Nucleofected Dendritic Cells for Multivirus CTL Generation
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Solution Overview
Problem
Existing methods for generating multivirus-specific and tumor-associated antigen (TAA)-specific cytotoxic T lymphocytes (CTLs) are limited by the need for large blood volumes, lengthy manufacturing processes, and the use of infectious virus material, making them expensive and time-consuming, and they often require complex procedures that are not universally applicable.
Innovation Solution
The use of dendritic cells (DCs) nucleofected with DNA plasmids encoding immunodominant and subdominant viral antigens, combined with specific cytokines and specialized cell culture environments, allows for the rapid generation of multivirus-specific CTLs and TAA-specific CTLs that target multiple epitopes within 10 days, using plasmids or peptide libraries and gas permeable culture vessels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional methods are used to generate multivirus-specific CTLs, then the CTLs can be produced with virus-specific immunity, but the manufacturing process requires large blood volumes and takes 10-12 weeks
Solution Approach 1:
The patent applies preliminary action by pre-differentiating dendritic cells and pre-transducing them with viral antigen expression vectors before the actual CTL generation process. This preparatory work is done in advance, allowing the CTL production to proceed more rapidly when needed, reducing the overall manufacturing time from 10-12 weeks to a shorter period while maintaining virus-specific immunity
Solution Approach 2:
The patent extracts the viral antigen expression capability from the conventional complex manufacturing process by using genetically modified dendritic cells that constitutively express viral antigens. This separation allows the antigen presentation function to be independently optimized and reused across multiple CTL generation batches, reducing both time and blood volume requirements
2Adaptability or versatility
If conventional methods are used to generate multivirus-specific CTLs, then the CTLs can target multiple viruses, but the process requires infectious virus material and is expensive
Solution Approach 1:
The patent uses copying by transducing dendritic cells with viral antigen expression vectors that contain viral gene sequences. These vectors serve as genetic copies of the viral antigens, allowing the dendritic cells to present viral antigens without requiring actual infectious virus material. This approach maintains multivirus specificity while simplifying manufacturing and reducing costs
Solution Approach 2:
The patent applies universality by creating dendritic cell lines that can present multiple viral antigens simultaneously through co-transduction with multiple expression vectors. These universal dendritic cell factories can be used to generate CTLs against multiple different viruses (EBV, CMV, adenovirus, etc.) using the same base methodology, reducing manufacturing complexity and increasing ease of production
3Reliability
If conventional methods are used to generate TAA-specific CTLs, then the CTLs can target tumor antigens, but the process requires lengthy culture periods and is not rapidly producible
Solution Approach 1:
The patent applies preliminary action by pre-differentiating dendritic cells and pre-loading them with tumor antigen expression vectors before CTL generation. This advance preparation creates ready-to-use antigen-presenting cells that can rapidly induce TAA-specific CTLs when needed, dramatically increasing production speed while maintaining tumor antigen specificity
Solution Approach 2:
The patent maintains continuity of useful action by using dendritic cells that constitutively express tumor antigens through stable genetic integration. This continuous expression allows for repeated CTL generation cycles without needing to re-differentiate or re-transduce the dendritic cells, enabling rapid, continuous production of TAA-specific CTLs for clinical use
Data Source
AI summary
The present invention encompasses methods and compositions for the generation and use of cytotoxic T lymphocytes that target multiple viruses or that are specific for multiple tumor antigens. In specific embodiments, the generation methods employ use of certain cytokines to promote proliferation and reduce cell death in an activated T cell population and/or that employ a particular bioreactor having a gas permeable membrane.