A protein binding to ny-eso-1 antigen

Abstract

The application discloses a protein combined with NY-ESO-1 antigen, and a preparation method thereof. The preparation method comprises the following steps: performing E. coli host codon optimization on an amino acid sequence of a precursor protein combined with NY-ESO-1 antigen, constructing a nucleotide sequence of the precursor protein on a pET21b plasmid to obtain plasmid 1; transforming the plasmid 1 into E. coli; inducing culture; purifying, collecting the protein, adding thrombin, dialyzing overnight, and cutting enzymes; and further purifying to obtain the protein combined with NY-ESO-1 antigen. Experiments prove that the protein combined with NY-ESO-1 antigen can recognize NY-ESO-1 antigen K D = 6.65 μM.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a protein that binds to the NY-ESO-1 antigen and its applications. Background Technology

[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 expressed in restricted amounts in 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 expression frequency of NY-ESO-1 varies considerably among these tumor types, with the most common expression occurring in neuroblastoma (82%), synovial sarcoma (80%), melanoma (46%), and ovarian cancer (43%). Expression of NY-ESO-1 protein in other tumor types ranges from 20% to 40%. Therefore, NY-ESO-1 is an important target for immunotherapy.

[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 exhibits restricted expression in healthy adult tissues, resulting in a relatively low risk of off-target toxicity from treatment. NY-ESO-1 has become one of the most promising targets for tumor immunotherapy. Targeting this protein can be achieved by modifying the binding protein that recognizes and binds to it. Current bispecific antibody drugs (such as Kimmtrak) also link the binding protein that recognizes and binds to this target protein to an antibody that targets the CD3 receptor on the surface of T cells.

[0005] However, there are currently few types of bispecific antibody protein drugs. Therefore, there is an urgent need for more proteins that bind to the NY-ESO-1 antigen to prepare bispecific antibody protein drugs. Summary of the Invention

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

[0007] A second objective of this invention is to provide a nucleotide sequence encoding the aforementioned protein.

[0008] A third objective of this invention is to provide a method for preparing a protein that binds to the NY-ESO-1 antigen.

[0009] A fourth objective of this invention is to provide the use of the above-mentioned protein in the preparation of drugs for treating tumors.

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

[0011] A protein that binds to the NY-ESO-1 antigen, the amino acid sequence of which 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 the NY-ESO-1 antigen includes the following steps:

[0014] 1) The amino acid sequence of a precursor protein that binds to the NY-ESO-1 antigen was optimized using the E. coli host codon to obtain the nucleotide sequence encoding the precursor protein. The nucleotide sequence encoding the precursor protein was then constructed onto the 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) Plasmid 1 was transformed into the expression bacterium E. coli BL21(DE3) and cultured on LB plates containing antibiotics;

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

[0018] 4) Purify and collect the protein, add thrombin and dialyze overnight for enzymatic digestion;

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

[0020] The aforementioned protein that binds to the 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 of this invention that binds to the NY-ESO-1 antigen can recognize and bind to the NY-ESO-1 antigen with high affinity. D =6.65μM. Attached Figure Description

[0023] Figure 1 A gel image of a protein that binds to the NY-ESO-1 antigen;

[0024] Figure 2 Gel images of the α chain (HLA-A*02:01) and β2m chain of the NY-ESO-1 antigen;

[0025] Figure 3 This is a gel image of NY-ESO-1 antigen after refolding.

[0026] Figure 4 This is a graph showing the results of a protein affinity assay for the NY-ESO-1 antigen. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0029] The dissociation equilibrium constant K between the protein and the NY-ESO-1 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.; the BLI detector was purchased from SARTORIUS.

[0033] 3. The buffer solutions involved in the following examples are 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, 20mM Tris-HCl (pH 7.5), 0.2M NaCl, 2mM EDTA, 1Mm DTT;

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

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

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

[0039] Refolding buffer 2: 100mM Tris (pH 8.0), 400mM arginine, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.5mM 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 media involved in the following examples are as follows:

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

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

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

[0047] I. Experimental Procedure

[0048] 1. Plasmid synthesis

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

[0050] 2. Plasmid 1 was transformed into the expression bacterium E.coli BL21(DE3) and allowed to grow overnight on LB plates containing the antibiotic ampicillin (100 μg / mL).

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

[0052] 4. Collect the bacterial culture by centrifuging at 4℃ and 4000 rpm for 20 min. Remove the supernatant, add 20 mL of gel filtration chromatography buffer 2, and resuspend the bacterial cells. Sonicate the bacterial cells to disrupt their structure, then centrifuge at 4℃ and 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. After incubation, the mixture was added to a gravity column and washed with gel filtration chromatography buffer 2. Then, eluted with 40 mL of elution buffer and the eluent was collected.

[0055] 7. Concentrate the protein to a volume of 0.5 mL using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. Use a Superdex 7510 / 300GL column at a flow rate of 0.5 mL / min and a pressure of 2.5 MPa. Load the sample loop with 0.5 mL and perform gel filtration chromatography purification using gel filtration chromatography buffer 2. Collect the protein (the protein concentration in the eluent is measured using a Nanodrop instrument).

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

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

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

[0059] II. Experimental Results

[0060] A purified protein that binds to the NY-ESO-1 antigen; results are shown below. Figure 1In the figure, M represents a 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] I. Experimental Procedure

[0064] 1. Plasmid construction

[0065] The amino acid sequence of the α chain of the NY-ESO-1 antigen (SEQ ID NO.3) was submitted to Qingke Biotechnology Co., Ltd. for E. 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 using Nde I / Hind III to obtain plasmid 2.

[0066] The amino acid sequence of the NY-ESO-1 antigen β2m chain (SEQ ID NO.5) was submitted to Qingke Biotechnology Co., Ltd. for E. coli host codon optimization, 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 using Nde I / Hind III to obtain plasmid 3.

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

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

[0069] 4. Collect the bacterial culture by centrifuging at 4℃ and 4000rpm for 20min, remove the supernatant, add 20mL of lysis buffer, resuspend the bacterial cells, sonicate the bacterial cells, centrifuge at 4℃ and 18000rpm for 20min, and remove the supernatant.

[0070] 5. Wash the precipitate with detergent buffer. Add 10 mL of detergent buffer to resuspend the precipitate, and use a homogenizer to resuspend the precipitate evenly. Add detergent buffer to a final volume of 30 mL. Centrifuge at 18,000 rpm for 20 min at 4°C to remove the supernatant.

[0071] 6. Wash the precipitate with Wash Buffer 1. Add 10 mL of Wash Buffer 1 to resuspend the precipitate, and homogenize it using a homogenizer. Add Wash Buffer 1 to a final volume of 30 mL. Centrifuge at 18000 rpm for 20 min at 4°C to remove the supernatant. Repeat this step twice.

[0072] 7. Wash the precipitate with washing buffer 2. Add 10 mL to resuspend the precipitate and homogenize it with a homogenizer. Add washing buffer 2 to 30 mL, centrifuge at 18000 rpm for 20 min at 4°C to remove the supernatant. 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. Use 10 μL of the protein sample for SDS-PAGE analysis.

[0074] II. Experimental Results

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

[0076] Example 3

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

[0078] I. Refolding 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 of DMSO, add it 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℃ for 24 hours.

[0084] 6. Concentrate the protein to 1 mL using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. Use a Superdex 20010 / 300GL column at a flow rate of 0.5 mL / min and a pressure of 2.0 MPa. Load with a 1 mL sample loop and purify by gel filtration chromatography using gel filtration chromatography buffer 2. The obtained protein is the NY-ESO-1 antigen protein. Collect the NY-ESO-1 antigen protein product and store it at 4°C. Use 10 μL of the sample for SDS-PAGE analysis.

[0085] II. Experimental Results

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

[0087] Example 4

[0088] Characterization of the binding of a protein to the NY-ESO-1 antigen

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

[0090] 1. Replace the NY-ESO-1 antigen protein solution with biotinylation buffer 1 (50 mM N-diglycine, pH 8.3) using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. Measure the protein concentration using a Nanodrop instrument.

[0091] 2. After changing the medium, concentrate the NY-ESO-1 antigen protein to approximately 2.4 mg / mL. Prepare the biotinylation reaction system according to the instructions of the biotinylation kit (Biotin-Protein Ligase, BI001). First, incubate the system in a 30°C water bath for 1 hour, then allow it to react overnight at room temperature to ensure complete biotinylation of the NY-ESO-1 antigen. Measure the protein concentration using a Nanodrop instrument.

[0092] II. BLI Affinity Determination

[0093] 1. The biotinylated NY-ESO-1 antigen was immobilized on the SA detector.

[0094] 2. Using an SA detector immobilized with biotinylated NY-ESO-1 antigen, the levels of a protein binding to NY-ESO-1 antigen at concentrations of 0.03 μM, 0.3 μM, 3 μM, 30 μM, and 300 μM were detected (see [link to SA detector]). Figure 4The concentrations of A, B, C, D, and E were 0.03 μM, 0.3 μM, 3 μM, 30 μM, and 300 μM, respectively. Kinetic parameters were calculated using Octet Analysis Studio software.

[0095] III. Experimental Results

[0096] The results of the affinity assay for a protein that binds to the NY-ESO-1 antigen are as follows: Figure 4 As shown, affinity K D =6.65 μM. Therefore, proteins that can bind to the NY-ESO-1 antigen show promise for the development of drugs to treat tumors.

Claims

1. A protein that binds to the 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. The gene encoding the protein of claim 1.

3. A method for preparing a protein binding to the NY-ESO-1 antigen according to claim 1, characterized in that: Includes the following steps: 1) The amino acid sequence of a precursor protein that binds to the NY-ESO-1 antigen was optimized using the E. coli host codon to obtain the nucleotide sequence encoding the precursor protein. The nucleotide sequence encoding the precursor protein was then constructed onto the 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) Plasmid 1 was transformed into the expression bacterium E. coli BL21(DE3) and cultured on LB plates containing antibiotics; 3) Select a single colony for induction culture; 4) Purify and collect the protein, add thrombin and dialyze overnight for enzymatic digestion; 5) Further purification yields a protein that binds to the NY-ESO-1 antigen.

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