N-terminal recombinant protein of CCR4 and use thereof

Abstract

The present invention provides a recombinant protein or a derivative thereof, wherein the recombinant protein has an amino acid sequence of the N-terminal portion of CC chemokine receptor 4 (CCR4). The invention also provides the use of the recombinant protein or a derivative thereof for treating or preventing a disease or condition associated with CCR4 signaling, such as an allergic disease, inflammatory enteritis, psoriasis, an inflammatory skin disease, vasculitis, spondyloarthropathy, scleroderma, asthma, a respiratory allergic disease, an autoimmune disease, graft rejection, leukemia, lymphoma, a blood-borne cancer, a disease requiring inhibition of undesirable inflammation, and a cancer.

Claims

1. A isolated recombinant protein or a derivative thereof, wherein the recombinant protein consists of the amino acid sequence of the N-terminal portion of CC chemokine receptor 4 (N-CCR4), as shown in SEQ ID NO: 1, wherein the derivative is a concatemer of the recombinant protein as shown by (N-CCR4).sub.n, wherein n is 2 or 3, or a fusion protein of the recombinant protein with an Fc fragment of an immunoglobulin as shown by (N-CCR4).sub.m-Fc, wherein m is 1, 2 or 3.

2. The isolated recombinant protein or a derivative thereof of claim 1, wherein the amino acid sequence of the derivative is shown in SEQ ID NO. 2.

3. The isolated recombinant protein or a derivative thereof of claim 1, wherein the amino acid sequence of the derivative is shown in SEQ ID NO: 3.

4. A pharmaceutical composition comprising the recombinant protein or a derivative thereof of claim 1.

5. A method of treating a subject having a disease or condition associated with CCR4 signaling, the method comprising administering to the subject the isolated recombinant protein or a derivative according to claim 1 or a pharmaceutical composition comprising the isolated recombinant protein or a derivative thereof of claim 1.

6. The method of claim 5, wherein the disease or condition associated with CCR4 signaling is selected from the group consisting of: an allergic disease, inflammatory enteritis, psoriasis, an inflammatory skin disease, vasculitis, spondyloarthropathy, scleroderma, asthma, a respiratory allergic disease, an autoimmune disease, graft rejection, leukemia, lymphoma, a blood-borne cancer, a disease requiring inhibition of undesirable inflammation, and a cancer.

7. The method of claim 5, wherein the disease or condition associated with CCR4 signaling is a cancer.

8. The method of claim 6, wherein the allergic disease is systemic allergic reaction or hypersensitivity reaction.

9. The method of claim 6, wherein the inflammatory enteritis is selected from the group consisting of Crohn's disease, ulcerative colitis, ileitis and enteritis.

10. The method of claim 6, wherein the inflammatory skin disease is selected from the group consisting of dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatomyositis and urticaria.

11. The method of claim 6, wherein the respiratory allergic disease is selected from the group consisting of allergic asthma, exercise-induced asthma, allergic rhinitis and hypersensitivity lung disease.

12. The method of claim 6, wherein the autoimmune disease is selected from the group consisting of arthritis, multiple sclerosis, systemic lupus erythematosus, diabetes, and nephritis.

13. The method of claim 6, wherein the graft rejection is selected from the group consisting of allograft rejection and graft-versus-host disease.

14. The method of claim 6, wherein the blood-borne cancer is selected from the group consisting of cutaneous T-cell lymphoma and acute lymphocytic leukemia.

15. The method of claim 6, wherein the disease requiring inhibition of undesirable inflammation is selected from the group consisting of atherosclerosis, myositis, neurodegenerative disease, encephalitis, meningitis, hepatitis and nephritis.

16. The method of claim 6, wherein the cancer is selected from the group consisting of a solid tumor, a metastatic tumor, a liver cancer, a renal cancer, an intestinal cancer, a pancreatic cancer, a lung cancer, a bladder cancer, a breast cancer, an ovarian cancer, a cervical cancer, a melanoma, a thyroid cancer, a prostate cancer and a nervous system tumor.

17. The method of claim 16, wherein the metastatic tumor is a gastric cancer.

18. An isolated recombinant protein comprising: a concatemer of the amino acid sequence of the N-terminal portion of CC chemokine receptor 4 (N-CCR4), as shown in SEQ ID NO: 1, wherein the concatemer has a structure of (N-CCR4).sub.n, wherein n is 2 or 3, or a fusion protein of the N-terminal portion of CC chemokine receptor 4 (N-CCR4), as shown in SEQ ID NO: 1, with an Fc fragment of an immunoglobulin as shown by (N-CCR4).sub.m-Fc, wherein m is 1, 2 or 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A shows a hydropathy plot and prediction of the N-terminal region of N-CCR4 of CCR4. FIG. 1B shows predicted transmembrane helices and predicted Gibbs free energy. FIG. 1C and FIG. 1D show the purification of its derived proteins (N-CCR4).sub.3, (N-CCR4).sub.3-Fc.

(2) FIG. 2 shows the experiment of interaction between (N-CCR4).sub.3 and CCL22.

(3) FIG. 3 shows an experiment in which (N-CCR4).sub.3 inhibits the chemotaxis of Treg cells.

SPECIFIC MODE FOR CARRY OUT THE INVENTION

Example 1: Prediction of N-Terminal Protein N-CCR4 of CCR4

(4) 1. The N-terminal protein N-CCR4 of CCR4 was predicted by hydrophobic alignment of CCR4 to CXCR4 protein sequence (located on the World-Wide Web at bioinfo.mpg.de/AlignMe/AlignMe_MSA.html) and by calculation of Gibbs free energy AG of CCR4 protein sequence (ΔG predictor sever v1.0, located on the internet at: dgpred.cbr.su.se) (FIG. 1A, B).

(5) The sequence of N-CCR4 protein is:

(6) TABLE-US-00004 (SEQ ID NO. 1) MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYS

(7) 2. Purification of N-CCR4 Derived Protein (N-CCR4).sub.3

(8) The codon-optimized sequence of (N-CCR4).sub.3 DNA was cloned into pET22b(+) vector (Novagen, Cat. number 69744-3) via Ndel and BamHI cleavage sites, and transformed into Transetta competent cells (TransGen, Cat. number CD801-01). The culture broth shaken overnight was inoculated into a new LB medium at a ratio of 1:100, and when the OD600 of the culture broth was 0.6, IPTG (amresco, cat: 0487-1G) with a final concentration of 1 mM was added, and the protein expression was induced at 25° C., 220 rpm/min for 10 hours. After resuspending the cells in PBS, the cells were disrupted by a high pressure cell homogenizer (JN-02C low-temperature ultrahigh-pressure continuous flow cell homogenizer), loaded on a nickel column (BBI, cat #C600793) and eluted with Tris-HCl (pH8.0) buffer containing 100 mM imidazole, and then dialyzed twice against 2 L PBS. A pre-induced control sample(ctrl), a post-induced lysis sample(lys), a flow through sample(Ft), a washing sample (w), a 30 mM imidazole-elution sample, a 100 mM imidazole-elution sample, and a 500 mM imidazole-elution sample are detected by 12% SDS-PAGE (FIG. 1C).

(9) The codon optimized sequence of (N-CCR4).sub.3 protein DNA is:

(10) TABLE-US-00005 (SEQ ID NO. 4) ATGAATCCGACCGATATTGCAGATACCACACTGGATGAAAGCATCTAT AGCAACTATTATCTGTATGAGAGCATTCCGAAACCGTGTACCAAAGAA GGTATTAAAGCATTTGGCGAACTGTTTCTGCCTCCGCTGTATAGCGGT GGTGGTGGTAGTGGTGGCGGTGGTTCAATGAACCCGACAGATATCGCC GACACAACCCTGGATGAATCAATTTATTCCAACTACTACCTGTACGAG TCAATCCCGAAACCTTGCACAAAAGAGGGCATCAAAGCCTTTGGTGAG CTGTTTTTACCGCCTCTGTATTCAGGCGGTGGCGGTAGCGGAGGTGGC GGAAGCATGAATCCTACAGACATTGCGGATACGACCCTGGACGAGAGC ATTTATTCAAATTACTATTTATACGAAAGCATCCCGAAGCCATGTACG AAAGAGGGAATTAAGGCGTTCGGTGAATTATTTCTGCCACCGTTATAT AGCTTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTC GAGCACCACCACCACCACCAC

(11) The sequence of (N-CCR4).sub.3 protein is:

(12) TABLE-US-00006 (SEQ ID NO. 2) MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYSG GGGSGGGGSMNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGE LFLPPLYSGGGGSGGGGSMNPTDIADTTLDESIYSNYYLYESIPKPCT KEGIKAFGELFLPPLYSLDPNSSSVDKLAAALEHHHHHH

(13) 3. Purification of N-CCR4-Derived Antibody (N-CCR4).sub.3-Fc

(14) The murine kappa III signal peptide-(N-CCR4).sub.3-Fc DNA sequence was cloned into the pCEP4 vector by KpnI and BamHI, wherein the murine kappa III signal peptide is a secretory signal peptide and Fc is a human IgG1 Fc. The constructed plasmid was transfected into 293FT cells using PEI (Shanghai Qifa Biological Reagent Co. Ltd., cat #24765-2), and the supernatant was collected on the third day after transfection and then purified with protein A prepacked gravity column (BBI, cat #C600951). The elution samples were taken and detected on a 10% SDS-PAGE (FIG. 1D).

(15) The sequence of (N-CCR4).sub.3-Fc DNA:

(16) TABLE-US-00007 (SEQ ID NO. 5) ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCA GGTTCCACTGGTGACTCGATGAACCCCACCGACATCGCCGACACCACC CTCGACGAGAGCATCTACAGCAACTACTACCTGTACGAAAGCATCCCC AAACCCTGCACCAAAGAAGGCATCAAAGCCTTCGGCGAGCTGTTCCTC CCCCCCCTGTACAGCGGCGGCGGTGGATCTGGCGGCGGAGGATCAATG AACCCCACAGACATCGCCGATACCACCCTGGACGAAAGCATTTACAGC AATTACTACCTGTATGAGTCCATCCCCAAACCTTGCACCAAAGAGGGC ATCAAGGCCTTCGGCGAACTGTTCCTCCCACCCCTGTACAGTGGCGGC GGCGGAAGCGGAGGCGGAGGAAGCATGAACCCCACTGACATCGCCGAC ACAACCCTCGACGAAAGCATATACAGCAATTATTACCTGTACGAGTCC ATCCCTAAACCCTGCACAAAGGAAGGCATCAAGGCATTCGGAGAGCTG TTCCTGCCCCCCCTGTATAGCAAGCTTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTC CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC TCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG GATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC TTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGGTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAATAA

(17) The sequence of (N-CCR4).sub.3-Fc protein:

(18) TABLE-US-00008 (SEQ ID NO. 3) MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYSG GGGSGGGGSMNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGE LFLPPLYSGGGGSGGGGSMNPTDIADTTLDESIYSNYYLYESIPKPCT KEGIKAFGELFLPPLYSKLDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSL SLSPGK

Example 2: The Experiment of Interaction Between (N-CCR4).SUB.3 .and CCL22

(19) The GST-pulldown experiment can verify whether two proteins interact with each other, so that it is verified that (N-CCR4).sub.3 interacts with CCL22 by GST-pulldown experiment.

(20) 1. The Expression of CCL22-GST Protein

(21) The CCL22 DNA sequence was codon optimized and CCL22 and GST were cloned into pET22b vector via the cleavage sites Ndel, BamHI and HindIII. Transetta competent cells were transformed. The culture broth shaken overnight was inoculated into a new LB medium at a ratio of 1:100, and when the OD600 of the culture broth was 0.6, IPTG (amresco, cat: 0487-1G) at a final concentration of 1 mM was added, and the protein expression was induced at 20° C., 210 rpm/min for 10 hours. After resuspending the cells in PBS, the cells were disrupted by a high pressure cell homogenizer, loaded on GST agarose (BBI, cat #C600031) overnight, washed with 150 mM NaCl, 1 mM DTT (amresco, cat #0281-5G) 10 mM Tris-HCl (pH8.0) buffer for 10 column volumes, and then eluted with 10 mM reduced glutathione (BBI, cat #70-18-8) 150 mM NaCl, 1 mM DTT Tris-HCl (pH8.0) buffer, and then dialyzed twice against 2 L PBS.

(22) The sequence of CCL22-GST DNA:

(23) TABLE-US-00009 (SEQ ID NO. 6) ATGGGTCCGTATGGTGCAAATATGGAAGATAGCGTTTGTTGCCGTGAT TATGTTCGTTATCGTCTGCCGCTGCGTGTTGTTAAACACTTTTATTGG ACCAGCGATAGCTGTCCGCGTCCGGGTGTTGTTCTGCTGACCTTTCGT GATAAAGAAATTTGTGCAGATCCGCGTGTTCCGTGGGTTAAAATGATT CTGAATAAACTGAGCCAGGATCCGGGTGGCCCGGGTATGTCCCCTATA CTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTT TTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGAT GAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTT CCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCT ATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGT TGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTG GATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAA ACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATG TTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTA ACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATAC ATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAA AAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGC AAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGT GGCGACCATCCTCCAAAATGA

(24) The sequence of CCL22-GST protein:

(25) TABLE-US-00010 (SEQ ID NO. 7) MGPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFR DKEICADPRVPWVKMILNKLSQDPGGPGMSPILGYWKIKGLVQPTRLL LEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQS MAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFE TLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGG GDHPPK

(26) 2. The Expression of GST Protein

(27) Transetta competent cells were transformed with pGEX-6P-1 plasmid (GE, cat #27-4597-01). The culture broth shaken overnight was inoculated into a new LB medium at a ratio of 1:100, and when the OD600 of the culture broth was 0.6, IPTG (amresco, cat: 0487-1G) at a final concentration of 1 mM was added, and the protein expression was induced at 25° C., 210 rpm/min for 10 hours. After resuspending the cells in PBS, the cells were disrupted by a high pressure cell homogenizer, loaded on GST agarose (BBI, cat #C600031) overnight, and washed with 10 mM Tris-HCl, 150 mM NaCl, 1 mM DTT (amresco, cat #0281-5G) buffer for 10 column volumes, and then eluted with 10 mM reduced glutathione (BBI, cat #70-18-8) 150 mM NaCl, 1 mM DTT Tris-HCl (pH8.0) buffer, and then dialyzed twice against 2 L PBS.

(28) 3. GST Pulldown

(29) 120 ng CCL22-GST and GST protein were mixed with 360 ng (N-CCR4).sub.3 respectively; 20 ul GST agarose was added respectively; and the liquid volume was made up to 600 ul with PBS; and then rotated to bind at 4° C. for 4 h; washed with PBS+0.1% Triton-100 for 3 times, then washed with PBS for 3 times; the proteins on the beads were dissolved with 30 ul of 1× loading buffer, boiled for 3 minutes, centrifuged at high speed; 1 ul of supernatant was taken to 12% SDS PAGE gel and silver stained (Biyunyan, Rapid silver staining kit, cat #P0017S) for detection. As shown in FIG. 2, (N-CCR4).sub.3 interacted with CCL22.

Example 3: Experiment Showing that (N-CCR4).SUB.3 .Inhibits the Chemotaxis of Treg Cell

(30) Chemotaxis experiment is a common model for observing the chemotactic migration of cells and therefore is used to verify the inhibitory effect of (N-CCR4).sub.3 on the chemotaxis of Treg cell.

(31) Naïve CD4+ T lymphocytes were sorted and stimulated with anti-CD3 (2 μg/mL), anti-CD28 (1 μg/mL), TGF β (1 ng/mL) and IL-2 (4 ng/mL) for 2 days; the induced Treg cells were harvested and placed in the upper chamber of the chemotactic chamber by 2×10.sup.5/well; the tumor cell culture supernatant and 0.05 μM, 0.1 μM and 1 μM (N-CCR4).sub.3 recombinant protein were added respectively to the lower chamber; after 4 hours, Treg cells in the lower chamber were harvested and counted for statistical analysis, as shown in FIG. 3, the chemotactic movement of Treg cells was significantly inhibited by adding (N-CCR4).sub.3.