CN118681005A - A bivalent IL-17 therapeutic vaccine and its preparation method and application - Google Patents

Abstract

The present invention provides an IL-17 therapeutic vaccine, and a preparation method and application thereof. Specifically, the present invention provides a bivalent IL-17 therapeutic vaccine targeting IL-17A and IL-17F, which comprises vaccine antigens formed by fusion expression of IL-17A and IL-17F antigenic active fragments with carrier proteins or coupling with carrier proteins. The IL-17 therapeutic vaccine can induce the production of high-titer IL-17A and IL-17F neutralizing antibodies in mice and rhesus monkeys after being used in combination with a liquid adjuvant, blocking the IL-17 signaling pathway, thereby inhibiting autoimmune inflammation, and achieving the purpose of treating autoimmune diseases such as psoriasis, ankylosing spondylitis, and rheumatoid arthritis.

Description

A bivalent IL-17 therapeutic vaccine and its preparation method and application

Technical Field

The present invention belongs to the field of biotechnology and medicine, and specifically relates to a bivalent interleukin-17 (IL-17) therapeutic vaccine and a preparation method and application thereof.

Background Art

Interleukin 17 (IL-17) is a key target for autoimmune diseases such as psoriasis and ankylosing spondylitis. IL-17 monoclonal antibody has been approved for the treatment of psoriasis and ankylosing spondylitis.

IL-17 includes six subtypes: A, B, C, D, E, and F, of which IL-17A and IL-17F are the key subtypes involved in autoimmune diseases. Currently available IL-17 monoclonal antibodies mainly target IL-17A, and can target both IL-17A and IL-17F. The therapeutic effect of monoclonal antibodies that can target both IL-17A and IL-17F is significantly better than that of monoclonal antibodies targeting IL-17A. Although there are reports of IL-17A polypeptide vaccines in the literature, the length of polypeptide fragments is less than 30 amino acid residues, but there are no reports of interleukin 17 vaccines containing IL-17A spatial epitopes, and there are no reports of vaccines containing both IL-17A and IL-17F antigenic epitopes.

There is an urgent need to develop an IL-17 therapeutic vaccine with higher stability and better therapeutic effect in this field

Summary of the invention

The object of the present invention is to provide a bivalent IL-17 therapeutic vaccine targeting IL-17A and IL-17F, wherein the vaccine antigens in the vaccine include an IL-17A fragment (containing at least 58-151, but not more than 39-155 amino acids) and an IL-17F fragment (containing at least 70-156, but not more than 11-158 amino acids), wherein the IL-17 fragment has low IL-17 activity but retains the antigenic spatial epitope, and is fused with a carrier protein for expression, or covalently cross-linked with a carrier protein to form a vaccine antigen, and the vaccine can break the immune tolerance of the immune body after being combined with a liquid adjuvant, and induce the body to continuously produce neutralizing antibodies against IL-17A and IL-17F.

In a first aspect of the present invention, there is provided an IL-17 therapeutic vaccine, the therapeutic vaccine comprising: (a) an IL-17A vaccine antigen having a structure as shown in formula (I); and (b) an IL-17F vaccine antigen having a structure as shown in formula (II);

Z1-L-Z3(I)

Z2-L-Z3(II)

Among them, Z1 is the antigen-active fragment of IL-17A;

Z2 is the antigen-active fragment of IL-17F;

Z3 is the carrier protein;

L is a linker, and the linker is selected from a covalent bond formed by cross-linking with a cross-linking agent, or a connecting peptide or peptide bond used for fusion protein expression;

"-" indicates a chemical bond connecting the above elements.

In another preferred embodiment, the amino acid sequence of the IL-17A antigen-active fragment is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of Z1 has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, preferably, at least 95% sequence identity, more preferably at least 96%, 97%, 98%, or 99% sequence identity.

In another preferred embodiment, the amino acid sequence of the IL-17F antigen-active fragment is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

In another preferred embodiment, the amino acid sequence of Z2 has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, preferably, at least 95% sequence identity, more preferably at least 96%, 97%, 98%, or 99% sequence identity.

In another preferred embodiment, the Z3 is a diphtheria toxin mutant CRM197, and its amino acid sequence is shown in SEQ ID NO:5.

In another preferred embodiment, the L is a covalent bond formed by cross-linking with a cross-linking agent.

In another preferred embodiment, the cross-linking agent is selected from the following group: glutaraldehyde, carbodiimide (EDC), SMCC (4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt), sulfo-SMCC, SATP (N-succinimidyl S-acetylthiopropionate), or a combination thereof, preferably glutaraldehyde.

In another preferred embodiment, the cross-linking agent is glutaraldehyde.

In another preferred embodiment, the cross-linking agent is a combination of sulfo-SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt) and SATP (N-succinimidyl S-acetylthiopropionate).

In another preferred embodiment, the IL-17A vaccine antigen is a fusion protein, and its amino acid sequence is shown in SEQ ID NO:6.

In another preferred embodiment, the IL-17F vaccine antigen is a fusion protein, and its amino acid sequence is shown in SEQ ID NO:7.

In another preferred embodiment, the IL-17A vaccine antigen and the IL-17F vaccine antigen are both fusion proteins.

In another preferred example, in the IL-17 therapeutic vaccine, the content of the IL-17A vaccine antigen (fusion protein) is 0.375 mg/mL; the content of the IL-17F vaccine antigen (fusion protein) is 0.375 mg/mL.

In another preferred embodiment, the IL-17A vaccine antigen and the IL-17F vaccine antigen are both conjugates.

In another preferred embodiment, the molar ratio of the IL-17A/IL-17F antigen active fragment to the CRM197 protein in the conjugate is (6-10):1, preferably, 8:1.

In another preferred example, in the IL-17 therapeutic vaccine, the content of the IL-17A vaccine antigen (conjugate) is 0.4 mg/mL; the content of the IL-17F vaccine antigen (conjugate) is 0.2 mg/mL.

In another preferred embodiment, in the IL-17 therapeutic vaccine, the content ratio of IL-17A vaccine antigen to IL-17F vaccine antigen is (0.5-4):1.

In another preferred embodiment, in the IL-17 therapeutic vaccine, the content ratio of IL-17A vaccine antigen to IL-17F vaccine antigen is 1:1.

In another preferred embodiment, in the IL-17 therapeutic vaccine, the content ratio of IL-17A vaccine antigen to IL-17F vaccine antigen is 2:1.

In another preferred embodiment, in the IL-17 therapeutic vaccine, the content ratio of IL-17A vaccine antigen to IL-17F vaccine antigen is 1:2.

In another preferred embodiment, in the IL-17 therapeutic vaccine, the content ratio of IL-17A vaccine antigen to IL-17F vaccine antigen is 4:1.

In another preferred embodiment, the IL-17 therapeutic vaccine has the following characteristics: it contains both IL-17A vaccine antigen and IL-17F vaccine antigen, and the IL-17A vaccine antigen and IL-17F vaccine antigen can stimulate the body to produce neutralizing antibodies against IL-17A and IL-17F, respectively.

The second aspect of the present invention provides a method for preparing the IL-17 therapeutic vaccine according to the first aspect of the present invention, the method comprising the following steps:

(i) preparing IL-17A vaccine antigen and IL-17F vaccine antigen respectively;

(ii) mixing the IL-17A vaccine antigen and the IL-17F vaccine antigen prepared in step (i) in a ratio of (0.5-4):1 to obtain the IL-17 therapeutic vaccine.

In another preferred embodiment, the IL-17A/IL-17F vaccine antigen is an IL-17A/IL-17F-CRM197 conjugate, which is prepared according to the following steps:

(1) Providing expressed and purified IL-17A/IL-17F protein and diphtheria toxin mutant CRM197 protein;

(2) after mixing the IL-17A/IL-17F protein and the CRM197 protein, adding a cross-linking agent to carry out a cross-linking reaction, thereby obtaining an IL-17A/IL-17F-CRM197 conjugate;

(3) The IL-17A/IL-17F-CRM197 conjugate obtained in step (2) is concentrated by ultrafiltration to obtain the IL-17A/IL-17F vaccine antigen.

In another preferred embodiment, the amino acid sequence of the IL-17A protein has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, preferably, at least 95% sequence identity, and more preferably at least 96%, 97%, 98%, or 99% sequence identity.

In another preferred embodiment, the amino acids of the IL-17A protein are as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of the IL-17F protein has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, preferably at least 95% sequence identity, more preferably at least 96%, 97%, 98%, or 99% sequence identity.

In another preferred embodiment, the amino acid sequence of the IL-17F protein is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

In another preferred example, the amino acid sequence of the CRM197 protein is shown in SEQ ID NO:5.

In another preferred embodiment, the cross-linking agent is glutaraldehyde, and its final working concentration in the cross-linking reaction is 0.05% to 0.15% (v/v).

In another preferred embodiment, the reaction temperature of the cross-linking reaction is room temperature (25°C±2°C), and the reaction time is 1 hour to 3 hours.

In another preferred embodiment, the molar ratio of IL-17A protein to CRM197 protein in the IL-17A-CRM197 conjugate is (6-10):1, preferably, 8:1.

In another preferred embodiment, the molar ratio of IL-17F protein to CRM197 protein in the IL-17F-CRM197 conjugate is (6-10):1, preferably, 8:1.

In another preferred embodiment, the IL-17A/IL-17F vaccine antigen is an IL-17A/IL-17F-CRM197 fusion protein, which is prepared according to the following steps:

(A) constructing an expression vector containing a gene encoding IL-17A/IL-17F-CRM197 fusion protein;

(B) transducing the expression vector into host cells, and inducing the host cells to express the IL-17A/IL-17F-CRM197 fusion protein;

(C) Purifying the IL-17A/IL-17F-CRM197 fusion protein to obtain the IL-17A/IL-17F vaccine antigen.

In another preferred example, the amino acid sequence of the IL-17A-CRM197 fusion protein is shown in SEQ ID NO: 6.

In another preferred embodiment, the amino acid sequence of the IL-17F-CRM197 fusion protein is shown in SEQ ID NO:7.

The third aspect of the present invention provides a composition, comprising: the IL-17 therapeutic vaccine described in the first aspect of the present invention, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the composition is a pharmaceutical composition or a vaccine composition.

In another preferred embodiment, the composition comprises nanoliposomes encapsulating the vaccine, and the components of the nanoliposomes are a composition of ionized lipids, cholesterol, a lipid-polyethylene glycol conjugate (PEG-lipid), and an auxiliary lipid.

In another preferred embodiment, the composition is a vaccine composition, and the vaccine composition further contains an adjuvant.

In another preferred embodiment, the adjuvant includes: granular adjuvant and non-granular adjuvant.

In another preferred embodiment, the particulate adjuvant is selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, immunostimulatory complexes, or a combination thereof;

In another preferred embodiment, the non-particulate adjuvant is selected from the following group: muramyl dipeptide and its derivatives, saponin, lipid A, cytokine, derived polysaccharide, bacterial toxin, microorganisms and their products such as mycobacteria (tuberculosis, bacillus Calmette-Guérin), bacillus brevis, Bordetella pertussis, propolis, or a combination thereof.

In another preferred embodiment, the adjuvant is selected from the following group: Montanide ISA 51VG, Montanide ISA720VG, aluminum phosphate adjuvant, MF59, AS04, or a combination thereof.

In another preferred embodiment, the composition is prepared as a liquid preparation.

In another preferred embodiment, the composition is prepared as an injection, a suspension, or a spray.

The fourth aspect of the present invention provides a use of the IL-17 therapeutic vaccine described in the first aspect of the present invention in the preparation of a medicament for treating and/or preventing a disease.

In another preferred embodiment, the disease is an autoimmune disease.

In another preferred embodiment, the disease is selected from the group consisting of rheumatoid arthritis, psoriasis or psoriatic arthritis, ankylosing spondylitis, Crohn's disease, or a combination thereof.

The fifth aspect of the present invention provides a method for treating and/or preventing a disease, comprising administering the IL-17 therapeutic vaccine described in the first aspect of the present invention, or the composition described in the third aspect of the present invention to a subject in need thereof.

In another preferred embodiment, the disease is an autoimmune disease.

In another preferred embodiment, the disease is selected from the group consisting of rheumatoid arthritis, psoriasis or psoriatic arthritis, ankylosing spondylitis, Crohn's disease, or a combination thereof.

In another preferred embodiment, the subject in need is a human or non-human mammal.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the purity identification diagram of IL-17A reduced SDS-PAGE.

FIG2 shows the purity identification diagram of IL-17F reduced SDS-PAGE.

FIG3 shows that after mice are immunized with the bivalent therapeutic vaccine (conjugate) of the present invention, the mice are induced to produce high titers of IL-17A antibodies and IL-17F antibodies.

FIG4 shows that after mice were immunized with the bivalent therapeutic vaccine (fusion protein) of the present invention, the mice were induced to produce high titers of IL-17A antibodies and IL-17F antibodies.

FIG5 shows that the IL-17 truncated form and the IL-17 truncated form and CRM197 fusion protein of the present invention induce a decrease in the activity of MC-3T3 cells expressing IL-6.

FIG6 shows that the serum after immunization with the bivalent therapeutic vaccine of the present invention blocks IL-17A from inducing IL-6 expression in MC-3T3 cells.

FIG. 7 shows that the serum after immunization with the bivalent therapeutic vaccine of the present invention blocks IL-17F from inducing IL-6 expression in MC-3T3 cells.

FIG8 shows a comparison of the effects of the bivalent therapeutic vaccine of the present invention and the use of IL-17A-CRM197 conjugate or IL-17F-CRM197 conjugate alone on improving psoriasis symptoms in mice.

FIG9 shows a comparison of the effects of the bivalent therapeutic vaccine of the present invention and the use of IL-17A-CRM197 conjugate or IL-17F-CRM197 conjugate alone on improving rheumatoid arthritis symptoms in mice.

DETAILED DESCRIPTION

After extensive and in-depth research, the inventors have developed a bivalent IL-17 therapeutic vaccine targeting IL-17A and IL-17F for the first time. The therapeutic vaccine uses IL-17A antigen active fragment-CRM197 conjugate and IL-17F antigen active fragment-CRM197 conjugate, or IL-17A antigen active fragment-CRM197 fusion protein and IL-17F antigen active fragment-CRM197 fusion protein as the main active ingredients. The IL-17A vaccine antigen and IL-17F vaccine antigen in the therapeutic vaccine are truncated, with high antigen stability, and can minimize the non-essential sequence of the antigen fragment, reduce the production of non-neutralizing antibodies, reduce lysine active groups, and reduce the molecular weight of the conjugate. After the therapeutic vaccine is applied to the body, it can break the body's immune tolerance and induce the body to continuously produce high-titer anti-IL-17A and IL-17F neutralizing antibodies, and the immune effect is significantly better than the single administration of IL-17A or IL-17F monovalent vaccine.

On this basis, the present invention has been completed.

the term

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

IL-17A and IL-17F

IL-17 includes six subtypes: A, B, C, D, E, and F. Among them, IL-17A and IL-17F are the key subtypes involved in autoimmune diseases.

As used in the present invention, the term "IL-17A vaccine antigen" refers to a vaccine antigen comprising an IL-17A antigen active fragment, wherein the IL-17A antigen active fragment comprises at least the 58th to 151st amino acid fragment of the full length of IL-17A (as shown in SEQ ID NO: 2), but not exceeding the 39th to 155th amino acid range (as shown in SEQ ID NO: 1). In another preferred embodiment, the amino acid sequence of the IL-17A antigen active fragment has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, preferably, at least 95% sequence identity, more preferably at least 96%, 97%, 98%, or 99% sequence identity.

As used in the present invention, the term "IL-17F vaccine antigen" refers to a vaccine antigen comprising an IL-17F antigen active fragment, wherein the IL-17F antigen active fragment comprises at least the 70-156 amino acid fragment of the full length of IL-17F (as shown in SEQ ID NO: 4), but does not exceed the 11-158 amino acid range (as shown in SEQ ID NO: 3). In another preferred embodiment, the amino acid sequence of the IL-17F antigen active fragment has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, preferably, at least 95% sequence identity, more preferably at least 96%, 97%, 98%, or 99% sequence identity.

IL-17A(39-155aa)(SEQ ID NO: 1)

KNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA

IL-17A(58-151aa)(SEQ ID NO: 2)

TNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGN VDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVH

IL-17F(11-158aa)(SEQ ID NO: 3)

MVKYLLLSILGLAFLSEAAARKIPKVGHTFFQKPESCPPVPGGSMKLDIGIINENQRVSMSRNIESRSTSPWNYTVTWDPNRYPSEVVQAQCRNLGCINAQGKEDISMNSVPIQQETLVVRRKHQGCSVSFQLEKVLVTVGCTCVTPV

IL-17F(70-156aa)(SEQ ID NO: 4)

MSRNIESRSTSPWNYTVTWDPNRYPSEVVQAQCRNLGCINAQGKEDISMNSVPIQQETLVVRRKHQGCSVSFQLEKVLVTVGCTCVT

CRM197

As used in the present invention, the term "CRM197" refers to the diphtheria toxin mutant CRM197, specifically the glycine at position 52 of the diphtheria toxin is mutated to glutamic acid, and has an amino acid sequence as shown in SEQ ID NO: 5. The mutant toxin A fragment cannot bind to the elongation factor II in the cell nucleus, so that it loses its cytotoxic effect, but the antigenicity and immunogenicity are still basically consistent with the natural diphtheria toxin.

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVI RDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGT NPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAAIDGDVTFCRPKSPVYVGNG VHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS(SEQ ID NO: 5)

IL-17 therapeutic vaccine of the present invention

In one aspect of the present invention, an IL-17 therapeutic vaccine is provided, wherein the vaccine is a bivalent vaccine comprising: (a) an IL-17A vaccine antigen having a structure as shown in formula (I); and (b) an IL-17F vaccine antigen having a structure as shown in formula (II);

Z1-L-Z3(I)

Z2-L-Z3(II)

Among them, Z1 is the antigen-active fragment of IL-17A;

Z2 is the antigen-active fragment of IL-17F;

Z3 is a carrier protein. In another preferred embodiment, the carrier protein is a diphtheria toxin mutant CRM197, whose amino acid sequence is shown in SEQ ID NO: 5;

L is a linker, and the linker is selected from a covalent bond formed by cross-linking with a cross-linking agent, or a connecting peptide or peptide bond used for fusion protein expression;

"-" indicates a chemical bond connecting the above elements.

In a preferred embodiment of the present invention, the IL-17A vaccine antigen and the IL-17F vaccine antigen are both conjugates, that is, the IL-17 therapeutic vaccine comprises an IL-17A-CRM197 conjugate and an IL-17F-CRM197 conjugate. The conjugates are formed by cross-linking the IL-17A antigen active fragment or the IL-17F antigen active fragment by a cross-linking agent, and the cross-linking agent includes (but is not limited to) glutaraldehyde, carbodiimide (EDC), SMCC (4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt), sulfo-SMCC, SATP (N-succinimidyl S-acetylthiopropionate), or a combination thereof, preferably glutaraldehyde.

In another preferred embodiment of the present invention, the IL-17A vaccine antigen and the IL-17F vaccine antigen are both fusion proteins, that is, the IL-17 therapeutic vaccine comprises IL-17A-CRM197 fusion protein and IL-17F-CRM197 fusion protein. In another preferred example, the amino acid sequence of the IL-17A-CRM197 fusion protein is shown in SEQ ID NO: 6, and the amino acid sequence of the IL-17F-CRM197 fusion protein is shown in SEQ ID NO: 7.

IL17A(58-151)-CRM197 fusion protein sequence

TNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHGGGGSMGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRD KTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITA ENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKV NSKLSLFFEIKS(SEQ ID NO: 6)

IL17F(70-156)-CRM197 fusion protein sequence

MSRNIESRSTSPWNYTVTWDPNRYPSEVVQAQCRNLGCINAQGKEDISMNSVPIQQETLVVRRKHQGCSVQLEKVLVTVGCTCVTGGGGSMGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPL MEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEH GPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIP GKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS (SEQ ID NO: 7)

As used in the present invention, the term "fusion protein" also includes variant forms of fusion proteins (such as the sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7) having the above-mentioned activity. These variant forms include (but are not limited to): deletion, insertion and/or substitution of 1-3 (usually 1-2, preferably 1) amino acids, and addition or deletion of one or more (usually within 3, preferably within 2, and more preferably within 1) amino acids at the C-terminus and/or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or more amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein.

The present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion proteins. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially maintain the function or activity of the fusion protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention can be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of an antigenic peptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6×His). According to the teachings of the present invention, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

A preferred class of active derivatives refers to polypeptides formed by replacing at most 3, preferably at most 2, and more preferably at most 1 amino acid with similar or similar properties compared to the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

Table A

Initial residue Representative replacement 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 present invention also provides analogs of the fusion protein of the present invention. The difference between these analogs and the polypeptides shown in any of SEQ ID NO.: 6 or 7 may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.

Preparation method of the present invention

The present invention also provides a method for preparing the IL-17 therapeutic vaccine according to the first aspect of the present invention, the method comprising the following steps:

(i) preparing IL-17A vaccine antigen and IL-17F vaccine antigen respectively;

(ii) mixing the IL-17A vaccine antigen and the IL-17F vaccine antigen prepared in step (i) in a ratio of (0.5-4):1 to obtain the IL-17 therapeutic vaccine.

In a preferred embodiment of the present invention, the IL-17A/IL-17F vaccine antigen is an IL-17A/IL-17F-CRM197 conjugate, wherein the IL-17A-CRM197 conjugate is prepared according to the following steps:

(1) Providing purified human interleukin-17 subtype A (IL-17A) protein and diphtheria toxin mutant CRM197 protein;

(2) diluting the IL-17A protein to a range of 1.0 to 3.0 mg/ml, diluting CRM197 to about 1 mg/ml, mixing, adding glutaraldehyde in a final concentration range of 0.05% to 0.15%, reacting at room temperature (25°C ± 2°C) for 1 to 3 hours, thereby obtaining an IL-17A-CRM197 conjugate.

(3) After the above conjugate is concentrated by ultrafiltration using a 50 kDa ultrafiltration membrane, the conjugate is separated by molecular sieve chromatography using Sephacryl S200 filler and the macromolecular components are collected, which is the IL-17A-CRM197 conjugate vaccine antigen stock solution.

The IL-17F-CRM197 conjugate is prepared by referring to the above-mentioned IL-17A-CRM197 conjugate preparation method.

In another preferred embodiment of the present invention, the IL-17A/IL-17F vaccine antigen is an IL-17A/IL-17F-CRM197 fusion protein, wherein the IL-17A-CRM197 fusion protein is prepared according to the following steps:

(1) The IL-17A encoding gene is connected to the CRM197 gene via a connecting peptide and constructed onto a vector containing a sumo tag, wherein the vector contains a gene that can co-express the Escherichia coli disulfide isomerase DsbC in the cytoplasm of Escherichia coli; the vector is transformed into an expression strain and induced for expression at 20-30°C.

(2) After the cells are broken, the His-tag affinity tag is used for affinity purification. After purification, the tag is removed using a SUMO tag-specific protease. The tag is then separated using affinity chromatography again.

(3) Using anion exchange chromatography at pH 8.0-8.5, use 5%-15% buffer B containing 1M NaCl to wash the impurities and 15-20% buffer B containing 1M NaCl to elute and collect the target protein fraction.

(4) Using gel filtration chromatography with Sephacryl S200 filler, the target protein component is collected after loading, which is the purified IL-17A-CRM197 fusion protein.

The IL-17F-CRM197 fusion protein is prepared by referring to the above-mentioned IL-17A-CRM197 fusion protein preparation method, wherein the difference is that in step (3), anion exchange chromatography is used to wash the impurities with 15-20% buffer B containing 1M NaCl at pH 8.0-8.5, and elute with 20-25% buffer B containing 1M NaCl to collect the target protein component.

Compositions of the present invention

The present invention also provides a composition. The composition contains the above-mentioned IL-17 therapeutic vaccine, and a pharmaceutically acceptable carrier, diluent, stabilizer and/or thickener, and can be prepared into a pharmaceutical type such as lyophilized powder, tablet, capsule, syrup, solution or suspension.

"Pharmaceutically acceptable carrier or excipient" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.

The composition may be a liquid or solid, such as a powder, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.

Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The composition may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration. The pharmaceutical composition is used for (a) inducing the production of neutralizing antibodies that block the binding of IL-17A and IL-17F to receptors; (b) for the treatment or prevention of autoimmune diseases.

The autoimmune disease includes, but is not limited to, rheumatoid arthritis, psoriasis or psoriatic arthritis, ankylosing spondylitis, Crohn's disease, or a combination thereof.

In addition, the composition can be administered to a subject in need alone or in combination with other pharmaceutical preparations for the treatment or prevention of the disease.

The composition provided by the present invention is preferably a vaccine composition. The vaccine composition comprises the IL-17 therapeutic vaccine according to the first aspect of the present invention and a vaccine-acceptable carrier, wherein the vaccine-acceptable carrier is preferably a pharmaceutically acceptable carrier.

In another preferred embodiment, the vaccine composition further contains an adjuvant, and the adjuvant is preferably a liquid adjuvant. After the vaccine composition of the present invention is mixed and emulsified with the liquid adjuvant, it can be inoculated into humans or non-human mammals to induce the production of anti-IL-17A and IL-17F neutralizing antibodies in vivo.

In another preferred embodiment, the adjuvant is selected from the following group: Montanide ISA 51VG, aluminum phosphate adjuvant, MF59, AS04, or a combination thereof.

Therapeutic/preventive applications

The present invention also provides a method for treating/preventing a disease, comprising administering the IL-17 therapeutic vaccine described in the first aspect of the present invention, or the composition described in the third aspect of the present invention to a subject in need thereof. Wherein, the disease is an autoimmune disease. In another preferred embodiment, the disease is selected from the group consisting of rheumatoid arthritis, psoriasis or psoriatic arthritis, ankylosing spondylitis, Crohn's disease, or a combination thereof.

The main advantages of the present invention are:

(1) The present invention couples or fuses the IL-17 antigen with a carrier protein, and the antigen induces the body to break through immune tolerance and produce anti-IL-17 antibodies, thereby blocking the IL-17 signaling pathway and the inflammatory signaling pathway, thereby achieving the effect of treating autoimmune diseases.

(2) The IL-17 therapeutic vaccine of the present invention is a bivalent vaccine, which contains both IL-17A vaccine antigen and IL-17F vaccine antigen. After administration to the human body, the IL-17A vaccine antigen and IL-17F vaccine antigen can stimulate the body to produce neutralizing antibodies against IL-17A and IL-17F, respectively. The immune effect is significantly better than the effect of using IL-17A vaccine antigen or IL-17F vaccine antigen alone.

(3) The IL-17A vaccine antigen and IL-17F vaccine antigen of the present invention are both truncated forms, which do not contain irregular structures at the N-terminus and C-terminus, have high antigen stability, and reduce IL-17 activity. At the same time, they can minimize non-essential sequences of antigen fragments and reduce the production of non-neutralizing antibodies; reduce lysine active groups and reduce the molecular weight of the conjugate.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

Example 1: Preparation of recombinant interleukin-17A (IL-17A)

According to the codon bias of Escherichia coli, an IL-17A (30-155aa) DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 1 was artificially synthesized (as shown in SEQ ID NO: 8). The DNA sequence was constructed into a pCDFDuet-1-DsbC vector containing a sumo tag, and the constructed vector was pCDF-Duet-1-sumoIL-17A-DsbC, wherein sumoIL-17A was in the first open reading frame and Escherichia coli disulfide isomerase DsbC was in the second open reading frame.

pCDF-Duet-1-sumoIL-17A-DsbC was transformed into E. coli Origami B (DE3) strain, and clones growing on streptomycin-resistant LB plates were screened. The clones were picked into LB medium and expanded in two stages to 20L of fermentation medium. The fermentation parameters were: stirring speed: 600rpm, dissolved oxygen control 20-50%, expansion culture temperature: 37°C, and post-induction temperature: 30°C. 8-10 hours after induction, the culture was terminated and the cells were collected by 7000g centrifugation.

The collected bacteria were crushed by high-pressure homogenization, and the supernatant was collected by centrifugation at 9000g after crushing. The target protein containing his-tag was captured using affinity filler chelated with nickel ions, and the 400mM imidazole eluate was collected. A sumo tag-specific protease containing 1/500 of the total amount of tagged protein was added to remove the tag. The tagged protein was captured again using affinity filler containing Ni ions, and the tagged protein was collected. Using Source 30S cationic chromatography filler, under the buffer condition of pH 7.4, isocratic gradient elution was performed, and 32% buffer B containing 1M NaCl was set to wash impurities, and 45% buffer B containing 1M NaCl was set to elute and collect IL-17A. The collected IL-17A was identified by reduced SDS-PAGE. The results are shown in Figure 1. The SDS-PAGE purity is greater than 95%.

Example 2: Preparation of recombinant interleukin-17F (IL-17F)

According to the codon bias of Escherichia coli, an IL-17A (11-158aa) DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 3 was artificially synthesized (as shown in SEQ ID NO: 9). The DNA sequence was constructed into a pCDFDuet-1-DsbC vector containing a sumo tag, and the constructed vector was pCDF-Duet-1-sumoIL-17F-DsbC, wherein sumoIL-17F was in the first open reading frame and Escherichia coli disulfide isomerase DsbC was in the second open reading frame.

The steps of expression, bacterial disruption, affinity chromatography and tag removal were the same as those in Example 1. Using Source 30S cationic chromatography filler, under the buffer condition of pH 6.8, isocratic gradient elution was performed, 22% buffer B containing 1M NaCl was set to wash impurities, and 32% buffer B containing 1M NaCl was set to elute and collect IL-17F. The collected IL-17F was identified by reduced SDS-PAGE, and the results are shown in Figure 2. The SDS-PAGE purity was greater than 95%.

Example 3: Preparation of recombinant IL-17A-CRM197/IL-17F-CRM197 conjugate

The recombinant IL-17A-CRM197 conjugate was prepared as follows:

(1) Binding reaction: Take an appropriate amount of 25% glutaraldehyde and prepare a concentration of 0.5% glutaraldehyde with phosphate buffer (PBS: 20mM PB, 150mM NaCl, pH 7.0). Take a certain amount of IL-17A and dilute it to 1.8mg/ml with PBS, take a certain amount of diphtheria toxin mutant CRM197 and dilute it to 1mg/ml with PBS, mix the two in equal volumes, and the molar ratio is about 8:1. Add 0.5% glutaraldehyde dropwise to a final concentration of 0.08%. Place the reaction container in a 25°C constant temperature shaker, set the shaker speed to 130rpm, and react for 2 hours.

(2) Ultrafiltration purification: After the reaction, ultrafiltration and concentration were performed using a 50 kDa ultrafiltration membrane.

(3) Sephacryl S200 molecular sieve chromatography was used with a loading volume of 2% of the column volume to separate the conjugate and collect the macromolecular component, which was the IL-17A-CRM197 conjugate stock solution.

The preparation of recombinant IL-17F-CRM197 conjugate refers to the above-mentioned method for preparing recombinant IL-17A-CRM197 conjugate, wherein in step (1), IL-17F is diluted with PBS to 2.1 mg/ml, and the remaining steps are the same as the method for preparing recombinant IL-17A-CRM197 conjugate.

Example 4: Preparation of bivalent IL-17 therapeutic vaccine (conjugate) and in vivo biological activity determination

The recombinant IL-17A-CRM197 conjugate stock solution was diluted to 1.6 mg/ml, and the recombinant IL-17F-CRM197 conjugate stock solution was diluted to 0.8 mg/ml. The two diluted conjugates were mixed at a volume ratio of 1:1 to obtain a bivalent IL-17 therapeutic vaccine (conjugate form).

Dilute the bivalent IL-17 therapeutic vaccine 10 times, take 0.5ml of the diluted vaccine into a 2ml syringe, take another 0.5ml of liquid adjuvant Montanide ISA 51VG into another 2ml syringe, connect the two syringes with a joint, push back and forth slowly for 15 rounds, and then push back and forth as quickly as possible for 30 times to complete the emulsification. After the emulsification is completed, push all the emulsion into the syringe on one side. The emulsion was injected into the subcutaneous part of the mouse at a volume of 0.1ml/mouse, for a total of 8 mice, and injected again once every 1 week. Blood was collected from the eye sockets 1, 3, and 5 weeks after the second injection, and the antibody titers against IL-17A and IL-17F were detected after blood collection.

Anti-IL-17A/IL-17F antibody titer detection method:

(1) Coating: Take an ELISA assay 96-well plate, dilute IL-17A or IL-17F protein to 0.5 μg/ml with Na ₂ CO ₃ -NaHCO ₃ , pH 9.6 coating buffer, add 100 μL of the diluted IL-17A or IL-17F protein to each well of the 96-well plate, and incubate at 2-8°C overnight.

(2) Blocking: The next day, discard the coating solution, add 150 μL of PBS blocking solution containing 0.05% (v/v) Tween-20 and 1% BSA to each well, and incubate at 37°C for 1 hour;

(3) Washing: After discarding the blocking solution, each well was washed three times with 200 μL of PBS containing 0.05% (v/v) Tween-20, and the liquid in the well was aspirated;

(4) Dilute the collected mouse serum, take 100 μL of the diluted serum and add it to the leftmost well of the 96-well plate, with each mouse as one row, dilute from left to right, and dilute 12 gradients in total. Use the serum of unimmunized mice as a control, incubate at 37°C for 1 h after adding the serum; after incubation, discard the incubation solution, and wash repeatedly three times as described above;

(5) Dilute the secondary antibody HRP enzyme-labeled anti-mouse antibody to 1:5000 with blocking solution, add 100 μL of HRP enzyme-labeled antibody to each well, incubate at 37°C for 1 h; discard the incubation solution, and wash repeatedly 5 times as described above;

(6) Color development: Add 100 μl TMB to each well and incubate at room temperature in the dark for 15 min;

(7) Termination: Add 100 μl of 0.5 mol/L sulfuric acid to each well to terminate color development;

(8) After termination, the light absorption value of each well OD450 was measured using a microplate spectrophotometer. The light absorption value was measured at 450 nm to determine the highest dilution factor at which the OD value of the serum sample/OD value of the saline control group sample was ≥ 2.1.

The test results are shown in Figure 3 and Table 1. After mice were immunized with the bivalent IL-17 therapeutic vaccine (conjugate), they were able to induce mice to produce high titer IL-17A antibodies and IL-17F antibodies, respectively. One week after the second immunization, the titers of IL-17A and IL-17F antibodies in the serum of all mice were higher than the positive conversion standard of 1:4000. Five weeks after the second immunization, the geometric mean titers of IL-17A and IL-17F antibodies in the serum of mice reached more than 400,000.

Table 1

Example 5: Preparation of bivalent IL-17 therapeutic vaccine (fusion protein) and in vivo biological activity determination

According to the codon bias of Escherichia coli, a DNA sequence encoding the IL17A (58-151) -CRM197 amino acid sequence as shown in SEQ ID NO: 6 was artificially synthesized. The DNA sequence was constructed into a pCDFDuet-1-DsbC vector containing a sumo tag, and the constructed vector was pCDF-Duet-1-sumoIL-17A-CRM197-DsbC, wherein sumoIL-17A-CRM197 was in the first open reading frame and Escherichia coli disulfide isomerase DsbC was in the second open reading frame.

pCDF-Duet-1-sumoIL-17A-CRM197-DsbC was transformed into E. coli Origami B (DE3) strain, and clones growing on streptomycin-resistant LB plates were screened. The clones were picked into LB medium and expanded in two stages to 20L of fermentation medium. The fermentation parameters were: stirring speed: 600rpm, dissolved oxygen control 20-50%, expansion culture temperature: 37°C, and post-induction temperature: 25°C. 10-16 hours after induction, the culture was terminated and the bacteria were collected by 7000g centrifugation.

The collected bacteria were crushed by high-pressure homogenization, and the supernatant was collected by centrifugation at 9000g after crushing. The target protein containing his-tag was captured using affinity filler chelated with nickel ions, and the 400mM imidazole eluate was collected. A sumo tag-specific protease containing 1/500 of the total amount of tagged protein was added to remove the tag. The tagged protein was captured again using affinity filler containing Ni ions, and the tagged protein was collected. Using Source 30Q anion chromatography filler, at pH 8.0, isocratic gradient elution was performed, and 10% buffer B containing 1M NaCl was set to wash, and 15% buffer B containing 1M NaCl was set to elute and collect, and then the IL-17A-CRM197 fusion protein component was collected by gel filtration chromatography using SephacrylS200 filler.

According to the above-mentioned IL17A (58-151) -CRM197 preparation method, a DNA sequence encoding the IL-17F (70-156) -CRM197 amino acid sequence as shown in SEQ ID NO: 7 was artificially synthesized, and the pCDF-Duet-1-sumoIL-17F-CRM197-DsbC vector was constructed. The difference in purification preparation is that under the conditions of pH 8.0-8.5, 15-20% impurities are washed and 20-25% are eluted to collect the target protein components, and then the target protein components are collected by gel filtration chromatography using Sephacryl S200 filler, which is the purified IL-17F-CRM197 fusion protein.

The recombinant IL-17A-CRM197 fusion protein and IL-17F-CRM197 fusion protein stock solutions were diluted to 1.5 mg/ml, and the two diluted fusion proteins were mixed at a volume ratio of 1:1 to obtain a bivalent IL-17 therapeutic vaccine (fusion protein form). The in vivo biological activity was detected as in Example 4. In this example, only the serum antibody titer was detected one week after the secondary immunization.

The results are shown in FIG4 . The bivalent IL-17 therapeutic vaccine (fusion protein) can simultaneously induce mice to produce high titer anti-IL-17A and anti-IL-17F antibodies after immunization.

Example 6: IL-17 activity assay of IL-17A(39-155), IL-17F(11-158), IL-17A(58-151)-CRM197 and IL-17F(70-156)-CRM197

The present invention uses truncated IL-17A and IL-17F as antigens, and the IL-17 activity of the truncated form is significantly lower than that of the full-length form. The IL-17 activity of the truncated form and the fusion protein of the truncated form is detected by the MC-3T3 cell method, and the principle and steps are as follows:

Under the condition of low concentration of tumor necrosis factor (TNFα), if IL-17A or IL-17F activity exists in the culture medium, cells can be induced to express IL-6. The biological activity of IL-17A or IL17F can be detected by detecting the expression level of IL-6 in the cell culture supernatant.

The specific steps are as follows (IL-17A and IL-17F methods are the same, hereinafter referred to as IL-17):

In a 96-well plate, 80,000 to 100,000 MC-3T3 cells were seeded into each well and cultured for 20 to 24 hours.

Prepare α-MEM culture medium containing 0.6% FBS and 4ng/ml TNFα, and dilute IL-17 (full-length control), IL-17 truncated form or IL-17 truncated form-CRM197 fusion protein to 10nmol/L with the culture medium, add to the cultured MC-3T3 cells, set up 3 replicates, continue to culture for 24 hours, and collect the culture supernatant. The negative control is the dilution buffer.

IL-6 in the culture supernatant was detected according to the instructions of the mouse IL-6 detection kit (Sigma-Aldrich, catalog number: RAB0311-1KT).

The results are shown in Figure 5. The activity of IL-17 truncation and IL-17 truncation-CRM197 fusion protein in inducing MC-3T3 cells to express IL-6 was significantly reduced, indicating that the biological activity of truncated IL-17A and IL-17F was significantly reduced.

Example 7: Mouse immunization serum can simultaneously inhibit the biological activities of IL-17A and IL17F

IL-17A and IL-17F have similar biological activities and can both be tested using MC-3T3 cells. Refer to the biological activity detection method of IL-17A or IL17F in Example 6.

The specific steps are as follows (IL-17A and IL-17F methods are the same, hereinafter referred to as IL-17):

In a 96-well plate, 80,000 to 100,000 MC-3T3 cells were seeded into each well and cultured for 20 to 24 hours.

Prepare α-MEM culture medium containing 0.6% FBS, 4ng/ml TNFα and 200ng/ml IL-17. Take the mouse serum 5 weeks after the second immunization of mice immunized with the bivalent IL-17 therapeutic vaccine in Example 4, and dilute the serum 50 times, 100 times and 200 times respectively with α-MEM culture medium (without FBS, TNFα and IL-17).

The culture medium diluted at different times was mixed with equal volumes of α-MEM culture medium containing 0.6% FBS, 4ng/ml TNFα and 400ng/ml IL-17, and then added to the cultured MC-3T3 cells. Three replicate wells were set up and cultured for 24 hours, and then the culture supernatant was collected. The negative control was the serum of non-immunized mice, and the positive control was the purchased IL-17 antibody.

IL-6 in the culture supernatant was detected according to the instructions of the mouse IL-6 detection kit (Sigma-Aldrich, catalog number: RAB0311-1KT).

The results are shown in Figures 6 and 7. The serum (diluted 50 times) after immunization with the bivalent IL-17 therapeutic vaccine can simultaneously block IL-17A and IL-17F from inducing MC-3T3 cells to express IL-6, showing the neutralizing activity of the induced serum against IL-17A and IL-17F, respectively.

Example 8: Bivalent IL-17 therapeutic vaccine can significantly inhibit psoriasis symptoms in mice

Eight-week-old C57BL/6 mice were selected and immunized with the bivalent IL-17 therapeutic vaccine on days 1, 8, 15, and 22, respectively, using the emulsification method described in Example 4. At the same time, adjuvant control, IL-17A-CRM197 conjugate, and IL-17F-CRM197 conjugate were selected as controls, respectively.

Psoriasis was induced on day 29 by removing hair from the back of mice by plucking and applying 0.125 g of 5% imiquimod cream [6.25 mg imiquimod (Perrigo)] topically daily for 5 consecutive days. Imiquimod was used to induce epidermal thickening to simulate human plaque psoriasis and increase IL-17 and other inflammatory cytokines, and epidermal thickness, which correlates with the expression of these cytokines, was selected as the outcome measure.

24 hours after the last drug application, the mice were sacrificed and the skin was removed for histological examination.

As shown in the results of Figure 8, both IL-17A-CRM197 conjugate and IL-17F-CRM197 conjugate can improve psoriasis symptoms to a certain extent, but the effect of bivalent IL-17 therapeutic vaccine in inhibiting psoriasis symptoms is significantly better than the effect of using IL-17A-CRM197 conjugate or IL-17F-CRM197 conjugate alone.

Example 9: Bivalent IL-17 therapeutic vaccine can significantly inhibit rheumatoid arthritis symptoms in mice Male DBA mice about 6 weeks old were selected, with 8 mice in each group, for a total of 4 groups;

Add 5 ml of complete Freund's adjuvant to a mixing tube, then place it on ice, immerse the emulsification head below the liquid level of the adjuvant, turn on the emulsifier (10,000 rpm to 30,000 rpm), then add 5 ml of chicken type II collagen dropwise, and move the emulsification tube up and down to ensure uniform emulsification. Emulsify for 2 minutes.

After the emulsion was placed in an ice bath for 5 minutes, the emulsifier was turned on again (10,000 rpm to 30,000 rpm), and the emulsification tube was moved up and down to ensure uniform emulsification. The emulsification was continued for 2 minutes.

After emulsification is completed, a water drop test can be performed to determine whether the emulsification is complete.

The emulsion was drawn into the Hamilton syringe through the three-way connector of the emulsification tube.

Type II collagen immunization: Type II collagen immunization was performed on the tail of mice, and the injection volume for each mouse was 100 μl.

The first type II collagen immunization was recorded as day 0, and another type II collagen booster immunization was performed on day 21. The type II collagen booster immunization operation was the same as the first type II collagen immunization.

After the completion of type II collagen booster immunization, the mice were scored for arthritis every week. The mice were expected to begin to develop the disease around the 28th day, reach the peak of disease on the 42nd day, and the symptoms would spontaneously alleviate after the 60th day.

Administration: The emulsification scheme described in Example 4 was used to immunize the bivalent IL-17 therapeutic vaccine on days 5, 12, 19, and 26, respectively. At the same time, adjuvant control, IL-17A-CRM197 conjugate, and IL-17F-CRM197 conjugate were used as controls.

Starting from day 21, the four paws of the mice were scored weekly according to the following scoring criteria, and the total score of each mouse was the sum of the scores of the four paws.

score standard 0 No change 0.5 Visible swelling and redness on one toe 1 Swelling and erythema of two toes 2 Slight swelling and redness of the paw or swelling of more than two toes 3 Visible swelling and redness of the paw 4 Swelling and redness greatest in the limb, spreading to the ankle

As shown in the results of Figure 9, both IL-17A-CRM197 conjugate and IL-17F-CRM197 conjugate can inhibit the progression of rheumatoid arthritis symptoms to a certain extent, but the effect of the bivalent IL-17 therapeutic vaccine in inhibiting arthritis symptoms is significantly better than the effect of using IL-17A-CRM197 conjugate or IL-17F-CRM197 conjugate alone.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (10)

1. An IL-17 therapeutic vaccine, characterized in that, the therapeutic vaccine comprises: (a) an IL-17A vaccine antigen having the structure shown in formula (I); and (b) an IL-17F vaccine antigen having the structure shown in formula (II);

Z1-L-Z3(I)

Z2-L-Z3(II)

wherein Z1 is an IL-17A antigen-active fragment;

Z2 is an IL-17F antigen-active fragment;

Z3 is a carrier protein;

L is a linker selected from a covalent bond formed by cross-linking by a cross-linking agent, or a linker peptide or peptide bond for fusion protein expression;

"-" means a chemical bond connecting the above elements.

2. The IL-17 therapeutic vaccine of claim 1, wherein the amino acid sequence of the IL-17A antigen-active fragment is as set forth in SEQ ID NO:1 or SEQ ID NO: 2.

3. The IL-17 therapeutic vaccine of claim 1, wherein the amino acid sequence of the IL-17F antigen-active fragment is as set forth in SEQ ID NO:3 or SEQ ID NO: 4.

4. The IL-17 therapeutic vaccine of claim 1, wherein Z3 is diphtheria toxin mutant CRM197 having an amino acid sequence as set forth in SEQ ID No. 5.

5. The IL-17 therapeutic vaccine of claim 1, wherein the IL-17A vaccine antigen is a fusion protein having an amino acid sequence as set forth in SEQ ID NO:6, and

The IL-17F vaccine antigen is fusion protein, and the amino acid sequence of the fusion protein is shown as SEQ ID NO: shown at 7.

6. The IL-17 therapeutic vaccine of claim 1, wherein the IL-17A vaccine antigen and the IL-17F vaccine antigen are both conjugates, and wherein the molar ratio of IL-17A/IL-17F antigen active fragment to CRM197 protein in the conjugates is (6-10): 1.

7. The therapeutic IL-17 vaccine according to claim 1, wherein the ratio of IL-17A vaccine antigen to IL-17F vaccine antigen in the therapeutic IL-17 vaccine is (0.5-4): 1.

8. A method of preparing the IL-17 therapeutic vaccine of claim 1, the method comprising the steps of:

(i) Preparing an IL-17A vaccine antigen and an IL-17F vaccine antigen respectively;

(ii) And (3) mixing the IL-17A vaccine antigen prepared in the step (i) and the IL-17F vaccine antigen according to the proportion of (0.5-4): 1, thus obtaining the IL-17 therapeutic vaccine.

9. A composition, characterized in that it comprises: the IL-17 therapeutic vaccine of claim 1, and a pharmaceutically acceptable carrier.

10. Use of the IL-17 therapeutic vaccine of claim 1 in the manufacture of a medicament for the treatment and/or prophylaxis of a disease.