Preparation method of separated immune cells and application of separated immune cells in cancer treatment

Abstract

The invention relates to the field of biology, in particular to a preparation method of separated immune cells and application of the separated immune cells in cancer treatment. The immune cell prepared by the invention can effectively kill ovarian cancer cells through combined application of the immune cell and an antibody specifically bound with HER2, so that a composition prepared from the immune cell and the antibody can be used for treating cancers, such as ovarian cancer.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a method for preparing isolated immune cells and an application thereof in treating cancer. Background Art

[0002] The mortality rate of ovarian cancer ranks first among gynecological malignancies, and it poses a serious threat to women's lives and health. 70% of ovarian cancer patients are already in the advanced stage when they seek medical treatment, and most of them seek medical treatment due to abdominal pain and gradually increasing ascites. The conventional treatment plan is adjuvant chemotherapy after tumor cell reduction surgery. Tumor cell reduction surgery can only remove visible tumor tissue to the maximum extent, and it is inevitable to retain residual lesions after surgery, which also causes the characteristic of ovarian cancer that it is easy to recur. The effect of adjuvant chemotherapy for patients with advanced ovarian cancer is not ideal. Due to drug resistance, toxic and side effects, low immunity of patients, etc., the effect is poor, resulting in a poor prognosis for patients with advanced ovarian cancer.

[0003] In recent years, tumor biotherapy has attracted much attention. Among them, adoptive cell immunotherapy, with cytokine-induced killer cells (CIK) and dendritic cells (DC) as the main research hotspots, is a relatively effective treatment method in tumor biotherapy and a new anti-tumor program that is gradually receiving attention from medical institutions. CIK cells have strong fecundity, histocompatibility complex restriction and cytotoxicity; DC cells are antigen-presenting cells that can stimulate immune responses mediated by B lymphocytes and T lymphocytes. DC cells recognize foreign antigens and thus activate the immune system, while CIK cells can kill tumor cells by exerting their own cytotoxicity and timely secreting related secretory factors. Combining DC cells and CIK cells can play a synergistic role in treating tumors. Therefore, the co-culture of DC-CIK cells gradually shows the common advantages of CIK cells and DC cells, with advantages such as high anti-tumor activity, increased cell surface antigens, and increased number of immune cells. Therefore, it has gradually become an important means of adjuvant treatment of tumors. DC-CIK combined therapy for tumors has been applied to many aspects including lymphoma, urinary system tumors, digestive system tumors, respiratory system tumors, etc., and has achieved good results.

[0004] HER2 is one of the four major members of the EGFR family and is involved in the regulation of cell survival, growth, and differentiation. HER2 encodes a transmembrane receptor-like protein with tyrosine kinase activity. Studies have found that HER2 gene amplification and / or HER2 protein overexpression are associated with a variety of malignant tumors, including 30%-80% esophageal adenocarcinoma and squamous cell tumors, 35%-45% pancreatic, 30% breast cancer, and 25%-30% ovarian cancer, and HER2 overexpression is associated with a poor prognosis of ovarian cancer. Therefore, HER2 is an important tumor-associated antigen for immunotherapy of ovarian malignancies.

[0005] Single cell immunotherapy has achieved good results in the treatment of tumors, and its synergy with antibody targeted therapy has also attracted more and more attention. In the exploration of treatment methods for ovarian cancer, there are few studies on the combination of cell immunotherapy and antibody targeted therapy, and more research is needed. Summary of the invention

[0006] In view of the prior art, the object of the present invention is to provide an antibody-cell therapy for the treatment of ovarian cancer to achieve efficient, safe and lasting therapeutic effects.

[0007] The first aspect of the present invention provides an antibody specific for HER2, comprising a heavy chain variable region (V H ) and light chain variable region (V L ), wherein the heavy chain\light chain variable region respectively includes three complementarity determining regions (CDRs), namely HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

[0008] In some embodiments, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0009] In some embodiments, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0010] In some embodiments, the V H The amino acid sequence of is shown in SEQ ID NO: 7, wherein V L The amino acid sequence is shown in SEQ ID NO:8.

[0011] The second aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned antibody specific for HER2, and a second agent or therapy. In some embodiments, the second agent or therapy is a standard or commonly used agent or therapy for treating a disease or condition. In some embodiments, the second agent or therapy includes immune cells. In some embodiments, the immune cells are DC-CIK cells.

[0012] The third aspect of the present invention provides the use of antibodies specific to HER2 and DC-CIK cells in the preparation of a pharmaceutical composition for killing ovarian cancer cells.

[0013] In some embodiments, the dosage of the antibody specific for HER2 is 200-300 μg / mL, and the effector-target ratio of the ovarian tumor cells to DC-CIK cells is 1:10-1:20.

[0014] In some embodiments, the above pharmaceutical composition can also be used to treat other tumors. Preferably, the tumor is selected from melanoma, pancreatic cancer, colorectal cancer, breast cancer, lung cancer, and breast cancer.

[0015] In some embodiments, the above pharmaceutical composition further contains other therapeutic agents.

[0016] In some embodiments, the therapeutic agent is a chemotherapeutic agent including, for example, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, doxycycline, dihydroxyanthraquinone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.

[0017] Beneficial Effects

[0018] The present invention extracts and prepares DC-CIK immune cells from blood, and the immune cells can effectively kill ovarian cancer cells when used in combination with antibodies that specifically bind to HER2, and the immune cells and antibodies are prepared into a composition that can be effectively used for the treatment of ovarian cancer. In addition, the antibodies that specifically bind to HER2 provided by the present invention can bind to the target antigen with high affinity and can effectively kill ovarian cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown are the CDR sequences of H2D8.

[0020] Figure 2 Shown are the heavy chain / light chain variable region sequences of anti-HER2 antibodies.

[0021] Figure 3 Shown is the killing effect of DC-CIK cells combined with anti-HER2 monoclonal antibody on ovarian cancer cells. DETAILED DESCRIPTION

[0022] In order to promote understanding of the present invention, some embodiments will be referred to below, and specific language will be used to describe the present invention. However, it should be understood that these specific embodiments are not intended to limit the scope of the present invention. Any changes and further modifications in the described embodiments, as well as any further applications of the present invention, are generally expected by those skilled in the art.

[0023] Example 1. Preparation and screening of anti-HER2 single-chain antibodies

[0024] RNA was extracted from 2000 human blood samples, and cDNA was obtained by reverse transcription. VH and VK specific primers were used to amplify VH and VK fragments. After gel recovery and purification, VH and VK were connected to construct scFv and cloned into the yeast display plasmid PYD1. Subsequently, the plasmid was electroporated into yeast to obtain the scFv antibody yeast display library. After several rounds of panning, scFvs that recognize the Her2 extracellular region were isolated from the yeast display library. The scFv antibody fragments obtained from the yeast library were PCR amplified using scFv-F and scFv-R primers, and the obtained scFvs antibody fragments were cloned into the phage display vector pDAN5 via SfiI. After connection, the plasmid was transformed into TG1 phage competent cells by electroporation to obtain the scFv antibody phage display library.

[0025] Isolate scFv antibodies that can specifically bind to Her2 from the phage library. Select monoclones for ELISA binding experiments and in vitro biological activity experiments. Obtain the most promising scFv antibodies. Construct them into IgG1 full-length antibodies with human IgG1 heavy chain constant regions and human kappa light chain constant regions. Through a series of experiments, obtain the anti-HER2 single-chain antibody H2D8 with strong binding activity, its amino acid sequence is as follows Figure 1 and Figure 2 shown.

[0026] Trastuzumab, the world's first Her2 antibody, has a heavy chain variable region sequence as shown in SEQ ID NO: 9 and a light chain variable region sequence as shown in SEQ ID NO: 10, and is used as a positive reference antibody in the following text.

[0027] Example 2. ELISA binding activity assay

[0028] Recombinant human Her2 extracellular domain antigen (ACRO Biosystems, Cat#: HE2-H5253) was dissolved in PBS solution and coated on a 96-well plate at 0.1 μg / well. After overnight incubation at 4°C, the plate was blocked with 5% milk at 37°C for 1-2 hours. The Her2 antibody was diluted in gradients and added to the corresponding wells and incubated at 37°C for 1 hour. 100 μL of secondary antibody (goat anti-human IgG-HRP, 1:1000) was added to each well and incubated at 37°C for 1 hour. 100 μL of TMB was added to each well and incubated at 37°C for 10-20 minutes. The reaction was terminated with a stop solution. The OD450nm value was read using an enzyme reader. Binding curves were generated using PRISM and the EC50 value was calculated.

[0029] The results are shown in Table 1 . The binding ability of H2D8 to Her2 antigen is better than that of the reference antibody trastuzumab.

[0030] Table 1

[0031] Antibody EC50(μg / mL) Antibody EC50(μg / mL) H2D8 0.259 Trastuzumab 0.596

[0032] Example 3. Antibody affinity determination

[0033] The binding affinity of anti-HER2 antibodies to human Her2 was measured by surface plasmon resonance (SPR) using a BiacoreTM 8K biosensor.

[0034] The Her2 antibody was diluted to 1 μg / mL and then injected into the BiacoreTM8K biosensor (10 μL / min, 50 seconds) to detect the binding affinity of the Her2 antibody to different concentrations of His-tagged human Her2 protein (hHER2, ACRO Biosystems, Cat#: HE2-H5225) (binding 180 seconds, dissociation 600 seconds).

[0035] The binding and dissociation rates of the antibodies were obtained and the binding affinity was determined. Table 5 lists the Kon, Koff and Kd values ​​of the anti-Her2 antibody H2D8 and the human Her2 antigen.

[0036] The results in Table 2 show that the anti-Her2 antibody H2D8 binds to the human Her2 antigen with superior affinity compared to the reference antibody trastuzumab.

[0037] Table 2

[0038]

[0039]

[0040] Example 4. Preparation of DC-CIK cells

[0041] Take 30mL of peripheral blood from a healthy person (voluntary donation), dilute it with 30mL PBS and put it into a centrifuge tube. Gently add Ficoll reagent from the bottom of the centrifuge tube, centrifuge at 2000rpm for 20min, and divide it into 4 layers from the liquid surface to the bottom of the tube. The second layer is peripheral blood mononuclear cells (PBMC). Carefully draw out the mononuclear cells in the second layer, add PBS to wash, centrifuge at 1000rpm for 5min, and repeat 3 times. Use AIM-V culture medium (Gibco Invitrogen) to adjust the cell concentration to 1×10 6 / mL, 37℃, 5% CO2, incubate for 2 hours.

[0042] Add cultured non-adherent cells (mainly T and B cells) to a 100 mL culture flask and adjust the cell concentration to 1×10 6 / mL, add 1000U / mL of rhIFN-γ (PeproTech) and culture for 24 hours. Coat 20ng / mL LCD3McAB (Beckman) in a 100mL culture flask, add 300U / mL rhIL-2 (Amoytop Biotech), and culture for 48 hours. Change the medium, count, observe the cell morphology, and adjust the cell number to 5×10 5 / mL, supplemented with rhIL-2 at 300U / mL, continued to culture, and harvested CIK cells after 7 days.

[0043] DCs were cultured with adherent cells in a culture flask, 10% FBS, 1000U / mL rhGM-CSF, 500U / mL rhlL-4 (Amoytop Biotech) were added, and cultured in a 37°C, 5% CO2 incubator. After 48 hours of culture, the medium was changed, the cells were counted, the cell morphology was observed, and cytokines were added. After 6 days, 200U / mL TNF-α (PeproTech) was added to induce DC cell maturation. The culture was continued, and DC cells were harvested after 7 days.

[0044] The mature DC cells and CIK cells after 7 days of culture were transferred into multiple culture flasks and serum-free culture medium was selected. The ratio of the two cells was 1:5 and co-cultured for 3 days. The cell concentration was adjusted to 3×10 5 / ml was used as the concentration for repeated culture.

[0045] Example 5. Detection of the killing effect of DC-CIK cells combined with monoclonal antibodies on ovarian cancer cells

[0046] CCK method was used to detect the killing effect of DC-CIK cells combined with monoclonal antibodies on SKOV3 cells. The experiment was divided into 3 groups: SKOV3 cells and DC-CIK cells were added to the DC-CIK cell group; SKOV3 cells and anti-Her2 antibody H2D8 were added to the monoclonal antibody group; SKOV3 cells, DC-CIK cells and anti-Her2 antibody H2D8 were added to the DC-CIK cell combined with monoclonal antibody group.

[0047] Dilute SKOV3 cells to 5 × 10 4 cells / mL, 100uL per well, as target cells. The DC-CIK cell group was added with a cell concentration of 5×10 5 100 μL of DC-CIK effector cells were added to each well, and the effector-target ratio was 10:1. The final concentration of the anti-Her2 antibody H2D was 100 μg / mL in the monoclonal antibody group. The cell concentration of the combined group was 5×10 5 cells / mL DC-CIK effector cells and the final concentration of 100 μg / mL anti-Her2 antibody H2D. At the same time, set the zeroing well (containing only the same amount of culture medium) and the control group (adding only SKOV3 cells and the same amount of culture medium). The 96-well plate was placed in an incubator for 24 hours, and 20 μL CCK reagent (Perfemiker, PM300075) was added to each well. The culture was continued for 5 hours, and the culture was terminated. The OD value at 450nm was detected by an ELISA instrument. The inhibition rate and killing rate (%) were calculated according to the formula = [1-(OD experiment-OD zeroing) / (OD control-OD zeroing)] × 100%, and the IC50 was calculated by linear regression.

[0048] The results are as follows Figure 3 As shown, the therapeutic effect of DC-CIK cells combined with monoclonal antibodies is better than that of monoclonal antibodies or DC-CIK cells alone, and there is a good synergistic effect.

Claims

1. An antibody specific for HER2, comprising a heavy chain variable region (V H ) and light chain variable region (V L ), wherein the V H comprising HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and the V L It comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively.

2. The antibody specific for HER2 according to claim 1, comprising: V H , the V H The amino acid sequence of V is shown in SEQ ID NO: 7; L , the V L The amino acid sequence is shown in SEQ ID NO:

8.

3. An antibody specific for HER2 according to any one of claims 1-2, wherein the antibody specific for HER2 comprises an Fc fragment.

4. The antibody specific for HER2 according to claim 3, wherein the antibody specific for HER2 is a full-length IgG1 antibody.

5. A pharmaceutical composition, characterized in that The invention comprises an antibody specific to HER2 according to any one of claims 1 to 4.

6. Use of the drug combination according to claim 5 in the preparation of a drug for treating ovarian cancer.

7. Use of a combination of an antibody specific for HER2 and DC-CIK cells in the preparation of a pharmaceutical composition for ovarian cancer cells, wherein the antibody specific for HER2 comprises the antibody specific for HER2 described in any one of claims 1 to 4.

8. The use according to claim 7, characterized in that The dosage of the antibody was 200 μg / mL, and the effector-target ratio of the ovarian cancer cells to the DC-CIK cells was 1:20.

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