CCR4 extracellular domain recombinant protein and application thereof

Abstract

The invention provides a recombinant protein combined with chemokines CCL22 and / or CCL17 or a derivative thereof, the recombinant protein is a fusion recombinant protein formed by any combination of extracellular segment parts of a CC chemokine receptor 4 (CCR4), and can also be a fusion recombinant protein obtained by any combination of the fusion recombinant protein and a CCR4 N-terminal recombinant protein. The invention also provides the use of the recombinant protein or derivatives thereof in the treatment, inhibition, amelioration or prevention of diseases or conditions associated with CCR4 signaling.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of extracellular domain recombinant proteins of CCR4 and any combination of their derivative compounds, fusion recombinant proteins alone or in combination with other compounds having related uses, such as CCR4 N-terminal recombinant proteins, etc., or compositions for the treatment, alleviation or prevention of cancer and diseases or conditions related to CCR4 signal transduction. Background Art

[0002] Chemokines are a subfamily of cytokines responsible for the transportation of immune cells and the development of lymphoid tissues. According to the positions of the first two cysteine (C) residues in their main protein structures, they can be divided into four major categories, namely C, CC, CXC, and CX3C chemokines. CXC chemokine receptor 4 (CXCR4) has an undisputed clinical correlation with the growth and spread of various malignant tumors, and CXCR4 inhibition has been proven to promote a strong anti-tumor T cell response. CC chemokine receptor 4 (CCR4), also known as CKR4, CMKBR4, ChemR13, K5-5, etc., belongs to the CC chemokine receptor family, contains 360 amino acid residues, is located in the region of chromosome 3 p24-p21.3, is a 7-transmembrane G protein-coupled receptor (GPCR), and is mainly expressed in various lymphocytes and tissues. The high expression of CCR4 is related to the infiltration, metastasis and prognosis of various inflammatory diseases, autoimmune diseases, hematological malignancies and solid tumors. There are two known high-affinity ligands of CCR4, namely thymus and activation regulated chemokine (TARC / CCL17) and macrophage-derived chemokine (MDC / CCL22). CCR4 mainly exerts immune effects by chemotaxing different types of T cells through the binding of CCR4 expressed on the surface of Th2 cells, Th17 cells and regulatory T cells (Treg) to their ligands CCL22 and / or CCL17. For example, Th2 cells play an important pro-inflammatory role in asthma and respiratory allergic diseases, etc.; Th17 cells induce inflammatory bowel diseases such as Crohn's disease, ulcerative colitis and autoimmune diseases, etc.; CCR4 on the surface of Treg cells chemotaxes Treg cells through the binding to its ligands CCL22 and / or CCL17, causing immune escape and thus leading to adverse clinical consequences. Therefore, compounds targeting CCR4 are expected to become a new strategy for the treatment of related diseases.

[0003] By synthesizing the extracellular domain recombinant protein of CCR4 and studying its recombinant protein function and developing its derivative compounds or antibodies, it is expected to competitively inhibit the CCL22 and / or CCL17-CCR4 signaling pathway by targeting CCR4, and be used alone or in combination with other compounds or compositions with related uses for the treatment, prevention, inhibition or improvement of cancer and diseases or conditions related to CCR4 signal transduction, such as inflammatory diseases including allergic diseases, inflammatory bowel disease, inflammatory skin diseases, asthma, autoimmune diseases, etc., graft rejection, hematological malignancies such as leukemia, lymphoma and other blood-derived cancers such as cutaneous T-cell lymphoma, acute lymphoblastic leukemia, etc. and cancers including solid tumors and metastatic diseases such as gastric cancer, liver cancer, kidney cancer, intestinal cancer, pancreatic cancer, lung cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, thyroid cancer, prostate cancer, nervous system tumors, etc., becoming a new strategy for immune-targeted therapy of related diseases and having good clinical application prospects.

[0004] The currently marketed CCR4 monoclonal antibody for treating tumors is mogamulizumab, which has good therapeutic effects in the treatment or research of diseases such as cutaneous T-cell lymphoma (CTCL), mycosis fungoides, Sézary syndrome, non-Hodgkin lymphoma, etc. And CCR4 receptor antagonists have good therapeutic effects in the research of diseases such as asthma, rhinitis, dermatitis, thrombotic diseases, autoimmune diseases, etc.

[0005] However, so far, there has been no report on the use of the extracellular domain recombinant protein of CCR4 and its derivative compounds in any combination of fusion recombinant proteins alone or in combination with other compounds or compositions with related uses for the treatment, prevention, inhibition or improvement of cancer and / or diseases or conditions related to CCR4 signal transduction. Summary of the Invention

[0006] The present invention discovers through research that the extracellular domain recombinant protein of CCR4 can bind to chemokines CCL22 and / or CCL17.

[0007] The present invention provides a fusion recombinant protein of the extracellular domain recombinant protein of chemokine receptor CCR4 and any combination of its derivative compounds, which can be used alone or in combination with other compounds including, for example, the recombinant protein of the N-terminus of CCR4, etc. or compositions, and has good clinical application prospects in the treatment, prevention, inhibition or improvement of cancer and diseases or conditions related to CCR4 signal transduction.

[0008] In one aspect, the present invention provides a fusion recombinant protein of an extracellular segment recombinant protein of CCR4 and any combination of its derivative compounds, which can be used alone or in combination with other compounds having related uses, including, for example, an N-terminal recombinant protein of CCR4, etc. or a composition, for the use of treating, preventing, inhibiting or improving cancer and diseases or conditions related to CCR4 signal transduction.

[0009] In another aspect, the present invention provides derivatives or compositions such as fusion recombinant proteins of an extracellular segment recombinant protein of CCR4 and any combination of its derivative compounds, including but not limited to extracellular segment recombinant proteins of CCR4 (EL1-CCR4, EL2-CCR4, EL3-CCR4) and fusion recombinant proteins ((ELn)n-CCR4) arranged in any combination thereof n , such as any permutation combination, order transformation and multiple repetitions of two sequences such as EL1-EL2-CCR4, EL1-EL3-CCR4, EL2-EL1-CCR, EL2-EL3-CCR4, EL3-EL1-CCR4, EL3-EL2-CCR4, etc., or any permutation combination, order transformation and multiple repetitions of three sequences such as EL1-EL2-EL3-CCR4, EL1-EL3-EL2-CCR4, EL2-EL1-EL3-CCR4, EL2-EL3-EL1-CCR4, EL3-EL1-EL2-CCR4, EL3-EL2-EL1-CCR4, etc., or a recombinant protein (N-(ELn)n-CCR4) fused with an N-terminal recombinant protein of CCR4 (represented by N- in the present invention) n Such as any permutation combination, order transformation and multiple repetitions of two sequences such as N-EL1-CCR4, N-EL2-CCR4, N-EL3-CCR4, EL1-EL2-CCR4, EL1-EL3-CCR4, EL2-EL3-CCR4, etc., or any permutation combination, order transformation and multiple repetitions of three sequences such as N-EL1-EL2-CCR4, N-EL1-EL3-CCR4, N-EL2-EL3-CCR4, etc., or any permutation combination, order transformation and multiple repetitions of four sequences such as N-EL1-EL2-EL3-CCR4, N-EL1-EL3-EL2-CCR4, N-EL2-EL1-EL3-CCR4, N-EL2-EL3-EL1-CCR4, N-EL3-EL1-EL2-CCR4, N-EL3-EL2-EL1-CCR4, etc., or a recombinant protein ((ELn)n-CCR4) fused with Fc n –Fc and (N-(ELn)n-CCR4) n –Fc or derivative compounds such as fusion proteins or antibodies fused with other functional protein analogs well known in the art.

[0010] The amino acid sequence of the N-terminal recombinant protein (N-CCR4) of CCR4 is as follows:

[0011] MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYS (SEQ ID NO. 1)

[0012] The amino acid sequence of the extracellular domain recombinant protein EL1-CCR4 of CCR4 is :

[0013] AADQWVFGLGLCKMISW (SEQ ID NO. 2)

[0014] The amino acid sequence of the extracellular domain recombinant protein EL2-CCR4 of CCR4 is:

[0015] STCYTERNHTYCKTKYSLNSTTWKVLSSLEINIL (SEQ ID NO. 3)

[0016] The amino acid sequence of the extracellular domain recombinant protein EL3-CCR4 of CCR4 is :

[0017] YNIVLFLETLVELEVLQDCTFERYLDYAIQAT(SEQ ID NO. 4)

[0018] The amino acid sequence of the N-EL2-CCR4 recombinant protein is:

[0019] MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYSGGGGSGGGGSSTCYTERNHTYCKTKYSLNSTTWKVLSSLEINIL(SEQ ID NO. 5)

[0020] The amino acid sequence of the N-EL3-CCR4 recombinant protein is:

[0021] MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYSGGGGSGGGGSYNIVLFLETLVELEVLQDCTFERYLDYAIQAT(SEQ ID NO. 6)

[0022] The amino acid sequence of the N-EL2-EL3-CCR4 recombinant protein is:

[0023] MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYSGGGGSGGGGSSTCYTERNHTYCKTKYSLNSTTWKVLSSLEINILGGGGSGGGGSYNIVLFLETLVELEVLQDCTFERYLDYAIQAT (SEQ ID NO.7)

[0024] The amino acid sequence of the N-EL1-EL2-EL3-CCR4 recombinant protein is as follows:

[0025] MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYSGGGGSGGGGSAADQWVFGLGLCKMISWGGGGSGGGGSSTCYTERNHTYCKTKYSLNSTTWKVLSSLEINILGGGGSGGGGSYNIVLFLETLVELEVLQDCTFERYLDYAIQAT (SEQ ID NO. 8)

[0026] In another aspect, the extracellular domain recombinant protein of CCR4 of the present invention and derivatives or compositions such as fusion recombinant proteins of any combination of its derivative compounds can be used alone or in combination with other compounds or compositions having related uses.

[0027] In another aspect, the extracellular domain recombinant protein of CCR4 of the present invention and derivatives or compositions such as fusion recombinant proteins of any combination of its derivative compounds can be administered by oral, parenteral such as intramuscular, intravenous, subcutaneous injection or implantation, inhalation, or topical administration, etc., and can be formulated alone or together into suitable dosage unit preparations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles suitable for each administration route.

[0028] In another aspect, other compounds or compositions for combined use can be administered by their common routes and dosages, and administered simultaneously, sequentially or separately with the compounds or compositions of the present invention. Therapeutic agents that can be used in combination with the compounds or compositions of the present invention simultaneously, sequentially or separately include but are not limited to conventional inflammatory disease treatment drugs such as anti-inflammatory drugs, hormonal drugs, immunosuppressants, antihistamine drugs, etc., conventional cancer treatment drugs such as radiotherapy and chemotherapy drugs, conventional first-line drugs (such as cisplatin, vincristine, sorafenib, etc.), immune checkpoint inhibitors (PD-1 / PD-L1, CTLA-4, 4-1BB, OX40 / OX40L, CD40 / CD40L, LAG3, Tim3, TIGIT, etc.) and other tumor immune targeting drugs (VEGF / VEGFR, EGF, etc.).

[0029] In another aspect, the compounds of the present invention can be used for treating, preventing, inhibiting or ameliorating cancer and diseases or conditions associated with CCR4 signaling.

[0030] In another aspect, the diseases or conditions associated with CCR4 signaling include but are not limited to: 1) allergic diseases, such as systemic anaphylactic or hypersensitivity reactions, etc.; 2) inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, ileitis and enteritis, etc.; 3) psoriasis and inflammatory skin diseases, such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatomyositis and urticaria, etc.; 4) vasculitis; 5) spondyloarthropathy; 6) scleroderma; 7) asthma and respiratory allergic diseases, such as allergic asthma, exercise-induced asthma, allergic rhinitis, hypersensitivity pneumonitis, etc.; 8) autoimmune diseases, such as arthritis, multiple sclerosis, systemic lupus erythematosus, diabetes, nephritis, etc.; 9) graft rejection, such as allograft rejection and graft-versus-host disease, etc.; 10) leukemia, lymphoma and other hematogenous cancers such as cutaneous T-cell lymphoma, acute lymphoblastic leukemia, etc.; 11) other diseases that require inhibition of adverse inflammation, such as atherosclerosis, myositis, neurodegenerative diseases, encephalitis, meningitis, hepatitis, nephritis, etc.; 12) cancer, including solid tumors and metastatic diseases such as gastric cancer, liver cancer, kidney cancer, colorectal cancer, pancreatic cancer, lung cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, thyroid cancer, prostate cancer, nervous system tumors, etc.

[0031] In another aspect, the compounds of the present invention and other therapeutic agents that can be used in combination can be administered simultaneously, sequentially or separately for treating, preventing, inhibiting or ameliorating cancer and diseases or conditions associated with CCR4 signaling.

[0032] In another aspect, the present invention provides a pharmaceutical composition, which can be formulated into an injection, tablet or capsule.

[0033] More specifically, the present invention provides the following:

[0034] 1. A recombinant protein or its derivative that binds to CCL22 and / or CCL17, wherein the recombinant protein is:

[0035] (A) A protein consisting of the amino acid sequence shown in any one of SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4;

[0036] (B) A protein derived from the protein of (A) by substitution and / or deletion and / or addition of one or several amino acid residues and having the activity of the protein of (A), or a protein having a sequence identity of 70% or more with the protein of (A).

[0037] 2. The recombinant protein or its derivative according to item 1, wherein the derivative is:

[0038] (a) A fusion protein formed by any combination of any combinations of the recombinant protein;

[0039] (b) A protein derived from the protein in (a) by substituting and / or deleting and / or adding one or several amino acid residues in the amino acid sequence of the fusion protein in (a) and having the activity of the protein in (a), or a protein having a sequence identity of 70% or more with the fusion protein in (a).

[0040] 3. The recombinant protein or its derivative according to item 1, wherein the derivative is:

[0041] (1) A fusion recombinant protein obtained by any combination of the recombinant protein defined in item 1 or the fusion protein defined in claim 2 and the CCR4 N-terminal recombinant protein;

[0042] (2) A protein derived from the protein in (1) by substituting and / or deleting and / or adding one or several amino acid residues in the amino acid sequence of the fusion recombinant protein in (1) and having the activity of the protein in (1), or a protein having a sequence identity of 70% or more with the fusion recombinant protein in (1);

[0043] The amino acid sequence of the CCR4 N-terminal recombinant protein is shown as SEQ ID NO. 1.

[0044] 4. The recombinant protein or its derivative according to item 1, wherein the derivative is:

[0045] (i) A fusion recombinant protein obtained by any combination of the recombinant protein defined in item 1 or the fusion protein defined in item 2 and the Fc fragment;

[0046] (ii) A protein derived from the protein in (i) by substituting and / or deleting and / or adding one or several amino acid residues in the amino acid sequence of the fusion recombinant protein in (i) and having the activity of the protein in (i), or a protein having a sequence identity of 70% or more with the fusion recombinant protein in (i).

[0047] 5. A pharmaceutical composition comprising the recombinant protein or its derivative according to any one of items 1-4.

[0048] 6. Use of the recombinant protein or its derivative according to any one of items 1-4 or the pharmaceutical composition according to item 5 in the preparation of a drug for inhibiting the binding of CCR4 to its ligand, wherein the ligand of CCR4 includes CCL22 and / or CCL17.

[0049] 7. Use of the recombinant protein or its derivative according to any one of Items 1-4 or the pharmaceutical composition according to Item 5 in the preparation of a drug for treating, inhibiting, improving or preventing a disease or condition associated with CCR4 signal transduction.

[0050] 8. The use according to Item 7, wherein the disease or condition associated with CCR4 signal transduction includes 1) allergic diseases, such as systemic allergic or hypersensitivity reactions; 2) inflammatory bowel diseases, preferably Crohn's disease, ulcerative colitis, ileitis and enteritis; 3) psoriasis and inflammatory skin diseases, preferably dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatomyositis and urticaria; 4) vasculitis; 5) spondyloarthropathy; 6) scleroderma; 7) asthma and respiratory allergic diseases, preferably allergic asthma, exercise-induced asthma, allergic rhinitis, hypersensitivity pneumonitis; 8) autoimmune diseases, preferably arthritis, multiple sclerosis, systemic lupus erythematosus, diabetes, nephritis; 9) graft rejection, preferably allograft rejection and graft-versus-host disease; 10) leukemia, lymphoma and other hematogenous cancers such as cutaneous T cell lymphoma, acute lymphoblastic leukemia; 11) other diseases requiring inhibition of adverse inflammation, preferably atherosclerosis, myositis, neurodegenerative diseases, encephalitis, meningitis, hepatitis, nephritis; 12) cancers, including solid tumors and metastases, preferably gastric cancer, liver cancer, kidney cancer, colorectal cancer, pancreatic cancer, lung cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, thyroid cancer, prostate cancer, nervous system tumors.

[0051] 9. The use according to Item 7, wherein the disease or condition associated with CCR4 signal transduction is cancer or tumor.

[0052] In the present invention, "extracellular segment", "extracellular region", "extracellular domain" and "extracellular domain" can be used interchangeably.

[0053] In the present invention, "CC chemokine receptor 4", "CCR4", "CC chemokine receptor 4", "CKR4", "CMKBR4", "ChemR13" and "K5-5" can be used interchangeably.

[0054] Technical effects of the present invention:

[0055] 1. All the different extracellular segment proteins of CCR4 provided by the present invention can bind to the CCR4 ligands chemokines CCL22 and / or CCL17, and can be used for treating, inhibiting, improving or preventing diseases or conditions associated with CCR4 signal transduction by competitively antagonizing CCL22 and / or CCL17 or other ligand signals with CCR4.

[0056] 2. The different extracellular segment protein arrangements of CCR4 provided by the present invention contribute to maximizing the competitive antagonism of CCL22 or other ligand signals, and are used in the treatment, inhibition, improvement, or prevention of diseases or conditions related to CCR4 signal transduction.

[0057] 3. The different extracellular segment proteins of CCR4 provided by the present invention can effectively delay tumor growth and can be used to develop new immunotherapy strategies for tumors.

[0058] 4. The different extracellular segment proteins of CCR4 provided by the present invention can be used in combination with other immunotherapy drugs for synergistic treatment, enhancing the combined anti-tumor effect, and laying a foundation for the development of a new generation of tumor treatment regimens. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 . Experiment on the interaction between extracellular segment proteins N-CCR4, EL1-CCR4, EL2-CCR4, EL3-CCR4 of CCR4 and CCL22.

[0060] Figure 2 . Experiment on the interaction between extracellular segment protein combinations EL3-CCR4, N-EL3-CCR4, N-EL2-EL3-CCR4, N-EL1-EL2-EL3-CCR4 of CCR4 and CCL22.

[0061] Figure 3 . Anti-tumor efficacy of extracellular segment proteins of CCR4 in vivo. DETAILED DESCRIPTION OF THE INVENTION

[0062] Example 1: Prediction of extracellular segment proteins EL1-CCR4, EL2-CCR4, and EL3-CCR4 of CCR4

[0063] Chemokine receptors respond to ligand signals mainly by binding ligands in the N-terminal and extracellular regions and transmitting signals to the intracellular region to activate downstream signals. Therefore, the N-terminal and extracellular regions are the main functional regions for chemokine receptors to exert their functions. However, since the structure of CCR4 has not been fully resolved, its specific ligand-binding region remains unknown. Therefore, in order to maximize the binding efficiency of CCR4 to ligands, it is still necessary to screen extracellular proteins and their different arrangements. Considering that expressing the full-length CCR4 protein may lead to protein folding errors and affect the binding of functional epitopes to ligands, the present invention predicts and preferably selects extracellular segment protein combinations of CCR4 in order to obtain better binding effects.

[0064] The extracellular domain proteins EL1-CCR4, EL2-CCR4, and EL3-CCR4 of CCR4 were predicted by hydrophobicity alignment of the CCR4 and CXCR4 protein sequences (http: / / www.bioinfo.mpg.de / AlignMe / AlignMe_MSA.html) and calculation of the Gibbs free energy ΔG of the CCR4 protein sequence (ΔG predictor sever v1.0, http: / / dgpred.cbr.su.se).

[0065] The amino acid sequence of EL1-CCR4 is:

[0066] AADQWVFGLGLCKMISW (SEQ ID NO. 2)

[0067] The amino acid sequence of EL2-CCR4 is:

[0068] STCYTERNHTYCKTKYSLNSTTWKVLSSLEINIL (SEQ ID NO. 3)

[0069] The amino acid sequence of EL3-CCR4 is:

[0070] YNIVLFLETLVELEVLQDCTFERYLDYAIQAT (SEQ ID NO. 4)

[0071] Example 2: Experiment on interaction with CCL22

[0072] 1. Expression of GST-tagged protein

[0073] The human DNA sequences of EL1-CCR4, EL2-CCR4, EL3-CCR4, N-EL1-CCR4, N-EL2-CCR4, N-EL3-CCR4, N-EL2-EL3-CCR4, N-EL1-EL2-EL3-CCR4 (all using the GGGGSGGGGS linker) and CCL22, as well as the DNA sequence of GST, were cloned into the pGEX6P-1 vector (D&B / Dipper, product number: S201150A) through the restriction enzyme sites NdeI, BamHI and HindIII. Transetta competent cells (TransGen Biotech, cat: CD801-02) were transformed. The overnight culture was inoculated into a new LB medium (Oxoid, cat: LP0042B, LP0021B) at a ratio of 1:100. When the OD600 of the culture reached 0.6, IPTG with a final concentration of 1 mM (amresco, cat: 0487-1G) was added, and the expression was induced at 20 °C and 210 rpm / min for 10 hours. After resuspending the cells with PBS, the cells were lysed by a high-pressure cell disruptor. After binding with GST agarose (BBI, product number C600031) overnight, it was washed with 150 mM NaCl, 1 mM DTT (amresco, product number 0281-5G), 10 mM Tris-HCl (pH 8.0) buffer for 10 column volumes, and eluted and purified with 10 mM reduced glutathione (BBI, product number 70-18-8), 150 mM NaCl, 1 mM DTT Tris-HCl (pH 8.0) buffer, and dialyzed twice with 2 L PBS.

[0074] CCL22-GST protein sequence:

[0075] MGPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVPWVKMILNKLSQDPGGPGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK(SEQ ID NO. 9)

[0076] GST protein sequence:

[0077] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK(SEQ ID NO. 10)

[0078] 2. ELISA experiment to verify the interaction between EL1-CCR4, EL2-CCR4, EL3-CCR4 and CCL22

[0079] Add 100 μL of purified CCL22 protein (1 μg / ml) to each well of the ELISA plate and coat the ELISA plate for more than 12 h. Remove the coating solution and wash the plate 3 times with washing buffer PBST for 5 minutes each time. After adding 100 μL of blocking solution (PBST containing 5% FBS) to each well, block at 37 °C for 2 h. After removing the blocking solution, wash the plate 3 times with washing buffer PBST for 5 minutes each time. Set eight gradient concentrations for the above EL1-CCR4-GST, EL2-CCR4-GST, and EL3-CCR4-GST proteins, add 100 μL of GST fusion protein solution to each well, and incubate at 37 °C for 1 h. After removing the GST fusion protein solution, wash the plate 3 times with washing buffer PBST for 5 minutes each time. Add 100 μL of GST mouse monoclonal antibody (diluted 1:3000, Beyotime Institute of Biotechnology, catalog number AF0174) to each well and incubate at 37 °C for 1 h. After removing the GST mouse monoclonal antibody solution, wash the plate 3 times with washing buffer PBST for 5 minutes each time. Add 100 μL of horseradish peroxidase-labeled goat anti-mouse IgG (diluted 1:250, Beyotime Institute of Biotechnology, catalog number A0216) to each well and incubate at 37 °C for 0.5 h. After removing the horseradish peroxidase-labeled goat anti-mouse IgG solution, wash the plate 3 times with washing buffer PBST for 5 minutes each time. Add 100 μL of TMB chromogenic substrate (TMB Substrate Chromogenic Kit, NOVON, catalog number SS0652) to each well and develop color at room temperature for 3 - 5 min. Add 100 μL of 2 M H 2 SO 4 to terminate the reaction. Measure OD450 - OD630 with an enzyme-linked colorimeter. The results are as Figure 1As shown, it is demonstrated that EL1-CCR4, EL2-CCR4, and EL3-CCR4 can all interact with CCL22. There are slight differences in the binding abilities of EL1-CCR4, EL2-CCR4, and EL3-CCR4 to CCL22. EL2-CCR4 has the strongest binding ability to CCL22, indicating that different extracellular domain recombinant proteins have different ligand-binding abilities. By designing the permutations and combinations of recombinant proteins covering different extracellular domains, their ligand-binding ability can be further enhanced to exert a competitive antagonistic inhibitory effect.

[0080] 3. ELISA experiment to verify the interaction between the combined fusion proteins EL3-CCR4, N-EL3-CCR4, N-EL2-EL3-CCR4, and N-EL1-EL2-EL3-CCR4 and CCL22

[0081] Add 100 μL of purified CCL22 protein (1 μg / ml) to each well of the ELISA reaction plate and coat the ELISA reaction plate for more than 12 h. Remove the coating solution and wash the plate 3 times with washing buffer PBST, 5 minutes each time. After adding 100 μL of blocking solution (PBST containing 5% FBS) to each well, block at 37 °C for 2 h. After removing the blocking solution, wash the plate 3 times with washing buffer PBST, 5 minutes each time. Set eight gradient concentrations (GST protein solution) for the above-mentioned EL3-CCR4, N-EL3-CCR4, N-EL2-EL3-CCR4, and N-EL1-EL2-EL3-CCR4 proteins. Add 100 μL of GST protein solution to each well and incubate at 37 °C for 1 h. After removing the GST protein solution, wash the plate 3 times with washing buffer PBST, 5 minutes each time. Add 100 μL of GST mouse monoclonal antibody (diluted 1:3000, Beyotime Institute of Biotechnology, product number AF0174) to each well and incubate at 37 °C for 1 h. After removing the GST mouse monoclonal antibody solution, wash the plate 3 times with washing buffer PBST, 5 minutes each time. Add 100 μL of horseradish peroxidase-labeled goat anti-mouse IgG (diluted 1:250, Beyotime Institute of Biotechnology, product number A0216) to each well and incubate at 37 °C for 0.5 h. After removing the horseradish peroxidase-labeled goat anti-mouse IgG solution, wash the plate 3 times with washing buffer PBST, 5 minutes each time. Add 100 μL of TMB chromogenic substrate (TMB substrate chromogenic kit, NOVON, product number SS0652) to each well and develop color at room temperature for 3 - 5 min. Add 100 μL of 2M H 2 SO 4 to terminate the reaction. Measure OD450 with an enzyme-labeled colorimeter. The results are as Figure 2As shown, it is demonstrated that EL3-CCR4, N-EL3-CCR4, N-EL2-EL3-CCR4, and N-EL1-EL2-EL3-CCR4 can all interact with CCL22. The binding ability of N-EL3-CCR4 is superior to the others, indicating that by screening different permutations and combinations of extracellular proteins with the strongest binding affinity for CCR4, its competitive antagonistic inhibitory effect can be maximized.

[0082] Example 3: Antitumor Efficacy of the Extracellular Segment Protein of CCR4 in Vivo

[0083] Inoculate 5×10 6 Mouse hepatoma cell line Hepa1-6 cells (ATCC, CRL-1830) subcutaneously into wild-type C57BL / 6 mice (Spearf Bio (Beijing) Biotechnology Co., Ltd.). On the fifth day, use vernier calipers to measure the long diameter (a) and short diameter (b) of the tumor in each mouse. Then, calculate the tumor volume according to the formula V = a×b 2 / 2. Divide the mice into four groups with the same volume, with 8 mice in each group. Each group of mice was intraperitoneally injected with N-CCR4-Fc, N-EL1-CCR4, N-EL2-CCR4, and N-EL3-CCR4 recombinant proteins dissolved in PBS at 2 mg / kg on days 5, 8, 11, 14, and 17 respectively. The Ctrl group was injected with PBS as a control. Measure the tumor size and record the tumor growth curve on days 5, 7, 10, 12, 16, and 19 respectively.

[0084] The Fc protein sequence is as follows:

[0085] KLDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK (SEQ ID NO. 11)

[0086] The results are as Figure 3As shown, therefore, the recombinant proteins N-CCR4-Fc, N-EL1-CCR4, N-EL2-CCR4, and N-EL3-CCR4 can all effectively inhibit tumor growth. Among them, the therapeutic effects of the recombinant proteins N-EL2-CCR4 and N-EL3-CCR4 are better than that of the recombinant protein N-CCR4-Fc, and there is no difference in the therapeutic effects between the recombinant proteins N-EL1-CCR4 and N-CCR4-Fc.

[0087] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A recombinant protein or a derivative thereof that binds to CCL22 and / or CCL17, wherein the recombinant protein is: (A) a protein consisting of the amino acid sequence shown in any one of SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4; (B) A protein derived from protein (A) having the activity of protein (A) by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of protein (A), or a protein having a sequence identity of 70% or more with protein (A).

2. The recombinant protein or its derivative according to claim 1, wherein the derivative is: (a) a fusion protein consisting of any combination of any combination of the recombinant proteins; (b) a protein derived from protein (a) which has the activity of protein (a) by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence of the fusion protein (a), or a protein having a sequence identity of 70% or more with the fusion protein (a).

3. The recombinant protein or its derivative according to claim 1, wherein the derivative is: (1) A fusion recombinant protein obtained by any combination of the recombinant protein defined in claim 1 or the fusion protein defined in claim 2 and a CCR4 N-terminal recombinant protein; (2) A protein derived from (1) protein that has the activity of (1) protein and has one or more amino acid residues substituted and / or deleted and / or added to the amino acid sequence of (1) fusion recombinant protein, or a protein that has a sequence identity of 70% or more with (1) fusion recombinant protein; The amino acid sequence of the CCR4 N-terminal recombinant protein is shown in SEQ ID NO.

1.

4. The recombinant protein or its derivative according to claim 1, wherein the derivative is: (i) a fusion recombinant protein obtained by any combination of the recombinant protein defined in claim 1 or the fusion protein defined in claim 2 and an Fc fragment; (ii) a protein derived from the protein (i) which has the activity of the protein (i) by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence of the fusion recombinant protein (i), or a protein with a sequence identity of 70% or more with the fusion recombinant protein (i).

5. A pharmaceutical composition comprising the recombinant protein or a derivative thereof according to any one of claims 1 to 4.

6. Use of the recombinant protein or its derivative according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for inhibiting the binding of CCR4 to a ligand of CCR4, wherein the ligand of CCR4 comprises CCL22 and / or CCL17.

7. Use of the recombinant protein or derivative thereof according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating, inhibiting, ameliorating or preventing a disease or condition associated with CCR4 signal transduction.

8. The use of claim 7, wherein the diseases or conditions associated with CCR4 signaling include 1) allergic diseases, such as systemic anaphylaxis or hypersensitivity reactions; 2) inflammatory bowel diseases, preferably Crohn's disease, ulcerative colitis, ileitis and enteritis; 3) psoriasis and inflammatory skin diseases, preferably dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatomyositis and urticaria; 4) vasculitis; 5) spondyloarthropathies; 6) scleroderma; 7) asthma and respiratory allergic diseases, preferably allergic asthma, exercise-induced asthma, allergic rhinitis, hypersensitivity lung disease; 8) autoimmune diseases, preferably arthritis, polyarthritis sclerosis, systemic lupus erythematosus, diabetes, nephritis; 9) transplant rejection, preferably allogeneic transplant rejection and graft-versus-host disease; 10) leukemia, lymphoma and other blood-borne cancers such as cutaneous T-cell lymphoma and acute lymphoblastic leukemia; 11) other diseases that require suppression of adverse inflammation, preferably atherosclerosis, myositis, neurodegenerative diseases, encephalitis, meningitis, hepatitis, nephritis; 12) cancer, including solid tumors and metastatic tumors, preferably gastric cancer, liver cancer, kidney cancer, intestinal cancer, pancreatic cancer, lung cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, thyroid cancer, prostate cancer, and nervous system tumors.

9. The use of claim 7, wherein the disease or condition associated with CCR4 signaling is cancer or tumor.

Images