CN114736861B - Mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella and preparation method and application thereof - Google Patents

Mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella and preparation method and application thereof Download PDF

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CN114736861B
CN114736861B CN202210268141.6A CN202210268141A CN114736861B CN 114736861 B CN114736861 B CN 114736861B CN 202210268141 A CN202210268141 A CN 202210268141A CN 114736861 B CN114736861 B CN 114736861B
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nanobody
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vnp
salmonella
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华子春
吴乐阳
李霖
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Targetpharma Laboratories Jiangsu Co ltd
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Abstract

The invention discloses a mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella, a preparation method and application thereof. The invention includes methods and compositions for treating a variety of solid tumors or metastases. Specifically, attenuated salmonella engineering bacteria capable of secreting immune checkpoint nanobodies including anti-PD 1 nanobodies are loaded in monocytes or macrophages, and the loaded cells are recruited to a tumor microenvironment, so that bacterial off-targeting to normal viscera is effectively avoided. The camouflage of the cell wrap also effectively avoids premature exposure of the bacteria. The anti-tumor therapeutic effect is excellent by directly/indirectly inhibiting tumors through activating/regulating an immune system by releasing intracellular engineering bacteria and immune checkpoint nanobodies such as anti-PD 1 nanobodies secreted and expressed by the engineering bacteria.

Description

Mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella and preparation method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella, a preparation method and application thereof.
Background
Since the 19 th century, welfare doctors (William Coley) tried to treat patients with tumors using heat-inactivated gram-positive bacteria (streptococci) and gram-negative bacteria (salmonella mucilaginosa), an increasing number of microorganisms have been developed and engineered for tumor treatment (Coley W B et al 1991,Clinical Orthopaedics and Related Research,262 (262): 3-11). These microorganisms, often referred to as "oncolytic bacteria" including salmonella, listeria, escherichia coli, etc., are capable of achieving high efficiency colonization of tumor sites after administration by injection due to their own facultative anaerobic nature and characteristic features of tumor microenvironments, including tumor internal hypoxic environment, immunosuppressive environment and nutrients released by large numbers of necrotic cells (Gurbatri CR, etc., 2020,Science Translational Medicine,12 (530); zhou S et al 2018,Nature Reviews Cancer,18 (12): 727-43; suh S et al 2019,Advanced Science,6 (3): 1801309.). However, such oncolytic bacteria often also cause different degrees of injury to the liver and spleen of the body due to their own toxicity. For example, VNP20009, an attenuated strain of Salmonella typhimurium (hereinafter abbreviated as VNP), has received much attention because of its lower toxicity and good preclinical tumor-inhibiting effects (Clairmont C et al, 2000,Journal of Infectious Diseases,181:1996-2002). Although the VNP toxicity is greatly reduced compared with the original salmonella by deleting two genes (purI and msbB), in the preclinical mouse experiment, tumor inhibition treatment by intravenous or intraperitoneal injection of VNP20009 still brings a certain degree of toxic and side effects, such as liver injury, splenomegaly, mouse weight collapse and the like, caused by the existence and accumulation of the strain in normal organs. Although the use of intratumoral injection can reduce the incidence of this injury (Chowdhury S et al, 2019,Nature Medicine,25 (7): 1057-1063; toso JF et al, 2002,Journal of Clinical Oncology,20 (1): 142-152), this clearly greatly limits the scope of application of microbiological therapies. In clinical phase I experiments, VNP exhibited favorable safety but was not well-behaved in terms of effectiveness (Toso J.F et al 2002,Journal of Clinical Oncology,20 (1): 142-52). Therefore, how to further improve tumor targeting and tumor inhibition effect of oncolytic bacteria such as VNP in vivo is a very challenging problem.
In recent years, various types of leukocytes, including macrophages, neutrophils, T cells, and the like, have been used as effective tumor drug delivery vehicles due to their unique homing effect to the tumor area (Xie Z et al, 2017, small,13 (10); xue J et al, 2017,Nature Nanotechnology,12 (7): 692-700; huang B et al, 2015,Science Translational Medicine,7 (291): 291ra 94). Such immune cells may reach the tumor area of the patient after crossing various barriers by sensing cues such as tumor associated chemokines or cytokines, e.g., colony stimulating factor 1 (CSF-1), tumor Necrosis Factor (TNF), chemokine ligand 5 (CCL 5), etc. (Salmon H, et al 2019,Nature Reviews Cancer,19 (4): 215-27).
Immune checkpoint blockers are a hotspot in the development of current international anti-malignant drugs. Tumor immune checkpoint inhibitor treatment regimens (Immune checkpoint inhibitor: ICI) to obtain good therapeutic effects also require high infiltration of tumors by T cells (Sznol et al., cancer J2014, 20 (4): 290-295), surface expression of high levels of inhibitory checkpoints such as PD1（Flemming et al., Nat Rev Drug Discov 2012, 11(8):601）,PD-L1,CTLA-4（Rowshanravan et al., Blood 2018, 131(1):58-67）,TIM-3（Das et al. Immunol Rev 2017, 276(1):97-111）,LAG3（Maruhashi et al., J Immunother Cancer 2020, 8）, etc. Many malignant tumors lack infiltrating T cells and respond poorly to ICI, resulting in inefficient clinical use of immune checkpoint blockers of only 10-30% (Sznol et al., cancer J2014, 20 (4): 290-295). Thus, how to induce more T cells to appear at the tumor site, initiates the T cell response of the tumor, which is a key factor in immune checkpoint blocker therapy, including PD1/PD-L1 inhibitors.
Programmed cell death protein 1 (also known as CD279 and PD 1) and its ligand PD1 ligand PD-L1 (CD 274) are critical immune checkpoints, functioning normally to prevent autoimmunity. Their interaction is also an important strategy for immune surveillance of many tumors (Ricklefs et al, 2018,Science Advances; 4 (3): eaar 2766). Despite promising results achieved by PD1/PDL1 blocking-based immunotherapy (Topalian et al, 2020, science; 367:525), most patients still lack a durable response to this therapy due to the low activity of effector immune cells, in particular CD 8T cells, in the tumor (Miller et al, 2019,Nature Immunology; 20:326). Thus, increasing the response rate of cancer patients to PD1/PDL1 immune blockers is a key challenge to overcome.
The invention can solve two problems of existing safety and treatment effect of attenuated salmonella to be improved, and meets the requirement of the field on a low-toxicity, high-efficiency and economic composition for treating solid tumors.
Disclosure of Invention
Aiming at the dilemma of the current tumor treatment, the invention provides a technology for performing tumor targeting treatment by using immune cells such as mononuclear cells or macrophages as a carrier for secreting engineered attenuated salmonella expressing immune checkpoint nanoantibodies, so as to realize 1) the tumor targeting enhancement of the engineered attenuated salmonella which is realized by depending on the presentation of the mononuclear cells or the macrophages, and the safety is improved; 2) The engineered attenuated salmonella released and proliferated in the tumor releases the intratumoral immunosuppression by secreting immune checkpoint nanobodies, enhances the anti-tumor immune response and improves the tumor suppression effect.
In order to solve the technical problems, the invention provides the following technical scheme: the invention relates to a monocyte/macrophage loaded with an engineered attenuated salmonella which secretes and expresses immune checkpoint nano-antibodies, wherein the monocyte/macrophage is a macrophage RAW264.7 or primary macrophages derived from abdominal cavity and blood, the attenuated salmonella is an attenuated salmonella typhimurium VNP20009 and derived or genetically modified strains thereof, the immune checkpoint nano-antibodies comprise but are not limited to PD1, PD-L1, CTLA-4, TIM-3 or LAG3 of each strain （ZL201410209851.7,ZL201610946268.3,ZL201610945015.4,ZL201610945021.X,202010182038.0;Acta Pharmaceutica Sinica B 2021,11(10):31653177;phoP/phoQ）; of the invention, and the immune checkpoint nano-antibodies are nano-antibody blocking agents capable of blocking immune checkpoints, including but not limited to PD1 nano-antibodies and PD-L1 nano-antibodies (ZL 201811065587.9, 202210011915.7, 202210182870.X, 202210182455.4) of the invention.
The invention relates to a preparation method of monocyte/macrophage loaded with engineering attenuated salmonella which secretes and expresses immune checkpoint nano antibody, which comprises the following steps: (1) Constructing attenuated salmonella which stably and constitutively expresses immune checkpoint nano antibody blocking agent. The constitutive strong promoter J23100 or NirB promoter or adhE promoter is used in the expression plasmid, so that the extracellular secretory expression of the nano antibody can be realized, and the secretory signal peptide is a conventional bacterial secretory signal peptide, including bacterial secretory signal peptide sseJ, bacterial secretory signal peptide MIS, bacterial secretory signal peptide Flic signal peptide, bacterial secretory signal peptide pelB, bacterial secretory signal peptide SOPE, bacterial secretory signal peptide SpA, bacterial secretory signal peptide OmpA or amplification plasmid loss prevention element AT.
(2) Mononuclear or macrophage loading secretes attenuated salmonella expressing immune checkpoint nanobodies. Primary monocytes/macrophages purified by RAW264.7 macrophage cell lines induced with 25-500 ng/mL LPS for 4-48 hours or by 2-4 days combined adherence culture stimulated by 5% starch broth intraperitoneal injection were co-cultured with attenuated Salmonella at a ratio of 1:5-1:100, respectively, for 30-150 minutes, and then treated with 50-100 μg/mL gentamicin for 30-60 minutes to kill extracellular bacteria.
(3) Assay of salmonella attenuated by single-core or macrophage loading secretion of nano-antibodies expressing immune checkpoints. The number of live attenuated salmonella secreting and expressing immune checkpoint nanobody after phagocytosis of macrophages is calculated by using a dilution plating plate so as to record the number and the cell activity of the attenuated salmonella secreting and expressing immune checkpoint nanobody in intracellular effective loading under different treatment conditions, and the number and the cell activity are used for determining the final co-culture time and calculating the actual administration dose of subsequent cells.
For example, when macrophages are selected to co-culture with attenuated salmonella typhimurium VNP20009 and attenuated salmonella typhimurium VNP20009 secreting PD1 nanobody expressed therein at a ratio of 1:10 for 60 minutes and 30 minutes, respectively, approximately equal amounts of intracellular viable bacteria numbers can be achieved, respectively: 510.+ -.13 CFU and 491.+ -.28.+ -. 28 CFU (mean.+ -. SEM)/100 cells.
Further, the PD1 nanobody sequence is a PD1 nanobody sequence (202210011915.7, 202210182870. X) including a nucleotide sequence shown in SEQ ID No.2, and the PD-L1 nanobody sequence is a PD-L1 nanobody sequence (ZL 201811065587.9, 202210182455.4) including a nucleotide sequence shown in SEQ ID No. 7-9; the sequence of the amplification plasmid loss prevention element AT is a nucleotide sequence shown in SEQ ID No. 1; the sequence of the bacterial secretion signal peptide PelB is a nucleotide sequence shown in SEQ ID No. 3; the sequence of the J23100 constitutive promoter is a nucleotide sequence shown as SEQ ID No. 4; the attenuated salmonella typhimurium VNP20009 and the derived or genetically modified strains thereof include, but are not limited to, the strains for which the invention has been applied （ZL201410209851.7,ZL201610946268.3,ZL201610945015.4,202010182038.0,ZL201610945021.X;Acta Pharmaceutica Sinica B 2021,11(10):31653177;phoP/phoQ）.
The invention relates to a detection method for loading mononuclear or macrophage of immune checkpoint nano antibody attenuated salmonella, which comprises the following steps: (1) Detecting the expression and secretion condition of attenuated salmonella of the secretory expression immune checkpoint nanobody, electrically converting the constructed secretory expression immune checkpoint nanobody plasmid into attenuated salmonella, randomly picking up a monoclonal of engineering bacteria, culturing the engineering bacteria to a growth platform stage by using a liquid LB culture medium, collecting total proteins in bacterial precipitation and total proteins in the liquid culture medium, and detecting the content of the immune checkpoint nanobody in the proteins by using an immunoblotting method. (2) Confirming that loading of a single core or macrophage secreting salmonella attenuated by expressing immune checkpoint nanobody releases salmonella attenuated by secreting by expressing immune checkpoint nanobody and effectively secreting by expressing immune checkpoint nanobody: standing mononuclear or macrophage loaded with salmonella attenuated by secreting and expressing immune checkpoint nanobody in a cell culture medium without antibiotics for culture; detecting the number of immune checkpoint nanobody attenuated salmonella secreted and expressed in the supernatant after culturing for different time points by using a dilution coating flat plate method; meanwhile, extracting the collected thalli and total protein in a culture medium supernatant, detecting the expression condition of immune checkpoint nanobodies in engineering bacteria by using a Western blot method, and secreting and expressing immune checkpoint nanobody attenuated salmonella to effectively secrete nanobodies at different time points, wherein the culture medium supernatant total protein is collected by adopting a TCA (trichloroacetic acid) -acetone precipitation method.
The invention relates to an evaluation and tracing method for bacterial loading efficiency and tissue distribution level of mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella, which comprises the following steps: the method comprises the steps of simultaneously loading fluorescent protein or luciferase tracer protein capable of being expressed in monocytes/macrophages, quantitatively detecting the fluorescent protein or the luciferase tracer protein, including red fluorescent RFP or luciferase LuxCDABE, and evaluating and tracing the bacterial loading efficiency and tissue distribution and level in the mononuclear cells or the macrophages secreting immune checkpoint nanobody-expressing attenuated salmonella.
The invention relates to a method for monitoring tissue distribution of mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella, which is characterized by comprising the following steps of: the mononuclear or macrophage secreting salmonella attenuated by expressing immune checkpoint nanobody is loaded with a common optical dye label including DiR near infrared dye, the optical dye is quantitatively detected, and the tissue distribution and level of the mononuclear or macrophage secreting salmonella attenuated by expressing immune checkpoint nanobody are evaluated and tracked.
Further, untreated M0 type macrophages RAW264.7, after 4-48 hours of induction with 25-500 ng/mL LPS, RAW264.7 was converted to M1 type macrophages; or primary monocytes/macrophages obtained by purification by combined wall-attached culture for 2-4 days stimulated by intraperitoneal injection of 5% starch broth. Co-culturing RAW264.7 or primary mononuclear cells/macrophages subjected to LPS induction treatment and attenuated salmonella simultaneously expressing tracer protein and therapeutic protein in a ratio of 1:5-1:100 for 30-150 minutes respectively, and quantitatively detecting the correlation of the quantity of the attenuated salmonella simultaneously expressing tracer protein or immune checkpoint nanobody phagocytized by mononuclear cells or macrophages and the co-culturing time after treating cells for 30-60 minutes by means of 50-100 mug/mL gentamicin to kill extracellular free or adherent engineered attenuated salmonella; obtaining a linear relation between the number of attenuated salmonella expressing the tracer protein or the immune checkpoint nanobody and the corresponding fluorescence intensity by a gradient dilution method, and effectively and indirectly calculating the number of attenuated salmonella expressing the tracer protein or the immune checkpoint nanobody in a macrophage cell; the enzyme label instrument detects the fluorescence intensity (550 nm,585 nm) of the tracer protein, and the total bacterial number loaded by each 100 macrophages is calculated by combining the linear relationship of the bacterial number and the fluorescence.
Further, transferring monocytes/macrophages loaded with attenuated salmonella secreting immune checkpoint-expressing nanobodies to 0.3% triton x-100 in PBS configuration for 10-15 min to perforate the macrophages to release intracellular attenuated salmonella; coating an LB plate with the resistance of the kanamycin after gradient dilution, and analyzing and detecting the correlation of the live loading secretion expression immune checkpoint nano antibody attenuated salmonella loaded by the mononuclear cells/macrophages and the loading efficiency and the co-culture time; detecting the activity state of the cells after 30, 60, 90, 120 and 150 minutes of co-culture of macrophages and attenuated salmonella by using a phenol blue assay; in order to further verify that macrophages are effectively loaded with the salmonella attenuated by loading and secreting the nano antibody expressing the immune checkpoint, after obtaining the macrophages loaded with the salmonella attenuated by loading and secreting the nano antibody expressing the immune checkpoint, the cells are fixed by using 4% paraformaldehyde, are protected from light at room temperature, are kept stand for 30min, are washed by PBS for 2-3 times, and are respectively dyed by using the phalloidin and DAPI according to manufacturer specifications, and are kept stand for 30min at room temperature, are washed by PBS for 2-3 times, and are observed and photographed by using an upright fluorescence microscope.
Given the wide range of current immune checkpoint antibody inhibitors to be combined with other drugs or therapies, such as chemotherapy, traditional Chinese medicine therapy, biological therapy, physical therapy, etc., thousands of combined treatment regimens with different drugs or therapies are currently under clinical study;
The invention relates to application of mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella expressed by secretion in preparation of antitumor drugs.
Furthermore, the medicine is mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella and is used for preparing an anti-tumor medicine independently, or is combined with the existing anti-tumor medicine to form a composition, or is combined with the existing chemical medicine treatment, traditional Chinese medicine treatment, biological treatment or physical treatment method to prepare the anti-tumor medicine.
The beneficial effects are that: the present invention describes a combination therapy established with engineered invisible bacteria camouflaged by macrophages. The invention has the advantages that three treatment methods of macrophage, attenuated salmonella and immune checkpoint nanobody are mutually supplemented for the first time, so as to form a unified system. Natural tumor-targeted macrophages released by trojan horses, bacteria exhibit (1) low inflammatory response and side effects; (2) low accumulation in normal organs; (3) the antitumor activity is unchanged. Along with the continuous expression and secretion of immune checkpoint nanobodies, engineering bacteria treatment shows (1) high immune activity, (2) remarkable tumor regression, and (3) stable and durable anti-tumor effect. In view of these unique advantages, the present invention predicts that this strategy has broad biomedical application prospects in tumor treatment.
Drawings
Brief description of the drawings:
VNP: attenuated mice injured salmonella VNP20009; PD1nb: anti-PD 1 nanobodies; VNP-PD 1nb: VNP carries anti-PD 1 nanobody gene plasmid; VNP-NC: VNP carries empty plasmid; PEMΦ (VNP-NC)/MΦ (VNP-NC): VNP-NC loaded mouse primary peritoneal macrophage PEMΦ; PEMΦ (VNP-PD 1 nb)/MΦ (VNP-PD 1 nb): a mouse primary peritoneal macrophage PEM Φ loaded with PD1 nb; DTIC: dacarbazine and a chemotherapeutic agent for melanoma treatment.
FIG. 1 shows the structure of the VNP-PD1nb plasmid and the secretion of PD1 nb. pJ23100-PD1nb plasmid schematic drawing carried by VNP-PD1nb engineering bacteria (left) and using Western Blot to detect precipitation after centrifugation of VNP-PD1nb engineering bacteria culture medium and PD1nb in supernatant (right). 1. Precipitating VNP-PD1nb-1 bacterial cells; 2. precipitating VNP-PD1nb-2 bacterial cells; 3. VNP-PD1nb-1 supernatant; 4. VNP-PD1nb-2 supernatant.
FIG. 2 shows the measurement of the loading of VNP-PD1nb by the primary peritoneal macrophage PEM phi according to the present invention.
FIG. 2A is a comparison of bacterial numbers of different bacteria of the present invention after 1 hour of culture in DMEM. 1. VNP-NC; 2. VNP-PD1nb (n=3).
FIG. 2B shows the loading of the primary peritoneal macrophages PEM phi of the present invention with different strains. 1. VNP-NC; 2. VNP-PD1nb; a. the intracellular viable count changes after the abdominal cavity macrophage PEM phi and the VNP are incubated together for different times; b. abdominal macrophage PEM phi and VNP were incubated for 1 hour and intracellular viable count was obtained. (n=3, < P < 0.0001)
FIG. 2C is a graph showing the change in phagocytic capacity of the primary peritoneal macrophages PEM phi of the present invention after loading with different strains. 1. No-load macrophages; 2. PEMΦ (VNP-NC); 3. PEMP Φ (VNP-PD 1 nb).
FIG. 3 shows the release profile of the VNP-PD1nb loaded macrophages PEM phi (VNP-PD 1 nb) of the present invention for intracellular strains.
FIG. 3A is a schematic representation of the experimental design of PEM phi (VNP-PD 1 nb) versus intracellular VNP release according to the present invention. The prepared PEM phi (VNP-PD 1 nb) was cultured in DMEM without antibiotics, and the number of viable bacteria in DMEM was detected by a dilution coating method at a specified time point.
FIG. 3B is a graph showing the quantity of viable bacteria in DMEM by dilution coating at various time points according to the invention, and the resulting release profile of PEM phi (VNP-PD 1 nb) against intracellular strains is plotted. (n=3)
FIG. 3C is a Western Blot of the present invention demonstrating expression and secretion of PD1nb by VNP-PD1nb released by PEM Φ (VNP-PD 1 nb). 1. Precipitating VNP-PD1nb bacterial cells; 2. VNP-PD1nb supernatant; a. detecting precipitation and supernatant PD1nb after culturing VNP-PD1nb in an LB culture medium; b. during release of intracellular VNP-PD1nb by PEM phi (VNP-PD 1 nb), PD1nb was detected in DMEM medium at different time points.
FIG. 4 shows in vivo attenuation, tumor targeting and tumor inhibition of VNP-PD1 nb-loaded macrophages PEMB phi (VNP-PD 1 nb) according to the present invention.
Fig. 4A shows the change in body weight of mice 1 day after treatment according to the present invention. 1. PBS; 2. VNP-PD1nb; 3. PEMP Φ (VNP-PD 1 nb). (n=11,/P < 0.001)
Fig. 4B is a comparison of liver injury 1 day after treatment according to the present invention. a. A representative picture of liver injury, black arrow is the place where the liver is obviously injured, and the scale is 5 mm; 1. VNP-PD1nb; 2. PEMP Φ (VNP-PD 1 nb); b. counting and comparing the number of the significant lesions of the liver; 1. PBS; 2. VNP-PD1nb; 3. PEMP Φ (VNP-PD 1 nb). (n=4, ns indicates no significant difference, P < 0.01)
Fig. 4C is a comparison of liver injury 1 day after treatment according to the present invention. a. Representative photographs of splenomegaly, scale 5 mm; 1. VNP-PD1nb; 2. PEMP Φ (VNP-PD 1 nb); b. spleen weight statistics and comparison; 1. PBS; 2. VNP-PD1nb; 3. PEMP Φ (VNP-PD 1 nb). (n=4, ns indicates no significant difference, P < 0.01)
Fig. 4D is a comparison of VNP titers in liver versus spleen for tumors at day 6 after treatment according to the invention. 1. VNP-PD1nb; 2. PEMP Φ (VNP-PD 1 nb); a. tumor/liver; b. tumor/spleen. (n=5,/P < 0.001)
FIG. 4E shows the therapeutic effect of different treatments of the invention on B16F10 tumor-bearing mice. 1. PBS; 2.2, VNP-PD1nb; 3. PEMP Φ (VNP-PD 1 nb). (n=7, < P <0.05, < P < 0.0001)
FIG. 5 shows the in vivo tumor-inhibiting effect assay of the loaded VNP-PD1nb macrophage PEM phi (VNP-PD 1 nb) of the present invention.
FIG. 5A is a schematic diagram showing experimental design of in vivo tumor suppression effect detection of VNP-PD1 nb-loaded macrophage PEM phi (VNP-PD 1 nb) according to the present invention. DTIC, PEM Φ+pd1nb, PEM Φ (VNP-NC) +pd1nb, PEM Φ (VNP-PD 1 nb) were injected into B16F10 tumor-bearing mice via the tail vein or intraperitoneally, with PBS as negative control.
FIG. 5B is a graph comparing tumor growth curves of VNP-PD1 nb-loaded macrophage PEM phi (VNP-PD 1 nb) of the present invention with tumor-bearing mice treated by other protocols. 1. PBS; 2. DTIC; 3. PEMΦ+PD1nb; 4. PEMΦ (VNP-NC); 5. PEMΦ (VNP-NC) +PD1nb; 6. PEMP Φ (VNP-PD 1 nb). (n=7)
Fig. 5C is a comparison of the survival of tumor-bearing mice after treatment with VNP-PD1 nb-loaded macrophages PEM Φ (VNP-PD 1 nb) of the present invention with other protocols. 1. PBS; 2. DTIC; 3. PEMΦ+PD1nb; 4. PEMΦ (VNP-NC); 5. PEMΦ (VNP-NC) +PD1nb; 6. PEMP Φ (VNP-PD 1 nb). (n=5,)
FIG. 5D is a comparison of the long term tumor inhibiting effect of the loaded VNP-PD1nb macrophages PEM phi (VNP-PD 1 nb) of the present invention. 1. PEMΦ (VNP-NC) +PD1nb; 2. PEMP Φ (VNP-PD 1 nb); a. tumor size photographs of each group at day 25 post-treatment, scale bar 10mm; b. statistical comparison of tumor weights for each group at day 25 post-treatment. (n=7, < P < 0.0001)
FIG. 6 shows the detection of the lung metastasis inhibition effect of the loaded VNP-PD1nb macrophages PEMB phi (VNP-PD 1 nb) of the present invention on B16F10 tumors.
FIG. 6A is a schematic diagram showing the experimental results of the detection of the lung metastasis inhibition effect of the loaded VNP-PD1nb macrophage PEMB phi (VNP-PD 1 nb) on B16F10 tumor according to the present invention. Tumor lung metastasis was dissected and examined by tail vein injection of 1X 10 6 B16F10 cells to establish a model of lung metastasis on day 6 by tail vein injection of PBS, PEM phi (VNP-NC), PEM phi (VNP-PD 1 nb).
FIG. 6B is a comparison of the lung metastasis inhibiting effect of the loaded VNP-PD1nb macrophages PEMB phi (VNP-PD 1 nb) of the present invention on B16F10 tumors. 1. Healthy mice; 2. PBS; 3. PEMΦ (VNP-NC); 4. PEMP Φ (VNP-PD 1 nb); a. lung photographs and sections after different treatments, black arrows indicate tumor infiltration sites; b. comparison of lung surface tumor area after different treatments. (n=3, < P <0.01, < P < 0.0001)
FIG. 7 shows the detection of tumor cell necrosis and proliferation levels of tumor-bearing mice of different groups according to the present invention
FIG. 7A shows comparison of necrotic cell duty cycle in different groups of tumors 1, 3, 5 days after treatment of the B16F10 tumor model of the present invention. a. Performing histogram statistical analysis; b. a representative flow chart is shown. 1. A PBS group; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line. (n=4, ns indicates no significant difference)
FIG. 7B shows the proportion of tumor necrosis in different treatment groups compared with H & E staining on day 3 after treatment of the B16F10 tumor model of the invention. a. Performing histogram statistical analysis; b. representative H & E images. Scale, 1 mm (whole tumor section) and 200 microns (enlarged tumor section). 1. A PBS group; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line. (n=4)
FIG. 7C shows comparison of the ratios of proliferation tumor cells in different groups on day 3 after treatment of the B16F10 tumor model of the present invention. 1. A PBS group; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line. (n=4)
FIG. 8 is a graph showing the detection of intratumoral macrophage phenotype in different groups of tumor-bearing mice according to the present invention
FIG. 8A is a comparison of the numbers of tumor infiltrating macrophages cells 1/3/5 day after treatment of the B16F10 tumor model of the invention. 1. A PBS group; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line. (n=4, ns indicates no significant difference)
FIG. 8B is a graph showing comparison of the ratio of tumor infiltrating macrophages (M1 or M2) of different phenotypes on day 3 after treatment of the B16F10 tumor model of the invention. 1. A PBS group; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line. (n=4)
FIG. 8C is a representative display of tumor-infiltrating macrophages of different phenotypes obtained by immunofluorescence detection at day 3 after treatment of the B16F10 tumor model of the present invention. 1. A PBS group; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line.
FIG. 9 shows the detection of CD 8T cell activation in tumor-bearing mice of different groups according to the invention
FIG. 9A is a comparison of tumor infiltrating CD8+ T cell numbers on day 3 after treatment of the B16F10 tumor model of the invention. 1. A PBS group; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line. (n=4, ns indicates no significant difference)
FIG. 9B is a comparison of the numbers of tumor-infiltrating CD8+ T cells that effectively produce IFNγ on day 3 post-treatment of the B16F10 tumor model of the invention. 1. A PBS group; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line. (n=4)
FIG. 9C is a comparison of the numbers of tumor-infiltrating CD8+ T cells that effectively produce TNF alpha on day 3 post-treatment of the B16F10 tumor model of the invention. 1. A PBS group; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line. (n=4)
Fig. 9D is a representative flow cytometry image of the above-described fig. a, B, and C of the present invention.
FIG. 10 shows the detection of serum inflammatory factors of tumor-bearing mice of different groups according to the present invention
FIG. 10A is a comparison of IFN gamma concentration in peripheral blood measured by CBA assay kit 3 days after treatment with the B16F10 tumor model of the present invention. 1. A PBS group; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line. (n=4)
FIG. 10B is a comparison of TNFα concentration in peripheral blood measured by CBA assay kit of the present invention in PBS group 1 after 3 days of B16F10 tumor model treatment; 2. m phi (VNP-NC) cell line; 3. m phi (VNP-PD 1 nb) cell line. (n=4)
FIG. 10C is a flow cytometry plot of representative CBA data from either of the above-described plots A or B of the present invention.
FIG. 11 is a schematic diagram showing the use of MΦ (VNP-PD 1 nb) cell therapy for tumor immunotherapy according to the present invention
Step 1: m phi (VNP-PD 1 nb) cells served as Trojan horse engineering strains and targeted tumors.
Step 2: (1) After release of VNP-PD1nb in tumors, it is loaded in mΦ (VNP-PD 1 nb) cells, activating intratumoral immunity, reversing dysfunctional cd8+ T cells, while up-regulating PDL1 in tumor cells, resulting in immunosuppression. (2) The PD1nb released by the VNP-PD1nb can block the PD1/PD-L1 interaction between CD8+T cells and tumor cells, thereby inhibiting immunosuppression and enhancing tumor killing effect.
Detailed Description
For a better understanding of the present invention, the content of the present invention will be further elucidated with reference to the examples and drawings, but the content of the present invention is not limited to the following embodiments. The experimental methods used therein are conventional methods unless otherwise specified.
PD1, PD-L1, CTLA-4, TIM-3, LAG3 are immune checkpoints and nanobodies have substantially similar properties. In the inventions already filed, we have demonstrated that PD1 nanobodies, PD-L1 nanobodies, whether nanobody proteins or secretion-expressed PD1 nanobodies, PD-L1 nanobody attenuated salmonella have substantially the same properties and similar antitumor activity (ZL 201811065587.9, 202210011915.7, 202210182870.X, 202210182455.4). The preparation method of mononuclear cells or macrophages loaded with attenuated salmonella secreting and expressing different immune checkpoint nanobodies is the same, and the antitumor activity is basically the same. Here, the present invention focuses on taking the PD1 nanobody as an example, and shows the mononuclear or macrophage loaded with the attenuated salmonella secreting and expressing the PD1 nanobody, and the preparation method and application thereof, to illustrate the mononuclear or macrophage loaded with the attenuated salmonella secreting and expressing different immune checkpoint nanobodies, and the preparation method and application thereof.
Example 1
Preparation and optimization of loading engineering attenuated salmonella macrophages
I) Method for constructing engineering attenuated salmonella VNP-PD1nb
Taking an anti-PD 1 nanobody as an example, the preparation method of the monocyte/macrophage loaded with the engineering attenuated salmonella which secretes and expresses the immune checkpoint nanobody comprises the following steps:
(1) Constructing VNP20009 strain VNP-PD1nb of stable constitutive expression anti-PD 1 nanobody (PD 1 nb). The constitutive strong promoter J23100 or NirB promoter or adhE promoter is used in the expression plasmid, so that the extracellular secretory expression of the nano antibody can be realized, and the secretory signal peptide is a conventional bacterial secretory signal peptide, including bacterial secretory signal peptide sseJ, bacterial secretory signal peptide MIS, bacterial secretory signal peptide Flic signal peptide, bacterial secretory signal peptide pelB, bacterial secretory signal peptide SOPE, bacterial secretory signal peptide SpA, bacterial secretory signal peptide OmpA or amplification plasmid loss prevention element AT. FIG. 1 (left) shows a block diagram of an expression plasmid for secretory expression of anti-PD 1 nanobody by selecting a constitutive strong promoter J23100, pelB secretory signal peptide, and linked to a FLAG tag at the N-terminus of PD1nb for subsequent protein detection analysis.
The PD1 nanobody sequence is a PD1 nanobody sequence (202210011915.7, 202210182870. X) comprising the nucleotide sequence shown in SEQ ID No.2, and the PD-L1 nanobody sequence is a PD-L1 nanobody sequence (ZL 201811065587.9, 202210182455.4) comprising the nucleotide sequence shown in SEQ ID No. 5; the sequence of the amplification plasmid loss prevention element AT is a nucleotide sequence shown in SEQ ID No. 1; the sequence of the bacterial secretion signal peptide PelB is a nucleotide sequence shown in SEQ ID No. 3; the sequence of the J23100 constitutive promoter is a nucleotide sequence shown as SEQ ID No. 4; the attenuated salmonella typhimurium VNP20009 and the derived or genetically modified strains thereof include, but are not limited to, the strains for which the invention has been applied （ZL201410209851.7,ZL201610946268.3,ZL201610945015.4,202010182038.0,ZL201610945021.X;Acta Pharmaceutica Sinica B 2021,11(10):31653177;phoP/phoQ）.
(2) The VNP-PD1nb strain was cultured in 40 mL LB medium supplemented with kanamycin until OD600 was between 0.6 and 0.8. The solution was then centrifuged at 5000 rpm and 4 ℃ for 10 minutes and the supernatant and precipitate were collected. The pellet was resuspended in 2 mL PBS, heated in a dry bath incubator at 110 ℃ for 20 minutes to destroy the bacteria and release the proteins, and then centrifuged at 13000 r.p.m. for 10 minutes, the total proteins in the bacteria contained in the supernatant. Total protein was collected from the bacterial LB medium supernatant obtained in the first step according to trichloroacetic acid (TCA) protein precipitation. Briefly, the supernatant was transferred to a 50 ml centrifuge tube and centrifuged at 10 min using an ultracentrifuge at 15,000g,4 degrees. The supernatant was transferred to a new 50 ml centrifuge tube, 10% TCA was added, vortexed to mix well, and left to stand on ice for 30: 30 min. Again 7,000 g,4 degrees, centrifuge 20 min, resuspend pellet at 300: 300 ml PBS and transfer to 1.5. 1.5 ml sterile EP tube. 1.2 ml pre-chilled acetone (pre-placed-20 degrees), 17,000 g,4 degrees, and centrifuged at 20: 20 min are added. The supernatant was removed, 300 ml PBS was added again, and the above procedure was repeated. Removing the supernatant, adding 40 ml PBS to resuspend, and obtaining the total protein secreted by the thalli in the supernatant. After the cells collected by centrifugation were added to Loading buffer, they were boiled at 100℃for 10 minutes to obtain total proteins of the cells. The total protein collected was examined for the production and secretion of the target protein by Western Blotting (WB). FIG. 1 (right) shows
II) fumbling of primary macrophage (PEM Φ) loading VNP conditions:
The macrophage is primary macrophage from abdominal cavity and blood source, and can also be macrophage RAW264.7. RAW264.7 macrophage cell line treated by induction of 25-500 ng/mL LPS for 4-48 hr; or preparing 5% starch broth (1.8% nutrient broth+5% soluble starch+redistilled water), heating and stirring, sterilizing with high pressure steam (115 deg.C for 30 min), packaging in 1.5 ml EP tubes, and storing at 4 deg.C, and heating and dissolving before use. The 7-10 week old female/male C57 mice are taken for intraperitoneal injection of 1 ml starch broth, 2-4 d, and then the peritoneal macrophages are killed and extracted, the high-purity primary peritoneal macrophages are obtained by purification through an adherence culture method, and the primary peritoneal macrophages are digested by lidocaine at 4 ℃ and standing for 5-8 min to obtain cell suspensions for subsequent experiments. Macrophages were co-cultured with VNP-NC or VNP-PD1nb at a ratio of 1:5-1:100 for different times (30-150 min), respectively, after treatment of the cells with 50-100 μg/mL gentamicin for 30-60 min to kill extracellular free or adherent VNP-NC or VNP-PD1nb, PEM Φ (VNP-NC) or PEM Φ (VNP-PD 1 nb) was transferred to 0.3% Triton X-100 in PBS configuration and allowed to stand for 10-15 min to perforate the macrophages to release intracellular VNP strain. LB plates with the resistance to Carna were coated after gradient dilution, and the number of active VNP-NC or VNP-PD1nb strains was calculated to obtain an average per 100 macrophage cells.
Example 2
Mouse peritoneal primary macrophage PEMF loaded VNP-PD1n
PEMF was co-cultured with VNP-NC (empty plasmid-loaded VNP strain) or VNP-PD1nb at a ratio of 1:5-1:100 for different times (30-150 min), respectively, and then cells were treated with 50-100 μg/mL gentamicin for 60min to kill extracellular bacteria. The present inventors found that there was no significant difference in the growth rates of VNP-NC and VNP-PD1nb in normal cell culture medium (fig. 2A). Notably, macrophages were loaded with more VNP-PD1nb strain than VNP-NC strain at the same time (FIG. 2B, left), and the number of intracellular VNP-PD1nb strains in the PEM reached a similar level at 30 min as VNP-NC at 60min (FIG. 2B, right). This suggests that VNP-PD1nb enhances phagocytic effects of macrophages (fig. 2C). Approximately equal numbers of intracellular viable bacteria (510.+ -.13 CFU and 491.+ -.28.+ -. 28 CFU (mean.+ -. SEM)/100 cells), respectively) were achieved when macrophages were co-incubated with VNP-NC and VNP-PD1nb at a 1:10 ratio for 60 minutes and 30 minutes, respectively.
Example 3
Feature detection of loading engineered attenuated salmonella macrophages
In vitro studies confirm that M.phi.VNP-PD 1nb releases loaded VNP-PD1nb and that efficient secretion of PD1nb proteins is expressed: after obtaining MΦ (VNP-PD 1 nb) according to the aforementioned method, the cells were allowed to stand in a cell culture medium to which no antibiotic was added, and cultured. The number of VNP-PD1nb strains in the supernatant after incubation at different time points was examined using the dilution-spread plate method. Simultaneously extracting the collected strain and total protein in the supernatant of the culture medium, and detecting the expression condition of PD1nb in the strain and the effective protein secretion condition in the supernatant corresponding to different time points by using an anti-FLAG antibody through Western blot. The total protein of the supernatant was collected using TCA (trichloroacetic acid) -acetone precipitation as described above.
Example 4
Loading efficiency and tissue distribution evaluation and tracking of loading engineered attenuated salmonella macrophages
The method comprises the steps of simultaneously loading fluorescent protein or luciferase tracer protein capable of being expressed in monocytes/macrophages, quantitatively detecting the fluorescent protein or the luciferase tracer protein, including red fluorescent RFP or luciferase LuxCDABE, and evaluating and tracing the bacterial loading efficiency, tissue distribution and level in the mononuclear cells or the macrophages of the attenuated salmonella secreting the immune checkpoint nanobody expressing PD1nb and the like.
Tissue distribution evaluation and tracking of macrophages loaded with engineered attenuated salmonella
The mononuclear or macrophage of salmonella attenuated by the immune checkpoint nanobody secreted and expressed by PD1nb and the like is marked and loaded by common optical dyes including DiR near infrared dyes, the optical dyes are quantitatively detected, and the tissue distribution and the level of the mononuclear or macrophage of salmonella attenuated by the immune checkpoint nanobody secreted and expressed by PD1nb and the like are evaluated and tracked and loaded.
Quantitative analysis of bacterial loading efficiency in loading engineered attenuated salmonella macrophages
Co-culturing RAW264.7 or primary mononuclear cells/macrophages subjected to LPS induction treatment and attenuated salmonella simultaneously expressing tracer protein and therapeutic protein in a ratio of 1:5-1:100 for 30-150 minutes respectively, and after treating cells for 30-60 minutes by means of 50-100 mug/mL gentamycin to kill extracellular free or adherent engineered attenuated salmonella, quantitatively detecting the correlation between the quantity of the attenuated salmonella of which mononuclear cells or macrophages phagocytose and simultaneously express immune checkpoint nanobodies such as tracer protein or PD1nb and the like and the co-culture time by fluorescence; obtaining a linear relation between the number of attenuated salmonella expressing immune checkpoint nanobodies such as tracer protein or PD1nb and the like and corresponding fluorescence intensity by a gradient dilution method, and effectively and indirectly calculating the number of attenuated salmonella expressing immune checkpoint nanobodies such as tracer protein or PD1nb and the like in macrophage cells; the enzyme label instrument detects the fluorescence intensity (550 nm,585 nm) of the tracer protein, and the total bacterial number loaded by each 100 macrophages is calculated by combining the linear relationship of the bacterial number and the fluorescence.
Transferring mononuclear cells/macrophages loaded with salmonella attenuated by secreting immune checkpoint nanobodies expressing PD1nb and the like into 0.3% TritonX-100 prepared by PBS (phosphate buffered saline), standing for 10-15 min, so as to perforate the macrophages and release the intracellular salmonella attenuated; after gradient dilution, an LB plate with the resistance of the kanamycin is coated, and the correlation of the loading efficiency and the co-culture time of live loading secretion expression PD1nb and other immune checkpoint nanobody attenuated salmonella loaded by mononuclear cells/macrophages is analyzed and detected; detecting the activity state of the cells after 30, 60, 90, 120 and 150 minutes of co-culture of macrophages and attenuated salmonella by using a phenol blue assay; in order to further verify that macrophages are effectively loaded with immune checkpoint nanobody attenuated salmonella loaded with and secreted to express PD1nb and the like, after obtaining the macrophages loaded with the immune checkpoint nanobody attenuated salmonella loaded with and secreted to express PD1nb and the like, the cells are fixed by using 4% paraformaldehyde, kept away from light at room temperature, kept stand for 30 min, washed by PBS for 2-3 times, and respectively stained macrophage frameworks and cell nuclei by means of phalloidin and DAPI according to manufacturer specifications, kept away from light at room temperature, kept stand for 30 min, washed by PBS for 2-3 times, and observed and photographed by using a positive fluorescent microscope.
Example 5
Attenuation effect comparison:
i) Intratumoral titer detection:
To examine the dynamic distribution of intracellular bacteria in vivo, PEM Φ (VNP-PD 1 nb) cells were prepared as described above, and injected into B16F10 tumor-bearing mice by tail vein injection of 100 μl PBS, 5×10 5 VNP-PD1nb, or 1×10 5 PEM Φ (VNP-PD 1 nb) cells. After the mice were sacrificed at the designed time point, the organs and tumor tissues of the mice were obtained, and after disruption by means of a tissue mill, 0.3% triton x-100 was added, and the cells were perforated by standing at room temperature for 15-20min to free VNP, during which time shaking was reversed several times. After dilution in appropriate proportions, the samples were applied to a carbaryl resistant LB plate assay to calculate the VNP titres in each organ in mice. The change in tumor/spleen targeting and tumor/liver targeting was calculated and compared.
The VNP-PD1nb loaded macrophage PEM Φ (VNP-PD 1 nb) was significantly less toxic than VNP-PD1nb (fig. 4A); in contrast to significant liver damage caused by the VNP-PD1nb group, PEM Φ (VNP-PD 1 nb) hardly causes damage to the liver (fig. 4B); likewise, PEM Φ (VNP-PD 1 nb) hardly produced enlargement of the spleen compared to the VNP-PD1nb group which caused significant spleen enlargement (fig. 4C). Tumor targeting of PEM Φ (VNP-PD 1 nb) was significantly improved over VNP-PD1nb (fig. 4D). VNP-PD1nb had good tumor therapeutic effect, and the tumor therapeutic effect of PEMP Φ (VNP-PD 1 nb) was significantly higher than that of the VNP-PD1nb group (FIG. 4E).
The macrophage PEM Φ (VNP-PD 1 nb) loaded with VNP-PD1nb has better long-term tumor-inhibiting effect than the PEM Φ (VNP-NC) +pd1nb combination treatment group, that is, the tumor treatment effect of the macrophage PEM Φ (VNP-PD 1 nb) loaded with attenuated salmonella secreting expressed PD1nb is significantly better than the treatment effect of the combined treatment strategy of the macrophage loaded with attenuated salmonella with PD1nb protein (fig. 5D).
II) histopathology, blood routine and blood biochemical analysis:
Blood is collected from the eyes for routine examination. After allowing the blood to stand at room temperature for 30 minutes, the supernatant was carefully aspirated by centrifugation at 4℃for 15 minutes at 3000 rpm. Serum obtained from blood was cryopreserved at-80 ℃ until blood biochemical index and ELISA were determined. Conventional blood tests, blood biochemical analysis, H & E staining of tumor, heart, liver, spleen, lung and kidney sections and fluorescent immunostaining of tumor-sectioned macrophages were prepared by the marshman service biosystems.
Example 6
Comparison of tumor Effect
I) Evaluation of melanoma-inhibiting solid tumor effect:
And constructing an in-situ melanoma tumor-bearing mouse model. Specifically, the experimental animal mice are female C57BL/6 mice of 6-8 weeks of age. Mice were randomly assigned to different groups. 100uL of cells were inoculated in the right forelimb axilla of mice, 100uL containing cells 2X 10 5 (C57 BL/6, B16F 10) for oncologic development. After the tumors of the mice grew to about 80-160mm 3, DTIC (80 mg/kg) and PD1nb (5 mg/kg) were intraperitoneally injected four times every other day, while VNP (5.0×10 5 cells per mouse) and cells (1.0×10 5 cells per mouse), including PEM Φ, PEM Φ (VNP-NC) and PEM Φ (VNP-PD 1 nb), were injected only once by tail vein. All strains treated by tail vein injection were activated twice prior to injection. Tumors were measured 3-4 times per week with calipers and tumor volumes were calculated according to the formula v=length×width 2 ×0.52. Tumor sizes were measured at intervals for mice of a particular group and used to plot tumor growth after calculation. Values are expressed as mean±sem. To draw survival curves, tumor-bearing mice were monitored daily and sacrificed when signs of adverse reaction were observed or when a human endpoint (tumor weight equal to 10% of the mice body weight) was reached (fig. 5A).
The result shows that compared with any single treatment mode, the PEMP phi (VNP-PD 1 nb) has obviously improved anti-tumor effect. Tumor fold growth time was significantly prolonged (fig. 5B, 5C, table 1). Tumor fold growth time of PBS group was 2.142.+ -. 0.1053 days, tumor fold growth time of DTIC group was 2.766.+ -. 0.0973 days, tumor fold growth time of PEM phi + PD1nb group was 2.540.+ -. 0.2911 days, tumor fold growth time of PEM phi (VNP-NC) group was 3.000.+ -. 0.0747 days, tumor fold growth time of PEM phi (VNP-NC) +PD1nb group was 4.898.+ -. 0.5797 days, and tumor fold growth time of PEM phi (VNP-PD 1 nb) group was 11.57.+ -. 1.764 days (FIG. 4, table 1). Compared with the PBS group, the DTIC group has 29.1 percent of tumor fold growth time, 18.6 percent of tumor fold growth time of the PEM phi+PD1nb group, 40.1 percent of tumor fold growth time of the PEM phi (VNP-NC) group, 128.7 percent of tumor fold growth time of the PEM phi (VNP-NC) +PD1nb group and 440.1 percent of tumor fold growth time of the PEM phi (VNP-PD 1 nb) group. Compared with the DTIC group, the tumor fold growth time of the PEM phi+PD1nb group is shorter than that of the DTIC group, the tumor fold growth time of the PEM phi (VNP-NC) group is prolonged by 8.5 percent, the tumor fold growth time of the PEM phi (VNP-NC) +PD1nb group is prolonged by 77.1 percent, and the tumor fold growth time of the PEM phi (VNP-PD 1 nb) group is prolonged by 318.3 percent; compared with the PEM phi+PD1nb group, the tumor fold growth time of the DTIC group is prolonged by 8.9 percent, the tumor fold growth time of the PEM phi (VNP-NC) group is prolonged by 18.1 percent, the tumor fold growth time of the PEM phi (VNP-NC) +PD1nb group is prolonged by 92.8 percent, and the tumor fold growth time of the PEM phi (VNP-PD 1 nb) group is prolonged by 355.5 percent; compared with PEM phi (VNP-NC), the tumor fold growth time of the PEM phi (VNP-NC) +PD1nb group is prolonged by 63.3 percent, and the tumor fold growth time of the PEM phi (VNP-PD 1 nb) group is prolonged by 285.7 percent; the tumor fold growth time was increased by 136.2% in the PEM phi (VNP-PD 1 nb) group compared to the PEM phi (VNP-NC) +PD1nb group. The extension of the tumor fold growth time (440.1%) in the PEM Φ (VNP-PD 1 nb) group was much greater than the theoretical value of the sum of the tumor fold growth time extension values of the two groups, PEM Φ+pd1nb and PEM Φ (VNP-NC) (18.6% +40.1% = 58.7%).
TABLE 1
The result shows that compared with any single treatment mode, the PEMP phi (VNP-PD 1 nb) has obviously improved anti-tumor effect. Tumor fold growth time was significantly prolonged (fig. 5B, 5C, table 1). The tumor delay time of PBS group was 2.090.+ -. 0.1183 days, the tumor delay time of DTIC group was 2.668.+ -. 0.1034 days, the tumor delay time of PEM [ phi ] +Pd1nb group was 2.667.+ -. 0.3121 days, the tumor delay time of PEM [ phi ] (VNP-NC) group was 3.071.+ -. 0.0644 days, the tumor delay time of PEM [ phi ] (VNP-NC) +P1nb group was 5.612.+ -. 0.6086 days, the tumor delay time of PEM [ phi ] (VNP-PD 1 nb) group was 10.90.+ -. 0.9538 days (FIG. 4), Table 1). Compared with the PBS group, the DTIC group has 27.7 percent of tumor delay time, the PEM phi + PD1nb group has 27.6 percent of tumor delay time, the PEM phi (VNP-NC) group has 46.9 percent of tumor delay time, the PEM phi (VNP-NC) +PD1nb group has 168.5 percent of tumor delay time, and the PEM phi (VNP-PD 1 nb) group has 421.5 percent of tumor delay time. Compared with the DTIC group, the tumor delay time of the PEM phi+PD1nb group is the same as that of the DTIC, the tumor delay time of the PEM phi (VNP-NC) group is prolonged by 15.1 percent, the tumor delay time of the PEM phi (VNP-NC) +PD1nb group is prolonged by 110.3 percent, and the tumor delay time of the PEM phi (VNP-PD 1 nb) group is prolonged by 308.6 percent; the tumor delay time of the PEM phi (VNP-NC) group is prolonged by 15.1 percent, the tumor delay time of the PEM phi (VNP-NC) +Pd1nb group is prolonged by 110.4 percent, and the tumor delay time of the PEM phi (VNP-PD 1 nb) group is prolonged by 308.7 percent compared with the PEM phi+P1nb group; the tumor delay time of the PEM phi (VNP-NC) +PD1nb group was increased by 82.7% compared to the PEM phi (VNP-NC), and the tumor delay time of the PEM phi (VNP-PD 1 nb) group was increased by 254.9%; the tumor delay time was increased by 94.2% in the PEM phi (VNP-PD 1 nb) group compared to the PEM phi (VNP-NC) +PD1nb group. The extension of tumor fold growth time (421.5%) in the PEM Φ (VNP-PD 1 nb) group is much greater than the theoretical value of the sum of the tumor fold growth time extension values of the two groups, respectively, the PEM Φ + PD1nb group and the PEM Φ (VNP-NC) group (27.6% +46.9% = 74.5%); the extension of tumor fold growth time (168.5%) in the PEM Φ (VNP-NC) +pd1nb group was also much greater than the theoretical value of the sum of the tumor fold growth time extension values of the two respective groups of PEM Φ+pd1nb and PEM Φ (VNP-NC) (27.6% +46.9% = 74.5%). The above results thus strongly demonstrate that the monocyte/macrophage or macrophage cell line loaded with attenuated salmonella engineering bacteria expressing PD1nb (RAW 264.7) produces a better, far superior synergistic therapeutic effect than the combined use of the attenuated salmonella loaded monocyte/macrophage cell with PD1 nb.
The results thus strongly demonstrate that the monocyte/macrophage or macrophage cell line loaded with attenuated salmonella engineering bacteria (RAW 264.7) produces a better, far superior synergistic therapeutic effect than the combined use of attenuated salmonella and monocyte/macrophage or macrophage cell line (RAW 264.7).
II) evaluation of the effect of inhibiting melanoma lung metastasis:
For experimental lung metastasis, 1×10 6 B16F10 cells in 100 μl PBS were injected into mice via the tail vein. Treatment started on day 6 and mice were sacrificed on day 18. The degree of lung metastasis was determined and compared by photographic observation with focal point counts and the total surface area of the tumor was analyzed using ImageJ.
In experiments with loading VNP-PD1nb macrophages PEM Φ (VNP-PD 1 nb) to inhibit B16F10 tumor lung metastasis (fig. 6A), PEM Φ (VNP-PD 1 nb) and PEM Φ (VNP-NC) can significantly inhibit lung metastasis of melanoma, and PEM Φ (VNP-PD 1 nb) has better efficacy than PEM Φ (VNP-NC) (fig. 6B).
Example 7
Analysis of tumor inhibition effect mechanism
Tumor-bearing mice were sacrificed, tumors were dissected, and then incubated in digestion medium (10U/mL collagenase I, 400U/mL collagenase IV, 30U/mL DNase I, all diluted in HBSS) for 30 min. The cell mass was removed by a 40 μm cell filter to obtain a single cell suspension. Cells were stained with fixable reactive dye (BD, 564407), incubated at room temperature for 10-15 min in the absence of light, and then ： CD45-PE-Cy7（clone 30-F11）、CD11b-APC（clone 561690）,CD11b-PE（clone M1/70）、F4/80-BV421（clone T45-2342）、CD86-PE（clone GL1）、CD3e-FITC（clone 145-2C11）、CD8-APC（clone 53-6.7）. intracellular proteins including CD206-APC (clone MR6F 3), ki67-BV421 (clone B56), IFN-PE (clone XMG 1.2) and TNF-PE (clone MP6-XT 22) were stained with the following anti-mouse antibodies, after using the membrane-disrupting fixative (BD, 565388). Blood was collected from the retroorbital sinus of the mice, left at room temperature for 1 hour, and then centrifuged at 4 ℃ and 3000 rpm to obtain serum. The CBA kit (BD, 560485) was used to analyze changes in inflammatory-related cytokines in serum, following the product instructions. All flow analyses were performed on a flow cytometer BD Canto II. Tumor H & E staining of mice and fluorescent immunostaining of macrophages on tumor sections were prepared by the company marchantia Servicebio.
The B16F10 tumor model was treated for days 1,3, and 5, with the mΦ (VNP-NC) and mΦ (VNP-PD 1 nb) groups leading to an increase in intratumoral necrotic cell duty cycle over time (fig. 7Aa, 7B) and an increase in immune cell fraction (fig. 7 Ab); mΦ (VNP-PD 1 nb) is better than MΦ (VNP-NC). Tumor cell proliferation was significantly inhibited in the mΦ (VNP-PD 1 nb) and mΦ (VNP-NC) treated groups on day 3 post-treatment with the B16F10 tumor model (fig. 7C). The B16F10 tumor model significantly increased the number of tumor-infiltrating macrophage cells from day 3 post-treatment and was time-dependent (fig. 8A). On day 3 after treatment of the B16F10 tumor model, M1-type tumor-infiltrating macrophages in both groups, mΦ (VNP-NC) and mΦ (VNP-PD 1 nb) were significantly reduced, M2-type tumor-infiltrating macrophages were significantly increased; moreover, the variation of the mΦ (VNP-PD 1 nb) group is larger than mΦ (VNP-NC) (fig. 8B). Immunofluorescent staining also showed the same results (fig. 8C).
After treatment of the B16F10 tumor model, the number of tumor-infiltrating CD 8T cells in tumor-bearing mice was unchanged (fig. 9A); however, the mΦ (VNP-NC) and mΦ (VNP-PD 1 nb) groups effectively produced significantly increased numbers of tumor-infiltrating cd8+ T cells of ifny (fig. 9B) or tnfα (fig. 9C), and mΦ (VNP-PD 1 nb) was better than mΦ (VNP-NC) (fig. 9D).
After 3 days of treatment with the B16F10 tumor model, ifnγ and tnfα concentrations in peripheral blood were significantly increased in the mΦ (VNP-NC) and mΦ (VNP-PD 1 nb) groups, and peripheral blood ifnγ and tnfα concentrations were higher in the mΦ (VNP-PD 1 nb) group than in the mΦ (VNP-NC) group (fig. 10A, 10B, 10C).
Given the wide range of current immune checkpoint antibody inhibitors to be combined with other drugs or therapies, such as chemotherapy, traditional Chinese medicine therapy, biological therapy, physical therapy, etc., thousands of combined treatment regimens with different drugs or therapies are currently under clinical study; therefore, mononuclear cells or macrophages loaded with immune checkpoint nano antibody attenuated salmonella for secreting and expressing PD1nb and the like can be independently applied to preparing antitumor drugs, can also be combined with the existing antitumor drugs to form a composition, or can be applied in antitumor treatment in combination with the existing methods of chemical drug treatment, traditional Chinese medicine treatment, biological treatment, physical treatment and the like. For example, the combined application of the mononuclear or macrophage loaded with the attenuated salmonella and the PD1nb nano antibody protein also generates very remarkable anti-tumor curative effect, so that the combined application of the mononuclear or macrophage loaded with the attenuated salmonella and other anti-tumor drugs or treatment methods can generate better anti-tumor curative effect.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
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Claims (9)

1. A loaded mononuclear or macrophage secreting salmonella attenuated to express immune checkpoint nanobody, comprising: loading and secreting immune checkpoint nano antibody-expressing attenuated salmonella by using macrophages, wherein the macrophages are primary macrophages of a macrophage line RAW264.7 or abdominal cavity and blood sources, and the attenuated salmonella is attenuated salmonella typhimurium VNP20009 and derivative or genetically modified strains thereof; the nano antibody of the immune checkpoint is PD1, PD-L1, CTLA-4, TIM-3 or LAG3, and the nano antibody of the immune checkpoint is a nano antibody blocking agent capable of blocking the immune checkpoint.
2. The method for preparing mononuclear or macrophage loaded with immune checkpoint nanobody-expressing attenuated salmonella of claim 1, comprising the steps of: (1) Constructing attenuated salmonella of a stable constitutive expression immune checkpoint nano antibody blocker, wherein a constitutive strong promoter J23100 or NirB promoter or adhE promoter is used in an expression plasmid, and the extracellular secretory expression of the nano antibody can be realized, wherein the secretory signal peptide is a conventional bacterial secretory signal peptide comprising a bacterial secretory signal peptide sseJ, a bacterial secretory signal peptide MIS, a bacterial secretory signal peptide Flic signal peptide, a bacterial secretory signal peptide pelB, a bacterial secretory signal peptide SOPE, a bacterial secretory signal peptide SpA, a bacterial secretory signal peptide OmpA or an amplification prevention plasmid loss element AT; the PD1 nanometer antibody sequence is a PD1 nanometer antibody sequence comprising a nucleotide sequence shown as SEQ ID No.2, and the PD-L1 nanometer antibody sequence is a PD-L1 nanometer antibody sequence comprising a nucleotide sequence shown as SEQ ID No. 7-9; the sequence of the amplification plasmid loss prevention element AT is a nucleotide sequence shown in SEQ ID No. 1; the sequence of the bacterial secretion signal peptide PelB is a nucleotide sequence shown in SEQ ID No. 3; the sequence of the J23100 constitutive promoter is a nucleotide sequence shown as SEQ ID No. 4; the upstream primer sequence of the J23100 constitutive promoter is a nucleotide sequence shown as SEQ ID No. 5; the downstream primer sequence of the J23100 constitutive promoter is a nucleotide sequence shown as SEQ ID No. 6;
(2) Mononuclear or macrophage is loaded with attenuated salmonella which secretes and expresses immune checkpoint nanobody, RAW264.7 macrophage line which is induced by 25-500ng/mL LPS for 4-48 hours, or primary mononuclear cells/macrophages which are obtained by purifying by 5% starch broth intraperitoneal injection stimulation for 2-4 days combined with an adherence culture method are respectively co-cultured with the attenuated salmonella in a ratio of 1:5-1:100 for 30-150 minutes, and then cells are treated with 50-100 mu g/mL gentamicin for 30-60 minutes to kill extracellular bacteria; (3) The method comprises the steps of measuring the attenuated salmonella of the secretory expression immune checkpoint nanobody of mononuclear or macrophage, calculating the quantity of the attenuated salmonella of the secretory expression immune checkpoint nanobody of the macrophage after phagocytosis by using a dilution plating plate, recording the quantity and the cell activity of the attenuated salmonella of the secretory expression immune checkpoint nanobody of the intracellular effective loading secretory expression immune checkpoint nanobody under different treatment conditions, and determining the final co-culture time and calculating the actual administration dosage of the subsequent cells.
3. The method for detecting mononuclear or macrophage loaded with immune checkpoint nanobody-expressing attenuated salmonella of claim 2, comprising the steps of: (1) Detecting the expression and secretion condition of attenuated salmonella of the secretory expression immune checkpoint nanobody, electrically converting the constructed secretory expression immune checkpoint nanobody plasmid into attenuated salmonella, randomly picking up a monoclonal of engineering bacteria, culturing the engineering bacteria to a growth platform stage by using a liquid LB culture medium, collecting total proteins in bacterial precipitation and total proteins in the liquid culture medium, and detecting the content of the immune checkpoint nanobody in the proteins by using an immunoblotting method; (2) Confirming that loading of a single core or macrophage secreting salmonella attenuated by expressing immune checkpoint nanobody releases salmonella attenuated by secreting by expressing immune checkpoint nanobody and effectively secreting by expressing immune checkpoint nanobody: standing mononuclear or macrophage loaded with salmonella attenuated by secreting and expressing immune checkpoint nanobody in a cell culture medium without antibiotics for culture; detecting the number of immune checkpoint nanobody attenuated salmonella secreted and expressed in the supernatant after culturing for different time points by using a dilution coating flat plate method; meanwhile, extracting the collected thalli and total protein in a culture medium supernatant, detecting the expression condition of immune checkpoint nanobodies in engineering bacteria by using a Western blot method, and secreting and expressing immune checkpoint nanobody attenuated salmonella to effectively secrete nanobodies at different time points, wherein the culture medium supernatant total protein is collected by adopting a TCA (trichloroacetic acid) -acetone precipitation method.
4. The method for evaluating and tracing the bacterial loading efficiency and tissue distribution level of a mononuclear or macrophage loaded with a secretion-expressing immune checkpoint nanobody attenuated salmonella of claim 2, comprising the steps of: the method comprises the steps of simultaneously loading fluorescent protein or luciferase tracer protein capable of being expressed in monocytes/macrophages, quantitatively detecting the fluorescent protein or the luciferase tracer protein, including red fluorescent RFP or luciferase LuxCDABE, and evaluating and tracing the bacterial loading efficiency and tissue distribution and level in the mononuclear cells or the macrophages secreting immune checkpoint nanobody-expressing attenuated salmonella.
5. The method for monitoring tissue distribution of mononuclear or macrophage loaded with immune checkpoint nanobody-expressing attenuated salmonella of claim 2, comprising the steps of: the mononuclear or macrophage secreting salmonella attenuated by expressing immune checkpoint nanobody is loaded with a common optical dye label including DiR near infrared dye, the optical dye is quantitatively detected, and the tissue distribution and level of the mononuclear or macrophage secreting salmonella attenuated by expressing immune checkpoint nanobody are evaluated and tracked.
6. The method of monitoring tissue distribution of mononuclear or macrophage loaded with immune checkpoint nanobody-expressing attenuated salmonella of claim 5, wherein: untreated M0 type macrophages RAW264.7, after 4-48 hours of LPS induction at 25-500ng/mL, RAW264.7 was converted to M1 type macrophages; or primary monocytes/macrophages obtained by purification by combined wall-attached culture for 2-4 days stimulated by intraperitoneal injection of 5% starch broth. Co-culturing RAW264.7 or primary monocyte/macrophage after induction treatment by LPS and attenuated salmonella simultaneously expressing tracer protein and therapeutic protein in a ratio of 1:5-1:100 for 30-150 minutes respectively, and quantitatively detecting the correlation of the quantity of the attenuated salmonella simultaneously expressing tracer protein or immune checkpoint nanobody phagocytized by mononuclear or macrophage and the time of co-culture after treating cells for 30-60 minutes by means of 50-100 mug/mL gentamicin to kill extracellular free or adherent engineered attenuated salmonella; obtaining a linear relation between the number of attenuated salmonella expressing the tracer protein or the immune checkpoint nanobody and the corresponding fluorescence intensity by a gradient dilution method, and effectively and indirectly calculating the number of attenuated salmonella expressing the tracer protein or the immune checkpoint nanobody in a macrophage cell; the enzyme label instrument detects the fluorescence intensity of the tracer protein, and the total bacterial number loaded by each 100 macrophages is calculated by combining the linear relationship of the bacterial number and the fluorescence.
7. The method for evaluating and tracing the bacterial loading efficiency and tissue distribution level of a mononuclear or macrophage loaded with a secretion-expressing immune checkpoint nanobody attenuated salmonella of claim 4, wherein: transferring monocytes/macrophages loaded with salmonella attenuated by secreting nano antibodies expressing immune checkpoints into 0.3% triton x-100 in PBS configuration for 10-15min to perforate the macrophages to release the intracellular attenuated salmonella; coating an LB plate with the resistance of the kanamycin after gradient dilution, and analyzing and detecting the correlation of the live loading secretion expression immune checkpoint nano antibody attenuated salmonella loaded by the mononuclear cells/macrophages and the loading efficiency and the co-culture time; detecting the activity state of the cells after 30, 60, 90, 120 and 150 minutes of co-culture of macrophages and attenuated salmonella by using a phenol blue assay; in order to verify that macrophages are effectively loaded with the salmonella attenuated by loading and secreting the nano antibody expressing the immune checkpoint, after obtaining the macrophages loaded with the salmonella attenuated by loading and secreting the nano antibody expressing the immune checkpoint, the cells are fixed by using 4% paraformaldehyde, are protected from light at room temperature, are kept stand for 30min, are washed by PBS for 2-3 times, and are respectively dyed by using the phalloidin and DAPI according to manufacturer specifications, are kept stand for 30min at room temperature, are kept away from light, are washed by PBS for 2-3 times, and are observed and photographed by using an upright fluorescence microscope.
8. Use of a mononuclear or macrophage loaded with a salmonella attenuated to secrete nano-antibody expressing an immune checkpoint according to any one of claims 1 to 7 for the manufacture of an anti-tumor medicament.
9. The use according to claim 8, characterized in that: the medicine is mononuclear or macrophage loaded with immune checkpoint nano antibody attenuated salmonella and is used for preparing an anti-tumor medicine alone, or is combined with the existing anti-tumor medicine to form a composition, or is combined with the existing chemical medicine treatment, traditional Chinese medicine treatment, biological treatment or physical treatment method to prepare the anti-tumor medicine.
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