Clinical-scale production of Aspergillus-specific T cells for the treatment of invasive aspergillosis in the immunocompromised host

Abstract

Invasive aspergillosis (IA) represents a leading cause of mortality in immunocompromised patients. Although adoptive immunotherapy with Aspergillus-specific T cells (Asp-STs) represents a promising therapeutic approach against IA, the complex and costly production limits its broader application. We generated Asp-STs from a single blood draw of healthy individuals or IA patients in only 10 days, by either Aspergillus fumigatus (AF) lysate or peptide stimulation of mononuclear cells. The cells were phenotypically and functionally characterized, and safety was assessed in xenografts. Healthy donorderived and lysate-or peptide-pulsed Asp-STs presented comparable fold expansion, immunophenotype, and Th1 responses. Upon cross-stimulation, only the lysate-pulsed Asp-STs were empowered to respond to peptide stimulation, although both cell products induced hyphal damage. Importantly, Asp-STs cross-reacted with other fungal species and did not induce alloreactivity in vivo. IA patient-derived T cells displayed an anergic phenotype that prohibited sufficient expansion and yield of meaningful doses of Asp-STs for autologous immunotherapy. Using a rapid and simple process, we generated, from healthy donors but not IA patients, functionally active Asp-STs of broad specificity and at clinically relevant numbers. Such an approach may form the basis for the effective management of IA in the context of allogeneic hematopoietic cell transplantation.

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

“…We also extended the repertoire of targeting to generate pentavalent T-cell products, including, in addition to viruses, the fungus AF, successfully addressing the challenge to effectively expand the less frequent in blood, as compared to VSTs, aspergillus-specific T cells ( 51 ). Importantly, as we have also shown previously ( 34 ), stimulation of AF-STs or multi-pathogen–STs with other fungi (Candida, Fusarium) results in expansion of cross-strain-immunity and a broader than reasonably anticipated, killing repertoire and overall fungal protection.…”

“…Similarly, after re-exposure of each T-cell product to its initial stimuli and IFN-γ and TNF-α secretion measurement by ELIspot assay, the functionality of Cb-STs was comparable to the corresponding specificity of Penta-STs, against all targeted pathogens ( Figures 3A, B ). As we have previously shown with Asp-STs ( 34 ) and multi-pathogen-specific T cells targeting AF ( 41 , in press), Cb-STs displayed broad anti-fungal cross-immunity showing specificity also against other Aspergillus genera (Aspergillus Flavus, Aspergillus Niger), and fungi species (Fusarium; Oxysporum and Solani; Figure 3C ). In addition, Cb-STs were capable of proliferating upon re-stimulation with PHA ( Figure 3D ), while inducing similar to penta-STs, strong and specific lysis of both autologous AdV-pulsed PHA blasts and AF hyphae ( Figures 3E, F ).…”

“…Towards increasing safety and simplifying Good manufacturing practice (GMP) production for future clinical trial, we proceeded to Cb-STs generation using an RNP complex for CRISPR/Cas9-sgRNA delivery by electroporation. In particular, pentavalent-specific T cells (penta-STs) targeting 4 viruses (AdV, CMV, EBV, and BKV) and the fungus AF were generated based on a previously optimized protocol for multivirus-specific or aspergillus-specific T cells (n = 5 different donors) and then edited using the optimal e2b sgRNA to generate Cb-STs ( Figure S1 ) ( 20 , 34 ). Unedited penta-STs from the same donor served as control group.…”

“…Aspergillus conidia were prepared from fresh, mature (2–5 days old) cultures of AF (A.T.C.C. 2004305) and the anti-fungal activity of T-cell products was determined on the basis of hyphal damage by the colorimetric assay with 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxyanilide sodium salt (XTT; Thermo Fisher), as previously described ( 34 ). Water-soluble antigens were prepared from clinical specimen isolates of filamentous fungi ( Aspergillus Niger, Aspergillus Flavus, Fusarium solani, Fusarium oxysporum ) as previously described ( 34 ).…”

The domestic pig as human‐relevant large animal model to study adaptive antifungal immune responses against airborne Aspergillus fumigatus

“…The adaptive antifungal immune response in humans, particularly CD4 + T cells recognizing Aspergillus antigens, have gained increasing attention for both, as noninvasive diagnostic targets to monitor invasive fungal growth and identifying patients at risk, and for potentially contributing to the protection against fungal infections [32, 33]. Evidence for the latter, however, is sparse [5,34].…”