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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/55—IL-2
• • A—HUMAN NECESSITIES
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  • C07K14/521—Chemokines
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  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/521—Chemokines
  • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
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  • C07K14/525—Tumour necrosis factor [TNF]
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5421—IL-8
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55522—Cytokines; Lymphokines; Interferons
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  • A61K2039/55533—IL-2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• Chemokine-cytokine fusion protein and its application
• the present invention relates to a fusion protein comprising a chemokine and a cytokine linked to the chemokine, wherein the chemokine and the cytokine have a common target cell, and which has enhanced cytokine activity and enhancement The role of chemokine activity.
• Cytokine is a group of proteins secreted by external stimulation of cells (only a few species of cytokines can be expressed in the cell membrane). Cytokines can be applied to target cells by cell or by blood transport. It has an effect and works at very low concentrations. It has a wide range of functions that promote the growth, differentiation or activation of target cells. Many cytokines can act on immune cells and participate in immune responses. Cytokines can be roughly classified into chemokines, interleukins, growth factors, transforming growth factors, and colony stimulating factors depending on their structural roles. Factors ), tumor necrosis factors and interferons.
• Chemokines are a group of cytokines that can chemotaxis to attract white blood cells. They are usually small, positively charged, secretable proteins. The main function is to attract immune cells to tissues that are injured or have pathogenic infections. The white blood cell can then undergo phagocytosis or inflammation in this particular site to protect against pathogens.
• the white blood cells attracted by chemokines may include neutral particles that are innate immunity. Cells (neutrophils), monocytes/macrophages, natural killer cells, dendritic cells, and other white blood cells, or are adaptive immunity T lymphocytes (T cells) or B lymphocytes (B cells) play a very important role in the immune system of an organism.
• chemokines have four highly retained cysteine (C) to form a disulfide bond to stabilize their structure. According to the difference in the number of amino acids between the first two C and the presence or absence of the first C, Divided into CXC (or ⁇ ), CC (or ⁇ ), C (or ⁇ ) and CX 3 C four subgroups (subfamilies).
• Stromal cell-derived factor-1 (SDF-1) is classified in the CXC subgroup of chemokines, also known as CXC ligand 12 (CXCL12), and is found in many species.
• SDF-1 spliced in different splicing mRNAs in mice and humans, so two SDF-1 isoforms (isoform) can be found: SDF -1 ⁇ and SDF-1 ⁇ 0 SDF-1 is widely distributed, including lymphoid tissues, kidneys, lungs, liver, brain and muscles (Shirozu et al, Genomics 28, 495-500).
• the receptor CXCR4 of SDF-1 in addition to its persistence in organs, can also be found in hematopoietic stem cells, endothelial cells, dendritic cells, B cells, and T cells, so these cells are attracted by SDF-1. Move to a site with a high concentration of chemokines (Bleul et al, Nature, 382: 829-833; Oberlin et al" Nature 382: 833-835; Read et al, Developmental and comparative immunology, 29, 143-152). Interleukin-8 (IL-8) is also classified in the CXC subgroup of chemokines, also known as CXCL8.
• IL-8 can chemotacticly multi-immune cells at low concentrations, including mononuclear cells, macrophages and lymphocytes, neutrophils and the like.
• CD40 ligand is a member of the tumor necrosis factor (TNF) superfamily. This cytokine has the function of promoting tumor necrosis and promoting leukocyte differentiation, proliferation and apoptosis. .
• TNF tumor necrosis factor
• the protein has a total of 261 amino acids. First, the amino terminal 22 amino acids are intracellular regions, then the 24 amino acids are transmembrane regions, and the carboxyl terminal is 215.
• the amino acid sequence is an extracellular (Exc) region, and the Exc region has a TNF homology (TNFh) region retained by a TNF superfamily protein at the carboxy terminus.
• CD40L exists mainly on the surface of activated CD4+ T cells in the form of transmembrane proteins, as well as CD8 + T cells, basophils, eosinophils > megapascals, natural killer cells, platelets, It was found even on the surface of cells expressing CD40.
• the receptor for CD40L is CD40, which is distributed in B cells, dendritic cells, macrophages and other antigen-presenting cells.
• antigen presenting cells APCs
• APCs antigen presenting cells surface.
• these antigen-presenting cells can be activated by CD40L expressed by helper T cells, promoting the expression of the second major histocompatibility complex.
• CD40L activates the information transmission pathway by binding to CD40 on the target cells. In addition to the above-mentioned effects of promoting antigen presentation, CD40L promotes B cell proliferation, isotype switching, and antibody secretion in B cells. Differentiation of memory B cells or avoidance of apoptosis; CD40L acts on macrophages to enhance their activation and promote the production of interleukin-12 (IL-12) to activate type 1 helper T cells. (T helper 1, Thl ) either secretes some chemokines or promotes nitric oxide
• Nictric oxide, NO is produced to enhance the ability of macrophages to defend microorganisms; in dendritic cells, it can mature and activate, and activated dendritic cells, in addition to expressing a large number of MHC-II molecules to promote antigen presentation, also secrete TNF- ⁇ and IL-8, macrophage inflammatory protein la (MIP-la) and other chemokines.
• DHBV duck hepatitis B virus
• HAV human immunodeficiency virus
• Interleukin-2 is classified in the hematopoietin family, which includes hormones or other cytokines associated with cell growth.
• the functions of IL-2 include: regulating the maturation and differentiation of T cells, stimulating the proliferation of B cells and antibody secretion, promoting the killing ability of natural killer cells, and activating monocytes and macrophages.
• IL-2 can also stimulate the continuous expression of MHC by T cells and B cells, and can also stimulate natural killer cells to produce a variety of different cytokines, including TNF- ⁇ , IFN- ⁇ and granulocyte/macrophage stimulating factor (granulocyte/macrophage). Colony stimulating factor, GM-CSF). Studies have shown that IL-2 has anti-tumor and vaccine-enhancing effects.
• a fusion protein comprising a chemokine and a cytokine linked to the chemokine has enhanced cytokine activity and enhanced chemokine activity.
• the present invention provides a fusion protein comprising a chemokine polypeptide which is a chemokine or a receptor binding domain thereof; and a cytokine polypeptide linked to the chemokine polypeptide, which is an interleukin a TNF-superfamily cytokine or a receptor binding domain thereof; wherein the chemokine polypeptide and the cytokine polypeptide share a common target cell, and the fusion protein has enhanced chemotaxis compared to the chemokine polypeptide Activity, and enhanced cytokine activity compared to the cytokine polypeptide.
• the chemokine is a CXC chemokine, CC chemokine, C chemokine and CX 3 C chemokine, preferably CXC chemokine.
• the chemokine may be a stromal cell-derived factor- 1 (SDF-1) or IL-8.
• the cytokine polypeptide is an interleukin, a TNF-superfamily cytokine or a receptor binding domain thereof.
• the cytokine polypeptide is an IL-2, CD40 ligand or a receptor binding domain thereof.
• the invention provides an isolated nucleic acid molecule encoding a fusion protein of the invention.
• the invention provides an expression vector comprising a nucleic acid molecule of the invention.
• the invention also provides a host cell comprising a nucleic acid molecule of the invention or an expression vector of the invention.
• Figure 1 shows chicken CD40L and its derived protein ⁇ 401 ⁇ ⁇ 3 ⁇ 4 schematic diagram
• Figure 2 shows the results of SDS-PAGE and western blot analysis of the expressed chicken recombinant fusion protein
• lane 1 (lane 1 ): IL8CD40L Exc , expected size 52 kDa
• lane 2 IL8IL2, expected size 44 kDa
• 3 channels SDFlCD40L Exc , expected size is 38 kDa
• lane 4 SDFlCDWLTWh, expected size is 44 kDa
• lane 5 SDF1IL2, expected size is 26 kDa;
• Figure 3 shows the results of SDS-PAGE and western blot analysis of purified single protein
• Lane 1 tagged protein, expected size 21 kDa; lane 2: IL-8, expected size 13 kDa; lane 3: SDF-1, expected size 11 kDa; lane 4: IL-2, expected Size is 32 kDa; Lane 5: CD40L Exc , expected size is 42 kDa; and lane 6: CD LTNFH, expected size is 33 kDa;
• Figure 4 shows the situation of IL-8-derived protein chemotactic PBMCs
• the cells are attracted by the chemotactic factors in the gel, which will swim from the center colloid to the surrounding area.
• the cells will appear cloudy at low magnification.
• the more cells around the colloid the greater the degree of chemotactic cells.
• IL8CD40L Exc or IL8IL2 The degree of chemotactic cells is significantly greater than that of IL-8 single protein;
• Figure 5 shows the situation of SDF-1 derived protein chemotactic PBMCs
• FIG. 6 shows that CD40L-derived protein activates macrophage to produce NO activity
• Figure 7 shows the results of the IL-2 fusion protein promoting lymphocytosis test
• SI Stimulation index
• Figure 8 shows that IL-2 fusion protein as a Newcastle disease (ND) vaccine adjuvant can significantly promote cellular immune response; administration of IL-2 fusion protein adjuvant and ND vaccine group chickens, and Newcastle disease virus (NDV) antigen re At the time of stimulation, the memory lymphocyte proliferation was significantly higher than that of the ND vaccine group;
• ND Newcastle disease
• Figure 9 shows that the fusion protein acts as an adjuvant for infectious bronchitis (IB) vaccine and can significantly promote cellular immune responses;
• IBV Infectious bronchitis virus
• chemokine polypeptide refers to a polypeptide which is a chemokine or a receptor binding domain thereof, wherein the chemokine includes, but is not limited to, CXC chemotaxis. , CC Chemokine, C Chemokine and CX 3 C Chemokine.
• cytokine polypeptide refers to a polypeptide which is a cytokine or a receptor binding domain thereof, wherein the cytokines include, but are not limited to, interleukin and TNF-superfamily cytokines.
• chemokine activity refers to the activity that a chemotactic hormone possesses or can exert in an organism, including but not limited to chemotaxis of various immune cells (including monocytes, macrophages, T cells). , B cells, natural killer cells, dendritic cells and neutrophils, etc.).
• cytokine activity refers to the activity possessed or achievable by a cytokine in a living being, including but not limited to promoting B cell proliferation, immunoglobulin type shift, antibody secretion; memory B cell Differentiate or avoid apoptosis; promote macrophage secretion of interleukin-12 to activate type 1 helper T cells or secrete chemokines; promote macrophage production of nitric oxide to enhance the ability to defend microorganisms; promote dendrites Cell maturation and activation; regulates T cell maturation and differentiation; promotes the killing ability of natural killer cells, produces a variety of different cytokines; activates monocytes and macrophages; and stimulates the continuous expression of T cells and B cells MHC and so on.
• the present invention provides a fusion protein comprising a chemokine polypeptide which is a chemokine or a receptor binding domain thereof; and a cytokine polypeptide linked to the chemokine polypeptide, which is an interleukin, a a TNF-superfamily cytokine or a receptor binding domain thereof; wherein the chemokine polypeptide and the cytokine polypeptide share a common target cell, and the fusion protein has a more enhanced chemokine activity than the chemokine polypeptide And a more enhanced cytokine activity compared to the cytokine polypeptide.
• the chemokine polypeptide and the cytokine polypeptide are linked by a polypeptide linker.
• a polypeptide linker In order to join the two proteins together and retain the original conformation and function, an appropriate polypeptide linker can be added between the two proteins to reduce mutual interference when the two proteins are folded.
• polypeptide linkers are, for example, an elastic polypeptide linker (Gly-Gly-Gly-Gly-Ser) n (usually n less than 6) having a degree of elasticity and hydrophilicity, or a hydrophilic helical polypeptide linker (Glu) -Ala-Ala-Ala-Lys) n (usually n is less than 6).
• the chemokine is a CXC chemokine.
• the chemokine is a stromal cell-derived factor (SDF- in another particular embodiment, the chemokine is IL-8.
• the chemokine polypeptide has one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and the same Homolog and analog ( analog ).
• the cytokine polypeptide is an IL-2, CD40 ligand; CD40L; or a receptor binding domain thereof.
• the cytokine polypeptide has one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and homologs thereof Things and analogues.
• the fusion protein of the invention has one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 40, 42, 44, 46 and 48.
• the invention provides an isolated nucleic acid molecule encoding a fusion protein of the invention.
• the isolated nucleic acid molecule comprises one of the nucleotide sequences encoding a chemokine polypeptide selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 15, 17 and its homologs and analogs.
• the isolated nucleic acid molecule comprises one of the nucleotide sequences encoding a cytokine polypeptide selected from the group consisting of: SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and their homologs and analogs.
• the isolated nucleic acid molecule has one of the following sequences: SEQ ID NO: 39, 41, 43, 45 and 47.
• the invention provides an expression vector comprising a nucleic acid molecule of the invention.
• the invention also provides a host cell comprising a nucleic acid molecule of the invention or an expression vector of the invention.
• a host cell comprising a nucleic acid molecule of the invention or an expression vector of the invention.
• the following examples are for illustrative purposes only and are not intended to be limiting of the invention.
• Example 1 Construction of prokaryotic expression vector
• chicken CD40L (chCD40L)-derived protein includes extracellular domain of CD40L, CD40L Exc or TNF homologous region (TNF homology domain of CD40L, 0040 ⁇ 3 ⁇ 4 ) (see Figure 1).
• conjugate protein or fusion protein such as SDF 1 CD40L Exc , SDF 1 CD40LTNFh , SDF 1 IL2 , IL8CD40L Exc , IL8CD40LxN Fh , IL8IL2 are amplified .
• the method is briefly described as follows. Two pairs of specific primer pairs are designed according to each gene sequence.
• the forward primer of the first pair of primers has a restriction enzyme site EcoR I cleavage and the N-terminal gene of the fusion protein in the anterior segment.
• the sequence, the reverse primer is provided with a helix polypeptide linker and a C-terminal gene sequence of the fusion protein located in the anterior segment, and the pair of primers can specifically amplify a DNA fragment encoding the fusion protein front segment and the polypeptide linker;
• the primer has a helical polypeptide linker and the fusion protein is located in the rear N-terminal gene sequence, and the reverse primer has a restriction enzyme site; the cleavage site and the fusion protein are located in the rear C-terminal gene sequence, this pair
• the primer can specifically amplify the DNA fragment encoding the polypeptide linker and the latter part of the fusion protein; using the two PCR products as a template, the first primer pair primer
• the two fragments will be joined for the purpose of ligation due to the overlapping partial sequence of the helical polypeptide linker, and the resulting product is the fusion gene sequence containing the helical polypeptide linker.
• the pET vector Novagen, Darmstad, Germany
• coR I and o I was subjected to T4 DNA ligase (Invitrogen) at 16 °C.
• the eukaryotic expression vectors were constructed as pETSDF1CD40L Exc , pETSDF1CD ⁇ LxNFh, pETSDFHL2, pETIL8CD40L Exc and pEm8IL2, respectively.
• Example 2 Expression of recombinant protein
• the constructed prokaryotic expression vector was transformed into E. coli expression strain BL21 (DE3) to 0.5 mM IPTG induces expression of the recombinant protein, and the culture medium is removed by centrifugation. All the cells were then resuspended in binding buffer, and the cells were lysed using a high pressure cell lysometer (French Pressure Cell Press, Thermo IEC, Needham, Height, MA, USA), and the water-soluble protein was centrifuged at high speed. It will be located in the supernatant and purified on a nickel ion affinity column. The insoluble protein is located in the bottom mass after centrifugation.
• the urea After treating the partially insoluble protein with 8 M urea, the urea will open the folded protein to dissolve it in the aqueous solution, and then centrifuge the 12000 rpm for 30 minutes to take the supernatant. The solution is dialyzed, the urea is slowly replaced, the protein is refolded into the original configuration, and finally the protein is dissolved in a phosphate buffer (H 7.3 ) containing 10% glycerol, and filtered through a 0.22 ⁇ m filter. Protein concentration was determined by BCA Protein Assay Kit (Pierce, ockford, IL, USA) and stored at -20 °C. The protein was purified and identified as the correct fusion protein by MALDI-TOF analysis.
• chemokine SDF-1 or IL-8
• peripheral blood mononuclear cells with Histopaque 1077 (Sigma, Saint Louis, Mo, USA) (eripheral blood) Mononuclear cells, PBMCs
• PBMCs peripheral blood mononuclear cells
• RPMI 1640 Gibco, Grand Island, NY, USA
• FBS fetal bovine serum
• Vegetable gum mix well, make the final concentration of the vegetable gum 0.3%, then take the mixture 2 ⁇ / hole in the center of the 48-well plate, put it into the water tank for five minutes to solidify the acacia gum, and fix the cells in Within the scope of the glue. Then, 250 ⁇ M of the culture medium containing the respective concentrations of the test protein was added to each well, and cultured overnight after 37 days of culture.
• MEC minimal effective concentration
• IL8CD40L Exc or IL8IL2 can significantly reduce MEC value and enhance chemotactic activity, and its fusion protein chemotactic activity is better than IL-8 (see Table 1); SDF-1 regardless of fusion CD40L-derived protein or IL-2
• CD40L activity after fusion of chemokines with CD40L-derived proteins was assessed based on the properties of CD40L-activated macrophages producing NO.
• Peripheral blood mononuclear cells were isolated and washed twice with PBS. The cells were adjusted to 2 ⁇ 10 6 /ml with RPMI 1640 containing 10% FBS plus 125 ng/ml chicken IL-2 and 4 ⁇ LPS in 24-well plates. Add 1 ml each, and add 1 ml of fresh medium (two 125 ng/ml chicken IL-2 and 4 ⁇ LPS) after two days. After five days, the mononuclear cells will differentiate into macrophages.
• the fusion protein IL8CD40L Exe showed significantly better activity than the group to which only CD40L Exe was added (5-30 nM, ⁇ ⁇ 0.05; 180 nM, p ⁇ 0.01).
• the effect of the fusion protein SDFlCD40L Exc was significantly better than that of the single CD40L Exc protein (5 ⁇ , ⁇ ⁇ 0.01; 30-180 ⁇ , ⁇ ⁇ 0.05).
• the combination of SDF-1 and CD40L TOFh has similar scoring, and the fusion protein SDFlCD ⁇ LxNFh is also significantly better than single protein (CD40LTNFh) (5 nM and 180 nM, /? ⁇ 0.01; 0.8 nM and 30 nM, ? ⁇ 0.05 ) See Figure 6).
• Example 5 IL2 fusion protein promotes lymphocyte proliferation
• IL-2 activity after fusion of chemokine with IL-2 was evaluated.
• the activity of intracellular acid phosphatase was proportional to the number of cells, and a chromogenic substrate p-nitrophenyl phosphate (pNpp) was added.
• pNpp chromogenic substrate p-nitrophenyl phosphate
• RPMI 1640 medium containing 10% FBS was added with different concentrations of protein, 10 ⁇ ConA (positive control group), or 10 nM tagged protein (negative control group), and cultured at 96.
• SI proliferation index
• IL-2 fusion protein is applied to chickens as a vaccine for Newcastle disease (ND) vaccine.
• Chicken blood is taken after administration of vaccine, lymphocytes are cultured and inactivated.
• ND Newcastle disease
• Newcastle disease virus (inactivated) Newcastle disease virus (NDV) is an antigen, and an antigen re-stimulation assay is performed.
• 10 g/ml ConA is added to the culture medium as a positive control group, and the chickens in each group are compared.
• the group inoculated with IL-2 fusion protein as ND vaccine adjuvant (SDF1IL2 + ND vaccine) was significant.
• Example 7 Fusion protein as an infectious bronchitis (IB) vaccine adjuvant promotes vaccine-induced immune response
• the IL-2 fusion protein or CD40L Exc fusion protein is used as an adjuvant for avian infectious bronchitis (IB) vaccine. After the vaccine is administered, the chicken blood is taken, the lymphocytes are cultured, and the infectious bronchitis virus is added.
• Virus, IBV is an antigen, and an antigen re-stimulation test is performed to compare the memory lymphocyte proliferation of IBV antigens in each group of chickens.
• the IL-2 fusion protein was IB vaccine adjuvant (SDF1IL2 + IB vaccine) (p ⁇ 0.01) or the CD40L Exc fusion protein was IB vaccine.
• the combination of the agents (SDFlCD40L Exc + IB vaccine ) (p ⁇ 0.05) significantly increased the memory lymphocyte proliferative activity with antigen recognition specificity during antigen re-stimulation (see Figure 9).

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