Method for preparing freezing shock-treated monocytes or macrophages loaded with attenuated salmonella and use thereof

Abstract

Provided are a method for preparing freezing shock-treated monocytes or macrophages loaded with attenuated Salmonella and the use thereof. The method comprises: loading engineering-modified attenuated Salmonella in monocytes or macrophages, and performed a freezing shock treatment on the monocytes or macrophages loaded with the strain by means of liquid nitrogen. The method for preparing the freezing shock-treated immune cells loaded with the strain is simple and easy to operate. The source problem of the monocytes or macrophages loaded with the engineering-modified attenuated Salmonella is solved, and the method has good application prospects. According to the strategy, by means of avoiding exposure and heterologous stimulation of bacteria, the biological safety of bacteria-based anti-tumor treatment is improved; and the anti-tumor immune response is promoted by means of the high titer of an intratumoral strain, so that a stronger anti-tumor efficacy is obtained.

Description

Preparation method and application of frozen shock treated monocytes or macrophages loaded with attenuated Salmonella Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of monocytes or macrophages loaded with attenuated Salmonella treated with freezing shock. Background Art

[0002] Bacterial therapy is changing the treatment paradigm for cancer. These therapeutic bacteria are able to preferentially colonize solid tumor sites after injection due to their own facultative anaerobic properties and the unique characteristics of the solid tumor microenvironment, including the hypoxic environment, immunosuppressive environment and nutrients released by a large number of necrotic cells in solid tumors (Gurbatri CR et al., 2020, Sci Transl Med, 12(530); Zhou S et al., 2018, Nat Rev Cancer, 18(12):727-43; Suh S et al., 2019, Adv Sci, 6(3):1801309), followed by significant intratumoral immune activation (Wu L et al., Adv Drug Deliv Rev 2022, 187:114363). However, direct injection of foreign microorganisms usually triggers a rapid immune response in the body, causing discomfort and potential adverse reactions. Studies have shown that direct administration of live bacteria can be toxic to the host, limiting the dose and effectiveness of bacterial tolerance in patients (Wu L et al., Adv Drug Deliv Rev 2022, 187:114-363; Gurbatri CR et al., Science, 2022, 378(6622):858-864). VNP20009 is an attenuated strain of Salmonella typhimurium (hereinafter referred to as VNP) that has attracted widespread attention due to its lower biotoxicity and excellent preclinical anti-tumor efficacy (Clairmont C et al., 2000, J Infect Dis, 181:1996-2002). Although the deletion / mutation of two genes (purI and msbB) significantly reduces the toxicity of the VNP20009 strain compared to the original Salmonella, in preclinical mouse studies, tumor-suppressing treatment with VNP20009 via intravenous or intraperitoneal injection still results in a degree of toxic side effects, such as liver damage, splenomegaly, and sudden weight loss in mice, due to the presence and accumulation of the strain in normal organs. Ideally, bacteria-based cancer treatments should minimize toxic effects caused by off-target effects or antigenic stimulation to ensure high biocompatibility.

[0003] In recent years, various types of white blood cells, including macrophages, neutrophils, and T cells, have been used as effective tumor drug delivery vehicles due to their unique chemotactic effects on tumor areas (Xie Z et al., 2017, Small, 13(10); Xue J et al., 2017, Nat Nanotechnol, 12(7):692-700; Huang B et al., 2015, Sci Transl Med, 7(291):291ra94). An ideal cell drug carrier should be easy to prepare and quickly available, which is difficult to achieve for immune cell carriers based on stem cells or primary cells. This is because these cells usually have complex in vitro culture conditions and low proliferation efficiency. Some immortalized immune cell lines, such as macrophage (MACS) lines RAW264.7, J774.1, Ana-1, iBMDM, U937, and monocyte (MC) lines THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, etc., have the characteristics of continuous proliferation and easy culture, which can theoretically solve the pain point of difficult cell acquisition in conventional cell therapy. However, these cell lines are accompanied by potential pathogenicity due to their ability to continuously proliferate. Generally, the structure of living cells will disintegrate when they die, resulting in the loss of proteins and cytokines (Green DR et al., Cold Spring Harb Perspect Biol., 2015; 7(12): a006080). In addition, external stimuli that may induce cell death, such as heat or radiation, can also inactivate proteins (Sanchez Y et al., Science, 1990; 248(4959): 1112-1115; Prise KM et al., Lancet Oncol., 2005; 6(7): 520-528). Studies have shown that freezing live tumor cells with liquid nitrogen can maintain the integrity of their cell structure and the biological activity of membrane proteins while losing their growth ability (Ci T et al., Sci Adv., 2020; 6(50): eabc3013; Meng J et al., Nat Commun., 2023; 14(1): 4505).

[0004] Therefore, is it possible to obtain a "dead" but "functional" immortalized immune cell line through liquid nitrogen cold treatment to eliminate the pathogenicity of the cell line while taking into account easy access? Furthermore, by loading the immune cell line with bacteria and then cold-treating it with liquid nitrogen, the off-target effect of bacteria and the toxic and side effects caused by direct and large-scale exposure to foreign substances can be avoided based on the cell's camouflage protection and cell-dependent tumor-targeted delivery. The stimulating effect of the strain in the immune cells further promotes the production of intracellular anti-tumor inflammatory factors and adhesion factors, corresponding to stronger anti-cancer efficacy and intratumor enrichment effect.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing monocytes or macrophages loaded with attenuated Salmonella treated with freeze shock and its application. Specifically, attenuated Salmonella is loaded in vitro with immune cells such as monocytes / macrophages, followed by rapid cold treatment with liquid nitrogen to obtain freeze-shocked monocytes / macrophages loaded with attenuated Salmonella, and a scheme for the combined use of the methods to treat tumors.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows: In a first aspect, the present application provides a method for preparing monocytes or macrophages loaded with attenuated Salmonella treated with freezing shock.

[0008] In a second aspect, the present application provides a method for preparing freeze-shocked monocytes or macrophages loaded with attenuated Salmonella, including freeze-shocked monocytes or macrophages loaded with attenuated Salmonella.

[0009] In a third aspect, the present application provides a method for preparing monocytes or macrophages loaded with attenuated Salmonella treated with freezing shock, and its application in the preparation of anti-tumor drug preparations.

[0010] The present invention provides a method for preparing freeze-shocked monocytes or macrophages loaded with attenuated Salmonella, comprising the following steps: co-culturing the freeze-shocked monocytes or macrophages loaded with attenuated Salmonella at an MOI value of 1-100 with an immortalized monocyte or macrophage line and attenuated Salmonella typhimurium to obtain engineered cells loaded with attenuated Salmonella, and then performing a cold shock treatment with liquid nitrogen to obtain freeze-shocked monocytes or macrophages loaded with attenuated Salmonella.

[0011] While the monocyte / macrophage cell lines lost their original pathogenic potential, the intracellular attenuated Salmonella maintained its biological activity and, after being released in vitro, reached a growth plateau within 24 hours. Compared to single injections of cold-shocked attenuated Salmonella, or a simple mixture of cold-shocked macrophages and cold-shocked attenuated Salmonella, the camouflaged protection and targeted delivery of the bacteria by freeze-shocked macrophages for tumor treatment significantly inhibited tumor growth and prolonged survival in mice.

[0012] Further, (1) any one of the monocyte cell line or macrophage cell line is co-incubated with attenuated Salmonella:

[0013] The monocytes or macrophages with good growth conditions were cultured at a rate of 1-100×10 5 Cells were inoculated into culture dishes at a ratio of 100 cells / well and cultured in antibiotic-free cell culture medium. Attenuated Salmonella monoclonal strains were picked from the agar plates and activated overnight in LB liquid medium. The bacterial solution grown to the logarithmic phase was centrifuged at 5000-8000 rpm for 5-10 minutes, the supernatant was discarded, and the pellet was resuspended in sterile saline. After adjusting the OD600 to 0.6-1.2, the bacteria were added to the culture dish containing the above cells at an MOI of 1-100 and incubated for 20-150 minutes.

[0014] (2) After staining the cell nucleus with Hoechst, observe the morphological changes of the cells under a microscope; record the changes in the percentage of disruption of the above-mentioned monocytes / macrophages at different time points of co-culture of cells and bacteria; after different time points, discard the supernatant, wash with sterile PBS 2 to 3 times, and then incubate with cell culture medium supplemented with 50-125μg / ml gentamicin for 20-60 minutes to kill the extracellular strain; discard the supernatant, wash with sterile PBS 2 to 3 times; collect the cells to obtain the live monocytes / macrophages of the above-mentioned type loaded with attenuated Salmonella strain; in order to detect the number of attenuated Salmonella effectively loaded in monocytes or macrophages, use a cell counter to detect the number of live cells of monocytes or macrophages loaded with attenuated Salmonella at different time points, and then use 0.5% Triton at room temperature. Lyse cells with X-100; dilute the lysate serially, plate on LB agar plates containing kanamycin, and incubate overnight at 37°C; count the number of viable bacteria in monocytes / macrophages;

[0015] (3) The living cells of the monocytes or macrophages loaded with attenuated Salmonella prepared above are resuspended in 500-1000 μl of serum-free cell freezing solution; the cell suspension is directly rapidly frozen in liquid nitrogen, placed for 6-18 hours, taken out, and thawed in a 37-42°C water bath to obtain freeze-shocked monocytes or macrophages loaded with attenuated Salmonella.

[0016] Rapid freezing shock treatment with liquid nitrogen for 6-18 hours can eliminate the pathogenicity of the cells, but does not affect the cells' tumor enrichment ability and the biological activity of intracellular attenuated Salmonella.

[0017] Furthermore, in step (1) or step (2), the attenuated Salmonella typhimurium is attenuated Salmonella typhimurium VNP20009 and its genetically modified strains and synthetic biology engineered strains capable of producing pharmaceutical proteins, including but not limited to the aforementioned strains for which inventions have been applied (ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201 610945015.4, ZL201610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 20221018 1929.9, 202210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ), the immortalized monocyte cell line / macrophage cell line includes any one of THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, RAW264.7, J774.1, Ana-1, iBMD or U937; wherein the monocytes include any one of the monocytic cell lines THP1, iBMMC, J-111, Mono-Mac-1 or JOSK-M, and the macrophages include any one of the macrophage cell lines RAW264.7, J774.1, Ana-1, iBMDM or U937.

[0018] Furthermore, in step (2), taking the macrophage cell line RAW264.7 as an example, after comprehensively considering the strain loading amount and cell integrity, a co-culture time of 60 minutes was selected as the optimal time point for preparing live CELL / VNP cells. At this time, the load of live bacteria in the cells was high, with a load of 257±27 strains per 100 cells, and the cell integrity was >90%.

[0019] The preparation method of the present invention is to prepare freezing shock treated monocytes or macrophages loaded with attenuated salmonella.

[0020] The invention discloses an application of the method for preparing monocytes or macrophages loaded with attenuated Salmonella subjected to freezing shock treatment in the preparation of anti-tumor drug preparations.

[0021] Furthermore, the therapeutic dose of frozen shock-treated monocytes or macrophages loaded with attenuated Salmonella is 0.4-40×10 6 cells, the unit is cells / mouse, corresponding to the actual amount of attenuated Salmonella is 0.1-10×10 7 CFU, the unit is CFU / mouse; the number of administrations of frozen shock-treated monocytes / macrophages loaded with attenuated Salmonella is a single administration. The main administration method is intravenous or intraperitoneal injection.

[0022] Furthermore, the pharmaceutical preparation includes at least one of an intravenous preparation, an intratumoral injection preparation or an intraperitoneal injection preparation. The frozen-shocked monocytes / macrophages loaded with attenuated Salmonella are mainly administered intravenously for treatment, and depending on the type of tumor, they can also be administered by intraperitoneal injection or intratumoral injection. Compared with a single injection of cold-shocked attenuated Salmonella, and a simple mixture of cold-shocked macrophages and cold-shocked attenuated Salmonella, the tumor growth of mice was more significantly inhibited and the survival time was significantly prolonged after being used for tumor treatment with the help of the camouflage protection and targeted delivery of the bacteria by frozen-shocked macrophages. Compared with a single injection of cold-shocked attenuated Salmonella, the camouflage protection and targeted delivery of the bacteria by frozen-shocked macrophages achieved an increase of 110.9% in the titer of Salmonella in the tumor, a 16.3% reduction in the off-target effect of normal organs, and a 90% reduction in the area of ​​liver lesions.

[0023] The attenuated Salmonella typhimurium VNP20009 and its genetically modified strains, which are delivered and released to tumor tissues via cells, are the key factors in exerting the anti-tumor efficacy of this type of drug.

[0024] The present invention is used in combination with other conventional anti-tumor drugs or methods.

[0025] Beneficial Effects: The freeze-shock-treated strain-loaded immune cell preparation method of the present invention is simple and easy to operate, with promising application prospects. This strategy avoids extensive in vivo exposure to bacterial xenobiotics, improving the biosafety of bacterial-based anti-tumor therapies. High intratumoral strain titers also promote anti-tumor immune responses, resulting in stronger anti-tumor efficacy.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) Cold treatment not only removes the potential pathogenicity of the original monocyte or macrophage cell lines, but also maintains their original tumor enrichment ability and has almost no effect on the activity and infectivity of the intracellular strains, thereby promoting the accumulation of bacteria in tumors. The facultative anaerobic bacteria Salmonella typhimurium VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins were selected (including but not limited to the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015. 4.ZL201610945021.X,ZL202210182222.4,202010182038.0,202210070594.8,202210181929. 9, 202210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ), because it can survive for a long time in immune cells such as monocytes / macrophages, liquid nitrogen cold shock treatment has no significant effect on its subsequent biological activity, and the strain itself has good tumor colonization and tumor inhibition effects.

[0028] (2) We selected immortalized monocyte or macrophage cell lines treated with liquid nitrogen cold shock, such as macrophage cell lines RAW264.7, J774.1, Ana-1, iBMDM, U937, etc., and monocyte cell lines THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, etc., because they can proliferate rapidly in vitro before cold treatment, have simple nutritional requirements, and have a clear research background, which allows for large-scale and rapid acquisition. After a simple liquid nitrogen cold shock treatment, the cell line loses its pathogenicity, achieving high biosafety.

[0029] (3) The method for preparing the immortalized monocyte cell line or macrophage cell line by freezing shock of the present invention can achieve the rapid acquisition and preparation of such engineered cells and the cells are non-pathogenic, and the attenuated Salmonella VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins (including but not limited to the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 202210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359;Acta Pharmaceutica Sinica B 2021,11(10):31653177;Signal Transduction and Targeted Therapy 2023,8:134;Frontier of Medicine,https: / / doi.org / 10.1007 / s11684-022-0925-2;phoP / phoQ) are low-toxic and easy-to-cultivate bacteria with low cost, so the present invention has the prospect of large-scale promotion and application. The immortalized monocyte cell line or macrophage cell line treated with liquid nitrogen cold shock was loaded with attenuated Salmonella VNP20009 and then used. This not only verified its significantly improved biosafety compared to the use of a single strain in a mouse model, but also demonstrated good anti-tumor effects.

[0030] (4) After administration, cold-treated bacterial strains loaded into monocytes / macrophages can be efficiently enriched inside the tumor, preventing the bacteria from being delivered to normal organs. The monocyte / macrophage encapsulation and camouflage also effectively prevent the premature exposure of bacteria. Ultimately, it indirectly inhibits tumors by releasing intracellular bacterial strains to activate / regulate the immune system. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a diagram showing the optimization of conditions for preparing monocytes or macrophages loaded with attenuated Salmonella by freeze-shock treatment according to the present invention; Figure 1a is a flow chart showing the preparation of monocytes / macrophages loaded with attenuated Salmonella by freeze-shock treatment according to the present invention. (Monocyte or macrophage cell lines include THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, RAW264.7, J774.1, Ana-1, iBMDM, and U937. The classical macrophage cell line (RAW264.7) is used as an example here, but the effects of using other cells are similar to those of RAW264.7.) Live macrophages RAW264.7 were first infected with attenuated Salmonella VNP20009 to obtain live VNP-loaded macrophages (live MACS / VNP), which were then frozen with liquid nitrogen for 12 hours to obtain liquid nitrogen-treated MACS / VNP cells (cold-treated MACS / VNP). Cold-treated MACS / VNP cells can deliver VNP strains to tumors. The resulting freeze-shocked monocytes / macrophages loaded with attenuated Salmonella were intravenously administered to tumor-bearing mice for targeted tumor therapy. Figure 1b shows microscopic observations of live RAW264.7 macrophages of the present invention before and after co-culture with attenuated Salmonella VNP20009. RAW264.7 and VNP20009 were co-cultured at an MOI of 20, and the nuclei were stained with the nuclear dye Hoechst for easier visualization. It can be observed that VNP20009 actively infects macrophages (red arrows); Figure 1c shows the total number of VNP20009 bacteria per 100 macrophages after co-culture of the live macrophages RAW264.7 of the present invention with the attenuated Salmonella VNP20009 at an MOI of 20 for different times (30 / 60 / 90 / 120 / 150 minutes), as well as the changes in the proportion of macrophages that maintain integrity observed by microscopy; Figure 1d shows microscopic observations of the live macrophages RAW264.7 of the present invention with the attenuated Salmonella VNP20009 at an MOI of 20 for different times (30 / 60 / 90 / 120 / 150 minutes). Living macrophages RAW264.7 were used as a control, and the nuclei were stained with the nuclear dye Hoechst for easy observation (blue). VNP20009 was observed around the macrophages (white arrows), as well as cells that were broken due to increased bacterial load as the co-culture time increased (red arrows).

[0032] Figure 2 is a graph evaluating the intracellular strain release performance of monocytes or macrophages loaded with attenuated Salmonella treated with freeze shock of the present invention; Figure 2a is a confocal fluorescence microscopy image of live macrophages RAW264.7 (live MACS), live macrophages loaded with attenuated Salmonella VNP20009 obtained after co-culture of live macrophages RAW264.7 and attenuated Salmonella VNP20009 (live MACS / VNP), and macrophages loaded with attenuated Salmonella VNP20009 treated with freeze shock (cold-treated MACS / VNP); RAW264.7 and VNP20009 were co-cultured at an MOI of 20, and the nuclei were stained with the nuclear dye DAPI (blue), and actin was labeled with FITC-podophyllin (green) for easy observation.It can be observed that neither strain loading nor low temperature shock significantly affects the overall integrity of macrophages, and intact strain VNP20009 macrophages can be observed in the cells (red arrow); scale bar = 20 μm; Figure 2b is the transmission electron microscopy of live macrophages RAW264.7 (live MACS) of the present invention, live macrophages loaded with attenuated Salmonella VNP20009 (live MACS / VNP), and macrophages loaded with attenuated Salmonella VNP20009 treated with freezing shock (cold-treated MACS / VNP). Microscope and scanning electron microscope observation; It can be seen that neither strain load nor low temperature shock significantly affects the overall integrity of macrophages, and strain VNP20009 macrophages can be observed in the cells (red arrow); Scale bar = 5 μm; Figure 2c is a live macrophage loaded with red fluorescent (RFP) attenuated Salmonella VNP-RFP of the present invention (live MACS / VNP-RFP) and a cryo-shocked macrophage loaded with red fluorescent (RFP) attenuated Salmonella VNP-RFP (cold-treated MACS / VNP-RFP) Figure 2d is a graph showing the changes in the number of live strains in the culture plates of live macrophages loaded with red fluorescent (RFP) attenuated Salmonella VNP-RFP (live MACS / VNP-RFP) and frozen-shock-treated macrophages loaded with red fluorescent (RFP) attenuated Salmonella VNP-RFP (cold-treated MACS / VNP-RFP) within 6 hours of the present invention; Figure 2e is a graph showing the changes in the red fluorescent (RFP) fluorescence intensity in the culture plates of live macrophages loaded with red fluorescent (RFP) attenuated Salmonella VNP-RFP (live MACS / VNP-RFP) and frozen-shock-treated macrophages loaded with red fluorescent (RFP) attenuated Salmonella VNP-RFP within 6 hours of the present invention; Transmission electron microscopy observations of the release process of attenuated Salmonella VNP20009 from macrophages loaded with attenuated Salmonella VNP20009 treated with freeze shock (cold-treated MACS / VNP) of the present invention (left and middle) and a schematic diagram of the intracellular bacterial release process (right), showing the movement of bacteria from the intracellular to the extracellular space (white curve); notches on the surface of cold-treated MACS / VNP cells (orange arrows); and extracellularly propagating bacteria (blue arrows). Scale bars (left = 1 μm, middle = 500 nm) (enlarged view).

[0033] FIG3 is a graph showing the biological activity detection of strains released from monocytes or macrophages loaded with attenuated Salmonella subjected to freeze shock treatment of the present invention; FIG3a is a graph showing the growth curve of VNPs released from macrophages loaded with attenuated Salmonella VNP20009 subjected to freeze shock treatment (cold-treated MACS / VNP) and untreated normal attenuated Salmonella VNP20009 in LB liquid culture medium, the culture temperature being 37° C.; FIG3b is a graph showing the growth curve of macrophages loaded with attenuated Salmonella VNP20009 subjected to freeze shock treatment of the present invention (cold-treated MACS / VNP); Figure 3c is a graph showing the number of bacteria internalized in tumor cells (B16F10, LLC, 4T1, A20, H22) after 1 hour of infection with VNPs released from macrophages loaded with attenuated Salmonella VNP20009 (cold-treated MACS / VNP) and untreated normal attenuated Salmonella VNP20009; the scale bar is 1 μm; Figure 3c is a graph showing the number of bacteria internalized in tumor cells (B16F10, LLC, 4T1, A20, H22) after 1 hour of infection with VNPs released from macrophages loaded with attenuated Salmonella VNP20009 (cold-treated MACS / VNP) and untreated normal attenuated Salmonella VNP20009; the tumor cells and different VNPs were all inoculated at an MOI of 100 for co-culture, washed with PBS and treated with gentamicin for 1 hour to remove extracellular residual VNPs, then lysed with 0.5% Triton X-100, diluted and plated to count the number of internalized bacteria; Figure 3d is a representative flow cytometric analysis of VNPs released from macrophages loaded with attenuated Salmonella treated with freeze shock of the present invention (cold-treated MACS / VNP) and untreated normal attenuated Salmonella after co-incubation with H22 cells for 4 hours, and the percentage of apoptotic cells (Annexin V+ cells) was quantitatively analyzed. H22 cells were co-cultured with different VNPs at an MOI of 100, ns=not significant, ****P<0.0001.

[0034] FIG4 is a graph showing the potential pathogenicity assessment of the monocyte or macrophage cell lines treated with freeze shock according to the present invention; FIG4a shows the cell viability of live macrophages RAW264.7 (live MACS), freeze shock-treated macrophages RAW264.7 (cold-treated MACS), and freeze shock-treated macrophages loaded with attenuated Salmonella VNP20009 (cold-treated MACS / VNP) at different time points (0 / 12 / 24 / 36 / 48 hours); the cell viability was detected by CCK8 assay, where au is an arbitrary unit; FIG4b shows the cell viability of live macrophages RAW264.7 (live MACS) according to the present invention; FIG4c shows the cell viability of live macrophages RAW264.7 (live MACS) according to the present invention; FIG4d shows the cell viability of live macrophages RAW264.7 (live MACS) according to the present invention; FIG4e shows the cell viability of live macrophages RAW264.7 (live MACS) according to the present invention; FIG4f ... Figure 4c is a comparison of the cell proliferation activity of live macrophages RAW264.7 (live MACS) of the present invention and freeze-shocked macrophages loaded with attenuated Salmonella VNP20009 (cold-treated MACS) 14 days after inoculation in vivo; the area within the dotted circle indicates the inoculation location.

[0035] Figure 5 shows an evaluation of the intratumoral enrichment performance of monocytes or macrophages loaded with attenuated Salmonella treated with the present invention; Figure 5a shows DIR fluorescence and bioluminescence observations of tumors in representative mice from each group (left) and statistical graphs of DIR fluorescence and bioluminescence intensity of tumors in each group (right) 8 hours after administration of different treatment methods (G#1-G#3) of the present invention to H22 tumor-bearing mice. DIR-labeled cells emit near-infrared fluorescence, and the VNP-LuxCDABE strain used can spontaneously generate bioluminescence. G#1: RAW264.7 macrophages treated with freezing shock and labeled with DIR were simply mixed with attenuated Salmonella VNP-LuxCDABE; G#2: RAW264.7 macrophages treated with freezing shock and loaded with attenuated Salmonella VNP-LuxCDABE were simply mixed with DIR; G#3: RAW264.7 macrophages fixed with paraformaldehyde and labeled with DIR were simply mixed with attenuated Salmonella VNP-LuxCDABE, ns=not significant, *P<0.05, ***P<0.001; Figure 5b is Confocal fluorescence microscopy images of live macrophages RAW264.7 (live MACS) of the present invention, live macrophages loaded with attenuated Salmonella VNP-RFP with red fluorescence (RFP) obtained after co-culture of live macrophages RAW264.7 and attenuated Salmonella VNP-RFP with red fluorescence (RFP) (live MACS / VNP-RFP), and freeze-shock-treated macrophages loaded with attenuated Salmonella VNP-RFP with red fluorescence (RFP) (cold-treated MACS / VNP-RFP). The expression of CD11b (yellow) and CCR2 (green) in each group of cells, as well as the morphology of intact bacteria in macrophages (white arrows) can be observed; scale bar = 10 μm; Figure 5c is a representative flow cytometric graph of the expression of CD11b protein (top) and CCR2 protein (bottom) in live macrophages RAW264.7 (live MACS), live macrophages loaded with attenuated Salmonella VNP20009 (live MACS / VNP), and freeze-shocked macrophages loaded with attenuated Salmonella VNP20009 (cold-treated MACS / VNP).

[0036] Figure 6 is an evaluation graph showing the enhanced tumor targeting performance of intracellular bacterial strains by freeze-shocked monocytes or macrophages loaded with attenuated Salmonella; Figure 6a is a graph showing the relationship between the number of intratumoral bacteria at different times (8 hours / 1 day / 3 days / 6 days / 12 days) after administration of different treatment methods (G2-G4) of the present invention to H22 tumor-bearing mice. G2: freeze-shocked attenuated Salmonella (cold-treated VNP); G3: freeze-shocked macrophages loaded with attenuated Salmonella (cold-treated MACS / VNP); G4: freeze-shocked RAW264.7 macrophages simply mixed with attenuated Salmonella (cold-treated MACS + cold-treated VNP); the following descriptions are identical for the groups. *P<0.05, **P<0.01; Figure 6b is a graph showing the relationship between the normal organs (including heart, liver, spleen, lung, and kidney) and the number of VNP20009 bacteria in each group of mice at specific time points (8 hours / 1 day / 3 days / 6 days / 12 days) after administration of different treatment methods of the present invention to H22 tumor-bearing mice; Figures 6c-g are statistical graphs of the VNP20009 bacterial titer in the heart (c), liver (d), spleen (e), lung (f), and kidney (g) of each group of mice at specific time points (8 hours / 1 day / 3 days / 6 days / 12 days) after administration of different treatment methods of the present invention to H22 tumor-bearing mice.

[0037] FIG7 is a diagram showing the in vivo biosafety assessment of monocytes or macrophages loaded with attenuated Salmonella treated with freeze shock according to the present invention; FIG7a is a schematic diagram showing the safety evaluation, tumor targeting, and treatment experiments of H22 tumor-bearing mice using different treatment methods (G0-G4) according to the present invention; G0: saline group; G1: freeze-shocked macrophages RAW264.7; G2: freeze-shocked attenuated Salmonella; G3: freeze-shocked macrophages loaded with attenuated Salmonella VNP20009; G4: Cryoshock-treated RAW264.7 macrophages were simply mixed with attenuated Salmonella; the following descriptions are the same for the groups; Figure 7b is a representative image of inflammatory lesions in the liver of H22 tumor-bearing mice in each group one day after administration of different treatment methods (G0-G4) of the present invention; the location of liver pathological damage can be observed (black arrow); scale bar = 10 mm; Figure 7c is a bar graph of the number of inflammatory lesions in the liver of H22 tumor-bearing mice in each group one day after administration of different treatment methods (G0-G4) of the present invention. ns = not significant, ***P < 0.001; Figure 7d is a close-up observation of representative H&E staining of liver inflammatory lesions in each group of mice after one day of administration of different treatment methods (G0-G4) of the present invention to H22 tumor-bearing mice; obvious liver damage sites can be observed (black arrows), scale bar = 40 μm; Figure 7e is the IL-6 (left) and IL-10 (right) concentrations in the peripheral blood serum of each group of mice after one day of administration of different treatment methods (G0-G4) of the present invention to H22 tumor-bearing mice, ns = not significant, *P < 0.05, **P < 0.01; Figure 7f is the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the peripheral blood of each group of mice after one day of administration of different treatment methods (G0-G4) of the present invention to H22 tumor-bearing mice, **P < 0.01.

[0038] Figure 8 shows an evaluation of the effects of freeze-shocked monocytes or macrophages loaded with attenuated Salmonella on common internal organs in vivo; Figure 8 shows representative H&E-stained close-up observations of heart, kidney, lung, and spleen sections from each group of H22 tumor-bearing mice after administration of different treatment methods (G0-G4) of the present invention. G0: saline group; G1: freeze-shocked RAW264.7 macrophages; G2: freeze-shocked attenuated Salmonella; G3: freeze-shocked macrophages loaded with attenuated Salmonella VNP20009; G4: freeze-shocked RAW264.7 macrophages simply mixed with attenuated Salmonella. Scale bar = 40 μm.

[0039] Figure 9 is a preliminary exploration of the mechanism of improving the biosafety performance of monocytes or macrophages loaded with attenuated Salmonella in vivo after freezing shock treatment of the present invention; Figure 9a is a representative flow cytometry comparison diagram (left) and bar chart (right) of the percentage changes of activated neutrophils (high expression of CD11b and low expression of CD62L) in the peripheral blood of H22 tumor-bearing mice 1 hour after administration of different treatment methods (G0-G4) of the present invention, G0: saline group; G1: macrophages RAW264.7 treated with freezing shock; G2: attenuated Salmonella treated with freezing shock; G3: freezing shock Treated macrophages loaded with attenuated Salmonella; G4: frozen shock treated macrophages RAW264.7 were simply mixed with attenuated Salmonella; ns = not significant, **P < 0.01, ***P < 0.001; Figure 9b is a schematic diagram of tumor-targeted delivery of VNP strains mediated by frozen shock macrophages RAW264.7 of the present invention; The protection of frozen shock treated macrophages RAW264.7 (cold-treated MACS) avoided neutrophil activation triggered by the exposed strain (red cross) and was able to target the release of intracellular strains in the tumor with the help of macrophages.

[0040] FIG10 is a graph showing the in vivo anti-tumor effect evaluation of monocytes or macrophages loaded with attenuated Salmonella treated with freezing shock of the present invention; FIG10a is a graph showing the statistical curves of tumor volume at specific time points (0 / 3 / 6 / 9 / 12 days) after administration of different treatment methods (G0-G4) of the present invention to H22 tumor-bearing mice, G0: saline group; G1: macrophages RAW264.7 treated with freezing shock; G2: attenuated Salmonella treated with freezing shock; G3: macrophages loaded with attenuated Salmonella treated with freezing shock; G4: macrophages RAW264.7 treated with freezing shock and attenuated Salmonella simply mixed; the following descriptions are the same for the groups; ns=not significant, *P<0.05; FIG10b is a graph showing the statistical curves of tumor volume at specific time points (0 / 3 / 6 / 9 / 12 days) after administration of different treatment methods (G0-G4) of the present invention to H22 tumor-bearing mice, G0: normal saline group; G1: macrophages RAW264.7 treated with freezing shock; G2: attenuated Salmonella treated with freezing shock; G3: macrophages loaded with attenuated Salmonella treated with freezing shock; G4: macrophages RAW264.7 treated with freezing shock and attenuated Salmonella simply mixed; the following descriptions are the same for the groups; ns=not significant, *P<0.05; FIG10b is a graph showing the statistical curves of tumor volume at specific time points (0 / 3 / 6 / 9 / 12 days) of different treatment methods (G0-G4) of the present invention Comparison of tumor doubling time of H22 tumor-bearing mice after administration of different treatment methods (G0-G4) of the present invention, *P<0.05; Figure 10c is a statistical graph of tumor weight of mice in each group 12 days after administration of different treatment methods (G0-G4) of the present invention to H22 tumor-bearing mice, ns=not significant, *P<0.05, **P<0.01; Figure 10d is a photograph of the tumor 12 days after administration of different treatment methods (G0-G4) of the present invention to H22 tumor-bearing mice; scale bar=10 mm; Figure 10e is a graph of mouse survival curves after administration of different treatment methods (G0-G4) of the present invention to H22 tumor-bearing mice. When the mice reached the humane endpoint, the mice were euthanized, ns=not significant, *P<0.05, ***P<0.001. DETAILED DESCRIPTION

[0041] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. However, it will be readily understood by those skilled in the art that the contents described in the examples are merely illustrative of the present invention and should not, and will not, limit the present invention described in detail in the claims. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall prevail.

[0042] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0043] The terms "first" and "second" are used solely for descriptive purposes to distinguish objects, such as substances, from one another and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the technical features being referred to. For example, a first XX could also be referred to as a second XX, and similarly, a second XX could also be referred to as a first XX, without departing from the scope of the embodiments of this application. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of such features.

[0044] In a first aspect, an embodiment of the present application provides a method for preparing freeze-shocked monocytes or macrophages loaded with attenuated Salmonella, comprising the following steps: the freeze-shocked monocytes or macrophages loaded with attenuated Salmonella are immortalized monocyte or macrophage cell lines (including THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, RAW264.7, J774.1, Ana-1, iBMDM, U937, etc.) and are co-cultured with attenuated Salmonella typhimurium at an MOI value of 1-100 to obtain engineered cells loaded with attenuated Salmonella, and then cold-shocked with liquid nitrogen to obtain freeze-shocked monocytes or macrophages loaded with attenuated Salmonella.

[0045] While the monocyte or macrophage cell lines lost their original pathogenic potential, the attenuated Salmonella inside the cells maintained biological activity and, after being released in vitro, reached a growth plateau within 24 hours. Compared to single injections of cold-shocked attenuated Salmonella, or a mixture of cold-shocked monocyte or macrophage cell lines and cold-shocked attenuated Salmonella, tumor growth was significantly suppressed and survival was significantly prolonged in mice after cryo-shocked monocytes or macrophages camouflaged and targeted delivery of the bacteria for tumor treatment.

[0046] The attenuated Salmonella typhimurium is attenuated Salmonella typhimurium VNP20009 and its genetically modified strains and synthetic biology modified strains capable of producing drug proteins (including but not limited to the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL20161094501 5.4, ​​ZL201610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929 .9, 202210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ); Monocyte / macrophage cell lines with immortalized proliferation include but are not limited to macrophage cell lines RAW264.7, J774.1, Ana-1, iBMDM, U937, etc., monocyte cell lines THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, etc.

[0047] Rapid freezing shock treatment with liquid nitrogen for 6-18 hours can eliminate the pathogenicity of monocyte or macrophage cell lines, but does not affect the tumor enrichment ability of the cells and the biological activity of intracellular attenuated Salmonella.

[0048] In some embodiments, a method for preparing freeze-shocked monocytes or macrophages loaded with attenuated Salmonella of the present invention comprises the following steps:

[0049] (1) Incubating either a monocyte cell line or a macrophage cell line with the attenuated Salmonella strain VNP20009:

[0050] The monocytes or macrophages with good growth conditions were cultured at a rate of 1-100×10 5Cells were inoculated into culture dishes at a ratio of 100 cells / well and cultured in antibiotic-free cell culture medium. Attenuated Salmonella monoclonal strains were picked from the agar plates and activated overnight in LB liquid medium. The bacterial solution grown to the logarithmic phase was centrifuged at 5000-8000 rpm for 5-10 minutes, the supernatant was discarded, and the pellet was resuspended in sterile saline. After adjusting the OD600 to 0.6-1.2, the bacteria were added to the culture dish containing the above cells at an MOI of 1-100 and incubated for 20-150 minutes.

[0051] (2) After staining the cell nucleus with Hoechst, observe the morphological changes of the cells under a microscope; record the changes in the percentage of disruption of the above-mentioned monocytes / macrophages at different time points of co-culture of cells and bacteria; after different time points, discard the supernatant, wash with sterile PBS 2 to 3 times, and then incubate with cell culture medium supplemented with 50-125μg / ml gentamicin for 20-60 minutes to kill the extracellular strain; discard the supernatant, wash with sterile PBS 2 to 3 times; collect the cells to obtain the live monocytes / macrophages of the above-mentioned type loaded with attenuated Salmonella strain; in order to detect the number of attenuated Salmonella effectively loaded in the monocytes / macrophages, use a cell counter to detect the number of live cells of the monocytes / macrophages loaded with attenuated Salmonella at different time points, and then use 0.5% Triton at room temperature. Lyse cells with X-100; dilute the lysate serially, plate on LB agar plates containing kanamycin, and incubate overnight at 37°C; count the number of viable bacteria in monocytes / macrophages;

[0052] (3) The live cells of the monocytes / macrophages loaded with attenuated Salmonella prepared above are resuspended in 500-1000 μl of serum-free cell freezing medium; the cell suspension is directly rapidly frozen in liquid nitrogen, placed for 6-18 hours, taken out, and thawed in a 37-42°C water bath to obtain freeze-shocked monocytes or macrophages loaded with attenuated Salmonella.

[0053] Rapid freezing shock treatment with liquid nitrogen for 6-18 hours can eliminate the pathogenicity of the cells, but does not affect the cells' tumor enrichment ability and the biological activity of intracellular attenuated Salmonella.

[0054] In a second aspect, the present application provides a method for preparing freeze-shocked monocytes or macrophages loaded with attenuated Salmonella, including freeze-shocked monocytes or macrophages loaded with attenuated Salmonella.

[0055] The third aspect of the embodiments of the present application provides a method for preparing a freeze-shock treated monocyte cell line or macrophage cell line loaded with attenuated Salmonella and its use in preparing an anti-tumor drug preparation.

[0056] The therapeutic dose of freeze-shocked monocytes or macrophages loaded with attenuated Salmonella is 0.4-40×10 6 The unit is cells / mouse, corresponding to the actual amount of attenuated Salmonella is 0.1-10×10 7 The unit is CFU / mouse. The freezing-shocked monocytes / macrophages loaded with attenuated Salmonella were administered only once, and the administration method was mainly intravenous injection.

[0057] The cryo-shock treated monocytes / macrophages loaded with attenuated Salmonella are mainly administered by intravenous injection for treatment. Depending on the type of tumor, they can also be administered by intraperitoneal injection or intratumoral injection.

[0058] Compared to single injections of cold-shocked attenuated Salmonella, or a combination of cold-shocked monocyte or macrophage cell lines and cold-shocked attenuated Salmonella, the use of cryo-shocked monocytes / macrophages for camouflaged protection and targeted delivery of the bacteria for tumor treatment significantly inhibited tumor growth and prolonged survival in mice. For example, using the macrophage RAW264.7 cell line, compared to single injections of cold-shocked attenuated Salmonella, cryo-shocked macrophages for camouflaged protection and targeted delivery of the bacteria achieved a 110.9% increase in tumor titer, a 16.3% reduction in off-target effects in normal organs, and a 90% reduction in liver lesion area.

[0059] The attenuated Salmonella typhimurium delivered and released to tumor tissues via the monocyte / macrophage cell lines is a key factor in exerting the anti-tumor efficacy of this type of drug.

[0060] VNP20009 and its genetically modified strains and synthetic biology engineered strains capable of producing pharmaceutical proteins (including but not limited to the aforementioned strains for which inventions have been applied for: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL20161 0945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 202 210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ), monocyte cell lines or macrophage cell lines with immortalized proliferation, including but not limited to macrophage cell lines RAW264.7, J774.1, Ana-1, iBMDM, U937, etc., monocyte cell lines THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, etc., are all suitable for such preparation and application schemes.

[0061] Example 1

[0062] Preparation of attenuated Salmonella loaded with monocyte or macrophage cell lines (cold-treated CELL / VNP) treated with liquid nitrogen freezing shock (hereinafter referred to as cold-treated)

[0063] (1.1) Preparation of Liquid Nitrogen Freezing Shock Treatment (hereinafter referred to as Cold Treatment) Attenuated Salmonella-Loaded Macrophages (Cold Treatment MACS / VNP)

[0064] To prepare macrophages loaded with attenuated Salmonella, the macrophage cell line RAW264.7 was co-incubated with the attenuated Salmonella strain. Specifically, RAW264.7 macrophages that have been well grown were cultured at a rate of 5×10 5Cells were inoculated into 6-well plates at a ratio of 100 cells / well and cultured in antibiotic-free cell culture medium. A monoclonal strain of attenuated Salmonella was picked from the agar plate and activated overnight in LB liquid medium. The bacterial suspension, which had grown to the logarithmic phase, was centrifuged at 8,000 rpm for 5 minutes. The supernatant was discarded and the pellet was resuspended in sterile saline. After adjusting the OD600 to 1.0, the bacteria were added to the 6-well plates containing the above cells (MOI 20) and co-cultured for different periods of time. The attenuated Salmonella strain can actively infect cells, and macrophages can also phagocytose the strain (Figures 1a, b).

[0065] To simultaneously observe morphological changes in macrophages during preparation, cell nuclei were stained with Hoechst (Solarbio, C0030, Beijing, China), and morphological changes in the cells were observed under a microscope. Changes in the percentage of macrophage disruption at different time points during co-culture of cells and bacteria were recorded. After different time points, the supernatant was discarded, the cells were washed 2-3 times with sterile PBS, and then incubated with cell culture medium supplemented with 75 μg / ml gentamicin for 30 minutes. Gentamycin can kill extracellular strains but has no significant effect on intracellular strains. The supernatant was discarded and the cells were washed 2-3 times with sterile PBS. Finally, the cells were collected to obtain live macrophages loaded with attenuated Salmonella strains (live MACS / VNP). Due to the self-protection mechanism of the VNP20009 strain, the strain loaded into the macrophages still maintained a certain degree of biological activity. To determine the number of attenuated Salmonella strains effectively loaded into macrophages, the number of viable MACS / VNP cells was measured at different time points using a cell counter. The cells were then lysed with 0.5% Triton X-100 at room temperature. The lysate was serially diluted, plated on LB agar plates supplemented with kanamycin, and incubated overnight at 37°C. The total number of viable bacteria loaded into the cells was counted. The number of viable strains loaded into the macrophages increased with increasing co-incubation time (Figure 1c). However, macrophage integrity was compromised with increasing bacterial load (Figures 1c, d), likely due to prolonged bacterial loading. Ultimately, after comprehensively considering strain loading and cell integrity, a co-incubation time of 60 minutes was selected as the optimal time point for preparing viable MACS / VNP cells, as this time achieved a high intracellular viable bacterial load (257 ± 27 strains per 100 cells) and high cell integrity (>90%) (Figure 1c).

[0066] Finally, the live MACS / VNP cells prepared above were resuspended in 500-1000 μL of serum-free cell freezing buffer. The cell suspension was quickly frozen directly in liquid nitrogen, placed for 6-18 hours, and then thawed in a 37-42°C water bath to obtain liquid nitrogen-cold-treated VNP20009 strain-loaded macrophages (cold-treated MACS / VNP cells) (Figure 1a).

[0067] The present invention also attempts to use VNP20009 and its genetically modified strains and synthetic biology modified strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0068] (1.2) Preparation of Liquid Nitrogen Freezing Shock Treatment (hereinafter referred to as Cold Treatment) Attenuated Salmonella-Loaded Macrophage Cell Line (Cold Treatment MACS / VNP)

[0069] To prepare macrophages loaded with attenuated Salmonella, follow the procedure described in (1.1) above and replace the macrophage cell line RAW264.7 with other macrophage cell lines J774.1, Ana-1, iBMDM, or U937 and co-incubate with attenuated Salmonella strains. Specifically, macrophages J774.1, Ana-1, iBMDM, or U937 that have good growth conditions were cultured at a rate of 5×10 5Cells were inoculated into 6-well plates at a ratio of 100 cells / well and cultured in antibiotic-free cell culture medium. A monoclonal attenuated Salmonella strain was isolated from the agar plate and activated overnight in LB liquid medium. Bacterial cultures grown to the logarithmic phase were centrifuged at 8,000 rpm for 5 minutes, the supernatant discarded, and the pellet resuspended in sterile saline. After adjusting the OD600 to 1.0, the bacteria were added to the 6-well plates containing the aforementioned cells (MOI 20) and incubated for varying periods of time. The attenuated Salmonella strain actively infects cells, and macrophages are also able to phagocytose the strain, with results similar to those in Figures 1a and 1b.

[0070] To simultaneously monitor macrophage morphological changes during preparation, cell nuclei were stained with Hoechst and observed under a microscope. Changes in the percentage of macrophage disruption were recorded at different time points during co-culture with bacteria. After various time points, the supernatant was discarded, the cells were washed two to three times with sterile PBS, and then incubated with cell culture medium supplemented with 75 μg / ml gentamicin for 30 minutes. The supernatant was discarded and the cells were washed two to three times with sterile PBS. The cells were harvested to obtain live macrophages loaded with attenuated Salmonella strains J774.1, Ana-1, iBMDM, or U937 (live MACS / VNP). The number of live MACS / VNP cells was determined at different time points using a cell counter. The cells were then lysed with 0.5% Triton X-100 at room temperature. The lysate was serially diluted, plated on LB agar plates supplemented with kanamycin, and incubated overnight at 37°C. The total number of live bacteria within the cells was counted. As the co-incubation time increased, the number of viable bacterial strains loaded into macrophages J774.1, Ana-1, iBMDM, or U937 cells increased. However, the integrity of the J774.1, Ana-1, iBMDM, or U937 macrophages was compromised as the bacterial load increased. The results were similar to those in Figures 1c, 1d in (1.1) above. A co-incubation time of 60 minutes was the optimal time point for preparing viable MACS / VNP cells, as this time achieved a high intracellular load of viable bacteria and maintained high cell integrity (>90%).

[0071] Resuspend the live MACS / VNP cells prepared above in 500-1000 μL of serum-free cell freezing buffer. Quickly freeze the cell suspension in liquid nitrogen, place it for 6-18 hours, remove it, and thaw it in a 37-42°C water bath to obtain liquid nitrogen-cold-treated attenuated Salmonella strain-loaded macrophages (cold-treated MACS / VNP cells).

[0072] The present invention also attempts to use VNP20009 and its genetically modified strains and synthetic biology modified strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0073] (1.3) Preparation of liquid nitrogen freezing shock treatment (hereinafter referred to as cold treatment) of attenuated Salmonella-loaded monocytic cell line (cold-treated MC / VNP)

[0074] To prepare monocytes loaded with attenuated Salmonella, the macrophage cell line RAW264.7 was replaced with the monocyte cell line THP1, or iBMMC, or J-111, or Mono-Mac-1, or JOSK-M and incubated with the attenuated Salmonella strain according to the above (1.1). Specifically, the monocyte THP1, or iBMMC, or J-111, or Mono-Mac-1, or JOSK-M cells that have been well grown were cultured at 5×10 5Cells were inoculated into 6-well plates at a ratio of 100 cells / well and cultured in antibiotic-free cell culture medium. A monoclonal strain of attenuated Salmonella was selected from the agar plate and activated overnight in LB liquid medium. The bacterial suspension, which had grown to the logarithmic phase, was centrifuged at 8000 rpm for 5 minutes. The supernatant was discarded and the pellet was resuspended in sterile saline. After adjusting the OD600 to 1.0, the bacteria were added to the 6-well plates containing the above cells (MOI 20) and co-cultured for different periods of time. The attenuated Salmonella strain actively infects cells, and monocytes can also phagocytose the strain, resulting in results similar to those in (1.1) and (1.2).

[0075] To simultaneously monitor morphological changes in monocytes during preparation, nuclei were stained with Hoechst and observed under a microscope. Changes in the percentage of monocyte disruption were recorded at different time points during co-culture with bacteria. After various time points, the supernatant was discarded, the cells were washed two to three times with sterile PBS, and then incubated with cell culture medium supplemented with 75 μg / ml gentamicin for 30 minutes. The supernatant was discarded and the cells were washed two to three times with sterile PBS. The cells were harvested to obtain viable monocytic THP1, iBMMC, J-111, Mono-Mac-1, or JOSK-M cells (viable MC / VNP) loaded with attenuated Salmonella strains. The number of viable MC / VNP cells was determined at different time points using a cell counter. The cells were then lysed with 0.5% Triton X-100 at room temperature. The lysate was serially diluted, plated on LB agar plates supplemented with kanamycin, and incubated overnight at 37°C. The total number of viable bacteria within the cells was counted. As the co-incubation time increased, the number of viable bacterial strains loaded into THP1 monocytic cells, or iBMMC, or J-111, or Mono-Mac-1, or JOSK-M increased. However, the integrity of THP1 monocytic cells, or iBMMC, or J-111, or Mono-Mac-1, or JOSK-M monocytic cells was destroyed as the bacterial load increased. The test results were similar to those in (1.1) and (1.2) above. A co-incubation time of 60 minutes was the optimal time point for preparing viable MC / VNP cells, at which the intracellular load of viable bacteria was high and the cell integrity was high (>90%).

[0076] Resuspend the viable MC / VNP cells prepared above in 500-1000 μL of serum-free cell freezing buffer. Quickly freeze the cell suspension in liquid nitrogen, place it for 6-18 hours, and thaw it in a 37-42°C water bath to obtain liquid nitrogen-cold-treated VNP20009 strain-loaded macrophages (cold-treated MC / VNP cells).

[0077] The present invention also attempts to use VNP20009 and its genetically modified strains and synthetic biology modified strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0078] Example 2

[0079] Evaluation of the bioactivity of cold-treated attenuated Salmonella virus loaded into monocyte / macrophage cell lines (CELL / VNP)

[0080] (2.1) Evaluation of the bioactivity of cold-treated attenuated Salmonella in macrophages

[0081] (2.1.1) Morphological observation of cold-treated MACS / VNP cells and intracellular strains

[0082] In order to compare the morphological changes of macrophages before and after liquid nitrogen cold treatment, the living MACS, living MACS / VNP and cold-treated MACS / VNP cells prepared above were photographed using fluorescence microscopy, scanning electron microscopy and transmission electron microscopy, respectively.

[0083] For observation of cell morphology under fluorescence microscopy, live MACS, live MACS / VNP, and cold-treated MACS / VNP cells were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, 648462, St. Louis, MO, USA). After washing three times with PBST, Actin-Tracker Green-488 (Biyuntian, C2201S) was added, incubated at 37°C for 1 hour, and stained with DAPI (Biyuntian, C1005). The VNP-RFP strain uses red fluorescent protein (RFP) for intracellular tracking, and images were obtained using a fluorescence microscope (Carl Zeiss, Axioplan 2, Oberkohen, Germany). For scanning electron microscopy (SEM) observation of cell morphology, live MACS, live MACS / VNP, and cold-treated MACS / VNP cells were prefixed in 2.5% isopropanol for 2 hours at room temperature and washed with 0.1 M phosphate buffer (pH 7.4). The samples were then suspended and embedded in 1% agarose, fixed with 1% osmium hydroxide in 0.1 M phosphate buffer (pH 7.4) for 2 hours at room temperature, and rinsed several times with 0.1 M phosphate buffer (pH 7.4) at room temperature. The samples were dehydrated through a series of graded ethanol solutions, dried in a critical point dryer, and finally coated with gold on a sputter coater for scanning electron microscopy. For transmission electron microscopy (TEM) observation of cell morphology, live MACS, live MACS / VNP, and cold-treated MACS / VNP cells were prefixed in 2.5% isopropanol for 2-4 hours at 4°C and washed with 0.1 M phosphate buffer (pH 7.4). The samples were then suspended and embedded in 1% agarose, fixed with 1% osmium hydroxide in 0.1 M phosphate buffer (pH 7.4) for 2 hours, and rinsed several times with 0.1 M phosphate buffer (pH 7.4) at room temperature. After fixation, the samples were dehydrated with a series of graded ethanol, embedded in Epon812, polymerized in a 60°C oven for 48 hours, and sliced ​​with an ultramicrotome to a thickness of 60-80 nm. TEM observations were performed using a double staining of 2% uranyl acetate and 2.6% lead citrate.

[0084] Fluorescence microscopy (Figure 2a) and scanning electron microscopy (Figure 2b) showed that neither intracellular bacterial strain loading nor liquid nitrogen cryotreatment significantly affected the integrity of the cells, and intact bacterial strains could be observed within the cells (Figure 2b). Therefore, it can be speculated that this cell-loading and then delivery strategy achieves effective protection because bacterial xenobiotics are no longer directly exposed to the environment in the organism.

[0085] The above-mentioned macrophages refer to RAW264.7, J774.1, Ana-1, iBMDM, U937 and other macrophages. The results of J774.1, Ana-1, iBMDM, and U937 are similar to those of RAW264.7.

[0086] The present invention also attempts to use VNP20009 and its genetically modified strains and synthetic biology modified strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0087] (2.1.2) Detection of intracellular bacterial strain activity and release in cold-treated MACS / VNP cells

[0088] Bacteria can acquire cold resistance by regulating their cell membrane composition and the expression of cold shock proteins. Next, the bioactivity of the intracellular strains in cold-treated MACS / VNP cells was examined. Equal numbers of live MACS / VNP cells prepared according to the above method and cold-treated MACS / VNP cells were lysed with 0.5% Triton X-100 at room temperature. The lysates were serially diluted and plated on LB agar plates supplemented with kanamycin. The plates were incubated overnight at 37°C. The number of monoclonal strains on each plate was counted and compared. The results showed that liquid nitrogen cold treatment did not affect the activity of the intracellular strains, which rapidly recovered their original bioactivity after the conditions were restored (Figure 2c). Subsequently, live MACS / VNP-RFP and cold-treated MACS / VNP-RFP cells were prepared according to the above method using the VNP20009 strain (VNP-RFP) expressing the RFP red fluorescent protein. The prepared cells were plated onto 96-well plates (1×10 4 cells / 200μL / well), and the RFP fluorescence intensity (excitation light 550nm, emission light 585nm) in each well was measured by a microplate reader as the incubation time changed. Generally, the total fluorescence intensity detected is positively correlated with the number of strains in the well. At the third hour of incubation, significant strain proliferation was detected in the cold-treated MACS / VNP-RFP cell group (Figure 2d). The release of the strain may be due to the continued proliferation and movement of the intracellular strain. Cold-treated MACS / VNP cells incubated in vitro for different times (0 / 2 / 4h) were obtained, fixed with 4% paraformaldehyde, and transmission electron microscopy was used to photograph the changes in the state of the intracellular strains according to the method described above. The "release of intracellular bacteria through cells" photographed at 2 hours of incubation and the "extracellular proliferation of bacteria" subsequently photographed at 4 hours of incubation can be more intuitively observed (Figure 2e).

[0089] These results confirm that the attenuated Salmonella strain within the cold-treated MACS / VNP cells retains its biological activity and can be released from the cells under appropriate conditions, rapidly proliferating. Compared to the live MACS / VNP group, the cold-treated MACS / VNP group exhibited earlier strain release, suggesting that anti-tumor therapy with the live MACS / VNP group may achieve more rapid strain release and efficacy (Figure 2c).

[0090] To examine the proliferation activity and morphological changes of attenuated Salmonella strains released from cold-treated MACS / VNP cells, cold-treated MACS / VNP cells were obtained and lysed with 0.5% Triton X-100 to release the intracellular strain. The VNP strain in the supernatant was collected and designated the released strain. Subsequently, the growth curves of the normal and released strains on LB medium were analyzed using Bioscreen C software (OY Growth 175 Curves Ab Ltd., Finland). Briefly, 1 mL of LB medium was infected with 10 μL of an attenuated Salmonella suspension (OD600 = 1.0), and 300 μL of the solution was added to each well of a Bioscreen C multiwell plate. The plates were incubated at 37°C for 30 hours. OD values ​​were measured every 30 minutes using a 600 nm brown filter. Simultaneously, fixed samples of the normal and released strains were obtained according to the preparation method described above and imaged under a scanning electron microscope. The results confirmed that the normal strain and the released strain had similar growth characteristics, reaching a plateau phase in approximately 24 hours (Figure 3a). There was also no significant difference in the appearance of the two strains (Figure 3b).

[0091] Salmonella can achieve cell invasion and motility through its flagella, thereby inducing apoptosis or pyroptosis in infected cells. Next, the ability of attenuated Salmonella strains released from cold-treated MACS / VNP cells to infect tumor cells and induce apoptosis was examined. To test bacterial infection ability, H22 tumor cells (mouse hepatoma cells) were infected with either the normal VNP strain or the released VNP strain at an MOI of 100 for 1 hour. After infection, the cells were washed with PBS and treated with 75 μg / mL gentamicin for 30 minutes to remove any extracellular residual strain. The cells were lysed with 0.5% Triton X-100, and the amount of internalized VNP strain was determined by diluting the cell lysate and plating it on LB plates. To test bacterial apoptosis-inducing ability, tumor cells (2.0 × 10⁵) were seeded onto 12-well plates and cultured adherently for 6–8 hours. Then, either the normal attenuated Salmonella strain or the released attenuated Salmonella strain was collected and co-incubated with the cells at an MOI of 100 for 4 hours. All cells in the plate were removed, washed, and resuspended in binding buffer, and then stained with 1 μg of laboratory-made APC-coupled Annexin V protein and incubated on ice for 30 minutes in the dark. Finally, 1 μL of propidium iodide (PI, 25g / mL) was added to all prepared samples, gently mixed, and detected by flow cytometry. The results showed that normal attenuated Salmonella or released attenuated Salmonella strains can effectively infect tumor cells, and there is no significant difference in the infection efficiency between the two (Figure 3c). In addition, compared with less than 5% apoptotic cells in the normal saline group, normal VNPs or released attenuated Salmonella strains can induce significant apoptosis in tumor cells, and the apoptosis level is higher than 15%, while there is no significant difference between the two (Figure 3d).

[0092] These results indicate that the attenuated Salmonella strains released intracellularly have no significant differences from normal attenuated Salmonella strains in terms of growth rate, strain morphology, infection, and induction of tumor cell necrosis. Therefore, these released attenuated Salmonella strains, when present in tumors, can reshape the tumor microenvironment in a classic manner and promote tumor regression. In addition, to achieve better delivery and therapeutic effects, it is recommended that the cold-treated MACS / VNP cells freshly obtained from liquid nitrogen be incubated in culture medium at 37°C for 10-20 minutes to simply restore the biological activity of the intracellular strains before use in subsequent research / treatment.

[0093] The above-mentioned macrophages refer to RAW264.7, J774.1, Ana-1, iBMDM, U937 and other macrophages. The results of J774.1, Ana-1, iBMDM, and U937 are similar to those of RAW264.7.

[0094] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0095] (2.2) Evaluation of the bioactivity of cold-treated attenuated Salmonella in mononuclear cells

[0096] (2.2.1) Morphological observation of cold-treated MC / VNP cells and intracellular strains

[0097] In order to compare the morphological changes of monocytes (MC) before and after liquid nitrogen cold treatment, the living MC, living MC / VNP and cold-treated MC / VNP cells prepared above were photographed using fluorescence microscopy, scanning electron microscopy and transmission electron microscopy, respectively.

[0098] For observation of cell morphology under fluorescence microscopy, live MC, live MC / VNP, and cold-treated MC / VNP cells were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 0.5% Triton X-100. After washing three times with PBST, Actin-Tracker Green-488 was added, incubated at 37°C for 1 hour, and stained with DAPI. The VNP-RFP strain uses red fluorescent protein RFP for intracellular tracking and is observed using a fluorescence microscope. For observation of cell morphology under scanning electron microscopy, live MC, live MC / VNP, and cold-treated MC / VNP cells were pre-fixed in 2.5% isopropanol for 2 hours at room temperature and washed with 0.1M phosphate buffer (pH 7.4). The samples were then suspended and embedded in 1% agarose, fixed with 1% osmium hydroxide in 0.1M phosphate buffer (pH 7.4) for 2 hours, and rinsed several times with 0.1M phosphate buffer (pH 7.4) at room temperature. After dehydration through a series of graded ethanol solutions, the samples were dried in a critical point dryer and finally coated with gold using a sputter coater for scanning electron microscopy. For transmission electron microscopy observation of cell morphology, live MC, live MC / VNP, and cold-treated MC / VNP cells were prefixed in 2.5% isopropanol at 4°C for 2–4 hours and washed with 0.1 M phosphate buffer (pH 7.4). The samples were then suspended and embedded in 1% agarose, fixed with 1% osmium hydroxide in 0.1 M phosphate buffer (pH 7.4) for 2 hours, and rinsed several times with 0.1 M phosphate buffer (pH 7.4) at room temperature. After fixation, the samples were dehydrated through a series of graded ethanol solutions, embedded in Epon 812, oven-polymerized at 60°C for 48 hours, and sectioned with an ultramicrotome at a thickness of 60–80 nm. TEM observations were performed using a double staining with 2% uranyl acetate and 2.6% lead citrate.

[0099] The results of fluorescence microscopy and scanning electron microscopy observations were similar to those in (2.1.1) and (2.1.2) above. Neither the loading of intracellular strains nor the liquid nitrogen cryotreatment significantly affected the integrity of the cells, and the complete strains could be observed in the cells.

[0100] The above-mentioned monocytes are THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, etc.

[0101] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that all VNP20009 genetically modified strains and synthetic biology engineered strains capable of producing pharmaceutical proteins exhibited the same results as the VNP20009 chassis strain. (2.2.2) Intracellular activity and release assays of the strains in cold-treated MC / VNP cells.

[0102] Bacteria can acquire cold resistance by regulating their cell membrane composition and the expression of cold shock proteins. Next, the bioactivity of the intracellular strains in cold-treated MC / VNP cells was tested. Equal numbers of live MC / VNP cells prepared according to the above method and cold-treated MC / VNP cells were taken and lysed with 0.5% Triton X-100 at room temperature. The lysates were diluted serially and plated on LB agar plates supplemented with kanamycin. The plates were incubated overnight at 37°C. The number of monoclonal strains on each plate was counted and compared. The results were similar to those in (2.1.2) above, indicating that liquid nitrogen cold treatment did not affect the activity of the intracellular strains. These intracellular strains quickly recovered their original bioactivity after the conditions were restored. Using an attenuated Salmonella strain expressing the RFP red fluorescent protein (VNP-RFP), live MC / VNP-RFP and cold-treated MC / VNP-RFP cells were prepared according to the above method. The two prepared cells were plated into 96-well plates (1×10 4cells / 200μL / well), and the RFP fluorescence intensity (excitation light 550nm, emission light 585nm) in each well was measured by a microplate reader as the incubation time changed. Generally, the total fluorescence intensity detected is positively correlated with the number of strains in the well. At the third hour of incubation, significant strain proliferation was detected in the cold-treated MC / VNP-RFP cell group, and the results were similar to those in (1.2) above. The release of strains may be due to the continued proliferation and movement of intracellular strains. Cold-treated MC / VNP cells incubated in vitro for different times (0 / 2 / 4h) were obtained, fixed with 4% paraformaldehyde, and transmission electron microscopy was used to photograph the changes in the status of intracellular strains according to the method described above. The "release of intracellular bacteria through cells" after incubation for 2 hours and the "extracellular proliferation of bacteria" after incubation for 4 hours were visually observed, and the results were similar to those in (2.1.2) above.

[0103] The results confirmed that the attenuated Salmonella strain within the cold-treated MC / VNP cells retained its bioactivity and could be released from the cells under suitable conditions, rapidly proliferating. The release of the strain from the cold-treated MC / VNP group was earlier than that from the live MC / VNP group.

[0104] To examine the proliferation activity and morphological changes of attenuated Salmonella strains released from cold-treated MC / VNP cells, cold-treated MC / VNP cells were obtained and lysed with 0.5% Triton X-100 to release the intracellular strains. The attenuated Salmonella strains in the supernatant were collected and designated the released strain. Subsequently, the growth curves of the normal and released strains on LB medium were analyzed using Bioscreen C software. Briefly, 1 mL of LB medium was infected with 10 μL of a VNP20009 suspension (OD600 = 1.0), and 300 μL of the solution was added to each well of a Bioscreen C multiwell plate. The plates were incubated at 37°C for 30 hours. OD values ​​were measured every 30 minutes using a 600 nm brown filter. Simultaneously, fixed samples of the normal and released strains were obtained using the preparation method described above and imaged under a scanning electron microscope. The results were similar to those in (2.1.2) above: the normal and released strains exhibited similar growth characteristics, reaching a plateau phase within approximately 24 hours. There was no significant difference in the appearance of the two strains.

[0105] Salmonella can achieve cell infection and motility through its flagella, thereby inducing apoptosis or pyroptosis of infected cells. Next, the changes in the ability of attenuated Salmonella strains released by cold-treated MS / VNP cells to infect tumor cells and induce their apoptosis were detected. For the detection of bacterial infection ability, normal attenuated Salmonella strains and released attenuated Salmonella strains were used to infect tumor cells (B16F10, LLC, 4T1, A20, H22) at an MOI of 100 for 1 hour. After infection, the cells were washed with PBS and then treated with 75μg / mL gentamicin for 30 minutes to remove extracellular residual strains. The cells were lysed with 0.5% Triton X-100, and the cell lysate was diluted and spread on LB plates to determine the number of internalized VNP strains. For the detection of bacterial apoptosis-inducing ability, tumor cells (2.0×10 5 ) were inoculated onto 12-well plates and cultured for 6–8 hours. Then, normal attenuated Salmonella or released attenuated Salmonella strains were collected and co-cultured with the cells at an MOI of 100 for 4 hours. All cells in the plates were removed, washed, and resuspended in binding buffer. They were then stained with 1 μg of laboratory-made APC-conjugated Annexin V protein and incubated on ice for 30 minutes in the dark. Finally, 1 μL of propidium iodide (PI, 25 μg / mL) was added to all prepared samples, gently mixed, and analyzed by flow cytometry. The results were similar to those in (1.2) above. Both normal attenuated Salmonella and released attenuated Salmonella strains effectively infected tumor cells, with no significant difference in infection efficiency between the two groups. Compared to the saline group, which had less than 5% apoptotic cells, both normal attenuated Salmonella and released attenuated Salmonella strains induced significant apoptosis in tumor cells, with apoptosis levels exceeding 15%, with no significant difference between the two groups.

[0106] These results are similar to those in (2.1.2) above, showing that the intracellularly released attenuated Salmonella strains showed no significant differences from normal attenuated Salmonella strains in growth rate, strain morphology, infection, and induction of tumor cell necrosis. Therefore, these released attenuated Salmonella strains, when present in tumors, can reshape the tumor microenvironment in a classic manner, promoting tumor regression.

[0107] The above-mentioned monocytes are THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, etc.

[0108] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0109] Example 3

[0110] Cellular bioactivity assay of cold-treated attenuated Salmonella loaded with monocyte or macrophage cell lines (CELL / VNP)

[0111] (3.1) Cellular bioactivity assay of macrophages loaded with cold-treated attenuated Salmonella

[0112] (3.1.1) Cell proliferation capacity detection in vivo and in vitro

[0113] Generally, immortalized cell lines have the ability to proliferate rapidly, so when using such cells as carriers, it is necessary to consider the potential toxic side effects of their strong proliferative ability on the body. Next, the in vivo and in vitro proliferation activity of liquid nitrogen-cold-treated macrophage cell lines was tested. For in vitro proliferation assays, freshly prepared, healthy, live MACS, cold-treated MACS, and cold-treated MACS / VNP cells were selected and plated at 5×10 cells per well. 3Cells were seeded into 96-well plates, 100 μL of DMEM complete medium was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator. Microplate reader (BioTek) was used for detection at specific time points. Five replicate wells were set up for each group at each time point. At a specific time, the original culture medium in the 96-well plate was replaced with freshly prepared DMEM medium containing 10% CCK-8 solution (Biyuntian Biotechnology Co., Ltd., C0039), and the culture was continued for 2 hours. The 96-well plate was removed and detected using a microplate reader. The absorbance value of each group of cells was measured at 450 nm, and the data results of each replicate well at 0h, 12h, 24h, 36h and 48h were recorded. The test results showed that compared with the rapid proliferation of live MACS cells (proliferation of ~75% within 24 hours and ~163% within 48 hours), cold-treated MACS and cold-treated MACS / VNP cells did not proliferate significantly within 48 hours. Therefore, it can be basically inferred that cold shock treatment causes the macrophage cell line to lose its proliferative activity (Figure 4a). Subsequently, BALB / c mice were used for in vivo pathogenicity experiments. Live MACS or cold-treated MACS cells were subcutaneously injected into the right armpit of BALB / c mice (1×10 6 cells), and photographs were taken every 1-2 days to observe whether a lump formed in the armpit. The results showed that a lump was observed on the 5th day after the injection of live MACS cells, and the lump continued to grow over time (Figure 4b). In contrast, no observable lump was found in mice injected with cold-treated MACS cells, and no obvious lump was observed after autopsy on the 14th day (Figure 4c). This shows that, consistent with the in vitro test results, live MACS cells can continue to proliferate in vivo, while cold-treated MACS cells do not have the activity of re-proliferation in vivo.

[0114] In traditional cell therapy, rapidly and conveniently obtaining sufficient cells for patient treatment remains a major challenge. Liquid nitrogen-treated macrophage cell lines, however, eliminate their pathogenicity while ensuring rapid and large-scale availability. Therefore, it is speculated that liquid nitrogen-treated macrophage cell lines will have broader application value.

[0115] The above-mentioned macrophages refer to RAW264.7, J774.1, Ana-1, iBMDM, U937 and other macrophages. The results of J774.1, Ana-1, iBMDM, and U937 are similar to those of RAW264.7.

[0116] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0117] (3.1.2) Detection of tumor-targeting enrichment ability in cells

[0118] In the inventors' previous publications and patent applications related to anti-tumor research using attenuated Salmonella, the most frequently used tumor models were B16F10 (melanoma), LLC (lung cancer cells), 4T1 (breast cancer cells), and A20 (lymphoma cells), respectively; H22 (hepatocellular carcinoma cells) was the least frequently used tumor model. The present invention utilizes five tumor models: B16F10, LLC, 4T1, A20, and H22.

[0119] Establishment of a subcutaneous tumor-bearing mouse model

[0120] After the tumor cells were grown in DMEM cell culture medium to the exponential growth phase, the cells were collected and injected intraperitoneally into Balb / c mice (6-week-old, female, 2×10 6 After 3-5 days of abdominal swelling, the mice were euthanized and H22 cells were collected from the peritoneal fluid. The obtained tumor cells were washed 2-3 times with PBS and then resuspended in PBS to adjust the final cell concentration to 1×10 7Each Balb / c mouse was inoculated with 100 μL of the virus into the axillary fat pad, i.e., 1×10 6 After inoculation, the mice were kept in a clean animal room until the tumor volume of the mice grew to about 80-120 mm. 3 Follow-up experiments were conducted.

[0121] Administration: Tumor-bearing mice were randomly divided into groups and treated with the near-infrared (NIR) fluorescent dye DIR (Abbkine, BMD0074, Wuhan, China) for 30 minutes to produce DIR-labeled cells. DIR-labeled cold-treated cells were fixed with 4% paraformaldehyde at room temperature for 1 hour to denature and inactivate cell surface proteins. Fixed cold-treated cells were used as controls. The above cells were administered once via tail vein injection at a dose of 1.0 × 10 7 cells / cells) and / or cells (4.0×10 6 The VNP20009 strain used the VNP-LuxCDABE strain constructed earlier in the laboratory, which can spontaneously generate bioluminescence for in vivo tracing.

[0122] The cells were injected into tumor-bearing mice via the tail vein. III, Waltham, MA, USA) detected the fluorescence signals of LuxCDABE and DIR in mouse tumors after treatment with different methods. Significant DIR signals were observed in the cold-treated MACS+VNP and cold-treated MACS / VNP cell groups 8 hours after injection, while the DIR signals in the cold-treated MACS / VNP cell group after fixation were weak. In addition, the LuxCDABE in the cold-treated MACS / VNP cell group was significantly higher than that in the other two groups, suggesting that it corresponds to a higher strain titer (Figure 5a). This shows that the tumor-targeted delivery of the strain by cold-treated macrophages does achieve more effective intratumoral enrichment of the strain through the tumor-targeting effect of macrophages.

[0123] The changes in protein content on the surface of cold-treated cells were then detected. Cell adhesion molecules such as CD11b and chemokine receptors such as CCR2 play a vital role in macrophage anchoring to tumor sites. To test whether CD11b and CCR2 still exist on the cell surface, live MAC, live MACS / VNP and cold-treated MACS / VNP cells were collected and analyzed for the expression of CD11b and CCR2 on the cell surface using fluorescence microscopy and flow cytometry. For fluorescence microscopy analysis, the above cells were resuspended in 1% BSA and incubated with protein-specific antibodies, including CD11b antibody (ABclonal, A1581, Wuhan, China) and CCR2 antibody (Proteintech, 16153-1-AP, Wuhan, China). After washing two to three times with PBST, the primary antibody was labeled with a FITC-conjugated fluorescent secondary antibody (Absin, Asp20004, Shanghai, China), and labeling was observed using a fluorescence microscope (Carl Zeiss, Axioplan 2, Oberkohen, Germany). For flow cytometric analysis, the cells were resuspended in cell staining buffer and stained with CD11b-APC (BD, 553312) and CCR2-AF647 (Biolegend, clone SA203G11), respectively, at 4°C in the dark for 30 minutes. The precipitate was collected by centrifugation and washed one to two times with PBS. After resuspending in PBS, cell surface fluorescence was analyzed by flow cytometry. The results showed that high levels of CD11b and CCR2 protein were detected on the surface of live MACS, live MACS / VNP, and cold-treated MACS / VNP cells (Figures 5b, c).

[0124] Surface integrins and chemokine receptors, including CD11b and CCR2, coupled with the enhanced capture effect of disordered and tortuous micrometer-diameter tumor capillaries, may be important factors in achieving the efficient accumulation of cold-treated MACS / VNP cells at tumor sites. In summary, these results confirm that the liquid nitrogen-cold-treated macrophages described in this invention can effectively enrich in tumor areas and achieve effective anti-tumor bacterial delivery.

[0125] The above-mentioned macrophages refer to RAW264.7, J774.1, Ana-1, iBMDM, U937 and other macrophages. The results of J774.1, Ana-1, iBMDM, and U937 are similar to those of RAW264.7.

[0126] The results of B16F10, LLC, 4T1, and A20 tumor models were very similar to those of the H22 tumor model.

[0127] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0128] (3.2) Cellular bioactivity assay of cold-treated attenuated Salmonella loaded into monocytes

[0129] (3.2.1) Cell proliferation capacity detection in vivo and in vitro

[0130] Generally, immortalized cell lines have the ability to proliferate rapidly. Therefore, when using such cells as carriers, the potential toxic side effects of their strong proliferative ability on the body must be considered. Next, the in vivo and in vitro proliferation activity of liquid nitrogen-cold-treated monocytic cell lines was tested. For in vitro proliferation assays, freshly prepared, healthy, live MCs, cold-treated MCs, and cold-treated MC / VNP cells were selected and plated at 5×10 cells per well. 3Cells were seeded into 96-well plates, and 100 μL of complete DMEM medium was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator and analyzed using a microplate reader at specific time points. Five replicate wells were set up for each group at each time point. At the specified time, the original medium in the 96-well plate was replaced with fresh DMEM medium containing 10% CCK-8 solution, and the culture was continued for 2 hours. The 96-well plates were removed and analyzed using a microplate reader. The absorbance of each cell group was measured at 450 nm. The data for each replicate well were recorded at 0, 12, 24, 36, and 48 hours. The results were similar to those in (3.1.1) above. Compared with the rapid proliferation of live MC cells (approximately 75% within 24 hours and 160% within 48 hours), cold-treated MC and cold-treated MC / VNP cells showed no significant proliferation within 48 hours. Cold shock treatment abolished the proliferative activity of the macrophage cell line.

[0131] BALB / c mice were used for in vivo pathogenicity experiments. Live MC or cold-treated MC cells were subcutaneously injected into the right axilla of BALB / c mice (1×10 6 Mice injected with cold-treated MACS cells showed no observable lumps, and no obvious lumps were observed after autopsy on day 14. While live MC cells can continuously proliferate in vivo, cold-treated MACS cells lack the ability to regenerate in vivo.

[0132] Liquid nitrogen-treated monocytic cell lines lose their original pathogenicity while ensuring easy, rapid, and large-scale availability. Therefore, liquid nitrogen-treated monocytic cell lines will have a wider range of applications.

[0133] The above-mentioned monocytes are THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, etc.

[0134] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0135] (3.2.2) Detection of tumor-targeting enrichment ability in cells

[0136] The present invention uses five tumor models: B16F10, LLC, 4T1, A20, and H22.

[0137] Establishment of a subcutaneous tumor-bearing mouse model

[0138] After the tumor cells were grown in DMEM cell culture medium to the exponential growth phase, the cells were collected and injected intraperitoneally into Balb / c mice (6-week-old, female, 2×10 6 After 3-5 days of abdominal swelling, the mice were euthanized and H22 cells were collected from the peritoneal fluid. The obtained tumor cells were washed 2-3 times with PBS and then resuspended in PBS to adjust the final cell concentration to 1×10 7 Each Balb / c mouse was inoculated with 100 μL of the virus into the axillary fat pad, i.e., 1×10 6 After inoculation, the mice were kept in a clean animal room until the tumor volume of the mice grew to about 80-120 mm. 3 Follow-up experiments were conducted.

[0139] Administration: Tumor-bearing mice were randomly divided into groups and treated with the near-infrared (NIR) fluorescent dye DIR for 30 minutes to produce DIR-labeled cells. The DIR-labeled cold-treated cells were fixed with 4% paraformaldehyde at room temperature for 1 hour to denature and inactivate cell surface proteins. Fixed cold-treated cells were used as controls. The different cells were administered once via tail vein injection at a dose of 1.0×10 7 cells / cells) and / or cells (4.0×10 6 The attenuated Salmonella strains used were laboratory-constructed VNP-LuxCDABE strains that can spontaneously generate bioluminescence for in vivo tracing.

[0140] The above cells were injected into tumor-bearing mice via the tail vein. The fluorescence signals of LuxCDABE and DIR in the mouse tumors after treatment in different ways were detected using an in vivo imaging system. 8 hours after injection, the results were similar to those in (3.1.2) above. Significant DIR signals were observed in the cold-treated MC+VNP and cold-treated MC / VNP cell groups, while the DIR signals in the corresponding cold-treated MC / VNP cell group after fixation were weak. The LuxCDABE in the cold-treated MC / VNP cell group was significantly higher than that in the other two groups, suggesting that it corresponds to a higher strain titer. The targeted delivery of strains to tumors by cold-treated macrophages indeed achieved more effective intratumoral enrichment of the strains through the tumor-targeting effect of monocytes.

[0141] Subsequently, the protein expression changes on the surface of cold-treated cells were examined using the same method as described above (3.1.2). Monocytes (such as THP1, iBMMC, J-111, Mono-Mac-1, or JOSK-M), like macrophages, also express cell adhesion molecules such as CD11b and chemokine receptors such as CCR2. To test whether CD11b and CCR2 are still present on the cell surface, live MCs, live MC / VNPs, and cold-treated MC / VNPs were collected and analyzed for cell surface expression of CD11b and CCR2 using fluorescence microscopy and flow cytometry. For fluorescence microscopy analysis, the cells were resuspended in 1% BSA and incubated with protein-specific antibodies, including CD11b and CCR2. After washing two to three times with PBST, the primary antibody was labeled with a FITC-conjugated fluorescent secondary antibody, and labeling was observed using fluorescence microscopy. For flow cytometric analysis, the cells were resuspended in cell staining buffer and stained with CD11b-APC and CCR2-AF647, respectively, at 4°C in the dark for 30 minutes. The pellet was collected by centrifugation and washed one to two times with PBS. After resuspending in PBS, cell surface fluorescence was analyzed by flow cytometry. The results were similar to those in (3.1.2) above, with high levels of CD11b and CCR2 protein detected on the cell surface of live MCs, live MC / VNPs, and cold-treated MC / VNPs.

[0142] These results demonstrate that the liquid nitrogen-cooled monocytes described in the present invention can be effectively enriched in tumor areas and achieve effective anti-tumor bacterial delivery.

[0143] The above-mentioned monocytes are THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, etc.

[0144] The results of B16F10, LLC, 4T1, and A20 tumor models were similar to those of the H22 tumor model.

[0145] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0146] Example 4

[0147] Anticancer Biosafety Assessment of Cold-treated Attenuated Salmonella Loaded with Monocytes / Macrophages (CELL / VNP)

[0148] (4.1) Anticancer Biosafety Assessment of Cold-treated Attenuated Salmonella Loaded Macrophages

[0149] The present invention uses five tumor models: B16F10, LLC, 4T1, A20, and H22.

[0150] The establishment of a subcutaneous tumor cell-bearing mouse model was carried out according to the description in Example 3.

[0151] Administration: Tumor-bearing mice were randomly divided into groups and injected with different cells once via tail vein. The dosage was cold-treated VNP strain (1.0×10 7 cells / cells) and / or cells (4.0×10 6 The mice were injected with cold-treated MACS, cold-treated VNP, cold-treated MACS / VNP, and cold-treated MACS + cold-treated VNP (a simple mixture of the two). Only saline was injected as a negative control.

[0152] Tissue distribution of bacteria in tumor-bearing mice

[0153] The above drugs were administered to tumor-bearing mice for 8 hours, 1 day, 3 days, 6 days, and 12 days. The mice were randomly sacrificed and the tumors and other organs (including the heart, liver, spleen, lungs, and kidneys) were removed from the tumor-bearing mice under a sterile environment. The organs were weighed and homogenized in 2 mL of PBS using a tissue homogenizer (frequency: 60 Hz, time: 100 seconds). Different tissues were diluted in different gradients and spread on LB plates and incubated in an inverted incubator at 37°C for 12 hours. Colony counts were performed and the number of VNP20009 strains in mouse tissues was compared and analyzed between the different groups.

[0154] In the present invention, the titer of bacteria in heart, liver, spleen, lung, kidney and tumor tissues was detected. High enrichment of strains in tumors was observed in the three groups of cold-treated MACS, cold-treated attenuated Salmonella strains, cold-treated MACS / VNP and cold-treated MACS+cold-treated attenuated Salmonella, and varying degrees of enrichment were also observed in normal organs. Among them, three groups reached the highest relevant intratumoral strain titer on the 6th day. Therefore, the 6th day is used as a representative time point for emphasizing the description. In the cold-treated VNP strain group, the bacterial titer in normal organs (including heart, liver, spleen, lung, and kidney) was 4.664, and the bacterial titer in tumors was 7.044; in the cold-treated MACS / VNP group, the bacterial titer in normal organs was 3.903, and the bacterial titer in tumors was 7.813; in the cold-treated MACS+cold-treated VNP group, the bacterial titer in normal organs was 4.474, and the bacterial titer in tumors was 6.938 (Figure 6a-g). It can be seen that there was no significant difference in bacterial titers in normal organs and tumor tissues between the cold-treated attenuated Salmonella strain group and the cold-treated MACS+cold-treated VNP group. Compared with the above two groups, the bacterial titer in normal organs of the cold-treated MACS / VNP group was significantly reduced, while the bacterial titer in tumor tissue was significantly increased. This difference between the three groups also existed at other time points. It can be seen that loading and re-delivery with the help of cold-treated MACS cells can indeed enhance the tumor targeting of the attenuated Salmonella strain and reduce off-target effects on normal organs. However, this effect cannot be achieved by simply mixing cold-treated MACS cells with cold-treated VNP strains. This highlights the effectiveness of the strategy of the present invention.

[0155] Acute toxicity assessment: Five groups of the drug (normal saline, cold-treated MACS, cold-treated attenuated Salmonella VNP strain, cold-treated MACS / VNP, and cold-treated MACS plus cold-treated attenuated Salmonella VNP) were administered intravenously to H22 tumor-bearing mice. One day later, orbital bleeding (approximately 200 μL) was collected. Whole blood was allowed to stand at room temperature for 30 minutes and then centrifuged at 3000 rpm for 15 minutes. The supernatant serum was collected for IL-6 / IL-10 cytokine analysis and blood biochemical analysis including ALT and AST. Mouse cytokine detection kits were used: IL-6 kit (BYabscience, BY-EM220188, Nanjing, China) and IL-10 kit (Liankebio, EK210, Hangzhou, China). Normal organs (including heart, liver, spleen, lungs, and kidneys) were dissected and photographed and stained with hematoxylin and eosin (Figure 7a).

[0156] The results showed that both the cold-treated VNP strain and the cold-treated MACS+cold-treated VNP group induced significant liver damage, as evidenced by the appearance of distinct foci on the liver surface (Figure 7b) and distinct necrotic areas observed by HE staining (Figure 7c). Cytokines (IL-6 / IL-10) and biochemical markers (ALT / AST), which are positively correlated with the degree of inflammation in the body, were significantly elevated (Figures 7e, f). In contrast, all of the above injury indicators were significantly alleviated in the cold-treated MACS / VNP group. Specifically, the average number of acute liver lesions in the normal saline group was 0, the average concentrations of IL6 and IL10 in serum were 11.3 pg / mL and 85.8 pg / mL, and the average concentrations of ALT and AST in serum were 34.5 U / L and 29.3 U / L; the average number of acute liver lesions in the cold-treated VNP strain group was 10, the average concentrations of IL6 and IL10 in serum were 27.4 pg / mL and 116.0 pg / mL, and the average concentrations of ALT and AST in serum were 134.9 U / L and 141.3 U / L; the cold-treated M The ACS+cold-treated VNP group had an average number of 11 acute liver lesions, average serum IL6 and IL10 concentrations of 22.1 pg / mL and 111.3 pg / mL, and average serum ALT and AST concentrations of 144.3 U / L and 153.1 U / L. The cold-treated MACS / VNP group had an average number of 1 acute liver lesion, average serum IL6 and IL10 concentrations of 10.9 pg / mL and 94.3 pg / mL, and average serum ALT and AST concentrations of 55.3 U / L and 59.2 U / L. No significant differences were observed in HE in other organs (including heart, spleen, lung, and kidney) between the above groups (Figure 8). This suggests that loading and re-delivery with cold-treated MACS cells can indeed reduce the toxic side effects caused by attenuated Salmonella strains and improve biosafety. This effect cannot be achieved by simply mixing cold-treated MACS cells with cold-treated attenuated Salmonella strains. This highlights the effectiveness of the strategy of the present invention.

[0157] To preliminarily investigate the potential mechanism by which cold-treated MACS cell loading and re-delivery can reduce the toxic side effects of the VNP strain and enhance its intratumoral accumulation, we examined immune cell activation in the mouse circulation following administration. Neutrophils are potent agents in clearing foreign bacteria and are also the primary immune cells responsible for triggering adverse reactions. Neutrophil activation was primarily assessed after mice were fully anesthetized following the different drug treatments. Whole blood was collected by enucleation and anticoagulant addition. Peripheral blood lymphocytes were isolated using a peripheral blood lymphocyte isolation kit (Solarbio, P8620). Cell clumps were removed by sieving through a 40 μm cell sieve to obtain a single-cell suspension. Cells were stained with the following anti-mouse antibodies: CD11b-APC (BD, 553312), Ly6G-BV421 (BD, 562737), and CD62L-PE (BD, 553151). Specifically, the collected peripheral blood lymphocytes were resuspended in Hanks buffer containing 1% BSA, and the above-mentioned CD11b, Ly6G, and CD62L antibodies were then added to detect activated neutrophils in the mouse peripheral blood. After incubation at 4°C for 30 minutes, the cells were washed 2-3 times with buffer to remove unbound antibodies, and then analyzed by flow cytometry. The test results showed that the percentage of activated neutrophils in the peripheral blood of the cold-treated MACS+cold-treated VNP group was higher (~23.5%), while the percentage of activated neutrophils in the peripheral blood of the cold-treated MACS / VNP group was significantly reduced (~7.6%) (Figure 9a). Activated neutrophils will rapidly eliminate free bacteria by phagocytosis and secretion of inflammatory factors, which will also have adverse effects on the body.

[0158] This demonstrates that the "camouflage protection" effect of cold-treated MACS cells can indeed reduce the strong activation of other immune cells in the peripheral blood by reducing the strain's exposure to large amounts of foreign substances. This not only prevents the rapid elimination of the strain, but also avoids the damage caused to the body by large numbers of rapidly activated immune cells (Figure 9b). This further demonstrates the great potential of this invention to enhance the clinical translational application of attenuated Salmonella.

[0159] The above-mentioned macrophages refer to RAW264.7, J774.1, Ana-1, iBMDM, U937 and other macrophages. The results of J774.1, Ana-1, iBMDM, and U937 are similar to those of RAW264.7.

[0160] The results of B16F10, LLC, 4T1, and A20 tumor models were similar to those of the H22 tumor model.

[0161] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0162] (4.2) Anticancer Biosafety Assessment of Cold-treated Attenuated Salmonella Loaded with Monocytes

[0163] The present invention uses five tumor models: B16F10, LLC, 4T1, A20, and H22. The subcutaneous tumor-bearing mouse model was established as described in Example 3.

[0164] Dosage

[0165] Tumor-bearing mice were randomly divided into groups and injected with different cells once via tail vein. The dosage of cold-treated VNP strain (1.0×10 7 cells / cells) and / or cells (4.0×10 6 The rats were injected with only saline as negative control.

[0166] Tissue distribution of bacteria in tumor-bearing mice

[0167] The above drugs were administered to tumor-bearing mice for 8 hours, 1 day, 3 days, 6 days, and 12 days. The mice were then randomly sacrificed. Tumors and other organs (including heart, liver, spleen, lung, and kidney) were removed from the tumor-bearing mice under a sterile environment. These organs were weighed and homogenized in 2 mL of PBS using a tissue homogenizer (frequency: 60 Hz, duration: 100 seconds). Different tissues were diluted in different gradients and spread onto LB plates. The plates were then incubated upside down at 37°C in a bacterial incubator for 12 hours. Colony counts were performed, and the number of attenuated Salmonella strains in mouse tissues from different groups was compared and analyzed.

[0168] In the present invention, bacterial titers were measured in heart, liver, spleen, lung, kidney, and tumor tissues, and the results were similar to those in (4.1) above. High levels of bacterial enrichment within tumors were observed in all three groups: cold-treated MC, cold-treated attenuated Salmonella VNP, cold-treated MC / VNP, and cold-treated MC + cold-treated VNP. Enrichment also occurred to varying degrees in normal organs. Three of these groups achieved the highest intratumoral bacterial titers on day 6. On day 6, in the cold-treated attenuated Salmonella VNP strain test group, bacterial titers in normal organs (including heart, liver, spleen, lung, and kidney) were approximately 4.5-4.8, and in tumors were 6.9-7.1. In the cold-treated MC / VNP group, bacterial titers in normal organs were 3.8-4.0, and in tumors were 7.7-7.9. In the cold-treated MC+cold-treated VNP group, bacterial titers in normal organs were 4.3-4.5, and in tumors were 6.8-7.0 (similar to the results in 4.1 above). There were no significant differences in bacterial titers in normal organs or tumor tissues between the cold-treated attenuated Salmonella VNP strain group and the cold-treated MC+cold-treated VNP group. While bacterial titers in normal organs in the cold-treated MC / VNP group were significantly reduced, bacterial titers in tumor tissues were significantly increased. This difference between the three groups was also present at other time points. This shows that loading and re-delivery of cold-treated MC cells can indeed improve the tumor targeting of attenuated Salmonella strains and reduce off-target effects on normal organs. This effect cannot be achieved by simply mixing cold-treated MC cells with cold-treated VNP strains.

[0169] Acute toxicity assessment

[0170] Five groups of H22 tumor-bearing mice were intravenously administered with normal saline, cold-treated MC, cold-treated VNP strain, cold-treated MC / VNP, and cold-treated MC plus cold-treated VNP. One day later, orbital bleeding (approximately 200 μL) was collected. Whole blood was allowed to stand at room temperature for 30 minutes and then centrifuged at 3000 rpm for 15 minutes. The supernatant serum was collected for IL-6 / IL-10 cytokine analysis and blood biochemical analysis including ALT and AST. At the same time, normal organs (including heart, liver, spleen, lungs, and kidneys) were dissected and photographed and stained with hematoxylin and eosin.

[0171] The results were similar to those in (4.1) above: obvious foci appeared on the liver surface of the cold-treated VNP strain and the cold-treated MC + cold-treated VNP group, both causing significant liver damage, and obvious necrotic areas were observed by HE staining; IL-6 / IL-10 and ALT / AST were significantly increased. In the cold-treated MC / VNP group, the above damage indicators were significantly alleviated. However, no significant differences were observed in the HE of other organs (including heart, spleen, lung, and kidney) of the above groups. Therefore, loading and re-delivery with cold-treated MC cells can indeed reduce the toxic side effects caused by VNP strains and improve biosafety. However, simply mixing cold-treated MC cells with cold-treated VNP strains cannot achieve this effect.

[0172] To investigate the potential mechanism by which cold-treated MC loading and re-delivery can reduce the toxic side effects of attenuated Salmonella strains and enhance their intratumoral accumulation, we examined immune cell activation in the mouse circulation following administration. Neutrophils are potent agents in clearing foreign bacteria and are also a major immune cell triggering adverse reactions. Neutrophil activation was primarily assessed. After complete anesthesia, mice treated with different medications were enucleated and whole blood was collected, followed by the addition of an anticoagulant. Peripheral blood lymphocytes were isolated using a peripheral blood lymphocyte isolation kit and passed through a 40 μm cell sieve to remove cell aggregates and obtain a single-cell suspension. The collected peripheral blood lymphocytes were resuspended in Hanks buffer containing 1% BSA. The CD11b, Ly6G, and CD62L antibodies described above were then added to detect activated neutrophils in the mouse peripheral blood. After incubation at 4°C for 30 minutes, the cells were washed two to three times with buffer to remove unbound antibodies and analyzed by flow cytometry. The test results were similar to those in (4.1) above. The percentage of activated neutrophils in peripheral blood was higher in the cold-treated MC + cold-treated VNP group (22-25%), while it was significantly lower in the cold-treated MC / VNP group (7.5-7.8%). Activated neutrophils rapidly eliminate free bacteria by phagocytosis and secreting inflammatory factors, which can also have adverse effects on the body.

[0173] This demonstrates that the "camouflage protection" of cold-treated MC cells can indeed reduce the strain's exposure to foreign substances, thereby reducing the potent activation of other immune cells in the peripheral blood. This prevents both the rapid elimination of the strain and the damage caused to the body by large numbers of rapidly activated immune cells. This further demonstrates the enormous potential of this invention to advance the clinical application of attenuated Salmonella.

[0174] The above-mentioned monocytes are THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, etc. The results of B16F10, LLC, 4T1, and A20 tumor models are similar to those of the H22 tumor model.

[0175] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0176] Example 5

[0177] Anticancer Biosafety Assessment of Cold-treated Attenuated Salmonella Loaded with Monocytes or Macrophages (CELL / VNP)

[0178] (5.1) Evaluation of the anticancer effect of cold-treated attenuated Salmonella loaded on macrophages

[0179] Six-week-old female BALB / c mice (Changzhou Animal Center) were used as experimental animals. Five tumor models, B16F10, LLC, 4T1, A20, and H22, were used in the present invention. The subcutaneous tumor-bearing mouse model of tumor cells was established by referring to the description in Example 3. When the mouse tumor grew to about 80-120 mm 3 Afterwards, mice were randomly assigned to different groups, with 7-8 mice in each group. Administration: Different cells were injected once via tail vein, with the dosage of cold-treated VNP strain (1.0×10 7 cells / cells) and / or cells (4.0×10 6 The tumor volume was calculated using the formula V = length × width. 2 Tumor size was measured and calculated using the % ± SD value for each group of mice at regular intervals. Tumor size was used to plot tumor growth curves. Values ​​are expressed as Mean ± SD.

[0180] The tumor doubling time of the normal saline group was 1.128 days, the tumor doubling time of the cold-treated MACS group was 1.344 days, the tumor doubling time of the cold-treated VNP20009 strain group was 1.403 days, the tumor doubling time of the cold-treated MACS / VNP group was 1.793 days, and the tumor doubling time of the cold-treated MACS+cold-treated VNP (a simple mixture of the two) group was 1.556 days (Figure 10a, b). Compared with the normal saline group, the tumor doubling time of the cold-treated MACS group was prolonged by 19.1%, the tumor doubling time of the cold-treated VNP20009 strain group was prolonged by 24.4%, and the tumor doubling time of the cold-treated MACS / VNP group was prolonged by 59.0%; compared with the cold-treated attenuated Salmonella group, the tumor doubling time of the cold-treated MACS+cold-treated VNP (a simple mixture of the two) group was prolonged by 10.9%, and the tumor doubling time of the cold-treated MACS / VNP group was prolonged by 27.8%; compared with the cold-treated MACS+cold-treated VNP (a simple mixture of the two) group, the tumor doubling time of the cold-treated MACS / VNP group was prolonged by 15.2%. The tumor doubling time after cold-treated MACS loaded with VNP strain was 1.15 times the theoretical value of the sum of the therapeutic effects of the two. Therefore, the cold-treated MACS / VNP group did produce a synergistic therapeutic effect.

[0181] After treatment, mice were sacrificed on day 12, and tumors were dissected, weighed, and photographed. The results showed that compared to the average tumor weight of 1.34 grams in the saline group, the average tumor weight in the cold-treated MACS group was 2.355 grams, the average tumor weight in the cold-treated VNP strain group was 0.91 grams, the average tumor weight in the cold-treated MACS / VNP group was 0.47 grams, and the average tumor weight in the cold-treated MACS + cold-treated VNP (a simple mixture of the two) group was 0.65 grams. The average tumor weights of the latter four groups all decreased to varying degrees. Compared to the cold-treated MACS + cold-treated VNP (a simple mixture of the two) group, the average tumor weight in the cold-treated MACS / VNP group was 72.3% (Figures 10c, d). Furthermore, compared to the cold-treated MACS + cold-treated VNP (a simple mixture of the two) group, the survival time of mice in the cold-treated MACS / VNP group was significantly prolonged (Figure 10e). This further demonstrates that the cold-treated MACS / VNP group indeed produces a synergistic therapeutic effect.

[0182] The above-mentioned macrophages refer to RAW264.7, J774.1, Ana-1, iBMDM, U937 and other macrophages. The results of J774.1, Ana-1, iBMDM, and U937 are similar to those of RAW264.7.

[0183] The results of B16F10, LLC, 4T1, and A20 tumor models were similar to those of the H22 tumor model.

[0184] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0185] (5.1) Evaluation of the anticancer effect of cold-treated attenuated Salmonella loaded on monocytes

[0186] Six-week-old female BALB / c mice (Changzhou Animal Center) were used as experimental animals. Five tumor models, B16F10, LLC, 4T1, A20, and H22, were used in the present invention. The subcutaneous tumor-bearing mouse model of tumor cells was established by referring to the description in Example 3. When the mouse tumor grew to about 80-120 mm 3 Afterwards, mice were randomly assigned to different groups, with 7-8 mice in each group. Administration: Different cells were injected once via tail vein, with the dosage of cold-treated VNP strain (1.0×10 7 cells / cells) and / or cells (4.0×10 6 The tumor volume was calculated using the formula V = length × width. 2Tumor size was measured and calculated using the % ± SD value for each group of mice at regular intervals. Tumor size was used to plot tumor growth curves. Values ​​are expressed as Mean ± SD.

[0187] The results were similar to those in (5.1) above. Compared with the saline group, the tumor doubling time in the cold-treated MC group was significantly prolonged by approximately 15%, the tumor doubling time in the cold-treated VNP20009 strain group was prolonged by approximately 20%, and the tumor doubling time in the cold-treated MC / VNP group was prolonged by approximately 50%. Compared with the cold-treated attenuated Salmonella group, the tumor doubling time in the cold-treated MC+cold-treated VNP (a simple mixture of the two) group was prolonged by approximately 10%, and the tumor doubling time in the cold-treated MC / VNP group was prolonged by approximately 30%. Compared with the cold-treated MC+cold-treated VNP (a simple mixture of the two) group, the tumor doubling time in the cold-treated MC / VNP group was prolonged by 20%, indicating that the cold-treated MACS / VNP group produced a synergistic therapeutic effect.

[0188] The treated mice were sacrificed on day 12, and the tumors were dissected, weighed, and photographed. The results were similar to those in (5.1) above. The cold-treated MC / VNP group did produce a synergistic therapeutic effect.

[0189] The above-mentioned mononuclear cells (MC) are THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, etc. The results of B16F10, LLC, 4T1, and A20 tumor models are similar to those of the H22 tumor model.

[0190] The above-mentioned Salmonella refers to VNP20009 and its genetically modified strains and synthetic biology engineered strains that can produce drug proteins, including the aforementioned strains that have been applied for invention: ZL201110232776.2, ZL201110220676.8, ZL201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201 610945021.X, ZL202210182222.4, 202010182038.0, 202210070594.8, 202210181929.9, 20 2210182870.X, 202210182455.4, 202210268084.1, 202210268141.6, 2023102136359; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; Signal Transduction and Targeted Therapy 2023, 8:134; Frontier of Medicine, https: / / doi.org / 10.1007 / s11684-022-0925-2; phoP / phoQ. The results showed that each VNP20009 genetically modified strain and the synthetic biology engineered strain capable of producing drug proteins showed the same results as the VNP20009 chassis strain.

[0191] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above-mentioned test examples. The above-mentioned test examples and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications are possible without departing from the spirit and scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims, the description, and their equivalents.

Claims

1. A method for preparing frozen shock treated monocytes or macrophages loaded with attenuated Salmonella, characterized in that The method comprises the following steps: the frozen shock treated monocytes or macrophages loaded with attenuated Salmonella are immortalized monocytes or macrophages and co-cultured with attenuated Salmonella typhimurium at an MOI value of 1-100 to obtain engineered cells loaded with attenuated Salmonella, and then cold shock treated with liquid nitrogen to obtain frozen shock treated monocytes or macrophages loaded with attenuated Salmonella.

2. The method for preparing the frozen-shock treated monocytes or macrophages loaded with attenuated Salmonella according to claim 1, characterized in that: (1) Co-incubating any cell of a monocyte cell line or a macrophage cell line with attenuated Salmonella: The monocytes or macrophages with good growth conditions were cultured at a rate of 1-100×10 5 The cells were inoculated into a culture dish at a ratio of 100 cells / well and cultured with a cell culture medium without antibiotics. The attenuated Salmonella monoclonal strain was picked from the agar plate and activated overnight in an LB liquid medium. The bacterial solution grown to the logarithmic phase was centrifuged at 5000-8000 rpm for 5-10 min, the supernatant was discarded and the precipitate was resuspended in sterile physiological saline. After adjusting the OD600 to 0.6-1.2, the bacteria were added to the culture dish containing the above cells at an MOI value of 1-100 and cultured together for 20-150 minutes. (2) After staining the cell nucleus with Hoechst, observe the morphological changes of the cells under a microscope; record the changes in the percentage of disruption of the above-mentioned monocytes or macrophages at different time points of co-culture of the cells and bacteria; after different time points, discard the supernatant, wash with sterile PBS 2 to 3 times, and then incubate with cell culture medium supplemented with 50-125 μg / ml gentamicin for 20-60 minutes to kill the extracellular strains; discard the supernatant, and wash with sterile PBS 2 to 3 times; Collect cells to obtain live monocytes or macrophages loaded with VNP20009 strain; in order to detect the number of attenuated Salmonella effectively loaded in monocytes or macrophages, use a cell counter to detect the number of live cells of monocytes or macrophages loaded with attenuated Salmonella at different time points, and then lyse the cells with 0.5% Triton X-100 at room temperature; dilute the lysate in multiple ratios, apply it on LB agar plates added with kanamycin, and incubate at 37°C overnight; count the number of live bacteria loaded in monocytes or macrophages; (3) resuspending the live cells of the monocytes or macrophages loaded with attenuated Salmonella prepared above in 500-1000 μl of serum-free cell freezing solution; the cell suspension is directly rapidly frozen in liquid nitrogen, taken out after being placed for 6-18 hours, and thawed in a 37-42° C. water bath to obtain freeze-shock-treated monocytes or macrophages loaded with attenuated Salmonella.

3. The method for preparing monocytes or macrophages loaded with attenuated Salmonella treated with freezing shock according to claim 1 or 2, characterized in that: In step (1) or step (2), the attenuated Salmonella typhimurium is attenuated Salmonella typhimurium VNP20009 and its genetically modified strains and synthetic biology modified engineering strains that can produce drug proteins, and the immortalized monocyte cell line / macrophage cell line includes any one of THP1, iBMMC, J-111, Mono-Mac-1, JOSK-M, RAW264.7, J774.1, Ana-1, iBMD or U937; wherein the monocytes include any one of the monocyte cell lines THP1, iBMMC, J-111, Mono-Mac-1 or JOSK-M, and the macrophages include any one of the macrophage cell lines RAW264.7, J774.1, Ana-1, iBMDM or U937.

4. The method for preparing the frozen-shocked monocytes or macrophages loaded with attenuated Salmonella according to claim 2, characterized in that: In step (2), after comprehensively considering the strain loading and cell integrity, a co-culture time of 60 minutes was selected as the optimal time point for preparing live CELL / VNP cells. At this time, the load of live bacteria in the cells was high, with a load of 257±27 strains per 100 cells, and the cell integrity was >90%.

5. The monocytes or macrophages loaded with attenuated Salmonella and subjected to freeze shock treatment and prepared by the preparation method according to claim 1.

6. Use of the method for preparing monocytes or macrophages loaded with attenuated Salmonella treated with freezing shock according to claim 1 in preparing anti-tumor drug preparations.

7. The use according to claim 6, characterized in that: The therapeutic dose of freeze-shocked monocytes or macrophages loaded with attenuated Salmonella is 0.4-40×10 6 cells, the unit is cells / mouse, corresponding to the actual amount of attenuated Salmonella is 0.1-10×10 7 CFU, the unit is CFU / mouse; the administration frequency of the frozen shock treated monocytes / macrophages loaded with attenuated Salmonella is single administration.

8. The use according to claim 6, characterized in that: The pharmaceutical preparation comprises at least one of an intravenous injection preparation, an intratumoral injection preparation or an intraperitoneal injection preparation.

9. The use according to claim 6 in combination with other conventional anti-tumor drugs or methods.

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