CAR T-Cell Isolation Using Labeled Peptides and Magnetic Separation
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Solution Overview
Problem
Current methods for isolating and purifying Chimeric Antigen Receptor (CAR) T-cells require the expression of inert Epidermal Growth Factor receptor domains, which can interfere with detection and isolation processes.
Innovation Solution
A method involving the use of labeled CAR-binding peptides and magnetic particles to form a labeled CAR T-cell-peptide complex, allowing for the separation of CAR T-cells from a cell population, thereby isolating and purifying the CAR T-cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If inert EGF receptor domains are expressed as part of lentiviral CAR expression product, then CAR T-cells can be isolated and detected, but the EGF receptor domains interfere with detection and isolation processes
Solution Approach 1:
The patent extracts and removes the inert EGF receptor domain from the CAR expression product, retaining only the essential CAR components (transmembrane domain, intracellular signaling domain, and antigen-binding domain). This eliminates the interfering EGF receptor domain while preserving the isolation and detection capabilities through the remaining functional domains.
Solution Approach 2:
The patent modifies the CAR expression product by removing the EGF receptor domain from specific locations where it causes interference, while preserving the essential functional domains. This creates a localized improvement where the CAR T-cells maintain detection and isolation capabilities without the harmful interference of the EGF receptor domain.
2Productivity
If lentivirus expresses CAR product, then CAR T-cells are generated, but inert EGF receptor domain is also expressed on cell surface causing interference
Solution Approach 1:
The patent extracts the harmful EGF receptor domain from the lentiviral CAR expression product while retaining the essential CAR components. This allows the lentivirus to continue expressing functional CAR T-cells without co-expressing the interfering EGF receptor domain, thus maintaining productivity while eliminating harmful factors.
Solution Approach 2:
The patent changes the composition parameters of the CAR expression product by removing the EGF receptor domain sequence from the expressed protein. This parameter change eliminates the harmful EGF receptor domain expression while preserving the CAR T-cell generation capability through the remaining essential domains.
3Quantity of substance
If bulk transduced T-cells are used, then CAR T-cell population is obtained, but purity and enrichment of CAR T-cells is low
Solution Approach 1:
The patent introduces a purified CAR expression product as an intermediary that specifically binds to and isolates CAR T-cells from the bulk transduced population. This intermediary enables the separation of pure CAR T-cells from non-CAR T-cells, achieving both sufficient quantity and high purity enrichment simultaneously.
Solution Approach 2:
The patent segments the bulk transduced T-cell population into pure CAR T-cells and non-CAR T-cells using the purified CAR expression product. This segmentation allows the isolation of highly enriched CAR T-cells while maintaining adequate population size for therapeutic use, resolving the contradiction between quantity and purity.
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 achieves high purity and enrichment of CAR T-cells, enhancing their therapeutic efficacy by increasing CAR expression and cytotoxic activity against target cells.
Implementation Method 1
contacting the labeled CAR T-cell-peptide complex with a label-binding magnetic particle thereby forming a bound labeled CAR T-cell-peptide complex; and separating said bound labeled CAR T-cell-peptide complex from the cell population
Data Source
AI summary
Provided herein, inter alia, are methods for the isolation and purification of CAR T-Cells from bulk transduced human T-cells. Also provided herein are chimeric peptide-CAR T-cell complexes useful for the isolation of CAR T-cells. In addition, pharmaceutical compositions and methods for treating cancer are described herein.