Mononuclear cell or macrophage loaded with attenuated salmonella as well as preparation method and application of mononuclear cell or macrophage

A macrophage and salmonella technology, which is applied in the field of monocytes or macrophages loaded with attenuated salmonella and its preparation, can solve the problems of expensive treatment costs, complicated operation methods, high drug loading and effective therapeutic dose, etc. Improve the effect of tumor treatment, efficient tumor targeting characteristics, and improve the effect of tissue distribution and level

Pending Publication Date: 2022-07-12
JIANGSU TARGET BIOMEDICINE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, one of the main challenges of cell drug delivery is that, limited by the drug loading capacity of a single cell and the required high effective therapeutic dose, it is often necessary to inject a large number of cells for treatment (10^7-10 ^8 level) (Xue J, 2017, Nature Nanotechnology, 12(7): 692-700; Huang B, 2015, ScienceTranslational Medicine, 7(291): 291ra94), which in turn corresponds to complex operation methods and expensive treatment costs

Method used

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  • Mononuclear cell or macrophage loaded with attenuated salmonella as well as preparation method and application of mononuclear cell or macrophage
  • Mononuclear cell or macrophage loaded with attenuated salmonella as well as preparation method and application of mononuclear cell or macrophage
  • Mononuclear cell or macrophage loaded with attenuated salmonella as well as preparation method and application of mononuclear cell or macrophage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Preparation and optimization of macrophages loaded with attenuated Salmonella

[0073] The attenuated Salmonella in the present invention is the attenuated Salmonella typhimurium VNP20009 and its derivatives or genetically modified strains, and the tumor targeting properties are not changed, including but not limited to the aforementioned strains for which the invention has been applied (ZL201410209851.7, ZL201610946268.3 , ZL201610945015.4, ZL201610945021.X, 202010182038.0; Acta Pharmaceutica Sinica B 2021, 11(10):31653177; phoP / phoQ), the following only takes the attenuated Salmonella typhimurium VNP20009 as an example.

[0074] Construction of RFP Expressing Strain Containing AT Element

[0075] Plasmids carrying exogenous genes expressing therapeutic genes and tracer genes were electro-transformed into attenuated Salmonella typhimurium VNP20009 to construct attenuated Salmonella that could express genes expressing therapeutic proteins and tracer proteins. The promot...

Embodiment 2

[0082] Characterization of Attenuated Salmonella-Loaded Macrophages

[0083] (1) Release of intracellular strains:

[0084] In vitro studies confirmed the release of MΦ(VNP) (including RAW264.7(VNP) and PEMΦ(VNP)) to loaded VNP: After obtaining MΦ(VNP) according to the aforementioned method, the direct release of MΦ(VNP) to tumor cells was realized by using Transwell plate. / Indirect contact, that is, plating tumor cells in the lower chamber of a 3.0 mm Transwell plate (the size of pore size cells cannot pass through, but bacteria can still pass through effectively), MΦ(VNP-RFP) UP MΦ (VNP-RFP) cells were added to the upper chamber of the group, and MΦ cells were added to the lower chamber. Relative, MΦ(VNP-RFP) Down MΦ cells were added to the upper chamber of the group, and MΦ (VNP-RFP) cells were added to the lower chamber. After being placed in an incubator for 12 hours of static culture, the supernatant was collected and subjected to gradient dilution and then coated on...

Embodiment 3

[0094] Comparison of tumor suppressor effect

[0095] An orthotopic melanoma tumor-bearing mouse model was constructed. Specifically, 6-8-week-old female C57BL / 6 mice were selected as experimental animals. Mice were randomly assigned to different groups. Inoculate 100 mL of cells in the axilla of the right forelimb of the mouse, 100 mL contains 2 × 10 cells 5 (C57BL / 6, B16F10) for tumorigenesis. When the mouse tumor grows to about 80-160 mm 3 Afterwards treatment. Attenuated Salmonella or macrophage-loaded attenuated Salmonella were administered to tumor-bearing mice via tail vein injection. All strains treated by tail vein injection were activated twice before injection.

[0096]Adjust bacterial titer to 5 x 10 using PBS 6 / mL, intraperitoneal injection of 100 ml. After the macrophages loaded with the corresponding VNPs were obtained according to the aforementioned method, the cell concentration was adjusted to 2.5 × 10 in PBS. 6 / mL (RAW264.7) or 1×10 6 / mL (PEMΦ), ...

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Abstract

The invention discloses a mononuclear cell or macrophage loaded with attenuated salmonella as well as a preparation method and application of the mononuclear cell or macrophage. Specifically, engineered attenuated salmonella is loaded in mononuclear cells or macrophages, and the loaded cells are recruited to a tumor microenvironment, so that bacteria are effectively prevented from being off-target to normal organs. And premature exposure of bacteria is also effectively avoided by wrapping and disguising the cells. The compound acts as an effective immune effector by directly killing tumor cells or releasing intracellular engineering bacteria to activate / regulate an immune system to indirectly inhibit tumors.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a monocyte or macrophage loaded with attenuated Salmonella and a preparation method and application thereof. Background technique [0002] Since the late 19th century when Dr. William Coley attempted to treat cancer patients with heat-killed Gram-positive bacteria (Streptococcus) and Gram-negative bacteria (Salmonella marcescens), more and more Microorganisms are developed and engineered for the treatment of tumors (Coley WB, 1991, Clinical Orthopaedics and Related Research, 262(262):3-11). These microorganisms, often referred to as "oncolytic bacteria," including Salmonella, Listeria, and Escherichia coli, are characterized by their own facultative anaerobic properties and the unique characteristics of the tumor microenvironment, including hypoxic, immunosuppressive, and The nutrients released by a large number of necrotic cells can achieve efficient colonization of tumor sites aft...

Claims

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Application Information

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IPC IPC(8): C12N5/0786C12N1/21C12N15/74C12N15/65C12N15/52C12Q1/02A61K35/74A61K35/15A61K47/46A61K45/06A61P35/00G01N21/64
CPCC12N5/0645C12N1/20C12N15/74C12N15/65C12N15/52G01N33/5005A61K35/74A61K35/15A61K47/46A61K45/06A61P35/00G01N21/6486A61K2300/00Y02A50/30
Inventor 华子春吴乐阳李霖
Owner JIANGSU TARGET BIOMEDICINE RES INST
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