“Cerberus” T Cells: A Glucocorticoid-Resistant, Multi-Pathogen Specific T Cell Product to Fight Infections in Severely Immunocompromised Patients

Abstract

Adoptive immunotherapy (AI) with pathogen-specific T cells is a promising alternative to pharmacotherapy for the treatment of opportunistic infections after allogeneic hematopoietic cell transplantation or solid organ transplantation. However, clinical implementation of AI is limited to patients not receiving high-dose steroids, a prerequisite for optimal T-cell function, practically excluding the most susceptible to infections patients from the benefits of AI. To address this issue, we here rapidly generated, clinical doses of a steroid-resistant T-cell product, simultaneously targeting four viruses (adenovirus, cytomegalovirus, Epstein Barr virus, and BK virus) and the fungus Aspergillus fumigatus, by genetic disruption of the glucocorticoid receptor (GR) gene using CRISPR/CAS9 ribonucleoprotein delivery. The product, “Cerberus” T cells (Cb-STs), was called after the monstrous three-headed dog of Greek mythology, due to its triple potential; specificity against viruses, specificity against fungi and resistance to glucocorticoids. Following efficient on-target GR disruption and minimal off-target editing, the generated Cb-STs maintained the characteristics of pentavalent-STs, their unedited counterparts, including polyclonality, memory immunophenotype, specificity, and cytotoxicity while they presented functional resistance to dexamethasone. Cb-STs may become a powerful, one-time treatment for severely immunosuppressed patients under glucocorticoids who suffer from multiple, life-threatening infections post-transplant, and for whom therapeutic choices are limited.

Reinforcing the Immunocompromised Host Defense against Fungi: Progress beyond the Current State of the Art

“…Ex vivo generated Rhizopus oryzae-specific T cells cross-reacted with either Aspergillus fumigatus or Rhizopus (microsporus, pusillus), Mucor circinelloides, Penicillium chrysogenum and Candida albicans [71,77]. Cross-protective immunity was also documented on multi-FSTs [59,76,79] and mp-STs targeting 3 viruses and Aspergillus fumigatus [81,82]. These data suggest an enhanced breadth of fungal recognition, however, whether the broad anti-fungal crossimmunity observed in vitro can provide global fungal protection in vivo, remains to be answered in clinical trials.…”

“…To further unleash the potential of adoptive immunotherapy, Khanna et al increased the range of targeting against 2-3 viruses and 1-2 fungi [80]. We have also recently shown the generation of GMP-compliant mp-STs simultaneously targeting 3-4 viruses and Aspergillus fumigatus [81,82]. These T cell products emerge as a powerful, one-time treatment for severely immunosuppressed patients who suffer from multiple, simultaneous or sequential, life-threatening infections post-transplant.…”

“…However, the variability in the quantity of antigens contained in the fungal lysates, along with the potential inclusion of molecules involved in immune evasion [118], makes the preparation of a GMP-compliant fungal lysate complex, demanding and profitless, therefore limiting its commercial availability. GMP-grade overlapping peptide pools from known immunogenic fungal antigens, such as Crf1, Gel1, Pmp20, catalase-1 and SHMT proteins of Aspergillus or mp65 for Candida albicans, offer an alternative source of antigens for the generation of FSTs for clinical use [72,75,81,82].…”

“…To overcome antigenic competition, Gottlieb's group has stimulated individual cultures of viral-and fungal-specific T cells, which they subsequently combined and expanded further after restimulation [62]. We have recently mitigated antigenic competition showing rapid and GMP-compliant generation of a multi-pathogen-specific T cell product as well as a CRISR/CAS9-edited glucocorticoid-resistant multi-pathogen-specific T cell product simultaneously targeting up to 4 viruses and Aspergillus fumigatus, to overcome pathogen-specific T cells' susceptibility to steroids [81,82].…”

“…To allow transplanted patients with opportunistic viral infections to enjoy the benefits of adoptive immunotherapy, regardless of the intensity of immunosuppression, groups have recently genetically modified VSTs to render them resistant to glucocorticoids [145][146][147]. In this setting, we edited the glucocorticoid receptor (GR) and developed a powerful T cell product, called "Cerberus" T cells, exhibiting multiple specificities against 4 viruses (adenovirus, CMV, EBV and BKV) and Aspergillus Fumigatus, along with resistance to glucocorticoids [82]. The genetic modification, was highly on-target with very low or meaningless off-target cutting while the generated GR-resistant mp-STs shared similar phenotypic and functional characteristics to their unedited counterparts.…”