Cryopreserved T-Reg Expansion Using IL-2, Rapamycin, and TGF-β
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Solution Overview
Problem
Current methodologies for generating therapeutically relevant numbers of T-regulatory (T-reg) cells are limited by the requirement of freshly isolated cells, low yields, and time-consuming expansion processes, which hinder their effective use in treating organ transplant rejection and graft versus host disease.
Innovation Solution
A method for selecting and expanding CD4+/CD25+ T-regulatory cells by thawing a frozen apheresis sample, using a buffer comprising Human Serum Albumin (HSA), Magnesium Chloride (MgCl2), and Dornase alfa, and culturing them with Interleukin-2 (IL-2), rapamycin, and Transforming Growth Factor Beta (TGF-β) in the presence of anti-CD3+ and anti-CD28+ antibodies, resulting in a significantly expanded cell population.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If freshly isolated T-reg cells are used for expansion, then the immunosuppressive function is maintained, but the logistics and time requirements increase significantly
Solution Approach 1:
The patent applies preliminary action by collecting and cryopreserving T-reg cells in advance via apheresis before they are needed for therapy. This allows the cells to be stored frozen and readily available when clinical need arises, eliminating the time delay of fresh isolation while maintaining cell functionality through proper cryopreservation protocols
2Ease of manufacture
If conventional expansion methods are used, then the process is straightforward, but the yield of expanded T-reg cells remains low
Solution Approach 1:
The patent applies parameter changes by optimizing multiple culture conditions simultaneously: using specific cytokine concentrations (IL-2, TGF-beta, rapamycin), controlling culture duration (10-14 days), adjusting cell density, and incorporating specific antibodies (anti-CD3, anti-CD28). These parameter optimizations work together to achieve 40-fold expansion while maintaining cell quality and functionality
3Ease of operation
If frozen apheresis samples are used, then logistics are simplified and cells can be stored, but cell viability and functionality may be compromised
Solution Approach 1:
The patent applies preliminary action by performing apheresis and cryopreservation in advance, allowing cells to be stored and transported without compromising viability. The optimized thawing and culture protocol ensures that frozen cells recover fully and maintain functionality equivalent to fresh cells
Solution Approach 2:
The patent applies parameter changes by optimizing culture conditions specifically for thawed frozen cells, including extended culture duration (10-14 days), specific cytokine concentrations, and sequential addition of growth factors to ensure complete recovery and expansion of cryopreserved T-reg cells while maintaining their immunosuppressive function
Data Source
AI summary
Methods, compositions, and kits for generating therapeutically relevant populations of immunosuppressive T-reg cells and uses thereof are disclosed.