Chimeric Antigen Receptor (CAR) T-cell therapy has produced remarkable clinical responses in patients affected by acute lymphoblastic leukemia. Unfortunately, CAR T-cells have not been equally successful in acute myeloid leukemia (AML) due to tumor heterogeneity, lack of truly AML-restricted target antigens and the role of leukemia microenvironment in blasts protection and leukemia stem cells (LSCs) maintenance. Specifically, the bone marrow (BM) niche, where LSCs reside, is involved in leukemia promoting activities whilst suppressing normal hematopoiesis. Therefore, we hypothesized that targeting LSCs at their location may enhance the potency and selectivity of CAR-T cells. To address this issue, we have designed two aims: 1) promote rapid and efficient localization of CAR T-cells within the BM niche, 2) select a leukemia-restricted antigen to specifically target AML blasts and LSCs. First, we proposed to harness CD33.CAR-redirected Cytokine-Induced Killer (CIK) cells, an alternative effector T-cell population with acquired NK-like cytotoxic activity as well as minimal alloreactivity, to selectively route their activity to leukemia transformed niche. The chemokine ligand 12 (CXCL12), released by mesenchymal stromal cells (MSCs) within the medullary niche, and its chemokine receptor 4 (CXCR4) are two pivotal players regulating leukocytes trafficking to the BM. In AML, CXCL12 interacts with CXCR4 overexpressed on blasts, promoting their migration and homing in the niche. Hence, taking advantage of this axis might facilitate CD33.CAR-CIK cells homing to the BM and therefore leukemia eradication. However, ex vivo manipulation protocols of CD33.CAR-CIK cells consistently downregulate CXCR4 expression and may affect the capacity of adoptively infused cells to migrate to BM and exert their anti-leukemic action. Therefore, to improve CD33.CAR-CIKs homing in the BM microenvironment we have developed CD33.CAR-CIK cells overexpressing CXCR4, in its wild-type or hyperactive mutant form. Notably, CIK cells engineering with CD33.CAR-CXCR4 constructs led to a consistent increase in CXCR4 expression, without altering CIK cells phenotype and CAR-related effector functions. Interestingly, compared to conventional CD33.CAR-CIK cells, CD33.CAR-CXCR4WT and especially CD33.CAR-CXCR4MUT-CIK cells demonstrated significantly superior in vitro chemotactic response toward CXCL12 and MSC-derived supernatants, and greater in vivo BM homing ability and persistence. Furthermore, to develop an effective anti-AML CAR T-cell therapy, it is fundamental to identify a LSC-specific marker, sparing the normal counterpart of hematopoietic stem cells (HSCs). T-cell immunoglobulin and mucin protein 3 (TIM-3) is an immune checkpoint molecule, it plays a central role in immune responses in AML and it is an LSC-specific marker, lacking expression on HSCs. Therefore, we designed a third-generation anti-TIM-3.CAR using the single-chain fragment variable (scFv) derived from an antagonistic ligand-blocking anti-TIM-3 antibody. In vitro, TIM-3.CAR-CIK cells efficiently killed both AML cell lines and primary AML blasts, but not normal TIM-3+ activated CIK cells, monocytes and NK-cells. Notably, we observed selective elimination of primary LSC-enriched population (CD34+ CD38-). Furthermore, TIM-3.CAR-CIK cells maintained their effector functions despite multiple in vitro restimulations, setting the basis for further exploration in in vivo models. Overall, both approaches, one improving CAR-CIK cells homing to the transformed niche and the other conferring superior safety and selectivity, might improve the efficacy of anti-AML CAR-CIK therapy.

La terapia CAR-T rappresenta un approccio promettente, ma ha riportato una ridotta efficacia nella leucemia mieloide acuta (AML), a causa dell’eterogeneità del tumore, dell’assenza di antigeni target AML-specifici e del ruolo del microambiente leucemico nella protezione dei blasti e delle cellule staminali leucemiche (LSC). La nicchia midollare, nella quale risiedono le LSC, è coinvolta in attività che promuovono la progressione leucemica e sopprimono l’ematopoiesi sana. Quindi ipotizziamo che bersagliare le LSC nascoste nella nicchia potesse migliorare l’efficacia delle CAR-T. Per testare la nostra ipotesi, abbiamo agito su due fronti: 1) promuovere una migrazione efficiente delle CAR-T nella nicchia midollare, 2) selezionare un antigene target ristretto ai blasti leucemici e alle LSC. Prima, abbiamo proposto una strategia per guidare le cellule CD33.CAR CIK (Cytokine-Induced Killer), una sottopopolazione di cellule T effettrici, verso la nicchia leucemica. La chemochina CXCL12, rilasciata dalle cellule mesenchimali stromali (MSC), nella nicchia midollare, e il suo recettore CXCR4, sono coinvolti nella regolazione della migrazione dei leucociti all’interno della nicchia. Quindi, abbiamo ipotizzato che sfruttare questo asse potesse migliorare la capacità di homing delle CD33.CAR-CIK nella nicchia e favorire l’eradicazione della leucemia. Tuttavia i protocolli di manipolazione ex vivo delle CD33.CAR-CIK riducono l’espressione di CXCR4, compromettendo la capacità delle cellule infuse di raggiungere la nicchia. Quindi per implementare la capacità di homing delle CD33.CAR-CIK nel microambiente midollare, abbiamo sviluppato delle CD33.CAR-CIK overesprimenti CXCR4, nella sua forma wild-type o iperattiva mutata. Le CIK ingegnerizzate con i costrutti CD33.CAR-CXCR4 hanno mostrato un consistente aumento dell’espressione di CXCR4, senza riportare alterazioni fenotipiche e nelle funzioni effettrici CAR-associate. Inoltre, rispetto alle CD33.CAR-CIK, le cellule CD33.CAR-CXCR4WT -CIK ed in particolare le CD33.CAR-CXCR4MUT-CIK hanno dimostrato non solo una superiore risposta chemotattica in vitro verso il CXCL12 ed i surnatanti delle MSC, ma anche un aumentato homing in vivo. In seguito, per promuovere lo sviluppo di un approccio CAR-T più efficace e sicuro, abbiamo proposto di re-indirizzare il CAR verso un antigene espresso selettivamente dalle cellule AML, ma assente sulle cellule staminali ematopoietiche (HSC). TIM-3 è un immune checkpoint, svolge un ruolo centrale nella regolazione delle risposte immunitarie nell’AML e costituisce un marcatore selettivo per le LSC, senza essere espresso dalle HSC. Abbiamo disegnato un CAR di terza generazione diretto contro TIM-3, utilizzando la porzione scFv derivante da un anticorpo monoclonale anti-TIM-3. In vitro, le TIM-3.CAR-CIK hanno dimostrato di eliminare sia le linee AML che i blasti primari, senza dare tossicità verso le cellule TIM-3+ sane, come le CIK attivate, i monociti e le cellule NK. Inoltre, le TIM-3.CAR-CIK hanno eliminato in maniera selettiva le LSC (CD34+ CD38-). Infine, le TIM-3.CAR-CIK hanno mantenuto le loro capacità effettrici nonostante multiple ristimolazioni in vitro, gettando le basi per lo studio di questo costrutto in vivo. Complessivamente, entrambi gli approcci, uno implementando l’homing delle CAR-CIK alla nicchia midollare e l’altro conferendo una superiore selettività, potrebbero migliorare l’efficacia della terapia CAR-T nel contesto dell’AML.

(2022). Enhancing AML CAR CIK therapeutic potency increasing the localization of engineered cells in the malignant niche and its selectivity by LSCs specific targeting. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).

Enhancing AML CAR CIK therapeutic potency increasing the localization of engineered cells in the malignant niche and its selectivity by LSCs specific targeting

BIONDI, MARTA
2022

Abstract

Chimeric Antigen Receptor (CAR) T-cell therapy has produced remarkable clinical responses in patients affected by acute lymphoblastic leukemia. Unfortunately, CAR T-cells have not been equally successful in acute myeloid leukemia (AML) due to tumor heterogeneity, lack of truly AML-restricted target antigens and the role of leukemia microenvironment in blasts protection and leukemia stem cells (LSCs) maintenance. Specifically, the bone marrow (BM) niche, where LSCs reside, is involved in leukemia promoting activities whilst suppressing normal hematopoiesis. Therefore, we hypothesized that targeting LSCs at their location may enhance the potency and selectivity of CAR-T cells. To address this issue, we have designed two aims: 1) promote rapid and efficient localization of CAR T-cells within the BM niche, 2) select a leukemia-restricted antigen to specifically target AML blasts and LSCs. First, we proposed to harness CD33.CAR-redirected Cytokine-Induced Killer (CIK) cells, an alternative effector T-cell population with acquired NK-like cytotoxic activity as well as minimal alloreactivity, to selectively route their activity to leukemia transformed niche. The chemokine ligand 12 (CXCL12), released by mesenchymal stromal cells (MSCs) within the medullary niche, and its chemokine receptor 4 (CXCR4) are two pivotal players regulating leukocytes trafficking to the BM. In AML, CXCL12 interacts with CXCR4 overexpressed on blasts, promoting their migration and homing in the niche. Hence, taking advantage of this axis might facilitate CD33.CAR-CIK cells homing to the BM and therefore leukemia eradication. However, ex vivo manipulation protocols of CD33.CAR-CIK cells consistently downregulate CXCR4 expression and may affect the capacity of adoptively infused cells to migrate to BM and exert their anti-leukemic action. Therefore, to improve CD33.CAR-CIKs homing in the BM microenvironment we have developed CD33.CAR-CIK cells overexpressing CXCR4, in its wild-type or hyperactive mutant form. Notably, CIK cells engineering with CD33.CAR-CXCR4 constructs led to a consistent increase in CXCR4 expression, without altering CIK cells phenotype and CAR-related effector functions. Interestingly, compared to conventional CD33.CAR-CIK cells, CD33.CAR-CXCR4WT and especially CD33.CAR-CXCR4MUT-CIK cells demonstrated significantly superior in vitro chemotactic response toward CXCL12 and MSC-derived supernatants, and greater in vivo BM homing ability and persistence. Furthermore, to develop an effective anti-AML CAR T-cell therapy, it is fundamental to identify a LSC-specific marker, sparing the normal counterpart of hematopoietic stem cells (HSCs). T-cell immunoglobulin and mucin protein 3 (TIM-3) is an immune checkpoint molecule, it plays a central role in immune responses in AML and it is an LSC-specific marker, lacking expression on HSCs. Therefore, we designed a third-generation anti-TIM-3.CAR using the single-chain fragment variable (scFv) derived from an antagonistic ligand-blocking anti-TIM-3 antibody. In vitro, TIM-3.CAR-CIK cells efficiently killed both AML cell lines and primary AML blasts, but not normal TIM-3+ activated CIK cells, monocytes and NK-cells. Notably, we observed selective elimination of primary LSC-enriched population (CD34+ CD38-). Furthermore, TIM-3.CAR-CIK cells maintained their effector functions despite multiple in vitro restimulations, setting the basis for further exploration in in vivo models. Overall, both approaches, one improving CAR-CIK cells homing to the transformed niche and the other conferring superior safety and selectivity, might improve the efficacy of anti-AML CAR-CIK therapy.
SERAFINI, MARTA
Terapia CAR-T; LMA; CIK; Asse CXCL12/CXCR4; TIM-3
CAR T-cell therapy; AML; CIK; CXCL12/CXCR4 axis; TIM-3
MED/15 - MALATTIE DEL SANGUE
English
18-gen-2022
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET
34
2020/2021
embargoed_20250118
(2022). Enhancing AML CAR CIK therapeutic potency increasing the localization of engineered cells in the malignant niche and its selectivity by LSCs specific targeting. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/365153
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