CAR-T Cell Preparation Without CD28 for Faster Expansion
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Solution Overview
Problem
Current CAR-T cell preparation methods require CD28 stimulation and prolonged ex vivo expansion, leading to increased costs, extended waiting periods, and compromised in vivo persistence due to differentiation, necessitating optimization to reduce ex vivo differentiation and shorten preparation cycles.
Innovation Solution
A method of preparing CAR-expressing cells without CD28 stimulation, using a CD3/TCR complex agonist and specific cytokines like IL-7 and IL-21, eliminating IL-2, and optimizing preparation duration to enhance in vivo efficacy and safety.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CD28 agonist is used for T cell activation, then T cell activation and CAR gene transfer efficiency is improved, but preparation costs increase and operation steps increase
Solution Approach 1:
The patent removes the CD28 agonist component from the traditional activation system, extracting only the necessary CD3/TCR complex agonist function. This simplifies the activation system by eliminating the need for dual co-stimulatory signaling, thereby reducing operation steps while maintaining essential activation functionality for CAR gene transfer.
Solution Approach 2:
The patent employs a multifunctional culture medium containing cytokines (IL-7, IL-21, and optionally IL-2) that simultaneously provide T cell activation support, proliferation signals, and survival factors. This single medium formulation replaces the need for separate CD28 agonist treatment and multiple culture conditions, consolidating multiple functions into one system.
2Reliability
If CD28 agonist is used for T cell activation, then T cell activation is improved, but preparation costs increase
Solution Approach 1:
The patent replaces expensive protein-based CD28 agonists with a cost-effective cytokine combination (IL-7, IL-21, and optionally IL-2) that can be produced at lower costs. The culture medium formulation uses readily available cytokines that are more economical than recombinant CD28 agonist proteins, thereby reducing preparation costs while maintaining activation efficacy.
3Quantity of substance
If prolonged ex vivo expansion is conducted, then CAR-T cell quantity is increased, but preparation time is extended and in vivo persistence is compromised
Solution Approach 1:
The patent optimizes cytokine concentrations and composition (IL-7 at 2.5-10 ng/ml, IL-21 at 12.5-50 ng/ml, and optionally IL-2 at 100-500 IU/ml) to achieve rapid T cell proliferation. This parameter optimization enables sufficient CAR-T cell expansion within 1-4 days, dramatically reducing preparation time from the traditional 7-14 days while maintaining adequate cell quantities for therapy.
Solution Approach 2:
The patent performs preliminary T cell activation and proliferation in the optimized culture medium before CAR lentiviral transfection. This preliminary expansion ensures sufficient cell numbers and optimal cellular state for subsequent gene transfer, reducing the need for prolonged post-transfection expansion and thereby shortening the overall preparation timeline.
4Quantity of substance
If prolonged ex vivo expansion is conducted, then CAR-T cell quantity is increased, but in vivo persistence is compromised due to differentiation
Solution Approach 1:
The patent performs partial expansion of T cells before CAR transfection, achieving sufficient cell numbers without excessive proliferation. By limiting the expansion duration to 1-4 days and using optimized cytokine concentrations, the patent obtains adequate cell quantities while preventing excessive differentiation that would compromise in vivo persistence. This partial action approach balances cell quantity generation with functional preservation.
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
The method results in rapid production of CAR-expressing cells within 1-4 days with improved in vivo efficacy and safety, reducing operation steps and costs while maintaining functional characteristics.
Implementation Method 1
contacting the cell population with a CD3/TCR complex agonist
Implementation Method 2
contacting the cell population with a nucleic acid molecule encoding the CAR, thereby providing cells containing the nucleic acid molecule, and step (iii): culturing the cell population for a period of time
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 2A~2B
AI summary
The present application provides a method for preparing a cell population expressing a chimeric antigen receptor (CAR), and a composition, a culture medium or a kit applicable to the method. The cell population has reduced in vivo cytotoxicity.