Acute Myeloid Leukemia (AML) is still associated with high relapse rates when treated with conventional chemotherapeutic and hematopoietic transplantation regimens. Thus, new treatment options are urgently needed. Immunotherapy adopting T cells engineered to express tumor-directed Chimeric Antigen Receptors (CARs) has shown striking results particularly in the context of B-cell malignancies, sparking a keen interest in extending this approach also to other hematological malignancies such as AML. Among the surface molecules identified, the CD33 and CD123 (IL-3 receptor α subunit) molecules have emerged as the main validated targets in AML and, being broadly expressed on both AML blasts and leukemic stem cells (LSCs), represent suitable antigens to be targeted with CAR-T cells. My PhD project has been focused on the characterization of non-viral Sleeping-Beauty engineered Cytokine-Induced Killer (CIK) cells with both anti-CD123 and CD33 CARs as a potential tool for the treatment of AML. In the context of CD123 targeting, we focused our attention on dissecting the effect of several variables involved in the CAR design, known to modulate CAR T-cell functional profiles, such as CAR binding affinity and expression in relation to the target antigen density. Indeed, while the “on target-off tumor” effect associated to the CD33 targeting is mostly limited to the hematological compartment, the CD123 targeting demands a higher level of caution, due to the potential recognition of low CD123-positive endothelial tissue. By using our model we were able to define both “lytic” and “activation” antigen thresholds showing that, while the early cytotoxic activity is not affected either by CAR expression or by CAR affinity tuning, the CAR expression represents the main variable impacting on later effector functions. Overall, the full dissection of all these variables offers additional knowledge for the proper design of a suitable anti-CD123 CAR for the treatment of AML which can grant a proper balance between efficacy and safety profiles. In parallel, a proper preclinical assessment of novel therapies also demands for accurate efficacy evaluation, particularly considering the AML disease complexity, such as the heterogeneity and the immunosuppressive myeloid microenvironment. Thus, regarding the CD33 targeting, we investigated the efficacy profiles of CD33.CAR CIK cells alone and in combination with conventional chemotherapeutic agents by exploiting a xenograft chemotherapy model to examine the CD33.CAR-CIK cell contribute in the elimination of the chemotherapy resistant/residual AML cells. We found that CD33.CAR CIK cells alone exhibited a potent anti-leukemic activity in vitro and in vivo, significantly reducing AML development when administered as an early treatment approach and delaying the progression of established disease in mice. Moreover, preliminary data showed that CD33.CAR-CIK cells were still capable to target chemotherapy resistant/residual AML cells in mice experiencing disease recurrence after chemotherapy.

La Leucemia Mieloide Acuta (LMA) è ancora associata ad alti tassi di ricaduta in seguito al trattamento con le terapie convenzionali che comprendono la chemioterapia e il trapianto di cellule staminali ematopoietiche. Da qui la necessità di identificare nuove strategie terapeutiche. L’immunoterapia con linfociti T ingegnerizzati per esprimere recettori chimerici artificiali (CARs) in grado di riconoscere specifici antigeni tumorali ha mostrato risultati sorprendenti in particolare nel contesto leucemie linfoblastiche di tipo B, suscitando un vivo interesse nell'estendere questo approccio anche ad altre neoplasie ematologiche come la LMA. Tra le molecole di superficie identificate nell’ambito della LMA, il CD33 e CD123 (la subunità α del recettore dell’IL-3), essendo ampiamente espressi sia dai blasti leucemici che dalle cellule staminali leucemiche, rappresentano dei buoni antigeni bersaglio per lo sviluppo di terapie CAR mediate. Il mio progetto di dottorato è stato dunque incentrato sulla caratterizzazione di cellule killer indotte da citochine (CIK) geneticamente modificate attraverso il sistema non virale “Sleeping Beauty” per esprimere CARs specifici per il CD123 e il CD33 quale strategia terapeutica per il trattamento della LMA. Nel contesto del targeting dell’antigene CD123, abbiamo focalizzato la nostra attenzione sull’ effetto di diverse variabili coinvolte nel CAR design, e note per la loro capacità di modulare i profili funzionali delle cellule CAR T, come l'affinità di legame e l'espressione del CAR in relazione alla densità dell'antigene bersaglio. Infatti, mentre l'effetto "off-target" associato al targeting del CD33 è per lo più limitato al compartimento ematologico, il targeting del CD123 richiede un livello più alto di cautela, a causa del potenziale riconoscimento da parte del CD123.CAR del tessuto endoteliale che esprime il CD123 a bassi livelli. Utilizzando il nostro modello in vitro siamo stati in grado di definire sia soglie di antigene "litiche" che di "attivazione" dimostrando che, mentre l'attività citotossica precoce non è influenzata né dall'espressione del CAR né dalla modifica dell’affinità del CAR, l’ espressione del CAR rappresenta la variabile principale che incide sulle funzioni effettrici. Complessivamente, la completa caratterizzazione di tutte queste variabili ha fornito ulteriori informazioni per la corretta progettazione di un CAR anti-CD123 per il trattamento della LMA che possa garantire un corretto equilibrio tra i profili di efficacia e sicurezza. In parallelo, una corretta valutazione preclinica di queste nuove terapie richiede anche un'accurata valutazione dell'efficacia, in particolare considerando le complessità intrinseca alla LMA, come l'eterogeneità e il microambiente mieloide immunosoppressivo. Pertanto, per quanto riguarda il targeting del CD33, abbiamo studiato i profili di efficacia di cellule CD33.CAR CIK da sole e in combinazione con agenti chemioterapici convenzionali, sfruttando un modello di xenotrapianto abbinato a chemioterapia per testare le cellule CD33.CAR-CIK nei confronti di cellule di LMA resistenti / residue alla chemioterapia. Abbiamo scoperto che le cellule CD33.CAR CIK da sole mostrano una potente attività anti-leucemica in vitro e in vivo, riducendo in modo significativo lo sviluppo della LMA quando somministrate come approccio di trattamento precoce, e ritardando la progressione di LMA in topi con una malattia conclamata. Inoltre, dati preliminari hanno mostrato che le cellule CD33.CAR-CIK sono ancora in grado di eliminare le cellule di LMA nei topi con recidive dopo la chemioterapia.

(2019). Characterization of Chimeric Antigen Receptors (CARs) as a potential tool for the treatment of Acute Myeloid Leukemia (AML). (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).

Characterization of Chimeric Antigen Receptors (CARs) as a potential tool for the treatment of Acute Myeloid Leukemia (AML)

ROTIROTI, MARIA CATERINA
2019

Abstract

Acute Myeloid Leukemia (AML) is still associated with high relapse rates when treated with conventional chemotherapeutic and hematopoietic transplantation regimens. Thus, new treatment options are urgently needed. Immunotherapy adopting T cells engineered to express tumor-directed Chimeric Antigen Receptors (CARs) has shown striking results particularly in the context of B-cell malignancies, sparking a keen interest in extending this approach also to other hematological malignancies such as AML. Among the surface molecules identified, the CD33 and CD123 (IL-3 receptor α subunit) molecules have emerged as the main validated targets in AML and, being broadly expressed on both AML blasts and leukemic stem cells (LSCs), represent suitable antigens to be targeted with CAR-T cells. My PhD project has been focused on the characterization of non-viral Sleeping-Beauty engineered Cytokine-Induced Killer (CIK) cells with both anti-CD123 and CD33 CARs as a potential tool for the treatment of AML. In the context of CD123 targeting, we focused our attention on dissecting the effect of several variables involved in the CAR design, known to modulate CAR T-cell functional profiles, such as CAR binding affinity and expression in relation to the target antigen density. Indeed, while the “on target-off tumor” effect associated to the CD33 targeting is mostly limited to the hematological compartment, the CD123 targeting demands a higher level of caution, due to the potential recognition of low CD123-positive endothelial tissue. By using our model we were able to define both “lytic” and “activation” antigen thresholds showing that, while the early cytotoxic activity is not affected either by CAR expression or by CAR affinity tuning, the CAR expression represents the main variable impacting on later effector functions. Overall, the full dissection of all these variables offers additional knowledge for the proper design of a suitable anti-CD123 CAR for the treatment of AML which can grant a proper balance between efficacy and safety profiles. In parallel, a proper preclinical assessment of novel therapies also demands for accurate efficacy evaluation, particularly considering the AML disease complexity, such as the heterogeneity and the immunosuppressive myeloid microenvironment. Thus, regarding the CD33 targeting, we investigated the efficacy profiles of CD33.CAR CIK cells alone and in combination with conventional chemotherapeutic agents by exploiting a xenograft chemotherapy model to examine the CD33.CAR-CIK cell contribute in the elimination of the chemotherapy resistant/residual AML cells. We found that CD33.CAR CIK cells alone exhibited a potent anti-leukemic activity in vitro and in vivo, significantly reducing AML development when administered as an early treatment approach and delaying the progression of established disease in mice. Moreover, preliminary data showed that CD33.CAR-CIK cells were still capable to target chemotherapy resistant/residual AML cells in mice experiencing disease recurrence after chemotherapy.
BIONDI, ANDREA
TETTAMANTI, SARAH
IMMUNOTHERAPY; CAR; AML; CD123; CD33
IMMUNOTHERAPY; CAR; AML; CD123; CD33
MED/38 - PEDIATRIA GENERALE E SPECIALISTICA
English
29-gen-2019
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
31
2017/2018
open
(2019). Characterization of Chimeric Antigen Receptors (CARs) as a potential tool for the treatment of Acute Myeloid Leukemia (AML). (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/241327
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