable for administration, for example in solid, semi-solid or liquid form, and may be in the form of an aqueous solution, non-aqueous solution or suspension, powder, tablet, capsule, granule, injection or infusion. Can be administered by intravascular, subcutaneous, intraperitoneal, intramuscular, inhalation, intranasal, airway instillation, or intrathoracic instillation. The pharmaceutical composition may also be administered in the form of an aerosol or spray, for example, nasally; alternatively, intrathecal, intramedullary or intraventricular administration may also be transdermal, percutaneous, topical, enteral, intravaginal, sublingual or rectal administration. The pharmaceutical composition can be prepared into various dosage forms according to the needs, and the dosage beneficial to the patient can be determined by doctors according to the type, age, weight and general disease condition of the patient, the administration mode and other factors.
In a seventh aspect, the present application provides the use of a fusion protein of the first aspect, an isolated nucleic acid of the second aspect, an expression vector of the third aspect, a host cell of the fourth aspect, or a pharmaceutical composition of the sixth aspect in the manufacture of a medicament for the prevention, treatment and/or diagnosis of an autoimmune disease.
In some embodiments, the autoimmune disease includes, but is not limited to juvenile dermatomyositis, atopic dermatitis, giant cell arteritis, polyarteritis, kawasaki disease, shingles, auto-inflammatory disease, aicarpi-Gouti res syndrome, alopecia areata, alopecia totalis, alopecia universalis, androgenetic alopecia, behcet's disease, diabetic kidney disease, dry eye, autoimmune uveitis, pruritis, psoriasis, plaque psoriasis, SVAI disease, scleroderma, vitiligo, chronic beryllium dust disease, palmoplantar pustule disease, autoimmune hepatitis, graves ' disease, graves-Barbis syndrome, hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, and inflammatory myopathy.
Preferably, the autoimmune disease is psoriasis.
Drawings
FIG. 1 shows a schematic structure of fusion protein A5/TAT-FC.
FIG. 2 shows a FITC-labeled fusion protein permeabilization assay using RAW264.7 as an example.
FIG. 3 shows qPCR detection of the effect of Annexin A5 derivatives on macrophages.
Figure 4 shows that R9-A5 is effective in alleviating imiquimod-induced psoriasis symptoms.
FIG. 5 shows the efficacy and weight change of R9-A5 compared to A5.
Detailed Description
The application provides a fusion protein of Annexin A5 and permeabilizing peptide, which can be used for treating autoimmune diseases and shows increased intracellular drug concentration, thereby enhancing the drug effect of the drug.
In some embodiments, the fusion protein comprises: an immunoglobulin Fc region having a hinge region, a CH2 domain, and a CH3 domain; annexin A5 and a permeant peptide, wherein Annexin A5 and permeant peptide can be connected at the same end of an immunoglobulin Fc region or respectively connected at two ends of the Fc region, or permeant peptide is connected at the N-terminal end of Annexin A5, the C-terminal end of Annexin A5 is connected at the hinge region or the CH3 region terminal end of the immunoglobulin Fc region, and one Fc region can be connected with one Annexin A5 or two Annexin A5 to form a dimer; the number of the permeabilizing peptides may be 1 or more.
The "N-terminus" referred to herein is the amino-terminus of a peptide or protein and, for the purposes of the present application, refers to a site capable of binding to a linker. For example, an amino acid residue near the N-terminus and a terminal-most amino acid residue at the N-terminus may be both included, and specifically, the first to tenth amino acid residues from the terminal-most region may be included, but the site of the amino acid residues is not particularly limited thereto.
The "C-terminal" referred to herein is the carboxy-terminal end of a peptide or protein and, for the purposes of the present application, refers to a site capable of binding a linker. For example, an amino acid residue near the C-terminus and a terminal-most amino acid residue at the C-terminus may be both included, and specifically, the first to tenth amino acid residues from the terminal-most may be included, but the site of the amino acid residues is not particularly limited thereto.
The "permeant peptides" referred to herein, alone or in combination with a drug, carry across most cell membranes and in certain embodiments include, but are not limited to, one or more of the following: TAT, R9, pennetrate, 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, hLock (35-47), K-FGF, mouse PrP.sup.c (1-28), synbl, HN-1, pSL.
In some embodiments, the permeant peptide is selected from, but is not limited to, any one of the following: SEQ ID NO:17, the TAT shown in SEQ ID NO:18, R9, SEQ ID NO:19, ARF (1-22) shown in SEQ ID NO. 20, P28 shown in SEQ ID NO. 21, CADY shown in SEQ ID NO. 22, R5 shown in SEQ ID NO. 24, R6 shown in SEQ ID NO. 25, R7 shown in SEQ ID NO. 26, R8 shown in SEQ ID NO. 27, R10 shown in SEQ ID NO. 28, R11 shown in SEQ ID NO. 29, BPrPr (1-28) shown in SEQ ID NO. 30, VT5 shown in SEQ ID NO. 31, bac7 shown in SEQ ID NO. 32, trasportan shown in SEQ ID NO. 33, pep-1 shown in SEQ ID NO. 34, trasportan 10 shown in SEQ ID NO. 36, VP22 shown in SEQ ID NO. 37, gH625 shown in SEQ ID NO. 38, INF shown in SEQ ID NO. 39, VP 5 shown in SEQ ID NO. 40, VP 7 shown in SEQ ID NO. 40, and clip NO. 48, P7 shown in SEQ ID NO. 48, P1-1 shown in SEQ ID NO. 33, pep-1, SEQ ID NO. 35 shown in SEQ ID NO. 48, hIV shown in SEQ ID NO. 48, P5, P1-48 shown in SEQ ID NO. 48, hC shown in SEQ ID NO. 48, hIV, 5, SEQ ID NO. 48, 5 shown in SEQ ID NO. 48, 5, SEQ ID NO. 35.
In certain embodiments of the application, "Fc fusion protein of Annexin A5" is used interchangeably with "Fc fusion protein" and "fusion protein".
The fusion proteins of the application are useful for the treatment of autoimmune diseases including, but not limited to, juvenile dermatomyositis, atopic dermatitis, giant cell arteritis, polyarteritis, kawasaki disease, shingles, auto-inflammatory disease, aicarpi-Gouti res syndrome, alopecia areata, alopecia totalis, alopecia universalis, androgenetic alopecia, behcet's disease, diabetic kidney disease, dry eye, autoimmune uveitis, pruritis, psoriasis, plaque psoriasis, SVAI disease, scleroderma, vitiligo, chronic beryllium dust disease, palmoplantar pustule disease, autoimmune hepatitis, graves ' disease, graves-Barbis syndrome, hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, and inflammatory myopathy.
Preferably, the autoimmune disease is psoriasis.
Therefore, the application has the advantages that the membrane-penetrating peptide is combined with Annexin A5 to form a novel fusion protein, thus the application has the advantages of increasing the membrane-penetrating efficiency of the medicine, increasing the medicine delivery quantity at the focus part and having better medicine effect than Annexin A5 under the condition of treating certain diseases. The fusion protein comprising the Fc domain can also prolong the half-life of the drug in vivo, prolong the administration time, improve the compliance of patients, and is particularly suitable for the treatment and prevention of chronic diseases and other diseases requiring long-term administration.
In the following specific examples, embodiments of the present application will be described in more detail. However, the following examples are merely illustrative, that is, the present application is not limited by the following examples.
Example 1 preparation of fusion proteins
In this example, the preparation of fusion proteins was performed using A5, R9-A5 and A5/TAT-FC as exemplary protocols, wherein R9-A5 was prepared by S.sp.sp.coli pET30a was constructed and cultured in a vector construction protocol comprising: the gene sequence and protein sequence of the fusion protein of the initiation codon, the HIS tag, the TEV cleavage site, the permeabilizing peptide and Annexin A5 are shown in Table 1.
The A5/TAT-FC entrusted aurora is prepared, the construction and culture system of the Fc fusion protein of Annexin A5 is HD CHO cells, and the carrier construction scheme comprises the following steps: ecoRI-Kozak sequence-artificial signal peptide-targeting protein-stop codon-HindIII, the specific structure is shown in figure 1; the gene sequence and protein sequence of the fusion protein are shown in Table 1 below.
TABLE 1 information on the preparation of fusion proteins
Example 2 Annexin A5 fusion protein in vitro transmembrane effects (peripheral System)
Since Annexin A5 regulates the external Zhou Kangyan to exert an effect by entering a signal pathway in an intracellular regulating cell, the embodiment is mainly aimed at RAW264.7 (macrophage), and compared with Annexin A5 fusion protein provided by the application, the membrane permeation effect is better than that of Annexin A5, so that the anti-inflammatory effect can be better exerted. The specific method and the results are as follows:
the method comprises the following steps: RAW264.7 cells were plated at a cell density of 3X 10 4 After cells are basically full, 1uM of A5, TAT-A5 and R9-A5 are respectively added, living cell nuclei are stained with hoechst 33342 (Biyun) after 24 hours, and finally fluorescence intensity in the cells is observed by a fluorescence microscope.
Results: as shown in FIG. 2, the fluorescence intensity of the FITC of TAT-A5 and R9-A5 in cells is obviously higher than that of the FITC of A5, which indicates that the permeabilizing peptide has a certain permeabilizing effect and is the increase of the amount of fusion protein in cells. Wherein, neither A5, TAT-A5 nor R9-A5 has an Fc region.
EXAMPLE 3 Effect of annexin A5 fusion proteins on macrophage transformation from M1 (pro-inflammatory) to M2 (anti-inflammatory)
The purpose of this example was to evaluate the anti-inflammatory effect of Annexin A5 fusion proteins. The specific method and results are as follows:
the method comprises the following steps: RAW264.7 cells (resting state is represented by N) are selected, after differentiation into M1 type and M2 type under certain conditions, 200nM TAT-A5, R9-A5 and A5/TAT-FC are respectively added, the cells are collected after 24 hours, and the relative expression amounts of IL-1 beta, IL-6 and TNF-alpha expressed by M1 type and CD206, fizz1 and Ym1 expressed by M2 type are detected by a qPCR method. Wherein the stimulation condition for differentiation into M1 type is that 10ng/mL LPS and 10ng/mL IFN-gamma are added into Raw264.7 cells for 6 hours; the stimulation conditions for differentiation into M2 form were that IL-4 was added to Raw264.7 cells at 20ng/mL for 6h. The primers used in the qPCR experiments were as follows:
IL-1β：
5'-CTTCAGGCAGGCAGTATCACTC-3' (SEQ ID NO: 57) (upstream)
5'-TGCAGTTGTCTAATGGGAACGT-3' (SEQ ID NO: 58) (downstream);
IL-6：
5'-ACAACCACGGCCTTCCCTAC-3' (SEQ ID NO: 59) (upstream)
5'-TCTCATTTCCACGATTTCCCAG-3' (SEQ ID NO: 60) (downstream);
TNF-α：
5'-CGAGTGACAAGCCTGTAGCCC-3' (SEQ ID NO: 61) (upstream)
5'-GTCTTTGAGATCCATGCCGTTG-3' (SEQ ID NO: 62) (downstream);
CD206：
5'-GCAGGTGGTTTATGGGATGT-3' (SEQ ID NO: 63) (upstream)
5'-GGGTTCAGGAGTGTTGTGG-3' (SEQ ID NO: 64) (downstream);
Fizz1:
5'-AGGAGCTGTCATTAGGGACATC-3' (SEQ ID NO: 65) (upstream)
5'-GGATGCCAACTTTGAATAGG-3' (SEQ ID NO: 66) (downstream);
Ym1:
5'-AGAAGGGAGTTTCAAACCTGGT-3' (SEQ ID NO: 67) (upstream)
5'-GTCTTGCTCATGTGTGTAAGTGA-3' (SEQ ID NO: 68) (downstream);
results: as shown in FIG. 3, TAT-A5, R9-A5 and A5/TAT-FC all have strong anti-inflammatory effects and can promote the conversion of M1 type to M2 type. Wherein, neither A5, TAT-A5 nor R9-A5 has an Fc region, and A5/TAT-FC has an Fc region.
EXAMPLE 4 anti-inflammatory mechanism of annexin A5 fusion protein efficacy against autoimmune diseases
Mice were counted: 25 pieces of
Animal model: BALB/c mice, 7-8 weeks of week old
The molding method comprises the following steps: shaving the back of the BALB/c mouse to remove most hair, removing Mao Gao and removing fine hair, cleaning with clear water, wiping, then dosing according to groups, uniformly coating about 62.5mg of imiquimod cream on the back of the mouse after 1h for molding psoriasis, weighing, photographing and PASI scoring respectively before dosing for 7 days;
the specific grouping is as follows:
1. control group: BALB/c mice, 5, 7-8 weeks of week old, were dosed: normal saline, 1 time a day, subcutaneously injected;
2. model group: BALB/c mice, 5, 7-8 weeks of week old, were dosed: normal saline, 1 time a day, subcutaneously injected;
a5 group: BALB/c mice, 5, 7-8 weeks of week old, were dosed: a5 5mg/kg,1 time a day, subcutaneously;
group r 9-A5: BALB/c mice, 5, 7-8 weeks of week old, were dosed: R9-A5 5mg/kg,1 time a day, subcutaneously;
A5/TAT-FC group: BALB/c mice, 5, 7-8 weeks of week old, were dosed: A5/TAT-FC 5mg/kg,1 day, subcutaneously.
Referring to FIG. 4, it was observed that R9-A5 and A5/TAT-FC were effective in alleviating imiquimod-induced skin thickening, erythema, scaling and other psoriasis-like symptoms in mice. Referring to FIGS. 4 and 5, it can be seen that R9-A5 and A5/TAT-FC have significantly increased efficacy over A5, and that R9-A5 is effective in reducing weight loss due to morbidity in terms of body weight. Wherein neither A5 nor R9-A5 has an Fc region, and A5/TAT-FC has an Fc region.
Claims (19)
1. A fusion protein comprising Annexin A5 and one or more permeant peptides.
2. The fusion protein according to claim 1, comprising a first domain comprising one or more Annexin A5, active fragments thereof, or variants thereof which remain active,
preferably, the plurality of Annexin A5, active fragments thereof, or variants thereof that remain active are linked in tandem, or are spaced apart from each other.
3. The fusion protein according to claim 1, further comprising a second domain comprising one or more permeabilizing peptides, active fragments thereof, or variants thereof which remain active,
the plurality of permeant peptides, active fragments thereof, or variants thereof that remain active may be the same or different from each other,
the plurality of permeant peptides, active fragments thereof, or variants thereof that remain active are linked in series, or are spaced apart from each other.
4. A fusion protein according to claim 2 or 3, wherein the first domain and the second domain are linked directly or via a linker peptide, or
The one or more Annexin A5, active fragments thereof, or variants thereof that remain active; and the one or more permeabilizing peptides, active fragments thereof, or variants thereof which remain active, are linked to each other either directly or through a linker peptide,
preferably, the linker peptide has an amino acid sequence selected from the group consisting of:
(GS) n n is 1-5;
(GGGGS) n n is 1-5;
(GGGGS) n (GS) m n is 1-5, and m is 1-5;
(EAAAK) n n is 1-5;
A(EAAAK) n a, n is 1-5;
(GGGGS) n (EAAAK) m (GGGGS) p the n is 1-5, the m is 1-5, and the p is 0-5；
(EAAAK) m (GGGGS) n (GGGGS) p M is 1-5, n is 1-5, and p is 0-5;
(GGGGS) n (PPPPP) n (GGGGS) p m is 1-5, n is 1-5, and p is 0-5;
EPKSSDKTHTPPPPP(SEQ ID NO:2)；
DKTHTCPPCP(SEQ ID NO:3)；
GGGGGDKTHTCPPCP(SEQ ID NO:4)；
EPKSSDKTHTPPPPPRT(SEQ ID NO:5)；
LGGGGSGGGGSGGGGSRT(SEQ ID NO:6)；
LEPKSSDKTHTPPPPPRT(SEQ ID NO:7)；
KLEPKSSDKTHTPPPPPRT(SEQ ID NO:8)；
RTGGGGSKL(SEQ ID NO:9)；
RTGGGGSGGGGSGGGGSKL(SEQ ID NO:10)；
RTGGGGSGGGGSGGGGSGGGGSGGGGSKL(SEQ ID NO:11)；
EPKSCDKTHTCPPCP(SEQ ID NO:12)；
EPKSSDKTHTCPPCP(SEQ ID NO:13)；
ERKCCVECPPCP(SEQ ID NO:14)；
ESKYGPPCPSCP (SEQ ID NO: 15); or (b)
ESKYGPPCPPCP(SEQ ID NO:16)。
5. The fusion protein according to claim 1, having a structure selected from the group consisting of:
R n1 -B n2 ，
R n1 -B n2 -R n3 ，
B n2 -R n1 -B n3 ，
wherein R is a permeabilizing peptide, an active fragment thereof, or a variant thereof that retains activity; b is Annexin A5, an active fragment thereof, or a variant thereof which retains activity, 0.ltoreq.n1.ltoreq.10, 0.ltoreq.n2.ltoreq.10, and 0.ltoreq.n3.ltoreq.10,
preferably, 0.ltoreq.n1.ltoreq.5, 0.ltoreq.n2.ltoreq.5, and 0.ltoreq.n3.ltoreq.5,
more preferably, 0.ltoreq.n1.ltoreq.3, 0.ltoreq.n2.ltoreq.3, and 0.ltoreq.n3.ltoreq.3,
more preferably, n3=1.
6. A fusion protein according to claim 2 or 3 or claim 5, wherein the permeant peptide is selected from any one of the following: TAT, R9, pennetrate, 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, hLock (35-47), K-FGF, mouse PrP.sup.c (1-28), synbl, HN-1, pSL;
the Annexin A5 is a human Annexin A5 or a mammalian ortholog, allelic or genetic variant or functional analogue of Annexin A5, or a variant having more than 70%, e.g. more than 80% or more than 90%, even more than 95% identity but retaining activity with human Annexin A5;
preferably, the Annexin A5 is an Annexin A5 having the amino acid sequence shown in SEQ ID NO. 1, or a variant having 70% or more, 80% or more, 90% or more, or 95% or more identity but retaining activity with an Annexin A5 having the amino acid sequence shown in SEQ ID NO. 1.
7. The fusion protein according to claim 1, further comprising a third domain comprising an Fc region of an immunoglobulin,
preferably, the immunoglobulin is selected from IgA, igG, igM, igD and IgE, preferably IgG, more preferably IgG1, igG2, igG3 or IgG4,
preferably, the Fc is that of human IgG1, or the Fc comprises an L234A, L235A and/or P329G mutation; or alternatively
The Fc is that of human IgG4, or the Fc comprises L234A and L235A mutations.
8. The fusion protein according to claim 1, which is a homodimer or a heterodimer,
the fusion protein comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain and the second polypeptide chain constituting a homodimer or a heterodimer,
the first polypeptide chain comprises a first Fc domain, the second polypeptide chain comprises a second Fc domain, thereby constituting a dimeric Fc region, and the first polypeptide chain and/or the second polypeptide chain comprises Annexin A5, an active fragment thereof, or a variant thereof that retains activity.
9. The fusion protein according to claim 8, wherein said first polypeptide chain further comprises said first domain and/or said second domain,
the second polypeptide chain further comprises the first domain, and/or the second domain,
preferably, the first domain and the second domain in the first polypeptide chain are linked directly or via a linker peptide, or
Said one or more Annexin A5 in said first polypeptide chain, an active fragment thereof, or a variant thereof that retains activity; and the one or more permeabilizing peptides, active fragments thereof, or variants thereof that remain active, are linked to each other either directly or through a linking peptide;
preferably, the first domain and the second domain in the second polypeptide chain are linked directly or via a linker peptide, or
Said one or more Annexin A5 in said second polypeptide chain, an active fragment thereof, or a variant thereof that retains activity; and the one or more permeabilizing peptides, active fragments thereof, or variants thereof that remain active, are linked to each other either directly or through a linking peptide;
preferably, in the first polypeptide chain, the C-terminus or N-terminus of the first domain, and/or the C-terminus or N-terminus of the second domain is linked directly or via a linking peptide to the N-terminus or C-terminus of the third domain;
preferably, in the second polypeptide chain, the C-terminus or N-terminus of the first domain, and/or the C-terminus or N-terminus of the second domain is linked directly or via a linking peptide to the N-terminus or C-terminus of the third domain.
10. The fusion protein according to claim 8, wherein said first polypeptide chain comprises a structure selected from the group consisting of:
(a) A first Fc domain;
(b) A first Fc domain and Annexin A5;
(c) A first Fc domain and a transmembrane peptide;
(d) A first Fc domain, annexin A5 and a permeabilizing peptide;
the parts are directly connected or connected through connecting peptide;
the second polypeptide chain comprises a structure selected from the group consisting of:
(a) A second Fc domain;
(b) A second Fc domain and Annexin A5;
(c) A second Fc domain and a transmembrane peptide;
(d) A second Fc domain, annexin A5 and a permeabilizing peptide;
the individual moieties are linked directly or via a linker peptide.
11. The fusion protein according to claim 8, wherein said first polypeptide chain comprises a structure selected from the group consisting of:
(a) A first Fc domain;
(b) Comprising, in order from the C-terminus to the N-terminus, a first Fc domain and Annexin A5, and vice versa;
(c) Comprising, in order from the C-terminus to the N-terminus, a first Fc domain and a permeabilizing peptide, and vice versa;
(d) Comprising, in order from the C-terminus to the N-terminus, a first Fc domain, annexin A5 and a permeabilizing peptide; or a first Fc domain, a permeabilizing peptide and Annexin A5; or a permeabilizing peptide, a first Fc domain and Annexin A5, and vice versa;
the parts are directly connected or connected through connecting peptide;
the second polypeptide chain comprises a structure selected from the group consisting of:
(a) A second Fc domain;
(b) Comprising, in order from the C-terminus to the N-terminus, a second Fc domain and Annexin A5, and vice versa;
(c) Comprising, in order from the C-terminus to the N-terminus, a second Fc domain and a permeabilizing peptide, and vice versa;
(d) Comprising, in order from the C-terminus to the N-terminus, a second Fc domain, annexin A5 and a permeabilizing peptide; or a second Fc domain, a permeabilizing peptide and Annexin A5; or a permeabilizing peptide, a second Fc domain and Annexin A5, and vice versa;
the individual moieties are linked directly or via a linker peptide.
12. The fusion protein according to claim 8, having a structure selected from the group consisting of:
(a) The first polypeptide chain comprises a first Fc domain, and the second polypeptide chain comprises, in order from C-terminus to N-terminus, a second Fc domain, a connecting peptide, annexin A5, and a permeabilizing peptide, thereby forming a heterodimer;
(b) The first polypeptide chain comprises, in order from the C-terminus to the N-terminus, a first Fc domain, a connecting peptide, annexin A5, and a transmembrane peptide, and the second polypeptide chain comprises a second Fc domain, thereby forming a heterodimer;
(c) The first polypeptide chain comprises a transmembrane peptide, annexin A5, a connecting peptide and a first Fc domain in sequence from the C-terminal end to the N-terminal end, and the second polypeptide chain comprises a second Fc domain, thereby forming a heterodimer;
(d) The first polypeptide chain comprises a first Fc domain, and the second polypeptide chain comprises a permeabilizing peptide, a linker peptide and a second Fc domain in sequence from the C-terminus to the N-terminus, thereby forming a heterodimer;
(e) The first polypeptide chain comprises a transmembrane peptide and a first Fc domain in sequence from the C-terminal end to the N-terminal end, and the second polypeptide chain comprises Annexin A5, a connecting peptide and a second Fc domain in sequence from the C-terminal end to the N-terminal end, so that a heterodimer is formed;
(f) The first polypeptide chain comprises Annexin A5, a connecting peptide and a first Fc domain from the C end to the N end in sequence, and the second polypeptide chain comprises a permeabilizing peptide and a second Fc domain from the C end to the N end in sequence, so that a heterodimer is formed;
(g) The first polypeptide chain comprises a first Fc domain and a transmembrane peptide in sequence from the C-terminal end to the N-terminal end, and the second polypeptide chain comprises a second Fc domain, a connecting peptide and Annexin A5 in sequence from the C-terminal end to the N-terminal end, so that a heterodimer is formed;
(h) The first polypeptide chain comprises a first Fc structural domain, a connecting peptide and Annexin A5 from the C end to the N end in sequence, and the second polypeptide chain comprises a second Fc structural domain and a transmembrane peptide from the C end to the N end in sequence, so that a heterodimer is formed;
(i) The first polypeptide chain comprises a transmembrane peptide and a first Fc domain in sequence from the C-terminal end to the N-terminal end, and the second polypeptide chain comprises a second Fc domain, a connecting peptide and Annexin A5 in sequence from the C-terminal end to the N-terminal end, so that a heterodimer is formed;
(j) The first polypeptide chain comprises, in order from the C-terminus to the N-terminus, a transmembrane peptide, a first Fc domain, a linker peptide and Annexin A5, and the second polypeptide chain comprises a second Fc domain, thereby forming a heterodimer;
(k) The first polypeptide chain comprises a transmembrane peptide, a first Fc domain, a connecting peptide and Annexin A5 from the C end to the N end in sequence, and the second polypeptide chain comprises a transmembrane peptide, a second Fc domain, a connecting peptide and Annexin A5 from the C end to the N end in sequence, so that a homodimer is formed;
(l) The first polypeptide chain comprises a transmembrane peptide, a first Fc domain, a connecting peptide and Annexin A5 from the C end to the N end in sequence, and the second polypeptide chain comprises Annexin A5, a connecting peptide, a second Fc domain and a transmembrane peptide from the C end to the N end in sequence, so that a heterodimer is formed;
(m) a first polypeptide chain comprising Annexin A5, a linker peptide, a first Fc domain and a transmembrane peptide in that order from the C-terminus to the N-terminus, and a second polypeptide chain comprising Annexin A5, a linker peptide, a second Fc domain and a transmembrane peptide in that order from the C-terminus to the N-terminus, thereby forming a homodimer;
(N) the first polypeptide chain comprises, in order from the C-terminus to the N-terminus, a first Fc domain, a linker peptide, annexin A5 and a permeabilizing peptide, and the second polypeptide chain comprises, in order from the C-terminus to the N-terminus, a second Fc domain, a linker peptide, annexin A5 and a permeabilizing peptide, thereby forming a homodimer;
(o) the first polypeptide chain comprises, in order from the C-terminus to the N-terminus, a first Fc domain, a linker peptide, annexin A5 and a permeabilizing peptide, and the second polypeptide chain comprises, in order from the C-terminus to the N-terminus, a permeabilizing peptide, annexin A5, a linker peptide and a second Fc domain, and thereby comprises a heterodimer;
(p) a first polypeptide chain comprising, in order from the C-terminus to the N-terminus, a permeabilizing peptide, annexin A5, a linker peptide and a first Fc domain, and a second polypeptide chain comprising, in order from the C-terminus to the N-terminus, a permeabilizing peptide, annexin A5, a linker peptide and a second Fc domain, thereby forming a homodimer;
(q) a first polypeptide chain comprising Annexin A5 and a first Fc domain in sequence from C-terminus to N-terminus, and a second polypeptide chain comprising a permeabilizing peptide and a second Fc domain in sequence from C-terminus to N-terminus, thereby forming a heterodimer;
(r) a first polypeptide chain comprising Annexin A5, a first Fc domain and a permeabilizing peptide in order from C-terminus to N-terminus, and a second polypeptide chain comprising Annexin A5, a second Fc domain and a permeabilizing peptide in order from C-terminus to N-terminus, thereby forming a homodimer;
(s) the first polypeptide chain comprises a permeant peptide, annexin A5 and a first Fc domain in order from the C-terminus to the N-terminus, and the second polypeptide chain comprises a permeant peptide, annexin A5 and a second Fc domain in order from the C-terminus to the N-terminus, thereby forming a homodimer.
13. An isolated nucleic acid encoding the fusion protein of any one of claims 1-12.
14. An expression vector comprising the isolated nucleic acid of claim 13,
the expression vector is eukaryotic cell expression vector or prokaryotic expression vector, preferably plasmid, adenovirus vector or lentiviral vector.
15. A host cell comprising the isolated nucleic acid of claim 13, or the expression vector of claim 14.
16. A method of preparing the fusion protein of any one of claims 1-12, said method comprising the step of preparing using the isolated nucleic acid of claim 13, the expression vector of claim 14 or the host cell of claim 15.
17. A pharmaceutical composition comprising the fusion protein of any one of claims 1-12, the isolated nucleic acid of claim 14 or the expression vector of claim 15, and a pharmaceutically acceptable carrier.
18. The pharmaceutical composition of claim 17, further comprising a second therapeutic agent.
19. The use of a fusion protein according to any one of claims 1 to 12, an isolated nucleic acid according to claim 13, an expression vector according to claim 14, a host cell according to claim 15, or a pharmaceutical composition according to claim 17 for the preparation of a medicament for the prophylaxis, treatment and/or diagnosis of autoimmune diseases,
preferably, the autoimmune disease is selected from: juvenile dermatomyositis, atopic dermatitis, giant cell arteritis, multiple arteritis, kawasaki disease, shingles, auto-inflammatory diseases, aicarpi-Gouti's syndrome, alopecia areata, alopecia totalis, alopecia universalis, androgenic alopecia, behcet's disease, diabetic kidney disease, dry eye, autoimmune uveitis, pruritis, psoriasis, plaque psoriasis, SVAI disease, scleroderma, vitiligo, chronic beryllium dust, palmoplantar pustulosis, autoimmune hepatitis, graves 'disease, graves-Barbis syndrome, hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, and inflammatory myopathy,
more preferably, the autoimmune disease is psoriasis.
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| CN1181099C (en) * | 2002-07-23 | 2004-12-22 | 中国人民解放军第二军医大学 | Anticoagulation and thrombolytic thrombus target fusion mA5UKB |
| CN105949326B (en) * | 2016-07-11 | 2020-08-14 | 北京师范大学 | Anticoagulant fusion protein EA and application thereof |
| CN106188309B (en) * | 2016-07-14 | 2023-05-02 | 北京师范大学 | Anticoagulation fusion protein HA and application thereof |
| EP3528832A1 (en) * | 2016-10-21 | 2019-08-28 | Westfälische Wilhelms-Universität Münster | Specific ac5 inhibitor |
| CN110684115A (en) * | 2018-07-06 | 2020-01-14 | 南京吉瑞康生物科技有限公司 | Fusion protein for identifying and marking apoptotic cells and preparation method and application thereof |
| US20230203104A1 (en) * | 2018-08-02 | 2023-06-29 | The Johns Hopkins University | Compositions comprising annexin v and hpv tumor antigen fusion polypeptides and methods for making and use |
| WO2020264181A1 (en) * | 2019-06-28 | 2020-12-30 | The Board Of Regents Of The University Of Oklahoma | Therapeutic annexin-drug conjugates and methods of use |
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