Bispecific antibody, T cell modified by bispecific antibody, and preparation method and application of bispecific antibody

Abstract

The invention discloses a bispecific antibody, a T cell modified by the bispecific antibody as well as a preparation method and application of the bispecific antibody. The preparation method of the bispecific antibody comprises the following steps: dissolving an anti-human CD155 antibody in a first buffer solution; adding 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic acid group succinimide ester sodium salt into the first buffer solution, uniformly mixing, and incubating at room temperature; free 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic acid group succinimide ester sodium salt is removed, and a first solution is obtained; replacing the buffer solution of the anti-CD3 antibody with a second buffer solution; adding 2-imino sulfane hydrochloride into the second buffer solution, uniformly mixing, and incubating at room temperature; free 2-imino sulfane hydrochloride is removed, and a second solution is obtained; and uniformly mixing the first solution and the second solution, concentrating, reacting and purifying to obtain the bispecific antibody. The bispecific antibody modified T cell provided by the invention can be used for preparing medicines for resisting malignant tumors such as lung cancer.

Description

Technical Field

[0001] The present invention relates to the field of drugs, and more particularly to bispecific antibodies and modified T cells thereof, and methods for preparing and using the same. Background Art

[0002] Surgery, radiotherapy, and chemotherapy are the three conventional methods for treating tumors. In recent years, biocellular immunotherapy has provided new options for cancer patients. The cellular immune response relied on by biocellular immunotherapy is mainly the specific anti-tumor immune response mediated by T lymphocytes. Obtaining tumor-specific T cells with anti-tumor activity is the key to achieving anti-tumor effects. As an emerging anti-tumor treatment method with remarkable curative effects, biocellular immunotherapy avoids the side effects of traditional surgery and radiotherapy and the drawbacks of chemotherapy, and it has been recognized as a promising treatment means in the comprehensive cancer treatment model in the 21st century. The combination of surgery, radiotherapy, chemotherapy, and tumor biocellular immunotherapy can accurately eliminate residual tumor cells, prevent recurrence and metastasis, thereby improving the quality of life and survival time of patients.

[0003] Chimeric Antigen Receptor T-cell (CAR-T) therapy has entered the clinical trial stage. In 2010, the US FDA approved the prostate cancer immunotherapy drug Sipuleucel-T for clinical use. In just a few years, immunotherapy has made a series of exciting progress and become the focus of attention. After decades of preclinical and clinical research, immunotherapy is rapidly moving towards maturity. The future development of immunotherapy is worthy of attention.

[0004] CAR-T technology and T cell receptor (TCR) chimeric T cell (TCR-T) technology, as the two latest immunocyte technologies in current adoptive cell therapy (ACT) technology, have received extensive attention and research because they can express specific receptors to target and recognize specific cells such as tumor cells, and have transformed from initial basic immunological research to clinical application. Based on synthetic biology, immunology, and genetic modification technologies, it has become possible to synthesize and transform T cells with enhanced specific functions. Existing studies have shown that CD19 antigen-specific CAR-T cells have shown continuous disease remission effects in clinical trials for treating B-cell leukemia and lymphoma. Due to the excellent performance and broad application prospects of CAR-T / TCR-T technology, it has entered the current highly competitive pharmaceutical industry and competed with traditional pharmaceuticals. However, CAR-T technology involves viral vectors, so corresponding safety control measures need to be configured.

[0005] Bispecific antibody (Bi-Ab)-mediated adoptive T cell therapy is a tumor treatment strategy mediated by T cells redirected by bispecific antibodies. Because of its lack of MHC restriction, target specificity and broad spectrum of antigen targets, and maximization of T cell killing, bispecific antibody therapy has great anti-tumor potential and has been applied to the anti-tumor treatment of various tumors. In recent years, Bi-Ab-mediated immunotherapy has achieved remarkable clinical results in tumor treatment experiments. So far, two bispecific antibodies have been approved for clinical use. One is Catumaxomab from Trion Pharma in Europe, which is used to treat malignant ascites and is the world's first bispecific antibody to be marketed. Catumaxomab recognizes the epithelial cell adhesion molecule (EpCAM) antigen on the surface of tumors, recruits T cells and can also bind to the receptor on the surface of killer cells through the Fc segment, mediating the direct killing of target cells by killer cells, and can achieve "trifunctional" antibody activity, improving the patient's immune activity against tumor cells. The other is Blinatumomab from Amgen in the United States, which is approved mainly for the treatment of hematological malignancies. One end targets the CD19 antigen on the surface of tumor cells, and the other end targets the CD3 receptor on the surface of cytotoxic T lymphocytes (CTL). Clinical trials have shown that Blinatumomab has durable anti-tumor treatment potential in specific subsets of patients; it has now been identified as an effective treatment for patients with relapsed or refractory ALL and is the first anti-leukemia drug approved for the treatment of minimal residual disease (MRD); however, Blinatumomab is ineffective when there is a lack of CD19 on the surface of tumor cells and recurrent acute lymphoblastic leukemia with extramedullary hematopoiesis occurs.

[0006] CD155, also known as poliovirus receptor (PVR), is a member of the nectin-like (Necl) protein family and is also called Necl-5. Functional analysis of CD155-related genes has shown that it is related to humoral immune responses, immune receptor activity, natural killer cell-mediated cytotoxicity, tumor malignant transformation pathways, autophagy regulation processes, and cytokine receptor interactions. CD155 is expressed at very low levels in normal human cells but is often overexpressed in malignant tumor cells. CD155 serves as a ligand for the co-stimulatory receptor CD226 (DNAX-associated molecule-1, DNAM-1) and the co-inhibitory receptors T cell immunoglobulin and ITIM domain (TIGIT) and CD96 on NK cells and T cells; therefore, CD155 may play a dual role in the progression of malignant tumors. The expression of CD155 on the surface of tumor cells mainly promotes the proliferation and migration of tumor cells in the early stage of cell malignant transformation; in the late stage, CD155 supports tumor progression and immune escape. Summary of the Invention

[0007] The present invention provides a method for preparing a bispecific antibody, comprising: dissolving an anti-human CD155 antibody in a first buffer; adding sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester to the first buffer, mixing well, and incubating at room temperature; removing free sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester to obtain a first solution; replacing the buffer of the anti-CD3 antibody with a second buffer; adding 2-iminothiolane hydrochloride to the second buffer, mixing well, and incubating at room temperature; removing free 2-iminothiolane hydrochloride to obtain a second solution; mixing the first solution and the second solution, concentrating, reacting, and purifying to obtain the bispecific antibody.

[0008] In some embodiments, removing free sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester to obtain a first solution comprises: removing free sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester using a PD-10 desalting column: loading the sample and discarding the effluent; then eluting with the first buffer and collecting the eluate to obtain the first solution.

[0009] In some embodiments, free 2-iminothiolane hydrochloride is removed to obtain a second solution, which includes removing free 2-iminothiolane hydrochloride using a PD-10 desalting column: loading the sample and discarding the effluent; then eluting with the first buffer and collecting the eluate to obtain the second solution.

[0010] In some embodiments, the concentration includes concentrating the mixed solution of the first solution and the second solution by 30 - 40 times, and the reaction includes reacting overnight at 4°C.

[0011] The present invention also provides a bispecific antibody, which is prepared according to the preparation method of the above-mentioned bispecific antibody.

[0012] The present invention also provides a preparation method of a bispecific antibody-modified T cell, including: obtaining activated T lymphocytes by amplifying peripheral blood mononuclear cells; adding the above-mentioned bispecific antibody to the activated T lymphocytes and incubating at room temperature; removing the bispecific antibody that has not bound to the activated T lymphocytes to obtain a bispecific antibody-modified T cell.

[0013] In some embodiments, adding the above-mentioned bispecific antibody to the activated T lymphocytes and incubating at room temperature includes: adding a 200 ng dose of bispecific antibody to 1×10 6 activated T lymphocytes and incubating at room temperature for 30 minutes.

[0014] The present invention also provides a bispecific antibody-modified T cell, which is prepared according to the preparation method of the above-mentioned bispecific antibody-modified T cell.

[0015] The present invention also provides the application of the bispecific antibody-modified T cell in the preparation of a drug for treating tumors.

[0016] In some embodiments, the tumor is lung cancer.

[0017] Verification through in vitro cell killing efficacy, RNA sequencing, and animal experiments shows that the bispecific antibody-modified T cells provided by the present disclosure can be used to prepare drugs for treating malignant tumors such as lung cancer. Description of the Drawings

[0018] Figure 1The expression of CD155 on various malignant tumor cells is shown. The results of evaluating the expression of CD155 on the cell surface of each tumor cell line by flow cytometry are presented. This includes lung cancer A549 cells, colon cancer HT29 cells, Colo 205 cells, breast cancer MDA-MB231 cells, SKBR3 cells, MCF-7 cells, pancreatic cancer BXPC3 cells, and brain astrocytic glioblastoma U87MG cells. The dark gray histograms represent the cells stained with anti-CD155 antibody, and the light gray histograms represent the cells stained with its isotype control antibody mouse IgG1. The values shown in the upper right quadrant of the histograms respectively indicate the mean fluorescence intensity (MFI) values of each tumor cell line obtained by dividing the staining with anti-CD155 antibody (dark gray part) by the staining with the control antibody (light gray part). The tumor cell lines are arranged in descending order according to the CD155 MFI values.

[0019] Figure 2a and Figure 2b The detection of the prepared bispecific antibody (CD155Bi-Ab) of anti-CD3 antibody x anti-CD155 antibody is shown. ( Figure 2a ) Flow cytometry-based binding assay of CD155Bi-Ab. T cells were incubated with CD155Bi-Ab (1 μg / mL, dark gray part) or a mixture of anti-CD3 antibody and anti-CD155 antibody (1 μg / mL, light gray part) respectively, and the binding of Bi-Ab was examined by detecting the anti-CD155 part of Bi-Ab with PE-labeled anti-mouse IgG1. ( Figure 2b ) Protocol for flow cytometry-based determination of the binding of CD155Bi-Ab.

[0020] Figure 3 The determination of the CD155Bi-Ab titer is shown. At an effector-to-target ratio (E:T) = 10:1, CD155Bi-Ab-modified T cells (1x10 5 / well, as effector cells) were incubated with A549-Luc cells stably expressing luciferase (1x10 4 / well, as target cells) in a 96-well microplate for 18 hours. The killing rate of T cells against A549-Luc cells at different bispecific antibody concentrations was detected by luciferase quantification analysis. The concentration of CD155Bi-Ab was designed to range from 5 ng to 2000 ng / 1x10 6 T cells.

[0021] Figure 4 The killing effect of CD155Bi-Ab-modified T cells on tumors under different effector-to-target ratio conditions is shown. The target cells (A549-luc: 1x10 4 / well) were co-incubated with CD155 Bi-Ab modified T cells (200 ng / Bi-Ab / 10 6 T cells) in a 96-well microplate for 18 hours. The killing rate of T cells against tumor cells was detected by luciferase quantification analysis at E / T ratios of 5:1, 10:1, and 20:1. T cells incubated with a mixture of anti-CD3 antibody and anti-CD155 antibody were used as control T cells. Data are presented as the mean ± SD of three determinations. **, P < 0.01.

[0022] Figure 5 The killing effect of CD155 Bi-Ab modified T cells on tumors under different action time conditions is shown. At E:T = 10:1, target cells A549-luc (1x10 4 / well were co-incubated with CD155 Bi-Ab-modified T cells (200 ng / Bi-Ab / 10 6 T cells) in a 96-well microplate. After incubation for 24 hours, 48 hours, and 72 hours, the killing effect of T cells in each group on A549-luc was detected by luciferase quantification analysis. T cells incubated with a mixture of anti-CD3 antibody and anti-CD155 antibody were used as control T cells. Data are presented as the mean ± SD of three determinations. **, P < 0.01.

[0023] Figure 6 The cytotoxic cytokines secreted by tumor cells by CD155 Bi-Ab modified T cells are shown. At E:T = 10:1, target cells (A549-luc, 2x10 5 / well) were co-incubated with CD155 Bi-Ab modified T cells (200 ng / Bi-Ab / 10 6 T cells) in a 24-well microplate for 48 hours. The supernatants of co-cultures with an E / T ratio of 10:1 were collected, and the production of cytokines by T cells in each group after incubation with tumor cells was detected by immunofluorescence using a cytokine multiplex detection kit. T cells incubated with a mixture of anti-CD3 antibody and anti-CD155 antibody were used as control T cells. Data are presented as the mean ± SD of three determinations. ***, P < 0.001.

[0024] Figure 7 The upregulation of signal pathway transcriptome sequencing by CD155 Bi-Ab modified T cells is shown. At E:T = 10:1, target cells (A549-luc, 2x10 5 / well) were co-incubated with CD155 Bi-Ab modified T cells (200 ng / Bi-Ab / 10 6T cells) were incubated together in 24-well microplates. T cells were collected at 48 hours, and transcriptome sequencing and GO functional enrichment analysis showed significantly enhanced biological processes related to T cell function and upregulated genes. T cells incubated with a mixture of anti-CD3 antibody and anti-CD155 antibody served as control T cells. P < 0.05.

[0025] Figure 8a and Figure 8b showed the antitumor effect of CD155 Bi-Ab-modified T cells in vivo. SCID mice were subcutaneously inoculated with tumor cells (A549-luc, 2 x 10 6 / mouse), and on the next day and the third day after inoculation, CD155 Bi-Ab-modified T cells (200 ng / Bi-Ab / 10 6 T cells) were injected at the tumor inoculation site at an E:T ratio of 5:1. On the day of tumor cell inoculation and on day 42, in vivo imaging of small animals was performed using IVIS ( Figure 8a ) and the fluorescence intensity was statistically analyzed ( Figure 8b ). T cells incubated with a mixture of anti-CD3 antibody and anti-CD155 antibody served as control T cells. n = 3 mice in each group, **, P < 0.01. Detailed implementation manners

[0026] The following implementation examples can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way. Unless otherwise specified, the materials, reagents, etc. used in the present disclosure can be obtained from commercial channels.

[0027] In the present disclosure, at two levels of cell experiments and animal experiments, T cells were modified with CD155 Bi-Ab, and its cytotoxic effect on tumor cells was detected by luciferase quantification experiments; the release of various T cell-derived cytokines was detected by multiplex immunofluorescence to evaluate its killing effect; through RNA sequencing (RNA-seq), its mechanism of action was explored. The present disclosure provides new strategies and experimental bases for the clinical treatment of CD155 + tumors.

[0028] 1. Determination of tumor cell lines and CD155 expression

[0029] Human lung cancer cell line A549, colon cancer cell lines HT29, Colo205, pancreatic cancer cell line BXPC3, breast cancer cell lines MDA-MB231, SKBR3, MCF-7 were purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences, and the brain astrocytic glioblastoma cell line U87MG was purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Lung cancer A549-Luc was purchased from Cliper life science company. Flow cytometry was used to detect the CD155 expression on the surface of tumor cells. SeeFigure 1 , the results of evaluating the expression of CD155 on the cell surface of each tumor cell line by flow cytometry are shown. The dark gray histogram represents the cells stained with anti-CD155 antibody, and the light gray histogram represents the cells stained with its isotype control antibody mouse IgG1. The values shown in the upper right quadrant of the histogram respectively show the mean fluorescence intensity (MFI) values of each tumor cell line obtained by dividing the staining with anti-CD155 antibody (dark gray part) by the staining with the control antibody (light gray part). The tumor cell lines are arranged in descending order of CD155 MFI value. From Figure 1 , it can be seen that CD155 is highly expressed on the human lung cancer cell line A549.

[0030] 2 Construction of CD155 Bi-Ab by chemical coupling method

[0031] (1) Dissolve 100 μg of anti-human CD155 mAb powder (R&D SYSTEMS) thoroughly in 50 μl of buffer A. Among them, buffer A (pH 7.2): Prepare 200 mL, Na 2 HPO 4 .12H 2 O 7.1628 g (0.1 mol / L), NaCl 1.754 g (0.15 mol / L), ethylenediaminetetraacetic acid (EDTA) 0.58 g (0.01 mol / L).

[0032] (2) Add 1 μl of sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester (Sulfo-SMCC powder 1 mg dissolved in 500 μl of dimethyl sulfoxide (DMSO)), gently mix evenly, and incubate at room temperature for 1 hour.

[0033] (3) Remove the free Sulfo-SMCC with a PD-10 desalting column: Load 2.5 ml of the sample and discard the effluent; then elute with 3.5 ml of buffer A and collect 3.5 ml of the eluate.

[0034] (4) Use a 10KD concentrator tube to replace the buffer of 100 μg of anti-CD3 antibody (OKT3) with 50 μl of buffer B. Among them, buffer B (prepare 200 mL, 50 mmol / L NaCl, 1 mmol / L EDTA, pH 8).

[0035] (5) Add 0.4 μl of 2-iminothiolane hydrochloride (Traut’s reagent powder 1 mg dissolved in 500 μl of DMSO), gently mix evenly, and incubate at room temperature for 1 hour.

[0036] (6) Remove the free Traut’s reagent using a PD-10 desalting column: Load 2.5 ml of the sample and discard the eluate; then elute with 3.5 ml of buffer A and collect 3.5 ml of the eluate.

[0037] Immediately mix 3.5 ml of the eluate obtained in steps (3) and (6), for a total of 7 ml of solution. Concentrate the solution to a volume of 200 μl using a 10 KD concentrator tube and incubate overnight at 4°C for reaction and purification to obtain CD155 Bi-Ab.

[0038] 3 Isolation of peripheral blood mononuclear cells (PBMC) from healthy donors and expansion of activated T lymphocytes (ATC)

[0039] (1) Routinely isolate the buffy coat PBMC layer and wash.

[0040] (2) Add RPMI1640 medium (Merck) and resuspend. The RPMI1640 medium should contain 5 μg / ml anti-human CD3 mAb, 5 μg / ml anti-human CD28 mAb, 100 IU / ml IL-2, and an appropriate amount of 10% fetal bovine serum. After resuspension, transfer the cell culture medium to a 24-well plate at a cell density of 1 - 2×10 6 / ml / well for expansion.

[0041] (3) Perform half-medium change every 2 - 3 days and supplement IL-2 (100 IU / ml) simultaneously to maintain the expansion of T cells. After culturing T cells for 14 days, collect them for subsequent experiments or freeze them in liquid nitrogen.

[0042] 4 Determination of the binding of Bi-Ab to T cells

[0043] For T cells, add CD155 Bi-Ab or a mixture of CD3 antibody and CD155 antibody (control group) respectively, incubate for 30 minutes at 4°C in the dark, wash, then add PE-anti-mouse IgG1 mAb and incubate for 30 minutes at 4°C in the dark. After washing the cells, add 300 μl of PBS, resuspend the cells, and detect them on the machine. Figure 2a and Figure 2b shows the detection of the bispecific antibody (CD155 Bi-Ab) of anti-CD3 antibody x anti-CD155 antibody prepared, where Figure 2aA flow cytometry-based binding assay of CD155Bi-Ab is shown. T cells were incubated with CD155Bi-Ab (1 μg / mL, dark gray) or a mixture of anti-CD3 antibody and anti-CD155 antibody (1 μg / mL, light gray), respectively, and the binding of Bi-Ab was examined by detecting the anti-CD155 portion of Bi-Ab with PE-labeled anti-mouse IgG1. Figure 2b A protocol for determining the binding of CD155Bi-Ab based on flow cytometry is shown.

[0044] 5. Preparation of effector cells by modifying ATC with CD155Bi-Ab

[0045] (1) The cryopreserved ATC was taken out of liquid nitrogen for cell resuscitation. After culturing with IL-2 (100 IU / ml) for 24 hours, the ATC was collected in a 15-ml centrifuge tube, centrifuged at 1500 rpm / min for 5 minutes, the supernatant was discarded, resuspended, and cell counting was performed.

[0046] (2) Usually, 1 × 10 6 ATC were modified with a 200-ng dose of CD155Bi-Ab and incubated at room temperature for 30 minutes to obtain experimental group CD155-T cells; meanwhile, a mixture of CD3 antibody, CD155 antibody and ATC was used as control group T cells.

[0047] (3) The unbound free antibody in the incubated ATC was eluted with serum-free RPMI 1640 medium, and an appropriate amount of RPMI1640 medium containing 10% fetal bovine serum was added to adjust the cell concentration to prepare effector cells.

[0048] 6. Luciferase quantitative analysis of in vitro cell killing efficacy

[0049] (1) Effector cells: CD155-T cells, control group T cells or T cells.

[0050] (2) Target cell preparation: Based on the results of FACS detection of CD155 expression in various malignant tumor cells in this study, it was shown that A549 cells had the highest expression, so A549-Luc cells were selected as target cells.

[0051] (3) According to the ratio of E:T = 5:1, 10:1, 20:1, 100 μl of the corresponding effector cells was added to the U-bottom 96-well plate, and three parallel control wells were set for each experimental group.

[0052] (4) Background group setting: Three wells containing only target cells were set in the background group.

[0053] (5) The effector cells and target cells were gently mixed evenly, and the total reaction volume reached 200 μl. Under the condition of 37 °C, containing 5% CO2 Co - incubate in an incubator for 24 hours, 48 hours, and 72 hours.

[0054] (6) The next day, add the substrate luciferin to each well at a final concentration of 150 μg / ml, and gently shake. Use an infrared video data imaging system (IVIS) to detect cell bioluminescence and calculate the average fluorescence intensity of the target cells.

[0055] (7) Calculation formula for cell killing rate:

[0056] Specific cell killing rate (%) = (fluorescence intensity of target cells in the background group - fluorescence intensity of target cells in the experimental group) / fluorescence intensity of target cells in the background group

[0057] (8) Use the two - sided unpaired t - test statistical method for data analysis.

[0058] Figure 3 Shows the determination of the CD155 Bi - Ab titer. With an effector - to - target ratio (E:T) = 10:1, CD155 Bi - Ab - modified T cells (1x10 5 / well, as effector cells), and A549 - Luc cells stably expressing luciferase (1x10 4 / well, as target cells) were co - incubated in a 96 - well microplate for 18 hours. By luciferase quantitative analysis, the killing rate of T cells against A549 - Luc cells at different bispecific antibody concentrations was detected. The concentration of CD155 Bi - Ab was designed from 5 ng to 2000 ng / 1x10 6 T cells. Figure 4 Shows the killing effect of CD155 Bi - Ab - modified T cells on tumors under different effector - to - target ratio conditions. The target cells (A549 - luc: 1x10 4 / well) and CD155 Bi - Ab - modified T cells (200 ng / Bi - Ab / 10 6 T cells) were co - incubated in a 96 - well microplate for 18 hours. By luciferase quantitative analysis, the killing rate of T cells against tumor cells was detected when E / T was 5:1, 10:1, and 20:1. T cells incubated with a mixture of anti - CD3 antibody and anti - CD155 antibody were used as control T cells. Data are the mean ± SD of three measurements. **, P < 0.01. It can be Figure 4 seen that the killing rate of CD155 Bi - Ab - modified T cells against tumors is higher. Figure 5 Shows the killing effect of CD155 Bi - Ab - modified T cells on tumors under different action time conditions. With E:T = 10:1, the target cells A549 - luc (1x10 4 / The pores were incubated with CD155Bi-Ab-modified T cells (200 ng / Bi-Ab / 10 6 T cells) in a 96-well microplate. After incubation for 24 hours, 48 hours, and 72 hours, the cytotoxicity of T cells in each group against A549-luc was detected using luciferase quantification analysis. T cells incubated with a mixture of anti-CD3 antibody and anti-CD155 antibody were used as control T cells. The data are the mean ± SD of three determinations. **, P < 0.01. It can be seen from Figure 5 that CD155Bi-Ab-modified T cells have a higher cytotoxicity rate against tumors.

[0059] 7 Cytokine detection

[0060] The cell-free culture supernatants co-cultured with tumor cells for 48 hours at an E:T ratio of 10:1 were collected, and multiplex cytokine concentration detection was performed using the corresponding kits (all purchased from Merck). Figure 6 It shows the cytotoxic cytokines secreted by CD155Bi-Ab-modified T cells against tumor cells. At an E:T ratio of 10:1, the target cells (A549-luc, 2 x 10 5 / well) were incubated with CD155Bi-Ab-modified T cells (200 ng / Bi-Ab / 10 6 T cells) in a 24-well microplate for 48 hours. The supernatants of the co-cultures at an E / T ratio of 10:1 were collected, and the production of cytokines after incubation of T cells in each group with tumor cells was detected using an immunofluorescence method with a cytokine multiplex detection kit. T cells incubated with a mixture of anti-CD3 antibody and anti-CD155 antibody were used as control T cells. The data are the mean ± SD of three determinations. ***, P < 0.001. It can be seen from Figure 6 that under the action of CD155Bi-Ab-modified T cells, the secretion of cytokines such as IFN-γ increased significantly.

[0061] 8. RNA-seq

[0062] Collection E: A reaction system cultured with tumor cells for 48 hours at E:T = 10:1 was collected, T cells were isolated, and transcriptome analysis was performed. Both CD155-T cells and control group T cells were three samples. RNA-seq was carried out with the assistance of Beijing Novogene Bioinformatics Technology Co., Ltd. Based on the MA-plot method, the random sampling model in the DEGseq package was used to determine Differentially Expressed Transcripts (DETs). The threshold for DETs was P < 0.05 and the Fold change ≥ 2 simultaneously. The functional and signaling pathway enrichment analysis of DETs was performed through Gene Ontology (GO) analysis. P < 0.05 and at least two associated genes were included, which was a significantly enriched pathway with significant differences. Figure 7 Shown is the transcriptome sequencing of the signal pathway upregulated by CD155 Bi-Ab modified T cells. At E:T = 10:1, target cells (A549-luc, 2x10 5 / well) were incubated with CD155 Bi-Ab modified T cells (200 ng / Bi-Ab / 10 6 T cells) in a 24-well microplate; T cells were collected at 48 hours, and transcriptome sequencing and GO functional enrichment analysis showed significantly enhanced biological processes related to T cell function and upregulated genes. T cells incubated with a mixture of anti-CD3 antibody and anti-CD155 antibody were used as control group T cells. P < 0.05.

[0063] 9. In vivo tumor suppression test

[0064] (1) Feeding of tumor-bearing mice: SPF-grade SCID 6-week-old male mice purchased from Beijing Huafukang Biotechnology Co., Ltd., with an average weight of about 20 g. Six mice were randomly divided into two groups, with three mice in each group. The feeding conditions mainly included: the temperature was controlled at 18°C - 29°C, and the temperature difference did not exceed 3°C; the humidity was controlled at 50% - 60%; the light cycle was set to 12 hours of light and 12 hours of darkness, and the light intensity was controlled at 150 - 300 Lux; fresh air was ensured, the air change rate was 10 times / hour, the air flow velocity ≤ 0.18 m / s, the pressure difference was 25 Pa, and the ammonia concentration did not exceed 15 mg / m 3 , and the noise level was below 60 dB.

[0065] (2) Inoculation of tumor cells: 100 μl of tumor cell suspension was subcutaneously injected into the mid-lateral back of each mouse, that is, each mouse was inoculated with 1×10 6 A549-Luc cells.

[0066] (3) Tumor detection: After inoculating tumor cells, 100 μl of fluorescein at a concentration of 1.5 mg / ml was intraperitoneally injected into each mouse, and then 200 μl of tribromoethanol at a concentration of 12.5 mg / ml was intraperitoneally injected to anesthetize the mouse. After the mouse was anesthetized, it was transferred to an IVIS Lumina II small animal in vivo imaging system for imaging. When the tumor bioluminescence signal was about to reach the plateau phase, the image was recorded. After imaging, the image was analyzed using small animal in vivo imaging analysis software, and the bioluminescence photon intensity at the tumor inoculation site was calculated. The maximum value in the numerical value represented the bioluminescence photon intensity of the corresponding tumor cells in the mouse body.

[0067] (4) T cell immunotherapy: On the day after inoculation and the 3rd day, the corresponding group of mice was treated with CD155-T cells or control group T cells. 5×10 6 T cells were injected in situ at the tumor injection site of each mouse, with a total volume of 100 μl, suspended in PBS.

[0068] (5) Monitoring of tumor size in mice: Bioluminescence imaging was performed on the 42nd day after tumor inoculation to monitor the tumors in the mice.

[0069] (6) Calculate the average value and standard deviation of the number of photons in each group, and perform statistical analysis using a two-tailed unpaired sample t-test to calculate the P value.

[0070] Figure 8a and Figure 8b shows the antitumor effect of CD155 Bi-Ab modified T cells in vivo. SCID mice were subcutaneously inoculated with tumor cells (A549-luc, 1x 10 6 / mouse). On the day after inoculation and the third day, CD155 Bi-Ab modified T cells (200 ng / Bi-Ab / 10 6 T cells) were injected in situ at the tumor inoculation site at an E:T ratio of 5:1. On the day of tumor cell inoculation and the 42nd day, small animal in vivo imaging was performed using IVIS ( Figure 8a ) and the fluorescence intensity was statistically analyzed ( Figure 8b ). T cells incubated with a mixture of anti-CD3 antibody and anti-CD155 antibody were used as control group T cells. n = 3 mice in each group, **, P < 0.01. It can be seen that the antitumor effect of CD155 Bi-Ab modified T cells in vivo is obvious.

[0071] Those skilled in the art should understand that the above embodiments are only exemplary embodiments, and various changes, substitutions, and alterations can be made without departing from the spirit and scope of the present invention.

Claims

1. A method for preparing a bispecific antibody, characterized in that: include: dissolving the anti-human CD155 antibody in a first buffer; Add 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt to the first buffer, mix well, and incubate at room temperature; removing free 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt to obtain a first solution; replacing the anti-CD3 antibody buffer with a second buffer; Add 2-iminothiolane hydrochloride to the second buffer, mix well, and incubate at room temperature; removing free 2-iminothiolane hydrochloride to obtain a second solution; The first solution and the second solution are mixed, concentrated, reacted, and purified to obtain a bispecific antibody.

2. The method for preparing a bispecific antibody according to claim 1, characterized in that: Removing free 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt to obtain a first solution comprising: The free sodium salt of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester is removed by using a PD-10 desalting column: the sample is loaded and the effluent is discarded; then the first buffer is used for elution, and the eluate is collected to obtain the first solution.

3. The method for preparing a bispecific antibody according to claim 1, characterized in that: Removing free 2-iminothiolane hydrochloride to obtain a second solution comprising: The free 2-iminothiolane hydrochloride is removed by using a PD-10 desalting column: the sample is loaded and the effluent is discarded; then the first buffer is used for elution, and the eluate is collected to obtain the second solution.

4. The method for preparing a bispecific antibody according to claim 1, characterized in that: The concentration includes concentrating the mixed solution of the first solution and the second solution by 30-40 times, and the reaction includes reacting at 4° C. overnight.

5. A bispecific antibody, wherein the bispecific antibody is prepared according to the method for preparing a bispecific antibody according to any one of claims 1 to 4.

6. A method for preparing bispecific antibody-modified T cells, characterized in that: include: Activated T lymphocytes were obtained by expanding peripheral blood mononuclear cells; adding the bispecific antibody according to claim 5 to the activated T lymphocytes and incubating at room temperature; The bispecific antibody that is not bound to the activated T lymphocytes is removed to obtain bispecific antibody-modified T cells.

7. The method for preparing bispecific antibody-modified T cells according to claim 6, characterized in that: Adding the bispecific antibody according to claim 5 to the activated T lymphocytes and incubating at room temperature comprises: A 200 ng dose of bispecific antibody was added to 1 × 10 6 The cells were incubated at room temperature for 30 minutes.

8. A bispecific antibody-modified T cell, wherein the bispecific antibody-modified T cell is prepared according to the method for preparing a bispecific antibody-modified T cell according to claim 6 or 7.

9. Use of the bispecific antibody-modified T cells according to claim 8 in the preparation of drugs for treating tumors.

10. The use according to claim 9, wherein: The tumor is lung cancer.

Images