Lymphocyte Retroviral Transduction With Regulated CAR Expression
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Solution Overview
Problem
Current methods for genetically modifying lymphocytes, particularly resting CD4 and CD8 cells, are inefficient and require extensive ex vivo manipulation, leading to issues with cell viability, sterility, scalability, and off-tumor on-target toxicity, as well as challenges in directing T cell engraftment and controlling propagation rates.
Innovation Solution
Development of replication-incompetent recombinant retroviral particles that can transduce and genetically modify lymphocytes, including resting T cells and NK cells, using engineered signaling polypeptides and membrane-bound cytokines to activate and proliferate these cells, with regulatory elements to control CAR expression and improve safety and efficacy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If resting lymphocytes are transduced with recombinant retroviral particles, then genetic modification efficiency is improved, but cell viability and sterility deteriorate due to extensive ex vivo manipulation
Solution Approach 1:
The patent applies preliminary action by pre-activating lymphocytes with anti-CD3/anti-CD28 antibodies and cytokines before transduction, preparing the cells in advance to be more receptive to viral transduction. This pre-preparation improves transduction efficiency while the standardized protocol maintains cell viability by avoiding excessive manipulation during the actual transduction process.
2Productivity
If lymphocytes are activated in vitro before transduction, then transduction efficiency is improved, but manufacturing complexity and time increase
Solution Approach 1:
The patent applies periodic action by using pulsing cytokine additions (IL-2, IL-7, IL-15) at specific time intervals during the 12-28 day expansion period. This periodic stimulation maintains high transduction efficiency by keeping cells in an activated state receptive to viral vectors, while the standardized timing protocol actually reduces manufacturing complexity compared to continuous stimulation approaches.
Solution Approach 2:
The activation and expansion steps are performed as preliminary actions before the critical transduction step. By preparing cells in advance with defined activation protocols and expanding them to optimal densities beforehand, the actual transduction process becomes simpler and more efficient, reducing overall manufacturing complexity.
3Productivity
If CAR expression is constitutively high, then therapeutic efficacy is improved, but off-target toxicity increases
Solution Approach 1:
The patent applies dynamics by implementing inducible CAR expression systems where CAR levels can be dynamically adjusted based on therapeutic needs. The system allows CAR expression to be turned on or off in response to specific stimuli, enabling high CAR expression when therapeutic efficacy is needed while minimizing expression when off-target toxicity is a concern, thus resolving the contradiction between efficacy and safety.
Data Source
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AI summary
The present disclosure provides methods for genetically modifying lymphocytes and methods for performing adoptive cellular therapy that include transducing T cells and/or NK cells. The methods can include inhibitory RNA molecule(s) and/or engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR), for example a microenvironment restricted biologic CAR (MRB-CAR). Additional elements of such engineered signaling polypeptides are provided herein, such as those that drive proliferation and regulatory elements therefor, as well as replication incompetent recombinant retroviral particles and packaging cell lines and methods of making the same. Numerous elements and methods for regulating transduced and/or genetically modified T cells and/or NK cells are provided, such as, for example, those including riboswitches, MRB-CARs, recognition domains, and/or pH-modulating agents.