NK Cell Production via CD3 Removal and Cytokine Activation
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Solution Overview
Problem
Current methods for producing NK cells for cancer therapy are inefficient and costly, requiring extensive time and resources, and existing methods struggle to achieve sufficient quantities of activated NK cells for clinical applications, particularly for solid cancers.
Innovation Solution
A method involving the removal of CD3-positive T cells from monocytes, followed by treatment with IL-15 and IL-21 cytokines, and subsequent culture to produce high-purity NK cells, which can be cryopreserved or cold-preserved for therapeutic use, allowing for efficient production and administration of NK cells for various cancers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used to produce NK cells for cancer therapy, then the production process is established and can be performed with existing techniques, but the methods are inefficient, costly, and require extensive time and resources to achieve sufficient quantities of activated NK cells
Solution Approach 1:
The patent applies preliminary action by pre-isolating and cryopreserving CD3-negative cells (NK cell precursors) before activation. This allows the critical isolation step to be performed in advance and stored, so that when clinical treatment is needed, only the final activation and expansion steps are required, dramatically reducing the time from treatment decision to therapeutic cell administration while maintaining high productivity
Solution Approach 2:
The patent segments the NK cell production process into distinct modules: (1) isolation of CD3-negative cells from peripheral blood, (2) cryopreservation of isolated cells, and (3) in vitro activation and expansion. This segmentation allows each step to be optimized independently and enables flexible scheduling where isolated cells can be banked and activated only when clinically needed, improving overall production efficiency
2Quantity of substance
If conventional methods are used to produce NK cells, then existing production protocols can be followed, but the methods struggle to achieve sufficient quantities of activated NK cells for clinical applications
Solution Approach 1:
The patent extracts and removes T cells (CD3-positive cells) from the peripheral blood mononuclear cell population before NK cell activation. This extraction of interfering T cells creates a purified starting population of CD3-negative cells that can be more efficiently expanded into NK cells, achieving sufficient quantities for clinical use while simplifying the overall manufacturing process by eliminating T cell contamination issues
Solution Approach 2:
The patent employs parameter changes by using specific cytokine combinations (IL-2, IL-7, IL-15, IL-21) at optimized concentrations and ratios to stimulate NK cell proliferation. This cytokine parameter optimization enables exponential expansion of NK cells from the isolated CD3-negative population, achieving the necessary clinical quantities while maintaining ease of manufacture through standardized culture conditions
3Productivity
If NK cells are produced using existing methods, then standard protocols can be applied, but the production is inefficient and costly requiring extensive resources
Solution Approach 1:
The patent applies preliminary action by pre-isolating and cryopreserving CD3-negative cells (NK cell precursors) before activation. This allows the critical isolation step to be performed in advance and stored, so that when clinical treatment is needed, only the final activation and expansion steps are required, dramatically reducing the time from treatment decision to therapeutic cell administration while maintaining high productivity
Solution Approach 2:
The patent utilizes peripheral blood as a readily available, low-cost starting material instead of requiring expensive stem cell sources or complex mobilization protocols. The use of standard cytokines for activation represents a cost-effective approach compared to more complex differentiation protocols, making NK cell production more economically viable while maintaining high productivity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables the rapid production of high-purity NK cells that exhibit significant anticancer effects against colorectal, lung, liver, pancreatic, and leukemia cancers, with preserved NK cells showing comparable efficacy to fresh cells, facilitating their use as a pharmaceutical composition for cancer treatment.
Implementation Method 1
allowing the CD3-positive T cells to crosslink to erythrocytes
Implementation Method 2
isolating the CD3-negative cells by density-gradient centrifugation
Implementation Method 3
culturing the CD3-negative cells by treating the CD3-negative cells with IL-15 and IL-21
Implementation Method 4
freezing the cultured CD3-negative cells in a cryopreservation medium containing 10% DMSO
Implementation Method 5
freezing the cultured CD3-negative cells
Implementation Method 6
cryopreservation medium containing 10% DMSO (dimethyl sulfoxide)
Data Source
Figure 1~2a
Figure 2b~2c
Figure 2d~2f
AI summary
The present invention relates to a method for producing a large amount of natural killer cells and the use of natural killer cells obtained by the method as an anticancer agent. The use of the method of the present invention can produce fresh NK cells with high purity within a short time compared to conventional method, and can also produce cold-preserved NK cells and thawed cryopreserved NK cells, which have efficacy comparable with that of the fresh NK cells. Furthermore, it can produce NK cells, which have efficacy comparable with that of the fresh NK cells, from cryopreserved CD3-negative cells. The fresh NK cells, cold-preserved NK cells and cryopreserved NK cells produced by the methods of the present invention can exhibit therapeutic effects against various cancers, including colorectal cancer, lung cancer, liver cancer, pancreatic cancer and leukemia, indicating that these NK cells can be effectively used as cellular therapeutic agents. In addition, the present inventors have established doses and methods of administration, which show excellent effects when the fresh NK cells, cold-preserved NK cells and cryopreserved NK cells of the present invention are used as pharmaceutical compositions for cellular therapy.