A kind of NK cell and its application in anti-tumor treatment

Abstract

The present invention discloses a B7-H3 single-domain antibody, wherein the B7-H3 single-domain antibody is B7-H3-1B, and the amino acid sequence of the VNAR of the B7-H3-1B is shown in SEQ ID NO.1. The present invention also discloses a pharmaceutical composition, which comprises the B7-H3-1B and NK cells. The present invention also discloses a NK cell, wherein the NK cell is a NK-B7-H3-1B cell, and the NK-B7-H3-1B cell contains a eukaryotic expression vector expressing the B7-H3-1B. The B7-H3 single-domain antibody (B7-H3-1B) prepared by the present invention has good activity. Based on B7-H3-1B, the present invention combines NK cells to prepare a pharmaceutical composition and NK-B7-H3-1B cells for the treatment of various tumors, and the pharmaceutical composition and NK-B7-H3-1B cells have good inhibitory effects on various tumors. This study provides a good reference for the study of the therapeutic effects of NK cells on various tumors.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a NK cell and an application thereof in anti-tumor treatment. Background Art

[0002] Natural killer cells (NK cells) originate from bone marrow lymphoid stem cells. Their differentiation and development depend on the bone marrow and thymus microenvironment. They are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs and lymph nodes. NK cells are different from T and B cells. They are a type of lymphocyte that can non-specifically kill tumor cells and virus-infected cells without prior sensitization.

[0003] Natural killer (NK) cells are an important type of cell in the innate immune system that has the ability to selectively lyse cells without prior activation. NK cells belong to innate lymphocytes (ILCs) and play a key role in antiviral and antitumor activities.

[0004] NK cells can recognize and kill cells infected by viruses or encapsulated by cancer cells, which makes them play a vital role in immune surveillance and host defense. In addition, NK cells can inhibit tumor growth through multiple mechanisms, such as direct mediated cytotoxicity.

[0005] However, although NK cells have strong anti-tumor functions in theory, these functions may be inhibited in the actual tumor microenvironment. Studies have shown that the anti-tumor efficacy of NK cells is significantly inhibited in the tumor microenvironment, which may be due to certain factors secreted by tumor cells, such as PD-L1. Therefore, it is necessary to study NK cells that can exert stronger anti-tumor effects for targeted tumor therapy. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a NK cell and its application in anti-tumor treatment.

[0007] Therefore, the present invention discloses a B7-H3 single-domain antibody on one hand, wherein the B7-H3 single-domain antibody is B7-H3-1B, and the amino acid sequence of VNAR of the B7-H3-1B is shown in SEQ ID NO.1.

[0008] Based on the screened B7-H3-1B, the present invention discloses a prokaryotic expression gene encoding the B7-H3-1B, and the nucleotide sequence of the prokaryotic expression gene is shown in SEQ ID NO.2.

[0009] The present invention also discloses a recombinant plasmid, wherein the recombinant plasmid is pET28a-B7-H3-1B, and the pET28a-B7-H3-1B contains the nucleotide sequence of SEQ ID NO.2.

[0010] The invention also discloses a recombinant expression bacterium, wherein the recombinant expression bacterium contains the pET28a-B7-H3-1B.

[0011] The present invention also discloses a pharmaceutical composition, which comprises the B7-H3-1B and NK cells.

[0012] The present invention also discloses a NK cell, wherein the NK cell is a NK-B7-H3-1B cell, and the NK-B7-H3-1B cell contains a eukaryotic expression vector for expressing the B7-H3-1B.

[0013] Preferably, the eukaryotic expression vector for expressing the B7-H3-1B of the present invention is pEE12.4-B7-H3-1B, and the pEE12.4-B7-H3-1B contains a gene encoding B7-H3-1B, and the nucleotide sequence of the gene encoding B7-H3-1B is shown in SEQ ID NO.3.

[0014] The present invention also discloses an application of the NK-B7-H3-1B cells in preparing drugs for treating tumors.

[0015] Preferably, the tumor described in the present invention includes one or more of small cell lung cancer, prostate cancer, esophageal squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, and breast cancer.

[0016] The B7-H3 single domain antibody (B7-H3-1B) screened and prepared by the present invention has good activity. Based on B7-H3-1B, the present invention combines NK cells to prepare a pharmaceutical composition for the treatment of various tumors and NK-B7-H3-1B cells. The pharmaceutical composition and NK-B7-H3-1B cells have good inhibitory effects on various tumors. This study provides a good reference for the study of the therapeutic effects of NK cells on various tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the detection of B7-H3-1B expression in NK cells of different generations. 0 to 5 are before NK cell transfection, 1st generation after transfection, 2nd generation after transfection, 3rd generation after transfection, 4th generation after transfection, and 5th generation after transfection.

[0018] Figure 2 Results of the inhibitory effects of different treatment groups on different cell lines.

[0019] Figure 3 The therapeutic effects of different treatment groups on nude mouse tumors. DETAILED DESCRIPTION

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0021] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0022] Example 1: Preparation of B7-H3 single domain antibody

[0023] 1. Research status of B7-H3

[0024] B7-H3, also known as CD276, is a member of the B7 family. It is an immunomodulatory protein that is overexpressed in a variety of solid tumors and is associated with disease progression and poor prognosis.

[0025] For example, in patients with small cell lung cancer (SCLC), approximately 65% ​​of patients have moderate to high expression of B7-H3. A phase 1 / 2 clinical study evaluated the efficacy and safety of the B7-H3-directed antibody-drug conjugate (ADC) Ifinatamab Deruxtecan (I-DXd) in the treatment of heavily pretreated SCLC patients. The study showed that I-DXd showed efficient and lasting efficacy and controllable and tolerable toxicity in these patients.

[0026] B7-H3 is expressed at higher levels in prostate cancer, which has prompted researchers to explore the potential of B7-H3 targeted therapy in prostate cancer. B7-H3 is also overexpressed in esophageal cancer, and studies have shown that high expression of B7-H3 is associated with poor prognosis in esophageal cancer. B7-H3 expression in colorectal cancer has been associated with tumor development and regulation of anti-tumor immunity. Although B7-H3 expression in non-small cell lung cancer (NSCLC) may not be as common as in SCLC, it is still a potential target for investigation. Overexpression of B7-H3 has also been found in other types of tumors, including but not limited to breast cancer, pancreatic cancer, head and neck squamous cell carcinoma, etc.

[0027] In summary, overexpression of B7-H3 makes it a promising therapeutic target, especially in tumor types that lack effective treatment options. Ongoing studies include the evaluation of B7-H3-targeted monoclonal antibodies, ADCs, bispecific antibodies, and other immunotherapeutic approaches.

[0028] 2. Preparation of B7-H3 single domain antibody

[0029] Shark single-domain antibody (also known as VNAR) is a new type of antibody with antigen-binding activity that is cloned from the serum of sharks and other cartilaginous fish and only retains the heavy chain variable region. Shark single-domain antibodies have many unique advantages, such as small molecular weight (about 12kDa), high affinity, strong stability, good solubility, strong tissue penetration, and the ability to recognize hidden antigenic epitopes. These characteristics make shark single-domain antibodies have broad application prospects in the fields of immune reagents and drug development. To this end, this study prepared shark single-domain antibodies against B7-H3. The steps are briefly described as follows:

[0030] (1) Immunization of sharks: Take 5-striped bamboo sharks, dilute the recombinant human CD276 protein (ab214154) to 20 μg / ml with shark physiological saline, and mix the diluted antigen with the adjuvant in a 1:1 ratio to emulsify. Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for subsequent immunizations.

[0031] (2) Sample collection: Peripheral blood or spleen tissue was collected from immunized sharks for RNA extraction.

[0032] (3) RNA extraction and cDNA synthesis: RNA is extracted from the sample using a specific kit and reverse transcribed to form the first strand of cDNA.

[0033] (4) Gene cloning and expression: The VNAR gene sequence is amplified using PCR technology and cloned into a suitable expression vector, which is then transformed into Escherichia coli or other host cells for expression.

[0034] (5) Antibody library construction: By constructing a phage or yeast display library, the VNAR gene fragments are displayed on the surface of microorganisms to facilitate subsequent screening.

[0035] (6) Screening of specific antibodies: Using the principle of antigen-antibody specific binding, single-domain antibodies that specifically bind to the target antigen are screened from the library through affinity chromatography, ELISA and other methods.

[0036] (7) Antibody purification: The positive clones screened out are cultured, and the expressed single-domain antibodies are collected and purified, usually using chromatography and other techniques.

[0037] (8) After the above steps, a good B7-H3 single domain antibody (denoted as B7-H3-1B) was screened. After sequencing, the amino acid sequence of VNAR of B7-H3-1B was shown in SEQ ID NO.1.

[0038] Example 2: Prokaryotic expression, preparation and testing of B7-H3-1B

[0039] 1. The codon optimization, gene synthesis and subcloning of the VNAR gene of B7-H3-1B into the Nco I and Xho I sites of pET28a and preservation in the DH5α strain were entrusted to Nanjing GenScript Biotechnology Co., Ltd. The nucleotide sequence of the codon-optimized VNAR is shown in SEQ ID NO.2 (including 6 His tags).

[0040] 2. Transform the prepared pET28a-B7-H3-1B plasmid into BL21 (DE3) competent cells, spread on LB agar plates containing kanamycin, and culture at 36-37°C, inverted for 16-20 hours. Observe the colony morphology. Pick a single colony from the plate, inoculate it into 3 ml of LB medium containing kanamycin, and culture it at 36-37°C, 220-240r / min with shaking until the OD600nm value reaches 0.4-0.6. Harvest part of the bacterial solution and add sterile glycerol to a final concentration of 25% for storage.

[0041] 3. Take the bacterial solution and culture it at 37℃, then cool it to 20℃, induce expression with IPTG, and collect the bacteria; resuspend the bacteria, break them, collect the supernatant by centrifugation, purify them through a nickel column, replace the liquid, filter and sterilize them through a 0.22μm low-protein adsorption filter membrane to obtain the recombinant protein, quantitatively package it, and store it in a -80℃ refrigerator for later use.

[0042] 4. The protein content was detected using the BCA kit. The recombinant protein content was 2.24 mg / ml. The expression level of the protein after purification was 300 mg / L, which has a high expression level and is suitable for large-scale applications.

[0043] 5. Dilute the recombinant Anti-CD276 antibody (ab134161) and the B7-H3-1B prepared above to a concentration of 1 μg / ml with 50 mM carbonate buffer (pH 9.6), spread 100 μl per well, and cover the plate overnight at 4°C. After washing 3 times with PBST, add PBST solution containing 2% BSA to each well and incubate at 37°C for 2 hours. After washing 3 times with PBST, dilute the recombinant human CD276 protein (ab214154) to 1 μg / ml with PBST solution containing 2% BSA, add 100 μl to each well, and incubate at 37°C for 1 hour. Use the recombinant HRP Anti-CD276 antibody (ab305813) as the secondary antibody, dilute 1:5000 with PBST, incubate at 37°C for 1 hour, wash 3 times with PBST, and pat dry. Add 100 μl TMB colorimetric solution to each well and react in the dark at room temperature for 10 min. After the reaction, add 50 μl 2M H 2 SO 4 The absorbance of the sample at 450 nm was measured by an ELISA instrument. The test results are shown in the table below. The single domain antibody can specifically bind to the CD276 protein, indicating that the single domain antibody has good activity and has better or equivalent activity than the recombinant Anti-CD276 antibody (ab134161).

[0044]

[0045] Example 3: NK cells expressing B7-H3 single domain antibody and preparation thereof

[0046] 1. Preparation of NK cells

[0047] NK cells were expanded from fresh or frozen peripheral blood mononuclear cells (PBMCs) with a viability of ≥ 90% using a commercial kit (Shenzhen Dakoway Biotechnology Co., Ltd., Cat#: 6113511 / 6113512). The specific process is briefly described as follows:

[0048] 1. Coating culture flask: Dilute the NK cell coating solution (10X) 10 times with PBS or saline and add it to the cell culture vessel. After coating overnight at 4°C or incubating at 37°C for 1.5 hours, aspirate the residual liquid.

[0049] 2. Prepare the culture medium: add 1 tube of 0.5 mL NK cell activator to every 1 L NK cell serum-free culture medium to form the NK cell expansion medium. The NK cell expansion medium is valid for three weeks.

[0050] 3. Heat-inactivated autologous plasma: After blood collection, separate the plasma, heat-treat at 56°C for 30 minutes, centrifuge at 1000g for 10 minutes, and obtain the supernatant to prepare heat-inactivated autologous plasma.

[0051] 4. On day 0, 3.0×10 NK cells were isolated and cultured in 10 mL of NK cell expansion medium (containing 10% heat-inactivated autologous plasma). 7 PBMCs were inoculated into coated T25 culture flasks (the recommended inoculation density is 3.0×10 6 / mL). 37℃, 5% CO 2 Culture in an incubator.

[0052] 5. On day 3, add 10 mL of fresh NK cell expansion medium (containing 10% heat-inactivated autologous plasma).

[0053] 6. Starting from the 5th day, samples were taken and counted every other day, and fresh NK cell expansion medium (containing 5% heat-inactivated autologous plasma) was added to adjust the NK cell density to 1.0-1.5×10 6 / mL. Choose to expand the flask or transfer to a cell culture bag according to the volume of the cell culture suspension. The maximum culture volume of a T75 culture flask is 40mL, and the maximum culture volume of a T175 culture flask is 200mL. When the culture medium volume exceeds 200mL, it can be transferred to a cell culture bag for culture.

[0054] 7. After the 7th day, the content of heat-inactivated autologous plasma in the fresh NK cell expansion medium can be reduced to 1%. Cell proliferation (rapid rehydration program is shown in the table below) and cell surface marker detection.

[0055] Days / d Original culture medium / mL Supplement medium / mL Added plasma / mL Total volume of culture medium / mL Culture flasks / culture bags 0 0 9 1 10 T25 culture flask 3 10 9 1 20 T25 culture flask 5 20 38 2 60 T175 culture flask 7 60 118.8 1.2 180 T175 culture flask 9 180 178.2 1.8 360 Transferable culture bag 11 360 356.4 3.6 720 13 720 277.2 2.8 1000

[0056] 8. After 13-14 days of culture, transfer the cell suspension from the culture bag into a large centrifuge bottle and centrifuge at 680g for 10 minutes to separate the cells and culture medium. The cells can be used for subsequent transfection experiments or directly applied. If used directly, they need to be processed as follows:

[0057] (1) Carefully remove the supernatant and resuspend all cell pellets in saline containing 0.1% human serum albumin and collect them in a centrifuge bottle. Repeat the centrifugation to wash the cells three times.

[0058] (2) Collect the cells, resuspend the cells with an appropriate amount of physiological saline containing 1% human serum albumin, filter through a disposable cell sieve, collect and inject back into the infusion solution of physiological saline containing 1% human serum albumin, with a total amount of 100 to 200 ml.

[0059] 2. Construction and transfection of vector expressing B7-H3 single domain antibody

[0060] The codon-optimized nucleotide sequence of B7-H3-1B (specific sequence as shown in SEQ ID NO.3) was cloned into a eukaryotic expression vector and connected with EcoRI and HindIII double restriction sites to construct pEE12.4-B7-H3-1B expression plasmid (containing 6His tag to facilitate antibody purification). The positive recombinant plasmid was sent to Sangon Biotech for sequence determination, and the determined nucleotide sequence and encoded amino acid sequence were analyzed and compared with software to check the correctness of the reading frame. After the plasmid was extracted, the plasmid was stored at -80°C for standby use.

[0061] The correctly identified pEE12.4-B7-H3-1B expression plasmid was transfected into the NK cells prepared above (the transfection quantity was adjusted according to the cell number). The brief description is as follows:

[0062] (1) Prepare two EP tubes, add 240 μL HBS and the plasmid to be transfected to one tube, and add 155 μL HBS and 25 μL PEI transfection reagent to the other tube. Mix the two tubes on a vortex mixer and let stand for 5 minutes.

[0063] (2) Mix the liquids in the two EP tubes into one tube, shake again, and let stand at room temperature for 15 minutes;

[0064] (3) In a clean bench, add the transfection system to the NK cells, mix the culture medium and transfection system, place in an incubator, and continue culturing.

[0065] (4) After 6 to 8 hours, fresh culture medium was replaced and cultured for 5 generations to detect whether B7-H3-1B single domain antibody was expressed. Figure 2 , Western blot detection was performed on the cell culture using an anti-His tag), and the NK cells (denoted as NK-B7-H3-1B cells) were able to express B7-H3-1B single domain antibody within 5 passages, with the best effect in the 2nd to 4th passages.

[0066] Example 4: Application of NK cells

[0067] 1. In vitro anti-tumor test

[0068] In order to verify the killing effect of B7-H3-1B, NK cells prepared in this study, and NK-B7-H3-1B cells on target tumors, small cell lung cancer NCI-H446 cell line, non-small cell lung cancer (NSCLC) line A549 cell line, prostate cancer PC-3 cell line, esophageal squamous cell carcinoma cell line EC9706, colorectal cancer HCT-8 cell line, and breast cancer cell line MCF-7 were selected as experimental subjects. The above cells were revived one by one, inoculated in RPMI 1640 medium containing 10% FBS, and incubated at 37°C and 5.0% CO2 The cells were cultured at a constant temperature until the logarithmic growth phase and used in experiments.

[0069] Various tumor cells were collected at 5×10 4 The 96-well culture plates were placed in a 37°C, 5% CO 2 Continue to incubate overnight in a cell culture incubator until the cells adhere to the wall and grow well. Each cell-positive well is divided into 3 groups (Group 1 is 5×10 5 NK cells / well, group 2: 10 μg / mL B7-H3-1B + 5×10 5 NK cells / well, group 3: 5×10 5 NK-B7-H3-1B cells / well), and the cell culture medium was used as a negative control. The cells were kept at 37°C with 5% CO 2 After incubation in a cell culture incubator for 48 hours, the 96-well plate was removed, the culture medium in the plate was discarded, and the culture medium containing 10% CCK8 reagent was added at a volume of 100 μL per well, and the plate was returned to the 37°C cell culture incubator to wait for color development. After the color development lasted for the required time, the OD450nm value of each well was measured with an ELISA reader. The cell proliferation rate was used to describe the effect of each group on the viability of different cells.

[0070] The results are as follows Figure 2 As shown, the three groups all had a certain inhibitory effect on the six cell lines, and group 3 was better than the other two groups. This shows that the B7-H3-1B, NK cells, and NK-B7-H3-1B cells provided by the present invention can effectively inhibit the proliferation of various tumor cells including small cell lung cancer, prostate cancer, esophageal squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, and breast cancer, thereby exerting an anti-tumor effect, and the inhibitory effects are NK-B7-H3-1B cells, B7-H3-1B+NK cells, and NK cells from strong to weak.

[0071] 2. In vivo anti-tumor assay

[0072] NCI-H446 small cell lung cancer cells in the logarithmic growth phase were selected and inoculated subcutaneously in the right axilla of 40 nude mice under sterile conditions. The cell inoculation volume was 5×10 6 Use a vernier caliper to measure the diameter of the transplanted tumor. When the tumor grows to 80 mm 3 40 nude mice with good growth and uniform tumor size were randomly divided into 4 groups, 10 mice in each group. Three groups were treated (group 1 was NK cells, group 2 was B7-H3-1B+NK cells, group 3 was NK-B7-H3-1B cells), and one group was the control group. 6 NK cells / mouse, group 2 was injected with 100 mg / kg B7-H3-1B antibody + 10 6NK cells / rat; Group 3 was injected with 10 NK cells per week through the tail vein 6 NK-B7-H3-1B cells / mouse; control group, equal volume of normal saline injected into tail vein every week. All nude mice were killed after 4 weeks of treatment. Subcutaneous tumors of killed nude mice were removed, weighed and recorded.

[0073] The results are as follows Figure 3 As shown, the tumors in the three groups were inhibited to a certain extent, and group 3 was better than the other two groups. This result is consistent with the results of cytological research.

[0074] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A B7-H3 single domain antibody, characterized in that: The B7-H3 single domain antibody is B7-H3-1B, and the amino acid sequence of VNAR of B7-H3-1B is shown in SEQ ID NO.

1.

2. A prokaryotic expression gene encoding the B7-H3-1B according to claim 1, characterized in that: The nucleotide sequence of the prokaryotic expression gene is shown in SEQ ID NO.

2.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the B7-H3-1B and NK cells according to claim 1.

4. A NK cell, characterized in that: The NK cells are NK-B7-H3-1B cells, and the NK-B7-H3-1B cells contain a eukaryotic expression vector expressing the B7-H3-1B according to claim 1.

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