The basis of Chimeric Antigen Receptor-based adoptive T cell immunotherapy in the murine cytomegalovirus infection model
Year: 2024
Type: dissertation
Abstract: The human cytomegalovirus (HCMV) is a widespread pathogen but usually causes mild and uncomplicated infection in healthy, immunocompetent individuals. However, in immunocompromised patients, especially stem cell transplant recipients, de novo infection or reactivation of latent infection can result in life-threatening complications. One cause of such severe complications is HCMV’s arsenal of viral effector proteins, which allow it to effectively evade the host immune system. Expression of human leukocyte antigen (HLA) molecules on the surface of infected cells is downregulated, rendering HCMV-infected cells invisible to effector T cells; however, these represent a crucial host defense mechanism to control infection. An elegant design to circumvent certain immune evasion mechanisms are chimeric antigen receptors (CARs). These receptors detect HCMV-infected cells in a HLA-independent manner and turn one of the virus’ strengths into a weakness: While the plethora of glycoprotein complexes found in the envelope of the virions directs infection of a variety of cell types, their expression on the infected cell’s surface makes them perfect targets for CARs. Engineered T cells equipped with CARs distinctively recognize and eliminate target cells displaying specific antigens on their surface; in this case: HCMV-specific glycoproteins. This study constitutes an initial investigation into the potential of CAR-T cell therapy as a treatment option for HCMV infections using the model system of the murine cytomegalovirus (MCMV). To evaluate the functionality of MCMV-specific CAR-T cells, a suitable in vitro model is required. Stable cell lines expressing different glycoprotein complexes of MCMV, consisting of glycoprotein B (gB), gN/gM or gH/gL, were generated. These cell lines were used to confirm the functionality of CAR-T cells in general, circumventing viral effector functions which interfere with T cell response. A novel molecular biosensor was invented to specifically indicate T cell-mediated cytotoxicity. By genetically engineering a fluorescent protein to undergo conformational changes, inducing green fluorescence upon cleavage by granzyme B (GZMB), the cleavage-responsive sensor for T cell activity level (CRSTAL) was generated and tested. This was complemented by the existing nano-luciferase (nLuc) system, which detects general cell damage. Thus, two individual readouts for CAR-T cell killing assays were established and used as a modular system. Target cells expressing one of the glycoprotein complexes and one of the two cytotoxicity reporters were generated to evaluate the efficacy of MCMV-specific CAR-T cells. Based on this framework, different CAR constructs targeting either gB, gH or gN were designed. Murine T cells were transduced via an optimized protocol and eight of the CARs exhibited strong surface expression. The resulting CAR-T cells were tested in killing assays. Out of eight expressed CARs, six constructs exerted successful lysis of reporter cells expressing the respective target antigen. Co-incubation assays with MCMV-infected cells showed a strong inhibition of viral replication by T cells transduced with one of six CAR constructs. In contrast to existing literature, T cells expressing one of five CARs were, in fact, able to specifically lyse MCMV-infected cells, of which gB-targeting M11-based CARs displayed the strongest cytolytic activity. This work presents an optimized transduction protocol for murine T cells with CAR constructs as well as a novel fluorogenic reporter for T cell activity (CRSTAL). The latter can be applied in settings where conventional cytotoxicity markers are limited by anti-apoptotic effector functions of certain viruses. By applying this and other reporters, we have shown that MCMV-specific CAR T cells are able to inhibit MCMV replication in vitro and to specifically lyse infected target cells, laying the foundation for investigation of CAR-T cell therapy as a viable treatment option for severe CMV infections.
Author: Bednar, Christopher
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
OpenAlex ID: W7081666608
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