BackgroundOpportunistic invasive fungal infections (IFI) are a major threat to immunocompromised populations such as patients with acute myeloid leukemia (AML) and allogenic hematopoietic stem cell transplant (HSCT) recipients(1,2). Specifically, Aspergillus fumigatus (AF) is responsible for high morbidity and mortality in cancer patients. As antifungal therapy has limited efficacy in immunocompromised patients, we sought to develop fungus-specific chimeric antigen receptor (CAR) T cells as a novel immune augmentation strategy to treat IFIs including invasive aspergillosis. To target fungal pathogens, we fused the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-ζ domains upon binding to β-1,3-glucan carbohydrates in the fungal cell wall(3). The generated Dectin-1 CAR+ T cells showed high specificity for β-1,3-gucan and inhibited the growth and branching of AF germlings in an in-vitro co-culture assay. However, we found poor efficacy of Dectin-1 CAR+ T cells against mature AF hyphae, likely due to changes in the fungal cell wall that hamper T-cellular binding to β-1,3-glucan carbohydrates. To overcome this limitation, we have recently developed an AF-specific CAR (AF-CAR) based on a monoclonal antibody which recognizes a surface epitope of mature AF hyphae.MethodsLentiviral vectors were used to generate AF-CAR expressing T cells from human peripheral blood mononuclear cells. Heat killed AF-293 hyphae was used for co-culture studies with No DNA T cells, and AF-CAR expressing T cells. Cell clusters, binding with AF hyphae were noticed in AF-CAR incubated wells whereas no such cell cluster were observed in NoDNA T cells incubated wells.ResultsWhen co-incubated with AF hyphae, AF-CAR+ T cells efficiently targeted mature hyphae and formed lytic synapses with hyphal filaments. The released cytolytic granules damage hyphae and controls branch node formation. Furthermore, exposure to AF hyphae stimulated significant upregulation of activation markers CD69 and CD154 on AF-CAR+ T cells. The activated CAR T cell secretes proinflammatory cytokines which can boost innate immune system to fight against IFI.ConclusionsIn summary, these results indicate that we have successfully generated a novel anti-Aspergillus CAR construct with good in-vitro targeting efficacy against mature AF hyphae. After thorough evaluation of fungicidal activity, cytokine response patterns, and release of cytotoxic mediators, we plan to embark on preclinical tolerability and efficacy studies in a murine model of invasive pulmonary aspergillosis. Thus, we report the production of Aspergillus specific CAR T cells to provide long term protection to immunocompromised patients, such as AML patients and HSCT recipients, from invasive Aspergillus infections.AcknowledgementsThis study was supported by NIAID-R33 AI127381.Ethics ApprovalThis study was approved by IBC committee, University of Texas MD Anderson Cancer Center, Houston, Texas, 77030.ReferencesPappas PG, Alexander BD, Andes DR, Hadley S, Kauffman CA, Freifeld A, Anaissie EJ, Brumble LM, Herwaldt L, Ito J, Kontoyiannis DP, Lyon GM, Marr KA, Morrison VA, Park BJ, Patterson TF, Perl TM, Oster RA, Schuster MG, Walker R, Walsh TJ, Wannemuehler KA, Chiller TM. Invasive fungal infections among organ transplant recipients: results of the transplant-associated infection surveillance network (TRANSNET). Clin Infect Dis 2010;50(8):1101–11.Bhatt VR, Viola GM, Ferrajoli A. Invasive fungal infections in acute leukemia. Ther Adv Hematol 2011;2(4):231–47.Kumaresan PR, Manuri PR, Albert ND, Maiti S, Singh H, Mi T, Roszik J, Rabinovich B, Olivares S, Krishnamurthy J, Zhang L, Najjar AM, Huls MH, Lee DA, Champlin RE, Kontoyiannis DP, Cooper LJ, Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection. Proc Natl Acad Sci U S A 2014;111(29):10660–5.
Monoclonal antibodies against cell wall chitooligomers as accessory tools for the control of cryptococcosis
