阅读说明：本技术 抑制眼部自身免疫性炎症反应的靶向生物肽的筛选及应用 (Screening and application of targeting biological peptide for inhibiting ocular autoimmune inflammatory reaction ) 是由 成璐 许迅 李京敬 牛田 *** 于 2019-02-25 设计创作，主要内容包括：本发明提供了一种抑制眼部自身免疫性炎症反应的靶向生物肽及其应用。本发明还涉及所述多肽的制法和应用以及含所述多肽的药物组合物。本发明多肽具有多种优点,例如分子量小、生产成本低、水溶性好、免疫源性小、毒副作用低及组织穿透性强等优势；并且,本发明通过EAE及EAU两种模型的研究表明,该生物肽具有显著的免疫调节、抗炎及改善视功能的作用。(The invention provides a targeting biological peptide for inhibiting an autoimmune inflammatory reaction of eyes and application thereof. The invention also relates to a preparation method and application of the polypeptide and a pharmaceutical composition containing the polypeptide. The polypeptide has multiple advantages, such as small molecular weight, low production cost, good water solubility, small immunogenicity, low toxic and side effects, strong tissue penetrability and the like; in addition, the research of two models of EAE and EAU shows that the biological peptide has obvious functions of immunoregulation, anti-inflammation and visual function improvement.)

1. A polypeptide shown as a formula I or a pharmaceutically acceptable salt thereof is characterized in that the polypeptide or the pharmaceutically acceptable salt thereof has the activity of inhibiting autoimmune inflammation;

X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16(I)

in the formula (I), the compound is shown in the specification,

x0 is nothing, or a peptide of 1-12 amino acids;

x1 is an amino acid selected from the group consisting of: cys, Ser

X2 is an amino acid selected from the group consisting of: arg, Lys, Gln, Asn;

x3 is an amino acid selected from the group consisting of: asn, Gln, His, Lys, Arg;

x4 is an amino acid selected from the group consisting of: pro, Ala;

x5 is an amino acid selected from the group consisting of: arg, Lys, Gln, Asn;

x6 is an amino acid selected from the group consisting of: gly, Pro, Ala;

x7 is an amino acid selected from the group consisting of: glu and Asp;

x8 is an amino acid selected from the group consisting of: glu and Asp;

x9 is an amino acid selected from the group consisting of: gly, Pro, Ala;

x10 is an amino acid selected from the group consisting of: gly, Pro, Ala;

x11 is an amino acid selected from the group consisting of: pro, Ala;

x12 is an amino acid selected from the group consisting of: trp, Tyr, Phe;

x13 is an amino acid selected from the group consisting of: cys, Ser;

x14 is an amino acid selected from the group consisting of: phe, Leu, Val, Ile, Ala, Tyr;

x15 is an amino acid selected from the group consisting of: thr, Ser;

x16 is nothing, or a peptide of 1-13 amino acids.

2. The polypeptide of claim 1, wherein the total length of the polypeptide is 80 amino acid residues or less, preferably 50 amino acid residues or less, and more preferably 35 amino acid residues or less.

3. The polypeptide of claim 1, wherein X0 and X16 are null.

4. The polypeptide of claim 1, wherein the amino acid sequence of said polypeptide is selected from the group consisting of: 1, 2, 3, 4,5 or 6.

5. The polypeptide of claim 1, wherein the amino acid sequence of said polypeptide is set forth in SEQ ID No. 1.

6. An isolated polynucleotide encoding the polypeptide of claim 1.

7. A vector comprising the polynucleotide of claim 6.

8. A host cell comprising the vector or genome of claim 7 integrated with the polynucleotide of claim 6.

9. A pharmaceutical composition, comprising:

(a) the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof; and

(b) a pharmaceutically acceptable carrier or excipient.

10. Use of the polypeptide of claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for inhibiting autoimmune inflammation.

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a targeting biological peptide for inhibiting an ocular autoimmune inflammatory reaction and application thereof.

Background

With the improvement of the sanitary environment, the proportion of infectious diseases in ocular inflammatory diseases is gradually reduced, and the incidence rate of autoimmune ocular diseases is increased year by year.

Autoimmune ocular diseases can be primary diseases of the eye, such as salix integra-protopanasis, white spot syndrome, etc., but are more commonly expressed as ocular manifestations of systemic autoimmune diseases, such as neuromyelitis optica (NMO) and Multiple Sclerosis (MS). Ocular autoimmune diseases can affect many parts including the ocular surface, the inner eye and the ocular appendages, with uvea and the optic nerve being most susceptible.

Autoimmune Uveitis (AU) and Autoimmune Optic Neuritis (AON) can cause severe damage to the posterior segment retina, choroid, and optic nerve due to their recurrent nature, posing a significant threat to vision. Epidemiological studies have shown that worldwide, patients with uveitis blindness account for the third of all blinding patients, while optic neuritis is the leading cause of blindness in young people. Autoimmune optic neuritis, either alone or as part of a central nervous system or systemic disease, is an important component of NMO and MS-related optic neuritis. Both AU and AON are extremely threatening to vision, so early and timely treatment is very important for improving the vision prognosis of patients, but the treatment of the AU and the AON faces some difficulties at present.

Glucocorticoids are the traditional classical treatment for the clinical treatment of autoimmune uveitis and optic neuritis, but their side effects are still not a trivial problem at present.

The application of the immunosuppressant alone or in combination is also a common means for autoimmune uveitis and optic neuritis, and the immunosuppressant which is clinically applied at present mainly comprises anti-cell metabolism medicaments, such as azathioprine, mycophenolate mofetil and the like; and calcineurin inhibitors such as cyclophosphamide, tacrolimus, cyclosporine, and the like. However, these immunosuppressive agents have inevitable toxic side effects when applied systemically or topically.

With the continuous and intensive research on the cellular and molecular mechanisms of AU and AON, bio-targeted drugs are beginning to be increasingly applied to the treatment of patients. In recent years, biological agents have been proven to have an effect of suppressing immune inflammatory responses through laboratory or clinical studies, and are used for treating various ocular autoimmune diseases to reduce the use of hormones and immunosuppressants.

However, most of these emerging targeted therapeutic drugs are biomacromolecules, which have the following disadvantages in specific applications: i) the molecular weight is large, the in vitro synthesis method is complex, the defects of complicated recombinant expression and purification process, endotoxin residue and the like exist in the preparation process, and the production cost is high; ii) strong antigenicity, which is easy to induce host immune response and aggravate immune disorder; iii) a loss of biological activity due to changes in protein conformation and modifying groups; iv) due to the particularity of the anatomical structure of the eyeball, the macromolecular compounds are difficult to pass through the blood-eye barrier and the blood-optic nerve barrier, and are required to be used by intravitreal injection or intrathecal injection, so that the popularization and the application are difficult in clinic.

Generally speaking, in the field of targeted therapy of autoimmune uveitis and optic neuritis, a therapeutic means with the characteristics of definite curative effect, low toxicity, small side effect, high cost performance and the like is still lacked. Therefore, the search for small molecule drugs with specific biological activity and biocompatibility, definite target, small toxic and side effects and low antigenicity is hopeful to penetrate various blood eye and blood optic nerve tissue barriers and improve the local bioavailability of the eyes, and is the key point for treating the autoimmune uveitis and optic neuritis of the eyes.

In summary, there is an urgent need in the art to develop a small molecule drug with specific biological activity and biocompatibility, clear target, low toxicity and side effects, low antigenicity, and hopefully penetrating various blood-eye and blood optic nerve tissue barriers to improve local bioavailability in the eye.

Disclosure of Invention

The invention aims to provide a small molecular medicine which has specific biological activity and biocompatibility, clear target, small toxic and side effect and low antigenicity, is hopeful to penetrate through various blood eye and blood optic nerve tissue barriers and improves the bioavailability of local parts of eyes.

In a first aspect of the invention, there is provided a polypeptide represented by formula I or a pharmaceutically acceptable salt thereof, wherein the polypeptide or the pharmaceutically acceptable salt thereof has an activity of inhibiting autoimmune inflammation;

X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16 (formula I)

In the formula (I), the compound is shown in the specification,

x0 is nothing, or a peptide of 1-12 amino acids;

x1 is an amino acid selected from the group consisting of: cys, Ser

X2 is an amino acid selected from the group consisting of: arg, Lys, Gln, Asn;

x3 is an amino acid selected from the group consisting of: asn, Gln, His, Lys, Arg;

x4 is an amino acid selected from the group consisting of: pro, Ala;

x5 is an amino acid selected from the group consisting of: arg, Lys, Gln, Asn;

x6 is an amino acid selected from the group consisting of: gly, Pro, Ala;

x7 is an amino acid selected from the group consisting of: glu and Asp;

x8 is an amino acid selected from the group consisting of: glu and Asp;

x9 is an amino acid selected from the group consisting of: gly, Pro, Ala;

x10 is an amino acid selected from the group consisting of: gly, Pro, Ala;

x11 is an amino acid selected from the group consisting of: pro, Ala;

x12 is an amino acid selected from the group consisting of: trp, Tyr, Phe;

x13 is an amino acid selected from the group consisting of: cys, Ser;

x14 is an amino acid selected from the group consisting of: phe, Leu, Val, Ile, Ala, Tyr;

x15 is an amino acid selected from the group consisting of: thr, Ser;

x16 is nothing, or a peptide of 1-13 amino acids.

In another preferred embodiment, the total length of the polypeptide is 80 amino acid residues or less, preferably 50 amino acid residues or less, more preferably 35 amino acid residues or less.

In another preferred embodiment, X0 is a peptide stretch of 1-12 amino acids derived from SEQ ID NO. 7.

In another preferred embodiment, 1 to 12 represent 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12.

In another preferred embodiment, 1-13 represents 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13.

In another preferred embodiment, X0 is a peptide stretch of SEQ ID NO 7 from position m to position 12, wherein m is a positive integer selected from 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11 or 12.

In another preferred embodiment, X16 is a peptide fragment derived from amino acids 1-13 of SEQ ID NO 8.

In another preferred embodiment, the peptide of SEQ ID NO 8 at position X16 is a peptide stretch from position 1 to n, wherein n is a positive integer selected from 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13.

In another preferred embodiment, said X0 and X16 are null.

In another preferred embodiment, the polypeptide is selected from the group consisting of:

(a) 1, 2, 3, 4,5 or 6, and the length of the polypeptide is 15-35 amino acids;

(b) 1, 2, 3, 4,5 or 6 amino acid sequence through 1-2 amino acid residue substitution, deletion or addition, and has the function of suppressing autoimmune inflammation.

In another preferred embodiment, the amino acid sequence of the polypeptide is selected from the group consisting of: 1, 2, 3, 4,5 or 6.

In another preferred embodiment, the amino acid sequence of the polypeptide is shown in SEQ ID NO 1.

In a second aspect of the invention, there is provided an isolated polynucleotide encoding a polypeptide according to the first aspect of the invention.

In a third aspect of the invention there is provided a vector comprising a polynucleotide as described in the second aspect of the invention.

In a fourth aspect of the invention, there is provided a host cell comprising a vector or genome according to the third aspect of the invention into which has been integrated a polynucleotide according to the second aspect of the invention.

In a fifth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) a polypeptide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof; and

(b) a pharmaceutically acceptable carrier or excipient.

In another preferred embodiment, the component (a) is 0.1-99.9 wt%, preferably 10-99.9 wt%, more preferably 70-99.9 wt% of the total weight of the pharmaceutical composition.

In another preferred embodiment, the pharmaceutical composition is a liquid, solid, or semi-solid.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form, an injection, or an external pharmaceutical dosage form.

In another preferred embodiment, the dosage form of the pharmaceutical composition comprises tablets, granules, capsules, oral liquid or injection.

In another preferred embodiment, the pharmaceutical composition is a liquid composition.

In another preferred embodiment, the pharmaceutical composition is an oral formulation.

In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of: an infusion solution carrier and/or an injection carrier, preferably, the carrier is one or more selected from the following group: normal saline, dextrose saline, or combinations thereof.

In another preferred embodiment, the pharmaceutically acceptable carrier may be a carrier comprising a nanomaterial.

In another preferred embodiment, the dosage form of the pharmaceutical composition is eye drops, injection solutions (such as periocular and intraocular injection solutions), ophthalmic gel or ophthalmic ointment.

In another preferred embodiment, the pharmaceutical composition is in a sustained release dosage form.

In another preferred embodiment, the pharmaceutical composition is used alone or in combination in the application for suppressing autoimmune inflammation.

In another preferred embodiment, the combined use comprises: in combination with other drugs for suppressing autoimmune inflammation.

In another preferred embodiment, the other drugs for suppressing autoimmune inflammation include: glucocorticoids, immunosuppressants, or macromolecular biological targeting drugs.

In another preferred embodiment, the glucocorticoid comprises: methylprednisolone, prednisolone, or dexamethasone.

In another preferred embodiment, the other drugs for suppressing autoimmune inflammation include: an antimetabolite, a calcineurin inhibitor, an alkylating agent, a novel immunosuppressive agent, or a combination thereof.

In another preferred embodiment, the antimetabolite comprises: azathioprine, mycophenolate mofetil, methotrexate, cyclophosphamide, or combinations thereof.

In another preferred embodiment, the calcineurin inhibitor comprises: tacrolimus, cyclosporine, or a combination thereof.

In another preferred embodiment, the alkylating agent includes: cyclophosphamide, busulfan, or a combination thereof.

In another preferred embodiment, the novel immunosuppressive agents include: rapamycin, FTY720, or a combination thereof.

In another preferred embodiment, the macromolecular biological targeting agent comprises: a TNF- α monoclonal antibody, an anti-CD 20 monoclonal antibody, an anti-VEGF monoclonal antibody, or a combination thereof.

In a sixth aspect of the invention, there is provided the use of a polypeptide according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in inhibiting autoimmune inflammation.

In another preferred embodiment, the autoimmune inflammation comprises: ocular autoimmune inflammation, systemic lupus erythematosus, autoimmune arthritis, autoimmune enteritis, autoimmune cranial neuritis, multiple sclerosis, or a combination thereof.

In another preferred embodiment, the ocular autoimmune inflammation comprises: autoimmune uveitis AU, autoimmune optic neuritis AON, neuromyelitis optica NMO, autoimmune keratoconjunctivitis, or combinations thereof.

In a seventh aspect of the invention, there is provided a method of inhibiting autoimmune inflammation in a mammal, comprising the steps of: administering to a subject in need thereof a polypeptide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the mammal comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal comprises: rodents (e.g., rats, mice), primates (e.g., monkeys).

In another preferred embodiment, the autoimmune inflammation is ocular autoimmune inflammation.

In another preferred embodiment, the administration comprises intraocular injection, eye drop infusion, retrobulbar injection, parabulbar injection, subconjunctival injection or intravenous injection.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a membrane-anchored ligand-receptor yeast two-hybrid system. Wherein (A) shows a schematic diagram of respectively constructing plasmids containing alternative Peptide (Peptide) and c-Met coding sequences and transferring the plasmids into two different yeast strains. (B) The working principle of the membrane-anchored ligand-receptor yeast two-hybrid system is shown. After hybridization of the two yeast strains, the candidate Peptide (Peptide) was chimeric on the cell membrane surface by transmembrane Peptide (TMP) and the membrane receptor c-Met was also expressed on the membrane surface. Once the candidate peptide interacts with c-Met, the ubiquitin detection system on the inner side of the membrane is activated, releasing GAL4 transcription factor to start the reporter gene.

FIG. 2 shows a flow chart of a plate screening experiment. Plasmids containing alternative Peptide (Peptide) and c-Met coding sequences are respectively constructed, the plasmids are transferred into two different yeast strains, and after the two yeast strains are hybridized, a plate screening experiment is carried out.

Figure 3 shows partial results of high throughput screening based on small peptide-receptor interactions. Wherein (A) shows that the interaction between small peptide and c-Met in the detection peptide library of the membrane anchoring ligand-receptor yeast two-hybrid system is detected, wherein the combination of H-CN and c-Met is stronger than that of other partial small peptide (the strain grows fastest and the color is deepest); (B) a partial three-dimensional structure of the interaction of H-CN with c-Met is shown.

FIG. 4 shows the effect of H-CN on EAE clinical scores. Mice in each group were treated intraperitoneally with PBS, H-CP, H-CN, or FTY720, starting on day 15 post-immunization. Mice were observed and scored for clinical performance on alternate days after immunization. There was a significant drop in clinical symptom score in the H-CN and FTY20 treated groups 2 days after the start of injection (day 17).

Figure 5 shows serum inflammatory factor levels in different EAE treated groups. EAE mice were treated with intraperitoneal injections of PBS, H-CP, H-CN, FTY720, and IFN-r, TNF-a, IL-2, IL-6, IL-17A, IL-10, and other cytokines were altered as shown, indicating P <0.05, indicating P <0.01, # > indicating P > 0.05.

FIG. 6 shows the effect of H-CN on Th cell subsets of spleen and lymph nodes in EAE mice. The proportions of Th0, Th1, Th17 and Treg in spleen and lymph node of EAE mouse are all increased, and H-CN can reduce the proportions of Th0, Th1 and Th17 cell populations and increase the proportion of Treg subgroup.

FIG. 7 shows the effect of H-CN on Th cell subsets of neural tissue in EAE mice. The upper panel shows the change of the ratio of CD4+ T in nerve tissue, normal mice have only a trace amount of CD4+ T, after EAE modeling, CD4+ T is increased, FTY720 can reduce the infiltration of CD4+ T in target organs, and H-CN has no influence on the change. The following figure shows the proportion of Th subpopulations in CD4+ T altered: the H-CN can obviously reduce the proportion of Th1 and Th17 cell populations in the nervous system of an EAE mouse, and simultaneously increase the proportion of Treg subgroups.

FIG. 8 shows the results of the EAE mouse flash Visual Evoked Potential (VEP) examination. The left panel shows the VEP waveforms in each group, with significant reduction in the normal amplitude of the EAE group, relative enhancement of the interfering wave, no improvement in the H-CP treatment group, and partial recovery of the accessible waveforms in the H-CN and FTY720 groups. The right panel shows a comparison of the amplitudes of the various groups N1-P1, with a sign of P <0.01, a sign of P <0.05 and a sign of # indicating no statistical significance of the differences.

Figure 9 shows fundus presentation at day 22 after immunization of each group of mice. As shown in the figure, the blood vessels of the normal mice have no thickening, no retinal lesion and the like; EAU mice can reach the blood vessel to increase thickness, dilate congestion and exude focus; the H-CP treatment group can reach large fusion focus around the optic disc; the H-CN treatment group can reduce the vascular inflammation and reduce the exudation.

FIG. 10 shows the effect of H-CN on EAU clinical scores. Mice in each group were treated with PBS, H-CP and H-CN intraperitoneal injections in combination with eye drops starting on day 12 after immunization. Mice clinical performance was observed and scored every other day, starting on day 10 after immunization. There was a significant drop in the H-CN treatment group clinical symptom score after 3 injections started (day 16).

FIG. 11 shows the effect of H-CN on EAU histopathological changes. On the 22 th day after immunization, the eyeballs of the mice are taken for HE staining, inflammatory infiltration and retinal fold can be achieved in the EAU group and the H-CP group, pathological changes can involve an outer nuclear layer and a photoreceptor cell layer, inflammatory granulomatosis foci around blood vessels can be achieved, and focal net collapse can occur in the H-CP group; the H-CN group can be infiltrated by inflammation of vitreous cavity and superficial retina, the retina is flat, and the outer layer structure is normal.

FIG. 12 shows the effect of H-CN on Th cell subsets of spleen and lymph nodes in EAU mice. The proportions of Th0, Th1, Th17 and Treg in spleen and lymph node of EAU mouse are all increased, and H-CN can reduce the proportions of Th0, Th1 and Th17 cell populations and increase the proportion of Treg subgroup.

FIG. 13 shows the effect of H-CN on subsets of Th cells of the eyeball of EAU mice. The upper panel shows the change in the ratio of CD4+ T in the eyeball, with only a trace amount of CD4+ T in the normal mouse eyeball, increased CD4+ T in the eyeball after EAU molding, and H-CN did not reduce the infiltration of CD4+ T in the eyeball. The following figure shows the proportion of Th subpopulations in CD4+ T altered: although H-CN has no effect on eyeball CD4+ T infiltration, it is able to modulate the grouping of eyeball local CD4+ T, and can significantly reduce the Th1 and Th17 cell population ratios while increasing the Treg subpopulation ratio.

FIG. 14 shows the results of MTS proliferation experiments for RAW264.7 cells (left) and HUVEC cells (right). The results show that the active peptide H-CN control peptide Pep17 with different concentrations has no obvious influence on the viability of RAW264.7 and HUVEC cells after being incubated for 24H, and the results show that the active peptide H-CN control peptide Pep17 has no cytotoxicity.

FIG. 15 shows that no significant changes are observed in the results of the mouse retinal slice electron microscopy, HE staining, Electroretinogram (ERG) and VEP between the H-CN intervention group and the control group.

Detailed Description

The inventor of the invention develops a small molecular polypeptide with the function of inhibiting autoimmune inflammation and the molecular weight of less than 4KD for the first time through extensive and intensive research and a large amount of screening. Specifically, the inventors applied bioinformatics methods to select several candidate polypeptide sequences based on homology analysis and analysis of biological properties, etc.; by utilizing a membrane anchoring ligand-receptor yeast two-hybrid system and screening candidate polypeptide sequences by using c-Met receptor protein, a small molecular polypeptide with a section of homologous sequence is screened out. In addition, the polypeptide is proved to be capable of relieving the clinical symptoms and obviously improving the visual function of an Experimental Autoimmune Encephaloneuritis (EAE) mouse model and an Experimental Autoimmune Uveitis (EAU) mouse model through the verification of the immunoregulation function. In addition, safety tests of the targeting biological peptide injected in a vitreous cavity show that the polypeptide has no influence on the structure of the retina of the mouse and no influence on the visual function of the mouse, namely the safety of the polypeptide for the vitreous cavity injection is proved. The present invention has been completed based on this finding.

Polypeptides

As used herein, the terms "polypeptide", "small molecule polypeptide", "short peptide" or "polypeptide of the invention" are used interchangeably and refer to a polypeptide having the structure of formula I as described in the first aspect of the invention which inhibits autoimmune inflammation.

In a preferred embodiment, the polypeptide of the invention has an amino acid sequence as shown in SEQ ID NO 1, 2, 3, 4,5 or 6.

In a more preferred embodiment, the polypeptide of the invention has the amino acid sequence shown in SEQ ID NO. 1. As used herein, the terms "H-CN polypeptide", "polypeptide H-CN", "H-CN small peptide" or "peptide H-CN" are used interchangeably to refer to a protein or polypeptide having the amino acid sequence of peptide H-CN (SEQ ID NO:1) that inhibits autoimmune inflammation.

Furthermore, the term "polypeptide" also includes the polypeptide of SEQ ID NO: 1.2, 3, 4,5 or 6 sequence variants. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 5 (usually 1 to 4, preferably 1 to 3, more preferably 1 to 2, most preferably 1) amino acids, and addition of one or several (usually up to 5, preferably up to 3, more preferably up to 2) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. For another example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein.

In another preferred example, the amino acid sequence added at the N-terminus is SSYRGKDLQENY (SEQ ID NO:7, from N-terminus to C-terminus); the amino acid sequence added at the C-terminus is represented by SNPEVRYEVCDIP (SEQ ID NO:8, from N-terminus to C-terminus).

The invention also includes active fragments, derivatives and analogs of the polypeptides of the invention. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of inhibiting autoimmune inflammation. The fragment, derivative or analogue of the polypeptide of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the polypeptide of the present invention is fused to another compound (such as a compound for increasing the half-life of the polypeptide, for example, polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (a protein which is then fused to a leader sequence, a secretory sequence or a tag sequence such as 6 His). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

A preferred class of reactive derivatives refers to polypeptides formed by the replacement of up to 5, preferably up to 3, more preferably up to 2, most preferably 1 amino acid with an amino acid of similar or analogous nature compared to the amino acid sequence of formula I. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

TABLE 1

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The invention also provides analogs of the polypeptides of the invention. These analogs may differ from the polypeptide of the invention by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

The polypeptides of the invention can also be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include, but are not limited to, salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include: salts with alkali or alkaline earth metals (such as sodium, potassium, calcium or magnesium), and in the form of esters, carbamates or other conventional "prodrugs".

Coding sequence

The present invention also relates to polynucleotides encoding the polypeptides of the invention.

The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be a nucleotide sequence encoding all degenerate variant forms of the amino acid sequence shown in SEQ ID NO 1, 2, 3, 4,5 or 6.

The full-length polynucleotide sequence or its fragment of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the polypeptides of the present invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to vectors comprising the polynucleotides of the invention, and to genetically engineered host cells produced using the vectors of the invention or the coding sequences for the polypeptides of the invention.

In another aspect, the invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the polypeptides encoded by the DNA of the polypeptides of the invention or fragments thereof.

Preparation method

The polypeptides of the invention may be recombinant polypeptides or synthetic polypeptides. The polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptides of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method.

A preferred method is to use liquid phase synthesis techniques or solid phase synthesis techniques, such as Boc solid phase method, Fmoc solid phase method or a combination of both. The solid phase synthesis can quickly obtain samples, and can select proper resin carriers and synthesis systems according to the sequence characteristics of target peptides. For example, the preferred solid support in the Fmoc system is Wang resin with C-terminal amino acid attached to the peptide, Wang resin is polystyrene in structure, and the arm between the Wang resin and the amino acid is 4-alkoxybenzyl alcohol; the Fmoc protecting group was removed by treatment with 25% piperidine/dimethylformamide for 20 minutes at room temperature and extended from the C-terminus to the N-terminus one by one according to the given amino acid sequence. After completion of the synthesis, the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol and the protecting groups are removed, optionally by filtration and isolated as a crude peptide by ether precipitation. After lyophilization of the resulting solution of the product, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography. When the solid phase synthesis is performed using the Boc system, it is preferable that the resin is a PAM resin to which a C-terminal amino acid in a peptide is attached, the PAM resin has a structure of polystyrene, and an arm between the PAM resin and the amino acid is 4-hydroxymethylphenylacetamide; in the Boc synthesis system, the protecting group Boc is removed with TFA/Dichloromethane (DCM) and neutralized with Diisopropylethylamine (DIEA)/dichloromethane in cycles of deprotection, neutralization, and coupling. After completion of the peptide chain condensation, the peptide chain was cleaved from the resin by treatment with p-cresol (5-10%) in Hydrogen Fluoride (HF) at 0 ℃ for 1 hour, while removing the protecting group. Extracting peptide with 50-80% acetic acid (containing small amount of mercaptoethanol), lyophilizing, separating and purifying with molecular sieve Sephadex G10 or Tsk-40f, and purifying with high pressure liquid phase to obtain the desired peptide. The amino acid residues may be coupled using various coupling reagents and coupling methods known in the art of peptide chemistry, for example direct coupling using Dicyclohexylcarbodiimide (DCC), hydroxy benzotriazole (HOBt) or 1,1,3, 3-tetraurea Hexafluorophosphate (HBTU). For the synthesized short peptide, the purity and the structure can be verified by reversed-phase high performance liquid chromatography and mass spectrometry.

In a preferred embodiment, the polypeptide of the present invention is prepared by a solid phase synthesis method according to the sequence thereof, and purified by high performance liquid chromatography to obtain high purity target peptide lyophilized powder, which is stored at-20 ℃.

Another method is to produce the polypeptide of the invention by recombinant techniques. The polynucleotides of the present invention may be used to express or produce recombinant polypeptides of the present invention by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide of the invention encoding a polypeptide of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

The recombinant polypeptide may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Because the polypeptide of the invention is short, a plurality of polypeptides can be considered to be connected in series, an expression product is obtained after recombinant expression, and then the required small peptide is formed by enzyme digestion and other methods.

Autoimmune Uveitis (AU)

AU is an ocular immune inflammatory disease which can affect the uveal tract, retina and vitreous body, has the characteristics of long course of disease, easy involvement of both eyes, repeated attack and the like, can cause serious complications such as complicated cataract, secondary glaucoma, secondary choroidal neovascularization and the like, and has the blindness rate of up to 30 percent.

The onset of AU and MS is associated with systemic and local immune disorders mediated mainly by helper T cells (Th cells).

For AU and MS, the target of the target therapy can be certain inflammatory factors, such as tumor necrosis factor (TNF-a), interleukin (IL-6, IL-17, IL-1) and the like; or soluble mediators such as interferons (interferons) and the like; it may also be some molecule on the cell surface, such as CD19, CD52, CD152, etc.

Neuromyelitis optica (NMO)

NMO is an autoimmune demyelinating disease in which the optic nerve is affected either sequentially or simultaneously with the spinal cord, with approximately 50% of NMO patients presenting with optic neuritis as the first manifestation.

NMO is particularly high in Asian population, and accounts for 20% -48% of demyelinating diseases in Asian population. In the past, NMO has been considered a particular phenotype of MS, and it has not been gradually isolated from MS as an independent disease until the last decade with the continued intensive research into its pathogenesis, particularly the discovery of the NMO-specific immunoglobulin, aquaporin 4(aquaporin4, AQP4) autoantibody.

AQP4 is the major aquaporin molecule located on the astrocyte foot processes, damaged in NMO by the force of the self AQP4 antibody and even lost vision. About half of NMO patients can exhibit isolated optic neuritis, 20% of which are both eye affected, with recurrent attacks that cause further vision loss once each attack, and thus persistent and severe vision impairment is a characteristic manifestation of NMO optic neuritis; compared with the traditional Chinese medicine, the MS has the advantages that the optic neuritis mostly happened in a single eye, and the visual acuity can be recovered to be close to a normal level after the happened optic neuritis, so the visual acuity prognosis is better.

For NMO, due to the existence of the characteristic antibody AQP4 autoantibody of NMO patients, the existing biological targeted therapy mainly includes B cell inhibition therapy, plasma replacement therapy and the like, and AQP4 antibody blocking therapy in preclinical experiments and the like aiming at the pathogenesis. However, the positive rate of the AQP4 antibody in NMO patients is only about 50% -60%, and the effect of the treatment measures is poor in patients with AQP4 antibody negativity. And because of the difficulty of NMO diagnosis, the patients who are negative to serum AQP4 antibody are easily confused with MS-related ON in early onset, and some novel biological treatment measures of MS, such as IFN-beta, natalizumab, oral fingolimod and the like, can aggravate the progress of NMO diseases, and bring more difficulty to the treatment.

Role of Teff/Treg imbalance in AU and AON

CD4⁺Helper T cells (Th cells) mediate the pathogenesis of AU and MS: peripheral initial T Cells (negative T Cells) are differentiated into Effector T cell subsets such as Th1 and Th17 under autoantigen stimulation, and inflammatory factors such as IFN-gamma and IL-17A are secreted, so that pathological damage to eye and nerve tissues is caused.

Regulatory T cells (Treg cells) are T cell subsets with immunosuppressive effect, can inhibit activation and proliferation of Teff, and secrete immunosuppressive factors such as IL-10 and the like, so that immune homeostasis in tissues is maintained, and autoimmune reaction is avoided. The imbalance of Teff/Treg in the periphery and tissues causes the onset of AU and MS. Regulating the balance of Teff/Treg has important significance for slowing the disease progression of AU and MS and improving vision prognosis.

In addition to humoral immunity, cellular immunity is also involved in the pathogenesis of NMO. Firstly, a large amount of CD3+ T cells infiltrate at the NMO focus; secondly, NMO patients have HLA-DR17 locus gene abnormalities encoding MHC class II molecules, which are mainly responsible for presenting processed antigen fragments of 12-15 amino acids in length to CD4+ T helper T cell (Th cell) cells at the initiation stage of immunity; again, AQP4 antibodies are IgG, whereas the conversion of antibodies from IgM to IgG requires the involvement of Th cells, and B cell suppression therapy does not reduce NMO-specific IgG titers, suggesting that it may play a therapeutic role by inhibiting T cell activation; in addition, a large number of animal experiments show that the AQP4 epitope injected into a mouse can activate cellular immune response and generate AQP4 specific T cells, and the AQP4 epitope AQP4281-300 can induce generation of Th1 and Th17 immune response responses in the mouse, which suggests that Teff cells are possibly used as an upstream link in the NMO autoimmune response process and participate in the generation and development of diseases.

In conclusion, the regulation of the balance of Teff/Treg is of great significance for slowing the progression of AU and AON diseases and improving vision prognosis.

In the invention, a small molecular targeted biological peptide capable of regulating Teff/Treg balance is provided, and a new strategy is provided for treating AU patients.

c-Met receptor targeting biological peptides

c-Met is a membrane protein receptor as a receptor tyrosine kinase family member, can activate tyrosine kinase activity of an intracellular segment after being combined with ligand Hepatocyte Growth Factor (HGF), and participates in processes such as tissue injury repair, immune regulation, anti-inflammation and the like. The Kringle 1 domain is one of the important functional domains of HGF, has high c-Met receptor affinity, and has been demonstrated to have anti-inflammatory and immunomodulatory effects. Research shows that after c-Met is activated by Kringle 1 structural domain, Treg differentiation can be induced, IL-10 factor secretion can be promoted, Teff proliferation and functions can be inhibited, IFN-gamma and IL-17A expression can be reduced, balance of Teff/Treg can be restored, and the immune regulation effect can be prominent in multiple autoimmune disease models such as Experimental Autoimmune Uveitis (EAU) and Experimental Autoimmune Encephaloneuritis (EAE).

The peptide drug is a small molecular active preparation separated and synthesized from endogenous functional protein, and compared with macromolecular protein, the biological peptide has the advantages of small molecular weight, low production cost, good water solubility, small immunogenicity, low toxic and side effects, strong tissue penetrability and the like while maintaining the function of parent protein.

However, the existing biological peptides have the problems of undefined action target, uncertain action mechanism and the like, so that potential side effects exist, and the clinical application of the biological peptides is limited; therefore, the research and development of the biological peptide with a definite target point have great significance for clinical transformation of research results.

In the invention, the c-Met receptor is selected to screen the small molecular peptide segment, the target point and the action mechanism are clear, and the defects of the macromolecular active preparation are overcome.

Membrane anchored ligand-receptor yeast two-hybrid system

The 'membrane anchoring ligand-receptor yeast two-hybrid system' is a high-flux detection system for the interaction of ligand and membrane receptor, and can be used for screening receptor targeting biological peptide. The system expresses membrane protein receptors on the surface of yeast cell membranes, and simultaneously anchors ligands on the surface of the cell membranes through fusion transmembrane peptides. The receptor and ligand modules are respectively coupled with the upper half region and the lower half region of the split ubiquitin detection system in the cell, and when the receptor and the ligand interact, the detection system is activated to release transcription factors and start reporter genes. Whether the ligand is combined with the membrane receptor can be known by detecting whether the reporter gene is expressed or not and the expression strength, and the affinity of the ligand and the receptor is pre-judged. The specific scheme and mechanism are shown in figure 1.

Based on the above knowledge, the present inventors screened biological peptides from the natural ligand HGF of c-Met receptor by bioinformatics technology and the above membrane anchored ligand-receptor yeast two-hybrid system, and examined the immunomodulatory biological activity and biological safety of the biological peptides by in vitro and in vivo experiments.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an effective amount (e.g., 0.1-99.9 wt%, preferably 10-99.9 wt%, more preferably 70-99.9 wt%) of a polypeptide of the invention (especially polypeptide H-CN), and a pharmaceutically acceptable carrier.

Generally, the polypeptides of the invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical composition of the invention contains a safe and effective amount of the polypeptide of the invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation is usually adapted to the mode of administration, and the pharmaceutical composition of the present invention may be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the invention can also be prepared into a sustained release preparation.

In another preferred embodiment, the dosage form of the pharmaceutical composition is eye drops, injection solutions (such as periocular and intraocular injection solutions), ophthalmic gel or ophthalmic ointment.

The effective amount of the protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the polypeptide of the present invention is administered at a daily dose of about 1 to 4mg/kg of animal body weight, preferably 2 to 3mg/kg of animal body weight. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The main advantages of the invention include:

1) compared with macromolecular protein, the small molecular peptide targeted drug has the advantages of small molecular weight, low production cost, good water solubility, small immunogenicity, low toxic and side effects, strong tissue penetrability and the like. These characteristics make the small molecular targeting biological peptide have potential advantages and wider application prospect in clinical treatment of the autoimmune inflammation of the eyes.

2) The novel biological peptide is screened by taking a c-Met receptor as a target through receptor targeting, and the parent protein HGF is endogenous macromolecular protein, and the safety of in-vivo and in-vitro application of the novel biological peptide is verified, so that the novel biological peptide has the advantages of clear target and small toxic and side effects compared with glucocorticoid and immunosuppressant.

3) Compared with targeted therapeutic drugs such as macromolecular monoclonal antibodies and the like, the targeted therapeutic drug is a micromolecule and hydrophilic biological peptide, has the advantage of strong tissue penetrability, and is hopeful to penetrate various blood eye and blood optic nerve tissue barriers to improve the local bioavailability of eyes; ② because the parent protein is endogenous protein, so it has the advantages of low immunogenicity and good biocompatibility; and the product can be obtained by in vitro solid phase synthesis, and the production cost is low.

4) Researches on two models of EAE and EAU show that the biological peptide has obvious functions of immunoregulation, anti-inflammation and visual function improvement, and effectiveness is verified.

5) The traditional immunosuppressant can inhibit the proliferation and activation of Treg cells while inhibiting the action of Teff subgroups such as Th1, Th17 and the like, and the target c-Met receptor pathway of the invention can promote the differentiation and function of Tregs while inhibiting Teff.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials and reagents used in the examples were all commercially available products unless otherwise specified.