Protein combined with NY-ESO-1 antigen

Abstract

The invention discloses a protein combined with an NY-ESO-1 antigen, and a preparation method of the protein comprises the following steps: carrying out escherichia coli host codon optimization on an amino acid sequence of a precursor protein combined with the NY-ESO-1 antigen, and constructing a nucleotide sequence of the precursor protein on a pET21b plasmid to obtain a plasmid 1; transforming the plasmid 1 into escherichia coli; performing induction culture; purifying, collecting protein, adding thrombin, dialyzing overnight and carrying out enzyme digestion; according to the present invention, the protein bound with the NY-ESO-1 antigen is obtained by further purification, and the experiment results show that the protein bound with the NY-ESO-1 antigen can achieve the high affinity recognition binding with the NY-ESO-1 antigen KD = 6.65 [mu] M;

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a protein binding to NY-ESO-1 antigen and its application. Background Art

[0002] New York esophageal squamous cell carcinoma-1 (NY-ESO-1) is a cancer-testis antigen 1B, encoded by the CTGAG1B gene located in the Xq28 region of chromosome X. NY-ESO-1 is restricted to germ cells, placental cells and tumor cells.

[0003] NY-ESO-1 is expressed in a variety of tumor types, including neuroblastoma, myeloma, melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and breast cancer. The frequency of NY-ESO-1 expression varies greatly among these tumor types, with the most common tumors being neuroblastoma (82%), synovial sarcoma (80%), melanoma (46%), and ovarian cancer (43%). The expression of NY-ESO-1 protein in other types of tumors ranges between 20-40%. Therefore, NY-ESO-1 is an important immunotherapy target.

[0004] NY-ESO-1 belongs to the cancer-testis antigen family and is one of the most immunogenic tumor-associated antigens in the family. NY-ESO-1 is restrictedly expressed in healthy adult tissues, and the risk of off-target toxicity caused by treatment is relatively low. NY-ESO-1 has become one of the most promising targets for tumor immunotherapy. For this target, the purpose of treating tumors can be achieved by modifying the binding protein that recognizes and binds to this target protein. The current dual-antibody protein drug (Kimmtrak) product also connects the binding protein that recognizes and binds to the target protein with an antibody that binds to the CD3 receptor on the surface of T cells.

[0005] However, at present, there are few types of bispecific protein drugs. Therefore, more proteins that bind to the NY-ESO-1 antigen are urgently needed for the preparation of bispecific protein drugs. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a protein that binds to the NY-ESO-1 antigen.

[0007] The second object of the present invention is to provide a nucleotide sequence encoding the above protein.

[0008] The third object of the present invention is to provide a method for preparing a protein that binds to NY-ESO-1 antigen.

[0009] The fourth object of the present invention is to provide the use of the above protein in the preparation of drugs for treating tumors.

[0010] The technical solution of the present invention is summarized as follows:

[0011] A protein that binds to NY-ESO-1 antigen, wherein the amino acid sequence of the protein that binds to NY-ESO-1 antigen is shown in SEQ ID NO.1.

[0012] The nucleotide sequence encoding the above protein.

[0013] A method for preparing a protein that binds to NY-ESO-1 antigen comprises the following steps:

[0014] 1) optimizing the amino acid sequence of a precursor protein that binds to the NY-ESO-1 antigen by codon optimization in Escherichia coli to obtain a nucleotide sequence encoding the precursor protein, and constructing the nucleotide sequence encoding the precursor protein into a pET21b plasmid to obtain plasmid 1;

[0015] The amino acid sequence of the precursor protein that binds to the NY-ESO-1 antigen is shown in SEQ ID NO.8; the nucleotide sequence encoding the precursor protein is shown in SEQ ID NO.9;

[0016] 2) Transform plasmid 1 into the expression bacteria E. coli BL21 (DE3) and culture on LB plates containing antibiotics;

[0017] 3) Pick a single colony for induction culture;

[0018] 4) Purification, protein collection, and overnight dialysis with thrombin;

[0019] 5) Further purification to obtain a protein that binds to the NY-ESO-1 antigen.

[0020] The above-mentioned protein binding to NY-ESO-1 antigen is used in the preparation of drugs for treating tumors.

[0021] The present invention has the following beneficial effects:

[0022] Experiments have shown that the protein binding to NY-ESO-1 antigen of the present invention can recognize and bind to NY-ESO-1 antigen with high affinity, K D =6.65μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a protein gel image that binds to NY-ESO-1 antigen;

[0024] Figure 2 It is the gel map of NY-ESO-1 antigen α chain (HLA-A*02:01) and β2m chain protein;

[0025] Figure 3 This is the protein gel image after NY-ESO-1 antigen renaturation;

[0026] Figure 4 This is a graph showing the results of affinity determination of a protein that binds to the NY-ESO-1 antigen. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The NY-ESO-1 antigen is a SLLMWITQC fragment presented by HLA-A*02:01.

[0029] Dissociation equilibrium constant K between protein binding to NY-ESO-1 antigen and antigen D =6.65μM.

[0030] Experimental materials and reagents

[0031] 1. Escherichia coli DH5α and Escherichia coli BL21 were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0032] 2. Tris-HCl, protease inhibitors, L-arginine, ethylenediaminetetraacetic acid (EDTA), reduced glutathione, oxidized glutathione, deoxycholic acid, NP-40, dithiothreitol (DTT), urea, guanidine hydrochloride, Triton-X100, DMSO, etc. were purchased from Sangon Biotech (Shanghai) Co., Ltd.; BLI detector was purchased from SARTORIUS.

[0033] 3. The buffer solution involved in the following embodiments is as follows:

[0034] Lysis buffer: 50 mM Tris-HCl (pH 8.0), 25% sucrose, 1 mM EDTA, 1 mM DTT;

[0035] Detergent buffer: 1% deoxycholic acid, 1% NP40, 20 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 2 mM EDTA, 1 Mm DTT;

[0036] Wash buffer 1: 0.5% Triton X-100, 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM DTT;

[0037] Wash buffer 2: 100 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 m DTT;

[0038] Denaturation buffer 2: 50 mM MES (pH 6.5), 0.1 mM EDTA, 1 mM DTT, 8 M urea;

[0039] Renaturation buffer 2: 100 mM Tris (pH 8.0), 400 mM arginine, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 0.5 mM PMSF;

[0040] Elution buffer: PBS containing 10 mM D-dethiobiotin;

[0041] Dialysis buffer: PBS

[0042] Gel filtration chromatography buffer 2: PBS.

[0043] 4. The culture medium involved in the following embodiments is as follows:

[0044] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, and the balance is water.

[0045] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 2%, and the balance is water.

[0046] Example 1: Expression and purification of a protein binding to NY-ESO-1 antigen

[0047] 1. Experimental steps

[0048] 1. Plasmid synthesis

[0049] The amino acid sequence of a precursor protein binding to NY-ESO-1 antigen (SEQ ID NO.8) was submitted to Qingke Biotechnology Co., Ltd. for Escherichia coli host codon optimization, and the nucleotide sequence encoding the precursor protein (SEQ ID NO.9) was synthesized and constructed into the pET21b plasmid to obtain plasmid 1.

[0050] 2. Transform plasmid 1 into the expression bacteria E. coli BL21 (DE3) and grow it overnight on LB plates containing the antibiotic ampicillin (100 μg / mL).

[0051] 3. Pick a single colony and inoculate it into 10 mL LB liquid culture medium, and culture it at 37°C and 220 rpm for 13 h to obtain seed solution; inoculate 10 mL of seed solution into 1 L LB culture medium with ampicillin (100 μg / mL) at a ratio of 1:100, and shake the culture at 37°C and 220 rpm for about 2.5 h until OD600 reaches 0.6-0.8, then add 1 mL of 1 M IPTG, and continue to induce the culture at 16°C for 12 h.

[0052] 4. Centrifuge at 4°C, 4000 rpm for 20 min to collect the bacterial solution. Remove the supernatant, add 20 mL of gel filtration chromatography buffer 2, and resuspend the bacterial cells. Ultrasonicate the cells, centrifuge at 4°C, 18000 rpm for 20 min, and collect the supernatant.

[0053] 5. Incubate the supernatant with 1 mL of Strep(II) tag protein agarose purification resin at 8°C for 2 hours.

[0054] 6. Add the incubated mixture to the gravity column and wash with gel filtration chromatography buffer 2. Then elute with 40 mL of elution buffer and collect the eluate.

[0055] 7. Use an ultrafiltration tube with a molecular weight cutoff of 3KDa to concentrate the protein to a volume of 0.5mL, select a Superdex7510 / 300GL column, a flow rate of 0.5mL / min, a pressure of 2.5mpa, a sample loop of 0.5mL, and perform gel filtration chromatography purification with gel filtration chromatography buffer 2 to collect the protein (use Nanodrop instrument to determine the protein concentration in the eluate);

[0056] 8. Add 3 μg of thrombin to the protein obtained in step 7 and dialyze overnight;

[0057] 9. The liquid obtained in step 8 was incubated with 1 mL of Strep(II)-tagged protein agarose purification resin at 8°C for 2 hours. The incubated mixture was added to the gravity column and the flow-through was collected. A protein (SEQ ID NO.1) binding to the NY-ESO-1 antigen was obtained, and the corresponding nucleotide sequence was SEQ ID NO.2.

[0058] 10. Take 10 μL of the sample for SDS-PAGE analysis.

[0059] 2. Experimental Results

[0060] A purified protein that binds to the NY-ESO-1 antigen. Figure 1In the figure, M represents protein marker, and 1 represents a purified protein that binds to the NY-ESO-1 antigen. After purification, SDS-PAGE clearly shows a protein that binds to the NY-ESO-1 antigen with high purity.

[0061] Example 2

[0062] Expression of NY-ESO-1 antigen (protein) heavy chain (α chain) and light chain (β2m chain)

[0063] 1. Experimental steps

[0064] 1. Plasmid construction

[0065] The amino acid sequence of the NY-ESO-1 antigen α chain (SEQ ID NO.3) was submitted to Qingke Biotechnology Co., Ltd. for Escherichia coli host codon optimization, and the corresponding nucleotide sequence (SEQ ID NO.4) was synthesized. The synthesized nucleotide sequence (SEQ ID NO.4) was constructed into the pET24a plasmid through Nde I / Hind III to obtain plasmid 2.

[0066] The amino acid sequence of the β2m chain of the NY-ESO-1 antigen (SEQ ID NO.5) was submitted to Qingke Biotechnology Co., Ltd. for codon optimization in the E. coli host, and the corresponding nucleotide sequence (SEQ ID NO.6) was synthesized. The synthesized nucleotide sequence (SEQ ID NO.6) was constructed into the pET24a plasmid through Nde I / Hind III to obtain plasmid 3;

[0067] 2. Plasmid 2 and plasmid 3 were transformed into expression bacteria E. coli BL21 (DE3) and grown overnight on LB plates containing kanamycin (50 μg / mL);

[0068] 3. Pick a single colony and inoculate it into 10 mL LB liquid medium, and culture it at 37°C and 220 rpm for 13 h to obtain seed solution; inoculate 10 mL seed solution into 1 L LB medium with kanamycin (50 μg / mL) at a ratio of 1:100, and shake the bacteria at 37°C and 220 rpm for about 2.5 h until OD600 reaches 0.6-0.8, then add 1 mL of 1 M IPTG at a final concentration, and continue induction culture at 37°C for 4 h;

[0069] 4. Centrifuge at 4°C, 4000 rpm for 20 min to collect the bacterial solution, remove the supernatant, add 20 mL of lysis buffer, resuspend the bacterial cells, disrupt the cells by ultrasonic wave, centrifuge at 4°C, 18000 rpm for 20 min to remove the supernatant;

[0070] 5. Wash the precipitate with detergent buffer. First add 10 mL to resuspend the precipitate, and use a homogenizer to evenly resuspend the precipitate, and add detergent buffer to 30 mL. Centrifuge at 4°C, 18,000 rpm for 20 minutes to remove the supernatant;

[0071] 6. Wash the precipitate with washing buffer 1. First add 10 mL, resuspend the precipitate, and use a homogenizer to evenly resuspend the precipitate, then add washing buffer 1 to 30 mL. Centrifuge at 4°C, 18,000 rpm for 20 min and remove the supernatant. Repeat this step 2 times;

[0072] 7. Wash the precipitate with washing buffer 2. First add 10 mL to resuspend the precipitate, and use a homogenizer to evenly resuspend the precipitate, add 2 to 30 mL of washing buffer, centrifuge at 4°C, 18,000 rpm for 20 min, remove the supernatant, and repeat this step once;

[0073] 8. Dissolve the precipitate from step 7 in 10 mL of denaturing buffer and determine the protein concentration using a Nanodrop instrument. Aliquot into 1.5 mL tubes, 1 mL per tube, and store at -80°C. Take 10 μL of protein sample for SDS-PAGE analysis.

[0074] 2. Experimental Results

[0075] The SDS-PAGE results of NY-ESO-1 antigen heavy chain (α chain) and light chain (β2m chain) proteins are shown in Figure 2 . Figure 2 The M represents protein marker, 1 represents the NY-ESO-1 antigen heavy chain (α chain), and 2 represents the NY-ESO-1 antigen light chain (β2m chain). After protein precipitation and washing, proteins of corresponding sizes of heavy and light chains were obtained with high purity.

[0076] Example 3

[0077] Renaturation and purification of NY-ESO-1 antigen protein

[0078] 1. Renaturation and purification of NY-ESO-1 antigen protein

[0079] 1. Prepare freshly prepared refolding buffer 2 and pre-cool it on ice.

[0080] 2. Dissolve 10 mg of NY-ESO-1 polypeptide (SEQ ID NO. 7) in 500 μL DMSO, add to 1 L of refolding buffer 2, and mix well.

[0081] 3. Add 23.8 mg of β2m obtained in Example 2 and mix well.

[0082] 4. Add 34 mg of the α chain obtained in Example 2 and mix well.

[0083] 5. Stir at 4°C for 24 hours.

[0084] 6. Use an ultrafiltration tube with a molecular weight cutoff of 10KDa to concentrate the protein to a volume of 1mL, select a Superdex20010 / 300GL column, a flow rate of 0.5mL / min, a pressure of 2.0mpa, a sample loop of 1mL, and perform gel filtration chromatography purification with gel filtration chromatography buffer 2. The obtained protein is NY-ESO-1 antigen protein, collect the product NY-ESO-1 antigen protein, and store it at 4°C. Take 10μL of the sample for SDS-PAGE analysis.

[0085] 2. Experimental Results

[0086] NY-ESO-1 antigen protein results are shown in Figure 3 . Figure 3 In the figure, M represents protein marker and 1 represents NY-ESO-1 antigen protein. The obtained monomers clearly show proteins of corresponding sizes of heavy and light chains, and have high purity.

[0087] Example 4

[0088] Characterization of a protein that binds to NY-ESO-1 antigen

[0089] 1. Biotinylated NY-ESO-1 antigen

[0090] 1. Use an ultrafiltration tube with a molecular weight cutoff of 10 KDa to replace the NY-ESO-1 antigen protein solution with biotinylation buffer 1 (50 mM N-bicine, pH 8.3). Use a Nanodrop instrument to measure the protein concentration.

[0091] 2. After the liquid was replaced, the NY-ESO-1 antigen protein was concentrated to about 2.4 mg / mL, and the biotinylation reaction system was prepared according to the instructions of the biotinylation kit (Biotin-Protein Ligase, BI001). The system was first placed in a 30°C water bath for 1 hour, and then reacted at room temperature overnight to make the NY-ESO-1 antigen completely biotinylated. The protein concentration was measured using a Nanodrop instrument.

[0092] 2. BLI Affinity Determination

[0093] 1. Immobilize the biotinylated NY-ESO-1 antigen on the SA detector.

[0094] 2. The SA detector with biotinylated NY-ESO-1 antigen fixed was used to detect 0.03 μM, 0.3 μM, 3 μM, 30 μM and 300 μM of a protein binding to NY-ESO-1 antigen (see Figure 4The concentration of A is 0.03 μM, the concentration of B is 0.3 μM, the concentration of C is 3 μM, the concentration of D is 30 μM, and the concentration of E is 300 μM). The kinetic parameters were calculated using Octet Analysis Studio software.

[0095] 3. Experimental Results

[0096] The results of the determination of the affinity between a protein binding to NY-ESO-1 antigen and NY-ESO-1 antigen are as follows: Figure 4 As shown, affinity K D =6.65 μM. Therefore, the protein that can bind to NY-ESO-1 antigen has the prospect of being used to prepare drugs for treating tumors.

Claims

1. A protein that binds to NY-ESO-1 antigen, characterized in that The amino acid sequence of the protein that binds to the NY-ESO-1 antigen is shown in SEQ ID NO.

1.

2. A nucleotide sequence encoding the protein according to claim 1.

3. A method for preparing a protein binding to NY-ESO-1 antigen according to claim 1, characterized in that The steps include: 1) optimizing the amino acid sequence of a precursor protein that binds to the NY-ESO-1 antigen by codon optimization in Escherichia coli to obtain a nucleotide sequence encoding the precursor protein, and constructing the nucleotide sequence encoding the precursor protein into a pET21b plasmid to obtain plasmid 1; The amino acid sequence of the precursor protein that binds to the NY-ESO-1 antigen is shown in SEQ ID NO.8; the nucleotide sequence encoding the precursor protein is shown in SEQ ID NO.9; 2) Transform plasmid 1 into the expression bacteria E. coli BL21 (DE3) and culture on LB plates containing antibiotics; 3) Pick a single colony for induction culture; 4) Purification, protein collection, and overnight dialysis with thrombin; 5) Further purification to obtain a protein that binds to the NY-ESO-1 antigen.

4. Use of the protein binding to NY-ESO-1 antigen according to claim 1 in the preparation of drugs for treating tumors.

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