Application of anti-human DR5 monoclonal antibody in preparation of cell immunotherapy enhancer

Abstract

The invention relates to the technical field of cellular immunity, and particularly discloses an application of an anti-human DR5 monoclonal antibody in preparation of a cellular immunotherapy enhancer, and the anti-human DR5 monoclonal antibody comprises a coramumab. The anti-human DR5 monoclonal antibody disclosed by the invention can be used as an agonist of a tumor necrosis factor receptor superfamily member 10B (DR5), and is applied to enhancement of cellular immunotherapy, so that the killing power to tumor cells is improved. According to the application, the coramumab and the natural killer NK cells are combined, so that a remarkable killing effect can be shown in a plurality of in-vitro cancer cell models. The application further finds that the combination of the CD133 CAR-T cell and the coramumab can show an obvious killing effect on the CD133 overexpressed SK-Hep1 cell, which indicates that the coramumab can obviously enhance the killing efficiency of the CAR-T cell on cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of cellular immunity technology, and in particular provides an application of an anti-human DR5 monoclonal antibody in the preparation of a cellular immunotherapy enhancer, and more particularly provides an application of a DR5 agonist in the preparation of a cellular immunotherapy enhancer. Background Art

[0002] T lymphocytes and natural killer (NK) cells are important components of the innate immune system and play an important role in controlling the development of malignant tumors. In recent years, various cancer treatments based on T lymphocytes and NK cells have attracted increasing attention and become a major area of ​​innovation in immunotherapy. These cells are able to spontaneously detect and kill infected or malignant cells. The development and application of T lymphocyte and NK cell therapy face many challenges. Malignant solid tumors are generally less sensitive to T lymphocyte and NK cell-mediated killing responses than hematological malignancies. The efficacy of T lymphocyte and NK cell infusion therapy for solid tumors is hampered by multiple factors, including insufficient tumor infiltration and persistent activation of the tumor microenvironment (TME). Researchers are testing various methods to enhance the toxicity and lifespan of T lymphocytes and NK cells, as well as adjuvant methods to enhance the therapeutic activity of T lymphocytes and NK cells in vivo. The combination of immune checkpoint inhibitors (such as PD-1) and cell therapy has shown good therapeutic prospects. The therapeutic effect of the PD-1 inhibitor pembrolizumab combined with NK cell therapy is almost twice that of pembrolizumab alone, and the overall survival and progression-free survival of the combination group are significantly prolonged.

[0003] In recent years, with the development of CRISPR / Cas9 technology, it has been widely used in the fields of gene knockout, knock-in, transcriptional activation and inhibition, DNA methylation, histone modification, and whole genome screening. Among them, CRISPR screening technology is used for whole genome screening, and has shown efficient and accurate results in the fields of immunotherapy target screening and drug development. When using CRISPR screening technology for high-throughput screening, a large number of gene mutant cells can be generated, and mutant cell libraries can be screened under different external environmental stimuli. The relationship between phenotype and genotype can be further determined by high-throughput sequencing and bioinformatics analysis. In order to systematically determine the molecular characteristics of human tumor cells, which usually determine their sensitivity to T lymphocytes and NK cells, gene editing screening using CRISPR is applied in tumor cell lines to functionally explore which genes regulate the response of tumor cells to T lymphocytes and NK cells, thereby determining the molecular characteristics associated with the sensitivity or resistance of T lymphocytes and NK cells.

[0004] Currently, there is no study using CRISPR-based genetic screening to identify anti-human DR5 monoclonal antibodies to enhance cellular immunotherapy. Summary of the invention

[0005] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide an application of an anti-human DR5 monoclonal antibody in the preparation of a cellular immunotherapy enhancer.

[0006] To achieve the above purpose, the technical solution adopted by this application is:

[0007] The present application provides an anti-human DR5 monoclonal antibody used as a DR5 agonist in the preparation of a cellular immunotherapy enhancer.

[0008] The anti-human DR5 monoclonal antibody of the present application can be used as an agonist of tumor necrosis factor receptor superfamily member 10B (DR5), which is mainly used to treat tumor metastasis. In the technical solution of the present application, it is used to enhance cellular immunotherapy and improve the killing power against tumor cells.

[0009] Moreover, after verification experiments in nude mouse models, it was shown that koningumab can significantly enhance the killing effect of cellular immunotherapy on tumor cells.

[0010] As a preferred embodiment of the application described in the present application, the anti-human DR5 monoclonal antibody includes Canatumumab.

[0011] In the technical solution of the present application, the present application adopts the DR5 agonist (Conatumumab) to enhance the killing ability of natural killer NK cells and T lymphocytes against tumor cells, thereby better treating tumors.

[0012] As a preferred embodiment of the application described in the present application, the cells include T lymphocytes or natural killer NK cells; the T lymphocytes include CD133 CAR-T cells, and the natural killer NK cells include NK-92 cells or PBMC-NK cells.

[0013] The present application also provides the use of anti-human DR5 monoclonal antibodies combined with T lymphocytes or natural killer NK cells in the preparation of a reagent for killing cancer cells, wherein the anti-human DR5 monoclonal antibodies include canatumumab.

[0014] Experiments have shown that the use of the konimumab of the present application can improve the killing efficiency of natural killer NK cells against cancer cells, which highlights the potential of using combination therapy to enhance the natural killer NK cell-mediated cancer cell killing ability.

[0015] As a preferred embodiment of the application described in the present application, the T lymphocytes include CD133 CAR-T cells, and the natural killer NK cells include NK-92 cells or PBMC-NK cells.

[0016] As a preferred embodiment of the application described in the present application, the cancer cells include at least one of lung cancer cells, liver cancer cells, breast cancer cells, prostate cancer cells and kidney cancer cells.

[0017] As a preferred embodiment of the application described in the present application, the lung cancer cells include A549 cells; the liver cancer cells include human liver cancer cells SK-Hep1 or human liver cancer cells SK-Hep1 overexpressing CD133;

[0018] The breast cancer cells include human breast cancer cells MDA-MB-231; the prostate cancer cells include human prostate cancer cells 22RV1; and the renal cancer cells include human renal cancer cells 786-O.

[0019] In the technical solution of the present application, the combination of konimumab and natural killer NK cells can show significant killing effects in multiple in vitro cancer cell models (lung cancer cells, liver cancer cells, breast cancer cells, prostate cancer cells and kidney cancer cells).

[0020] In addition, the present application further discovered that the combination of CD133 CAR-T cells and konimumab can show a significant killing effect on SK-Hep1 cells overexpressing CD133, indicating that konimumab can significantly enhance the killing efficiency of CAR-T cells against cancer cells.

[0021] As a preferred embodiment of the application described in the present application, the mass concentration of the konimumab when used is 0.25 to 4 μg / mL.

[0022] The present application also provides an application of an anti-human DR5 monoclonal antibody in the preparation of a reagent for inhibiting cellular Fas-associated death domain protein-like β-interleukin-converting enzyme inhibitory protein, wherein the anti-human DR5 monoclonal antibody includes canatumumab.

[0023] This application uses a high-throughput whole-genome screening method to simulate molecular regulation in tumors and screen key regulatory molecules. The human whole-genome plasmid knockout library (Brunello, #73179) purchased from addgene was packaged with lentivirus to obtain recombinant lentivirus; the recombinant lentivirus was infected with the lung cancer cell line A549, and the culture medium containing puromycin was screened and cultured for 3 to 4 days, and doxycycline was used to induce the A549 knockout cell library. After co-culture with NK-92 cells for one week, the surviving cells were collected; the DNA of the cultured cells was extracted, PCR amplified, and the amplified products were sequenced by the second generation; the data of the second generation sequencing was compared with the sequence of the purchased sgRNA library, and the sgRNA enrichment number was statistically analyzed and standardized, and the loss of the cell-type Fas-associated death region protein-like beta interleukin-converting enzyme inhibitory protein (cFLIP) was screened to increase the killing sensitivity of NK-92 cells. cFLIP prevents the activation of caspase 8 (CASP8) in the extracellular death pathway, is a death receptor inhibitor, and is an anti-apoptotic gene target of NF-κB.

[0024] The present application inhibits cFLIP through the DR5 agonist (canatumumab), which can further enhance the activation of the extracellular death pathway, promote the apoptosis of tumor cells, and enable T lymphocytes and natural killer NK cells to effectively kill a variety of cancer cells.

[0025] The present application also provides a composition, which comprises kanalitumumab and T lymphocytes, or the composition comprises kanalitumumab and natural killer NK cells.

[0026] The present application adopts the above-mentioned composition to achieve a tumor inhibition effect that is better than using koningomab, T lymphocytes or natural killer NK cells alone, indicating that the use of the above-mentioned composition can better enhance the killing efficiency of T lymphocytes or natural killer NK cells on cancer cells.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present application provides an application of an anti-human DR5 monoclonal antibody in the preparation of a cell immunotherapy enhancer. The anti-human DR5 monoclonal antibody (Conatumumab) of the present application can be used as an agonist of tumor necrosis factor receptor superfamily member 10B (DR5), which is mainly used to treat tumor metastasis. In the technical scheme of the present application, it is applied to enhance cell immunotherapy, which improves the lethality of tumor cells. The present application combines Conatumumab with natural killer NK cells, which can show significant killing effects in multiple in vitro cancer cell (lung cancer cells, liver cancer cells, breast cancer cells, prostate cancer cells and renal cancer cells) models. In addition, the present application further found that the combination of CD133 CAR-T cells and Conatumumab can show significant killing effects on SK-Hep1 cells overexpressing CD133, indicating that Conatumumab can significantly enhance the killing efficiency of CAR-T cells against cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The screening flow chart of the test in Example 1;

[0030] Figure 2 This is a diagram showing the screening gene results for NK cell sensitization in Example 1;

[0031] Figure 3 This is a diagram showing the experimental results of combining DR5 agonist conatumumab with NK-92 cells in Example 2;

[0032] Figure 4 The figure is a graph showing the killing results of PBMC-NK cells combined with conatumumab in Example 3 in multiple in vitro cancer cell models;

[0033] Figure 5 This is a graph showing the killing results of the combination of CD133 CAR-T cells and conatumumab on CD133-overexpressing SK-Hep1 cells in Example 4;

[0034] Figure 6 This is a diagram showing the results of Example 5 verifying that conatumumab significantly enhances the killing of tumor cells by NK cells in a nude mouse model. DETAILED DESCRIPTION

[0035] For better explanation of the purpose, technical scheme and advantage of the present invention, the present invention will be further described below in conjunction with specific embodiment and accompanying drawing, but embodiment does not limit the present invention in any form.Unless otherwise specified, the reagent, method and equipment adopted in the present invention are conventional reagents, methods and equipment in the art.Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0036] Example 1

[0037] In this example, CRISPR-based genetic screening identified Fas-associated death domain (FADD)-like interleukin (IL)-1β converting enzyme inhibitory protein (cFLIP) as a natural killer cell killing enhancer.

[0038] In order to determine which genes' inactivation can increase the killing efficacy of natural killer cells (NK cells), the present application used a doxycycline-inducible CRISPR / Cas9 vector system to perform genome-scale inactivation genetic screening, and used A549 lung cancer cells as a screening model (e.g. Figure 1 ).

[0039] Specifically, the following steps are included: A549 lung cancer cells are inoculated into a 15 cm culture dish, cultured for 20 to 26 hours to allow the cells to adhere to the wall and become 70 to 90% confluent before being infected with recombinant lentivirus (for specific virus packaging steps, refer to the plasmid instructions (from addgene, plasmid catalog number #73139, reference

[0040] https: / / www.addgene.org / search / catalog / pooled-libraries / ?q=73179)(MOI=0.3), after culturing for 2-4 days, replace with fresh complete cell culture medium, add puromycin-free dual-antibody culture medium with a final concentration of 0.5-1.5 μg / mL, change the medium every day, and observe the cell status; after screening and culturing with puromycin culture medium for 3-4 days, add doxycycline-free dual-antibody culture medium with a final concentration of 0.5-1.5 μg / mL, and after screening and culturing with doxycycline culture medium for 4-6 days, collect the knockout cells for verification and subsequent experimental screening.

[0041] In the screening, the present application used an allogeneic, activated, interleukin-2 (IL-2)-dependent natural killer cell line NK-92 to kill A549 cells.

[0042] The constructed A549 cell knockout library was divided into a group without NK-92 cells and a group with NK-92 cells. The A549 knockout cells were cultured with medium containing NK92 cells and medium without NK92 cells for 5 days, and the surviving cells were collected for genomic DNA extraction. The total genomic DNA was extracted using QIAGEN Puregene cell core kit.

[0043] Refer to the instructions for specific extraction steps; PCR was used to analyze the sgRNA information, and the specific steps were referred to the plasmid instructions, and the target fragment was purified by gel recovery; the purified product library was constructed for second-generation sequencing, and Annoroad was commissioned to perform second-generation sequencing; MaGeck software was used to compare and analyze the sequencing data with the sgRNA library sequence, and the sgRNA was counted and standardized; the MAGeCK-RRA algorithm was used to evaluate the importance of the gene by calculating the RRA score of the target gene site corresponding to the sgRNA in the sequencing results, and the target genes were ranked to identify gRNAs and their corresponding target genes that were significantly reduced in the presence but not absence of NK-92 cells.

[0044] The present application identified cFLIP (encoded by the CFLAR gene), an inhibitor of death receptor-mediated apoptosis, as the most common hit target (e.g. Figure 2 ).

[0045] Example 2

[0046] This application studies the use of the DR5 agonist conatumumab in combination with NK-92 cells (results are shown in Figure 2). Figure 3 ).

[0047] The specific experiment includes the following steps:

[0048] 5×10 4 A549 cells were seeded in 12-well plates and cultured overnight at 37°C. On the second day, NK92 cells were added to A549 at effector-target ratios (E:T) of 1:2, 1:1, and 2:1, with or without 4ug / mL conatumumab, and co-cultured for 5 days. The killing of A549 cells was investigated using a crystal violet staining kit.

[0049] Results Figure 3 As can be seen from the figure, conatumumab can enhance the killing efficiency of NK cells against cancer cells, which highlights the potential of using combination therapy to enhance NK cell-mediated cancer cell killing ability.

[0050] Example 3

[0051] NK-92 cells present significant safety concerns due to their potential tumorigenicity, as they are an immortalized cell line derived from a patient with non-Hodgkin lymphoma. In contrast, PBMC-derived natural killer cells (PBMC-NK) from the peripheral blood of the same cancer patient donor present a lower risk of immune rejection and tumorigenicity. These cells can be expanded in vitro within a week using a cocktail of cytokines such as IL-2, IL-15, and IL-21. PBMC-NK cells represent a more natural population of NK cells that have diverse activating and inhibitory receptors and are able to respond to a wide range of stimuli, making them more suitable for clinical applications.

[0052] In order to evaluate the effect of this method, this example expanded and purified CD56+, CD3- NK cells from PBMC of healthy donors. PBMC cells purchased from Shanghai Ruiyu Biotechnology Co., Ltd. were cultured according to NK cell expansion kit P01 was used for in vitro activation. The specific steps were referred to the kit instructions for activation and expansion. After culturing for 14 days, the cells were sorted according to the Stemcell EasySep Human CD56 Pos Sel Kit II to select the activated NK cells for subsequent experiments.

[0053] The specific experiment includes: 5×10 4 A549 cells, human liver cancer cell SK-HEP1, human breast cancer cell MDA-MB-231, human prostate cancer cell 22RV1 and human renal cancer cell 786-O were plated in 6-well plates, and prepared PBMC-NK cells were added in different ratios (for example, the effector-target ratios of A549 cells and PBMC-NK cells were 2:1, 4:1 and 8:1 respectively; the effector-target ratios of human liver cancer cell SK-HEP1 and PBMC-NK cells were 2:1, 4:1 and 8:1 respectively; the effector-target ratios of human breast cancer cell MDA-MB-231 and PBMC-NK cells were 2:1, 4:1 and 8:1 respectively). The effector-target ratios were 1:4, 1:2, and 1:1, respectively; the effector-target ratios of human prostate cancer cells 22RV1 and PBMC-NK cells were 1:2, 1:1, and 2:1, respectively; the effector-target ratios of human renal cancer cells 786-O and PBMC-NK cells were 1:2, 1:1, and 2:1, respectively), and a certain concentration (specifically 0.25μg / ml, 0.5μg / ml, 1μg / ml, 2μg / ml) of conatumumab was added or not, and the cells were co-cultured for 5 days. The crystal violet staining kit was used to investigate the killing of each tumor cell.

[0054] The present application found that PBMC-NK cells combined with conatumumab showed significant killing effects in multiple in vitro cancer cell models (such as Figure 4 shown).

[0055] Example 4

[0056] The present application further found that the combination of CD133 CAR-T cells and conatumumab can show significant killing effect on SK-Hep1 cells overexpressing CD133 (such as Figure 5 shown).

[0057] Specific experiments include:

[0058] The purchased PBMC cells were resuspended and mixed in X-VIVO medium containing 10 ng / mL IL-2, anti-CD3 / CD28 magnetic beads were added to activate T cells, and the cells were cultured in a 37°C, 5% CO2 incubator for 48 h to obtain activated T cells. The activated T cells were transfected with lentivirus, and then cultured in X-VIVO medium containing 10 ng / mL IL-2 for 3 days to obtain CD133CAR-T cells.

[0059] The following experiments were conducted using the human liver cancer cell line SK-Hep1 overexpressing CD133 as target cells:

[0060] Prepare a single cell suspension of SK-Hep1, a human hepatoma cell line overexpressing CD133, add 2 ml PBS and centrifuge and wash twice to remove the serum. Resuspend the cells in 1 ml serum-free medium and adjust the cell density to 1×10 6 / ml. Add 1 μl of 10 μM CFSE reagent, vortex the cells, and incubate at 37°C in the dark for 15 minutes; add 4-5 times the volume of pre-cooled 10% serum complete medium to wash once. CFSE-labeled human liver cancer cell line SK-Hep1 overexpressing CD133 was cultured at 4×10 4 / well were inoculated into ultra-low adsorption cell culture 96-well plates; the prepared CD133 CAR-T cells were inoculated into target cells according to the effector-target ratio (E:T) of 1:2, 1:1, and 2:1, and conatumumab was added to make its final concentration 0, 0.5, and 1.5 μg / ml, for a total of 12 treatments, each treatment had 3 replicates (wells), and each well was replenished to 200 μl; the culture plate after cell mixing was placed in a 37°C incubator and cultured for 24 hours; after 24 hours, all cells in each well were collected, the cells were transferred to a flow tube, and the change in the target cell ratio was detected by flow cytometry. The results are as follows Figure 5 As shown in Figure 2, conatumumab can significantly enhance the killing efficiency of CAR-T cells against cancer cells.

[0061] Example 5

[0062] This application verifies in a nude mouse model that conatumumab can significantly enhance the killing of tumor cells by NK cells (such as Figure 6 shown).

[0063] First, SK-Hep1 cells (5×10 6 / mouse) to construct a tumor-bearing mouse model. When the tumor grows to 100 mm 3 At the time of the study, the tumor-bearing mice were randomly divided into 4 groups, with 6 mice in each group. 5 μg / mouse conatumumab was injected intraperitoneally, and NK92 cells and the combination group were injected into the tail vein. The PBS injection group served as the negative control group. 1E7 / mouse NK92 cells were given once every 3 days and continued for 3 times. Conatumumab was given 5 days a week. The diameter of the mouse tumor was measured with a vernier caliper, and its volume was calculated according to the formula: V=d2×D / 2, where d represents the shortest diameter of the tumor, and D represents the longest diameter of the tumor, and the measurement unit is mm. Figure 6 As shown, the tumor inhibition effect of the combination group in mice was significantly superior to that of the single-dose group.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of an anti-human DR5 monoclonal antibody as a DR5 agonist in the preparation of a cell immunotherapy enhancer.

2. The use according to claim 1, characterized in that The anti-human DR5 monoclonal antibodies include canatumumab.

3. The use according to claim 1, characterized in that The cells include T lymphocytes or natural killer NK cells; The T lymphocytes include CD133 CAR-T cells, and the natural killer NK cells include NK-92 cells or PBMC-NK cells.

4. Use of anti-human DR5 monoclonal antibody combined with T lymphocytes or natural killer NK cells in the preparation of a cancer cell killing agent, characterized in that: The anti-human DR5 monoclonal antibodies include canatumumab.

5. The use according to claim 4, characterized in that The T lymphocytes include CD133CAR-T cells, and the natural killer NK cells include NK-92 cells or PBMC-NK cells.

6. The use according to claim 4, characterized in that The cancer cells include at least one of lung cancer cells, liver cancer cells, breast cancer cells, prostate cancer cells and kidney cancer cells.

7. The use according to claim 6, characterized in that The lung cancer cells include A549 cells; the liver cancer cells include human liver cancer cells SK-Hep1 or human liver cancer cells SK-Hep1 overexpressing CD133; The breast cancer cells include human breast cancer cells MDA-MB-231; the prostate cancer cells include human prostate cancer cells 22RV1; and the renal cancer cells include human renal cancer cells 786-O.

8. The use according to any one of claims 1 to 7, characterized in that: The mass concentration of the konamizumab when used is 0.25-4 μg / mL.

9. Use of an anti-human DR5 monoclonal antibody in the preparation of a reagent for inhibiting cellular Fas-associated death domain protein-like beta interleukin-converting enzyme inhibitory protein, characterized in that: The anti-human DR5 monoclonal antibodies include canatumumab.

10. A composition, characterized in that The composition comprises canatumumab and T lymphocytes, or the composition comprises canatumumab and natural killer (NK) cells.

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