Acute myeloid leukemia (AML) is the most frequently diagnosed leukemia in adults (25%) and accounts for 15-20% cases in pediatric patients. Conventional chemotherapy employing anthracycline and cytarabine represents the gold standard treatment for AML, with rates of complete remission from 60% to 80% in children and from 40% to 60% in adults (>60 years). Despite these high rates, relapse after conventional therapy is common and the estimated five-year survival of AML patients is still below 30%. Indeed, there is an urgency to find alternative therapeutic strategies for relapsed and refractory patients. The recent clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in the context of B-cell malignancies has opened a new route of investigation also towards AML. However, the development of CAR T cell therapy in the context of AML is still in its infancy due to heterogeneity of the disease, the lack of a suitable target antigen and the leukemia protective role of the tumor microenvironment (TME) and no approved CAR T cells study exists for AML treatment yet. Non-viral Sleeping-Beauty (SB) transposon platform was employed to redirect cytokine-induce killer (CIK) cell. In this scenario, we firstly characterize non-viral SB engineered CIK cells with anti-CD146.CAR as a potential tool for the targeting of the bone marrow (BM) microenvironment. We optimized the CAR design structure by testing 6 different CAR molecules, achieving a specific and efficient CD146 expression in the VLVH Long variant. CD146.CAR-CIK cells were subsequently tested in vitro, showing an optimal activation of effector functions (in terms of killing activity, cytokines production and proliferation) when they were engaged against CD146+ target cells. Consequently, we developed a bispecific Tandem CAR (CD33xCD146.CAR-CIKs), which displayed anti-leukemic activity in vitro. It has been extensively proven that BM niche contribute to establish a sanctuary in which leukemic stem cells (LSCs) are able to acquire drug-resistant phenotype, therefore, to better mimicking the human BM niche we tested CD33xCD146.CAR-CIK cells against CD146+ stromal cell lines (HS-27A and HS-5) and primary derived healthy (HD-) and patient-derived (AML-) mesenchymal stromal cells (MSCs). Results showed inhibition of the redirected CAR-CIK cells effector functions, resulting in a drastic decrease of cytokines production and proliferation. The balance between pro- and anti- inflammatory cytokines showed that Th1/Tc1 cytokines production by CD146.CAR-CIK cells was inhibited by the co-culture with stromal cells, while increase Th2/Tc2 cytokines was detected when CD146.CAR-CIK cells were co-cultured with stromal target cells. These results suggest a potential immunosuppressive role of the stromal compartment against CAR-CIK cells. According to these results, we hypothesized that BM stromal cells can potentially exert an immunomodulatory effect on T cells, suggesting that the niche microenvironment may be involved in the regulation of CAR T cells therapy effectiveness. Indeed, the targeting of CD146 on stroma represents a “proof-of-principle” that stromal components of leukemic microenvironment may be attractive targets for CAR T based immunotherapy. To minimize “off-tumor” toxicity, we are looking for a specific surface target antigen selectively overexpressed on AML stromal cells, with minimal expression in healthy stroma and possibly involved in leukemia/niche interactions. The newly marker of interest will be coupled to the CD33.CAR and this bispecific CAR will be compared with CD33xCD146.CAR construct, evaluating their efficacy and safety profiles both in vitro and in vivo.

La leucemia mieloide acuta (LMA) è la neoplasia ematologica maggiormente diagnosticata nei pazienti adulti (25%) e mentre rappresenta il 15-20% dei casi nei pazienti pediatrici. La chemioterapia convenzionale, che impiega antraciclina e citarabina, rappresenta il trattamento standard per l’LMA, con tassi di remissione completa dal 60% all'80% nei bambini e dal 40% al 60% negli adulti (>60 anni). Sfortunatamente, la ricaduta dopo tale terapia è comune e la sopravvivenza dei pazienti stimata a 5 anni è ancora inferiore al 30%. Risulta quindi di primaria importanza trovare alternative terapeutiche per i pazienti recidivanti e refrattari. Il recente successo clinico, ottenuto nelle leucemie di tipo B, dell'immunoterapia con cellule CAR (chimeric antigen receptor) T ha portato allo sviluppo di nuove strategie terapeutiche nell’ambito dell’LMA. Tuttavia, lo sviluppo del trattamento con cellule CAR T nel contesto dell'LMA è ancora agli albori a causa dell'eterogeneità della malattia, della mancanza di un antigene bersaglio adatto e del ruolo protettivo del microambiente tumorale (TME). Infatti, non esiste ancora un protocollo clinico approvato per il trattamento della leucemia mieloide. Per creare le cellule CAR T abbiamo scelto di utilizzare la piattaforma non virale Sleeping-Beauty (SB) per ingegnerizzare le cellule CIK (cytokine-induced killer). In primo luogo, abbiamo scelto di utilizzare come potenziale strumento per il targeting del TME le cellule CIK ingegnerizzate con anti-CD146.CAR. Di conseguenza, abbiamo ottimizzato 6 diverse molecole CAR aventi un design differente, ottenendo un'espressione ottimale di CD146 nella variante VLVH Long. Abbiamo quindi testato le cellule CD146.CAR-CIK in vitro, ottenendo l’attivazione specifica delle funzioni effettrici (in termini di capacità di killing, produzione di citochine e proliferazione) contro cellule target CD146+. In seguito, abbiamo progettato un Tandem CAR bispecifico (CD33xCD146.CAR-CIKs) che ha mostrato una significativa attività antileucemica in vitro. È stato ampiamente dimostrato che la nicchia midollare contribuisce al supporto e alla protezione delle cellule staminali leucemiche (CSLs). Quindi, per mimare al meglio l’azione del CAR nella nicchia midollare umana, abbiamo testato le cellule CD33xCD146.CAR-CIK contro le linee cellulari stromali CD146+ e le cellule mesenchimali (MSC) primarie sane (HD-) e di derivazione mieloide (LMA-). I dati mostrano una inibizione delle funzioni effettrici delle cellule CAR-CIK e una drastica diminuzione della produzione di citochine e della proliferazione. Inoltre, l'equilibrio tra citochine pro e antinfiammatorie è risultato alterato, infatti la produzione di citochine Th1/Tc1 da parte delle cellule CD146.CAR-CIK è stata inibita dalla co-coltura con cellule stromali, mentre è stato rilevato un aumento delle citochine Th2/Tc2. Questi risultati suggeriscono un potenziale ruolo immunosoppressivo del compartimento stromale nei confronti delle cellule CAR-CIK. Sulla base dell’ effetto immunomodulatorio delle MSC sui linfociti T, abbiamo ipotizzato che la nicchia midollare possa influenzare le funzioni effettrici delle cellule CAR T. Di conseguenza, il targeting del CD146 rappresenta una "proof-of-principles" del fatto che aggredire il microambiente leucemico possa migliorare la terapia CAR T nell’ambito dell’LMA. Per ridurre al minimo la tossicità "off-target ", stiamo cercando di selezionare un antigene bersaglio specifico ed overespresso sulle cellule stromali dell'LMA, che abbia un'espressione minima nello stroma sano e che sia coinvolto nelle interazioni leucemia/nicchia. Il nuovo marker di interesse sarà accoppiato al CD33.CAR nella creazione di un CAR bispecifico, che verrà confrontato con il costrutto CD33xCD146.CAR, valutandone i profili di efficacia e sicurezza sia in vitro che in vivo.

(2022). Evaluation of a Tandem CD33-CD146 Chimeric Antigen Receptor (CAR) for the simultaneous targeting of Acute Myeloid Leukemia (AML) blasts and stromal cells in the niche. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).

Evaluation of a Tandem CD33-CD146 Chimeric Antigen Receptor (CAR) for the simultaneous targeting of Acute Myeloid Leukemia (AML) blasts and stromal cells in the niche

ALBERTI, GAIA
2022

Abstract

Acute myeloid leukemia (AML) is the most frequently diagnosed leukemia in adults (25%) and accounts for 15-20% cases in pediatric patients. Conventional chemotherapy employing anthracycline and cytarabine represents the gold standard treatment for AML, with rates of complete remission from 60% to 80% in children and from 40% to 60% in adults (>60 years). Despite these high rates, relapse after conventional therapy is common and the estimated five-year survival of AML patients is still below 30%. Indeed, there is an urgency to find alternative therapeutic strategies for relapsed and refractory patients. The recent clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in the context of B-cell malignancies has opened a new route of investigation also towards AML. However, the development of CAR T cell therapy in the context of AML is still in its infancy due to heterogeneity of the disease, the lack of a suitable target antigen and the leukemia protective role of the tumor microenvironment (TME) and no approved CAR T cells study exists for AML treatment yet. Non-viral Sleeping-Beauty (SB) transposon platform was employed to redirect cytokine-induce killer (CIK) cell. In this scenario, we firstly characterize non-viral SB engineered CIK cells with anti-CD146.CAR as a potential tool for the targeting of the bone marrow (BM) microenvironment. We optimized the CAR design structure by testing 6 different CAR molecules, achieving a specific and efficient CD146 expression in the VLVH Long variant. CD146.CAR-CIK cells were subsequently tested in vitro, showing an optimal activation of effector functions (in terms of killing activity, cytokines production and proliferation) when they were engaged against CD146+ target cells. Consequently, we developed a bispecific Tandem CAR (CD33xCD146.CAR-CIKs), which displayed anti-leukemic activity in vitro. It has been extensively proven that BM niche contribute to establish a sanctuary in which leukemic stem cells (LSCs) are able to acquire drug-resistant phenotype, therefore, to better mimicking the human BM niche we tested CD33xCD146.CAR-CIK cells against CD146+ stromal cell lines (HS-27A and HS-5) and primary derived healthy (HD-) and patient-derived (AML-) mesenchymal stromal cells (MSCs). Results showed inhibition of the redirected CAR-CIK cells effector functions, resulting in a drastic decrease of cytokines production and proliferation. The balance between pro- and anti- inflammatory cytokines showed that Th1/Tc1 cytokines production by CD146.CAR-CIK cells was inhibited by the co-culture with stromal cells, while increase Th2/Tc2 cytokines was detected when CD146.CAR-CIK cells were co-cultured with stromal target cells. These results suggest a potential immunosuppressive role of the stromal compartment against CAR-CIK cells. According to these results, we hypothesized that BM stromal cells can potentially exert an immunomodulatory effect on T cells, suggesting that the niche microenvironment may be involved in the regulation of CAR T cells therapy effectiveness. Indeed, the targeting of CD146 on stroma represents a “proof-of-principle” that stromal components of leukemic microenvironment may be attractive targets for CAR T based immunotherapy. To minimize “off-tumor” toxicity, we are looking for a specific surface target antigen selectively overexpressed on AML stromal cells, with minimal expression in healthy stroma and possibly involved in leukemia/niche interactions. The newly marker of interest will be coupled to the CD33.CAR and this bispecific CAR will be compared with CD33xCD146.CAR construct, evaluating their efficacy and safety profiles both in vitro and in vivo.
SERAFINI, MARTA
TETTAMANTI, SARAH
AML; CAR T cell therapy; CIK cells; TME; immunomodulation
AML; CAR T cell therapy; CIK cells; TME; immunomodulation
MED/38 - PEDIATRIA GENERALE E SPECIALISTICA
English
24-mag-2022
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET
34
2020/2021
embargoed_20250524
(2022). Evaluation of a Tandem CD33-CD146 Chimeric Antigen Receptor (CAR) for the simultaneous targeting of Acute Myeloid Leukemia (AML) blasts and stromal cells in the niche. (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/10281/382304
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