US20150307577A1 - Chemokine-cytokine fusion proteins and their applications - Google Patents

Chemokine-cytokine fusion proteins and their applications Download PDF

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US20150307577A1
US20150307577A1 US14/649,714 US201214649714A US2015307577A1 US 20150307577 A1 US20150307577 A1 US 20150307577A1 US 201214649714 A US201214649714 A US 201214649714A US 2015307577 A1 US2015307577 A1 US 2015307577A1
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chemokine
polypeptide
fusion protein
cytokine
cells
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Shiow-Her CHIOU
Kuan-Chih Chow
Jui-Hung Shien
Yi-Hsin FAN
Pei-Hua LIN
Pei-Shan Wu
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National Chung Hsing University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5421—IL-8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K2039/55527—Interleukins
    • A61K2039/55533—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to fusion protein, comprising a chemokine and a cytokine connected thereto, wherein the chemokine and the cytokine have a mutual target cell, and the fusion protein has an improved cytokine activity and an improved chemokine activity.
  • Cytokines are a group of proteins that cells release upon excitation (only very few cytokines are expressed on cell membranes). Cytokines produced by cells can affect target cells nearby or through blood circulation at very low concentration. They have broad functions on promoting growth, differentiation and activation of target cells. Many cytokines can target immune cells and play a role in immune response. Based on structural and functional differences, cytokines may be broadly divided into chemokines, interleukins, growth factors, transforming growth factors, colony stimulating factors, tumor necrosis factors, and interferons, etc.
  • Chemokines are a group of cytokines being able to attract leukocytes, which are generally positively charged, secretory proteins having small molecule weights. Their main function is to attract immune cells to a region having tissue injuries or pathogen infection, allowing leukocytes to subsequently perform phagocytosis or elicit inflammation against pathogens at this specific site.
  • Leukocytes attracted by chemokines may include neutrophils, monocytes/macrophages, natural killer cells, dendritic cells and other leukocytes, which are of innate immunity; and T lymphocytes (T cells) or B lymphocytes (B cells) of adaptive immunity. Accordingly, chemokines play a very important role in the immune system of living organisms.
  • chemokines have four highly conserved cysteine (C) forming disulfide bonds to stable their structure. Based on different numbers of amino acids between the first two Cs and the procession of the first C or not, they may be classified into four subfamilies of CXC (or ⁇ ), CC (or ⁇ ), C (or ⁇ ) and CX 3 C.
  • Stromal cell-derived factor-1 (SDF-1) is classified into the CXC subfamily of chemokines, and is also known as CXC ligand 12 (CXCL12).
  • SDF-1 receptor CXCR4 not only constantly presents in organs, but can also be seen in hematopoietic stern cells, endothelial cells, dendritic cells, B cells and T cells. Therefore, these cells are attracted by SDF-1 to migrate to the site with high concentration of the 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 into the CXC subfamily of chemokines (also known as CXCL8). After initial discovery in humans, it was successively observed in economic animals of pigs, cows and chickens. IL-8 at low concentration is able to attract several immune cells, including monocytes, macrophages, lymphocytes, neutrophils, etc.
  • CD40 ligand is a member of tumor necrosis factor (TNF) superfamily, which is a cytokine having functions on tumor necrosis and promoting differentiation, proliferation and apoptosis of white blood cells.
  • CD40L is synthesized as a transmembrane protein. Take human CD40L as an example, the protein has a total of 261 amino acids, with first 22 amino-terminal amino acids being intracellular region, followed by 24 amino acids being transmembrane region, and 215 carboxy-terminal amino acids being extracellular (Exc) region, wherein the Exc region has at its carboxy terminus a TNF homology (TNFh) region conserved for all TNF superfamily proteins.
  • TNF tumor necrosis factor
  • CD40L presents mainly in the form of a transmembrane protein on the surface of activated CD4 + T cells, and also presents on CD8 + T cells, basophils, eosinophils, mast cells, natural killer cells, platelets, and even on the surface of CD40-expressing cells.
  • CD40 receptor of CD40L
  • APCs antigen presenting cells
  • B cells dendritic cells
  • macrophages etc.
  • CD40L activated by CD40L expressed by T helper cells, promoting the expression of major histocompatibility complex class II (MHC-II) molecules and B7 molecules to assist in antigen presentation.
  • MHC-II major histocompatibility complex class II
  • CD40L can promote B cell proliferation, isotype switching of immunoglobulins, antibody secretion, memory B cell differentiation, or prevention of apoptosis; effecting on macrophages, CD40L can enhance their activation, production of interleukin-12 (IL-12) to activate T helper 1 (Th1), or secretion of chemokines, or the production of nitric oxide (NO) to promote microorganism defense ability of macrophages; effecting on dendritic cells, it can make them mature and activated, wherein the mature dendritic cells not only express a large amount of MHC-II molecules to promote antigen presentation, but also secrete chemokines of TNF- ⁇ and IL-8, macrophage inflammatory protein 1a (MIP-1a), etc.
  • IL-12 interleukin-12
  • Th1 T helper 1
  • NO nitric oxide
  • IL-2 is classified into the hematopoietin family, the family including a number of cell growth-related hormones or other cytokines, etc.
  • Functions of IL-2 include: regulating the maturation and differentiation of T cells, stimulating proliferation and antibody secretion of B cells, promoting cytotoxicity of natural killer cells, and activating monocytes and macrophages, etc.
  • IL-2 can also stimulate T cells and B cells to continue expressing MHC, and also stimulate natural killer cells to produce several different cytokines, including TNF- ⁇ , IFN- ⁇ and granulocyte/macrophage colony stimulating factor (GM-CSF), etc.
  • GM-CSF granulocyte/macrophage colony stimulating factor
  • a fusion protein comprising a chemokine and a cytokine connected to the chemokine has an improved cytokine activity and an improved chemokine activity.
  • the present invention provides a fusion protein, comprising a chemokine polypeptide, which a chemokine or a receptor binding domain thereof; and a cytokine polypeptide connected 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 have a common target cells, and the fusion protein has an improved chemokine activity as compared to the chemokine polypeptide, and an improved cytokine activity as compared to the cytokine polypeptide.
  • the chemokine is a CXC chemokine, CC chemokine, C chemokine, and chemokine CX 3 C, preferably CXC chemokine.
  • the chemokine may be a stromal cell derived factor (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 IL-2, CD40 ligand, or a receptor binding domain thereof.
  • the present invention provides an isolated nucleic acid molecule, which encodes a fusion protein of the present invention.
  • the present invention provides an expression vector, comprising a nucleic acid molecule of the invention.
  • the present invention also provides a host cell, comprising an expression vector of the invention or a nucleic acid molecule of the invention.
  • FIG. 1 is a schematic diagram for chicken CD40L and its derivative proteins, CD40L Exc andCD40L TNFh .
  • FIG. 2 shows the results of SDS-PAGE and western blot analysis of the expressed chicken recombinant proteins.
  • Lane 1 IL8CD40L Exc , expected size being 52 kDa;
  • Lane 2 IL8IL2, expected size being 44 kDa;
  • Lane 3 SDF1CD40L Exc , expected size being 38 kDa;
  • Lane 4 SDF1CD40LTNFh, expected size being 44 kDa;
  • Lane 5 SDF1IL2, expected size being 26 kDa.
  • FIG. 3 shows the results of SDS-PAGE and western blot analysis for purified single proteins.
  • Lane 1 tagged protein, expected size being 21 kDa;
  • Lane 2 IL-8, expected size being 13 kDa;
  • Lane 3 SDF-1, expected size being 11 kDa;
  • Lane 4 IL-2, expected size being 32 kDa;
  • Lane 5 CD40L Exc , expected size being 42 kDa;
  • Lane 6 CD40L TNFh , expected size being 33 kDa.
  • FIG. 4 shows the chemotaxis of PBMCs by IL-8 derivative proteins.
  • Cells attracted by the chemokine outside the agar would travel from the center of the agar to the surrounding. Cells are seen cloudy at low magnification. More cells at the surrounding indicates greater degree of cell chemotaxis.
  • the chemotaxis extent of IL8CD40L Exc or IL8IL2 was significantly higher than single IL-8 protein.
  • FIG. 5 shows the chemotaxis of PBMCs by SDF-1 derivative proteins. At 2 ⁇ M, the chemotaxis extent of SDF1CD40L Exc , SDF1CD40L TNFh , or SDF 1IL2 was significantly higher than single SDF-1 protein.
  • FIG. 6 shows the activities of CD40L derivative proteins on activating macrophages to produce NO.
  • * represent significantly higher activity as compared to single proteins (*p ⁇ 0.05, **p ⁇ 0.01).
  • FIG. 7 shows the results of test on IL-2 fusion proteins' promotion of lymphocyte proliferation.
  • Stimulation index (SI) OD of test groups/OD of those cultured with RPMI 1640 only. At a concentration of 0.625-160 nM, the SI value of SDF1IL2 fusion protein was significantly higher than the IL-2 alone group (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001).
  • FIG. 8 shows that IL-2 fusion protein as an adjuvant of Newcastle disease (ND) vaccine can significantly promote cell immune response.
  • NDV Newcastle disease virus
  • FIG. 9 shows that the fusion proteins as adjuvants of infectious bronchitis (IB) vaccine can significantly promote cell immune response.
  • Antigen re-stimulation tests were performed for infectious bronchitis virus (IBV).
  • Chickens of the group administered with IL-2 fusion protein adjuvant and ND vaccine exhibited a significantly (p ⁇ 0.01) higher proliferation of memory lymphocytes, as compared to the IB vaccine group.
  • Chickens of the group administered with CD40L Exc fusion protein adjuvant and ND vaccine exhibited a significantly (p ⁇ 0.05) higher proliferation of memory lymphocytes, as compared to the IB vaccine group.
  • 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 chemokines, CC chemokines, C chemokines and CX 3 C chemokines.
  • cytokine polypeptide refers to a polypeptide, which is a cytokine or a receptor binding domain thereof, wherein the cytokine includes but is not limited to interleukins and cytokines of TNF-superfamily.
  • chemokine activity refers to the activities which chemokines possess or are able to exert in vivo, including but not limited to, chemotaxis of a variety of immune cells (including monocytes, macrophages, T cells , B cells, natural killer cells, dendritic cells and neutrophils, etc.).
  • cytokine activity refers to the activities which cytokines possess or are able to exert in vivo, including but not limited to the promotion of proliferation, immunoglobulin class switching and antibody secretion of B cells; differentiation of memory B cells, or prevention of their apoptosis; promoting macrophages' secretion of interleukin-12 to activate type I helper T cells or secrete chemokines; promoting macrophages to produce nitric oxide to enhance the defense capability against microorganisms; promoting the maturation and activation of dendritic cells; regulation of the maturation and differentiation of T cells; promoting the cytotoxicity and the production of a variety of different cytokines of natural killer cells; activation of monocytes and macrophages; and stimulation of T cells and B cells to continuously express MHC, etc.
  • 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 connected to the chemokine polypeptide, which is a interleukin, a TNF-superfamily cytokine or a receptor binding domain thereof; wherein the chemokine polypeptide and the cytokine polypeptide have a common target cell, and the fusion protein has an improved chemokine activity as compared to the chemokine polypeptide, and an improved cytokine activity as compared to the cytokine polypeptide.
  • the chemokine polypeptide and the cytokine polypeptide are connected by a peptide linker.
  • an appropriate peptide linker may be added between the two proteins to reduce the interference with each other when the proteins fold.
  • peptide linker may be a flexible peptide linker (Gly-Gly-Gly-Gly-Ser) n (usually n is less than 6) with a certain extent of flexibility and hydrophilicity, or a hydrophilic helical peptide linker (Glu-Ala-Ala-Ala-Lys) n (usually n is less than 6).
  • the chemokine is a CXC chemokine.
  • the chemokine is stromal cell-derived factor is (SDF-1).
  • the chemokine is IL-8.
  • the chemokine polypeptide has an amino acid sequence selected from the following: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, and a homolog thereof and an analog thereof.
  • the cytokine is IL-2, CD40 ligand (CD40L) or a receptor binding domain thereof.
  • the cytokine polypeptide has an amino acid sequence selected from the following: SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and a homolog thereof and an analog thereof.
  • the fusion protein of the present invention has an amino acid sequence selected from the following: SEQ ID NO: 40, 42, 44, 46 and 48.
  • the present invention provides an isolated nucleic acid molecule, which encodes a fusion protein of the present invention.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a chemokine polypeptide, selected from the following: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, and a homolog thereof and an analog thereof.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a chemokine polypeptide, selected from the following: SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and a homolog thereof and an analog thereof.
  • the isolated nucleic acid molecule has a sequence of one selected from the following: SEQ ID NO: 39, 41, 43, 45 and 47.
  • the present invention provides an expression vector, comprising a nucleic acid molecule of the invention.
  • the present invention also provides a host cell comprising an expression vector of the invention or a nucleic acid molecule of the invention.
  • Forward primer of the first pair of primers has a gene sequence of an EcoR I restriction enzyme site and a front N-terminus of the fusion protein
  • the reverse primer has a gene sequence of a helical peptide linker and a front C-terminus of the fusion protein.
  • This pair of primers can specifically amplify DNA fragments encoding the front section of the fusion protein and the peptide linker.
  • Forward primer of the second pair of primers has a gene sequence of a helical peptide linker and a rear N-terminus of the fusion protein
  • the reverse primer has a gene sequence of a Xho I restriction enzyme site and a rear C-terminus of the fusion protein.
  • This pair of primers can specifically amplify DNA fragments encoding the peptide linker and the rear section of the fusion protein.
  • an additional PCR was performed using the forward primer of the first pair of primers and the reverse primer of the second pair of primers, and accordingly the two fragments were connected due to partial overlapping sequences of the helical peptide linker.
  • the products obtained are fusion gene sequence comprising helical peptide linker gene therein.
  • the constructed prokaryotic expression vector was transformed into E. coli expression strain BL21 (DE3), 0.5 mM IPTG was used to induce the expression of the recombinant protein, and collected bacteria cells by centrifugation with the removal culture medium. Subsequently, all of bacteria cells were resuspended in binding buffer, a high pressure cell lysis instrument (French Pressure Cell Press, Thermo IEC, Needham, Height, MA, USA) was used to lysis the bacteria cells, and soluble proteins located in the supernatant after high speed centrifugation were isolated using nickel ion affinity column.
  • Insoluble protein located at the bottom pellet after centrifugation were treated with 8 M to unfold the proteins and they were dissolved in an aqueous solution, which was then subjected to a centrifugation of 12,000 rpm 30 minutes and dialysis of the supernatant was performed to slowly displace the urea, so that the proteins may refold to their original configurations.
  • the proteins were dissolved in phosphate buffer containing 10% glycerol (H 7.3), filtered through 0.22 ⁇ m membrane, concentration was determined by BCA protein assay kit (Pierce, Rockford, Ill., USA), and stored at ⁇ 20 ° C. The isolated proteins were identified to be correct fusion proteins using MALDI-TOF mass spectrometer.
  • chemokines SDF-1, or IL-8
  • CD40L derivative proteins or IL-2-fused chemokines were accessed.
  • Peripheral blood mononuclear cells were isolated using Histopaque 1077 (Sigma, Saint Louis, Mo., USA), washed twice with PBS, suspended with 10% FBS in RPMI 1640 (Gibco, Grand Island, N.Y., USA), and then added into 0.6% liquid agar, mixed well, resulting in a final concentration of 0.3% of the agar.
  • IL-8 fused either with CD40L derivative protein or IL-2 (IL8CD40L Exc or IL8IL2) exhibited a smaller MEC value and a better chemotactic activity.
  • the fusion proteins have a better chemotactic activity than IL-8 (see Table 1).
  • SDF-1 fused either with CD40L derivative protein or IL-2 (SDF1CD40L Exc or SDF1IL2) exhibited a smaller MEC value and a better chemotactic activity.
  • the fusion proteins have a better chemotactic activity than SDF-1 (see Table 1).
  • the chemotactic effects are better in higher concentrations of proteins.
  • the chemotactic extent of IL8CD40L Exc (with the best chemotactic activity) or IL8IL2 (with the second best chemotactic activity) were clearly higher than single IL8 protein (see FIG. 4 ).
  • the chemotactic extent of SDF1CD40L Exc , SDF1CD40L TNFh or SDF1IL2 were clearly higher than simple mixture or single SDF-1 protein (see FIG. 5 ).
  • MECs Minimum effective concentrations (MECs) of chemotactic activity Group Protein MEC A IL-8 125 nM B IL8CD40L Exc 62.5 nM C IL8IL2 62.5 nM D SDF-1 125 nM E SDF1CD40L Exc 62.5 nM F SDF1CD40L TNFh 62.5 nM G SDF1IL2 62.5 nM
  • CD40L activities of a chemokine fused with a CD40L derivative protein were assessed.
  • Peripheral blood mononuclear cells were isolated, washed twice with PBS, suspended in RPMI 1640 containing 10% FBS supplemented with 125 ng/ml chicken IL-2 and 4 ⁇ g/ml LPS at 2 ⁇ 10 6 cells/ml.
  • One ml of cells were added to each well of a 24-well plates.
  • One ml of fresh medium also supplemented with 125 ng/ml chicken IL-2 and 4 ⁇ g/ml LPS
  • IL8CD40L Exe fusion protein exhibited a significantly better activity as compared to the group added CD40L Exe alone (5-30 nM, p ⁇ 0.05; 180 nM, p ⁇ 0.01).
  • the effects of the fusion protein SDF1CD40L Exc were significantly better than CD40L Exc single protein (5 nM, p ⁇ 0.01; 30-180 nM, p ⁇ 0.05).
  • SDF1CD40L TNFh For the combination of SDF-1 and CD40L TNFh , similar results were obtained that SDF1CD40L TNFh fusion protein had a significantly better effects than a single CD40LTNFh protein (5 nM and 180 nM, p ⁇ 0.01; 0.8 nM and 30 nM, p ⁇ 0.05) (see FIG. 6 ).
  • IL-2 activities of a chemokine fused with IL-2 were accessed.
  • activity of intracellular acid phosphatase are proportional to cell number
  • chromogenic substrate p-nitrophenyl phosphate pNpp
  • Peripheral blood mononuclear cells were isolated, and then cultured in RPMI 1640 containing 10% FBS supplemented with different concentrations of proteins, 10 ⁇ g/ml ConA (positive control group), or 10 nM tagged protein (negative control group) on 96-well plate at 2 ⁇ 10 5 /well.
  • SI stimulation index
  • IL-2 fusion protein was used as an adjuvant of avian Newcastle disease (ND) vaccine and administered to chickens.
  • ND Newcastle disease
  • the blood of the chickens was drawn for the culture of lymphocytes, and inactivated Newcastle disease virus (NDV) were added as antigen to perform antigen re-stimulation assay. 10 ⁇ g/ml ConA were added to the culture medium as the positive control group.
  • the proliferation of memory lymphocytes that can recognize NDV antigen of each group of chickens was compared. The methods for determining proliferation state are the same as described in Example 5.
  • Proliferation rate (OD of test groups/OD of RPMI 1640 culture only) ⁇ 100%.
  • the groups vaccinated with IL-2 fusion proteins as ND vaccine adjuvant had a significantly enhanced proliferation of antigen-specific memory lymphocytes upon antigen re-stimulation (see FIG. 8 ).
  • IL-2 fusion proteins or CD40L Exc fusion proteins were used as an adjuvant of avian infectious bronchitis (IB) vaccine.
  • IB avian infectious bronchitis
  • the blood of the chickens was drawn for the culture of lymphocytes, and inactivated infectious bronchitis virus (IBV) were added as antigen to perform antigen re-stimulation assay.
  • IBV infectious bronchitis virus
  • the proliferation of memory lymphocytes that can recognize IBV antigen of each group of chickens was compared.
  • the methods for determining proliferation state are the same as described in Example 5.
  • Proliferation rate (OD of test groups/OD of RPMI 1640 culture only) ⁇ 100%.
  • the groups vaccinated with IL-2 fusion proteins as IB vaccine adjuvant (SDF1IL2+IB vaccine) (p ⁇ 0.01) or vaccinated with CD40L Exc fusion proteins as IB vaccine adjuvant (SDF1CD40L Exc +IB vaccine) (p ⁇ 0.05) had a significantly enhanced proliferation of antigen-specific memory lymphocytes upon antigen re-stimulation (see FIG. 9 ).

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020150553A1 (en) * 1999-10-29 2002-10-17 The Regents Of The University Of California Compositions containing C-terminal polypeptides of angiogenic chemokines and methods of use
US20050053579A1 (en) * 2001-10-23 2005-03-10 Jacques Galipeau Novel synthetic chimeric fusion transgene with immuno-therapeutic uses

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU778611B2 (en) * 1999-08-09 2004-12-16 Merck Patent Gmbh Multiple cytokine-antibody complexes
WO2001040311A1 (fr) * 1999-11-30 2001-06-07 Shionogi & Co., Ltd. Proteine fusionnee de chimiokine slc-il2
US20050287118A1 (en) * 2003-11-26 2005-12-29 Epitomics, Inc. Bacterial plasmid with immunological adjuvant function and uses thereof
CN101445560A (zh) * 2008-12-31 2009-06-03 河南省生物工程技术研究中心 一种畜禽用融合蛋白的制备方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020150553A1 (en) * 1999-10-29 2002-10-17 The Regents Of The University Of California Compositions containing C-terminal polypeptides of angiogenic chemokines and methods of use
US20050053579A1 (en) * 2001-10-23 2005-03-10 Jacques Galipeau Novel synthetic chimeric fusion transgene with immuno-therapeutic uses

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Arai et al. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng. 2001 Aug; 14(8):529-32. *
Nomura et al. Enhancement of anti-tumor immunity by tumor cells transfected with the secondary lymphoid tissue chemokine EBI-1-ligand chemokine and stromal cell-derived factor-1alpha chemokine genes. Int J Cancer. 2001 Mar 1; 91(5):597-606. *
Robbins et al. Interleukin-2-induced chemotaxis of human T-lymphocytes. J Lab Clin Med. 1986 Oct; 108(4):340-5. *
Stebler et al. Primordial germ cell migration in the chick and mouse embryo: the role of the chemokine SDF-1/CXCL12. Dev Biol. 2004 Aug 15;272(2):351-61. *
Tsai et al. Q5UB38_CHICK, 12/7/2004. *
Wilkinson et al. Chemoattractant activity of IL-2 for human lymphocytes: a requirement for the IL-2 receptor beta-chain. Immunology. 1994 May; 82(1):134-9. *

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