Colorectal cancer (CRC) is the 2nd cause of cancer-related death. Despite standard therapies, more than 50% of patients experience relapse, eventually with metastatic disease. The CRC microenvironment is densely infiltrated by T cells, which have a role in immune surveillance and modulation of tumor progression, and their presence correlates with improved overall survival. To counteract immune exhaustion, inhibitory receptors (IRs) blockade has been exploited, but efficacy was limited to a small group of CRC patients characterized by high mutational burden. Adoptive T cell therapy (ACT) with genetically engineered T cells could represent an innovative strategy to harness T cell function and specificity. Initially relying on isolation, ex vivo expansion and re-infusion of tumor-infiltrating T cells (TILs), the development of ACT cellular products has now been prompted by the newest gene transfer and genome editing techniques. However, the widening of ACT with genetically engineered T cells is still limited from by paucity of anti-tumoral T cell receptors (TCRs) and by the need to counteract the immune-suppressive tumor microenvironment (TME). This work aims at setting the basis for the development of effective T cell products for the ACT of CRC, endowed with the capacity to specifically recognize cancer cells and counteract the immune-suppressive CRC TME. We employed high-dimensional flow cytometry coupled with an advanced pipeline of data handling by dimensionality reduction and clustering algorithms to describe the phenotype and the exhaustion features of TILs retrieved from the healthy, peritumoral and neoplastic tissue of treatment-naïve primary CRC patients and from the peritumoral and tumoral tissue of CRC patients undergoing surgery for liver metastasis. Unsupervised analyses highlighted the co-expression of multiple IRs and activations markers in T cells within the tumors. Populations of TILs described by a peculiar IRs signature were enriched both in primary CRC and liver metastasis. Of note, the signatures retrieved from primary and metastatic CRC overlapped for the upregulation of PD1 and CD39 thus underlining these molecules as relevant targets for T cells engineering. With the aim of exploiting this exhaustion signature to retrieve new anti-tumor specificities, we performed TCRαβ sequencing on PD1+CD39+ T cells isolated from primary CRC samples, obtaining different results from MSS and MSI tumors, where the repertoire was more oligoclonal. Of note, a small but consistent subpopulation of PD1+CD39+ T cells was also enriched in the peripheral blood of CRC patients compared to healthy donors (HDs), suggesting that exhausted tumor-specific T cells might circulate although at low frequencies. This signature could thus be used to isolate CRC-specific T cells and TCRs. As an alternative approach, we exploited a strategy to isolate CRC-specific T cells starting from HD peripheral blood mononuclear cells (PBMCs). We repetitively stimulated HD PBMCs with autologous antigen-presenting cells loaded with a pool of peptides selected to be immunogenic and expressed by CRC. We efficiently expanded T cells specific for tumor-associated antigens and neoantigen epitopes and we sequenced their TCR. To set up a T cell genetic modification pipeline, we employed a published MUC-1 TCR. We used CRISPR/Cas9 to render T cells completely devoid of the endogenous TCR, and we redirected T cells specificity by lentiviral transduction, obtaining MUC-1 specific T cells functionally able to kill target cells. Moreover, we set up the efficient disruption of PD1 and CD39 by CRISPR/Cas9 with the aim of rendering T cells selectively resistant to CRC TME. Overall, by coupling these findings we aim at generating a library of T cell products able to specifically recognize tumor antigens and to counteract the immune-suppressive TME, to be tested in adoptive T cell therapy trials for the treatment of CRC.

Il tumore del colon-retto (CRC) rappresenta la II causa di morte per cancro. Nonostante le terapie standard, più del 50% dei pazienti va incontro a recidiva, spesso con l’insorgenza di metastasi. Il microambiente del CRC è densamente infiltrato da linfociti T, che hanno un ruolo nella sorveglianza immunologica e nel modulare la progressione tumorale, e la cui presenza correla con un aumento della sopravvivenza dei pazienti. Una strategia per contrastare l’esaurimento funzionale delle cellule immunitarie è il blocco dei recettori di inibizione (IRs), ma l’efficacia è limitata ad un piccolo gruppo di pazienti caratterizzati da alto tasso mutazionale. La terapia cellulare adottiva (ACT) con linfociti T inizialmente si basava sull’isolamento, espansione ex vivo e reinfusione dei linfociti T infiltranti il tumore (TILs), ma ora ha avuto un impulso dalle innovative tecniche di modificazione genetica che permettono di migliorarne la funzionalità e la specificità. Il suo utilizzo, però, è ancora limitato dalla scarsità di recettori dei linfociti T (TCRs) antitumorali, e dalla necessità di contrastare il microambiente tumorale (TME) immuno-soppressivo. Questo lavoro ha lo scopo di porre le basi per lo sviluppo di un prodotto cellulare efficace per la ACT del CRC, che abbia la capacità di riconoscere il tumore in maniera specifica e sopravvivere al suo TME. Tramite citofluorimetria multiparametrica, seguita da una strategia complessa di analisi dei dati tramite algoritmi di dimensionality reduction e clustering, abbiamo descritto il fenotipo e le caratteristiche di esaurimento funzionale dei TILs isolati dal tessuto sano, peri-tumorale e neoplastico di pazienti con CRC primitivo mai sottoposti ad alcun trattamento, e dal tessuto peri-tumorale e tumorale epatico di pazienti con CRC metastatico al fegato. Dall’analisi è emersa la co-espressione di molteplici recettori di inibizione e molecole attivatorie nei TILs. È rilevante che i TILs del tumore primario e della metastasi siano accomunati dalla sovra-espressione di PD-1 e CD39. I linfociti T che esprimono PD1+CD39+, seppure in bassa percentuale, sono arricchiti anche nel sangue periferico di pazienti con CRC confrontati con donatori sani (HD), sottolineando l’importanza di queste molecole come bersagli rilevanti per l’ingegnerizzazione delle cellule T. Per sfruttare le caratteristiche di esaurimento dei linfociti T nel selezionare nuove specificità antitumorali, abbiamo sequenziato i geni α e β dei TCR delle cellule co-esperimenti PD1 e CD39 isolate da CRC primitivi. Abbiamo inoltre utilizzato una strategia per isolare linfociti T specifici contro il CRC a partire da cellule mononucleate del sangue periferico (PBMCs) di HD. I PBMCs degli HD sono stati stimolati ripetutamente con un insieme di peptidi selezionati per essere immunogenici per il CRC. Abbiamo espanso in maniera efficiente popolazioni di cellule specifiche per epitopi di antigeni sovra-espressi nel tumore o neo-antigeni e abbiamo sequenziato il loro TCR. Per stabilire un protocollo di manipolazione genetica dei linfociti T, abbiamo utilizzato un TCR pubblicato in letteratura, specifico per l’antigene MUC-1. Utilizzando le nucleasi CRISPR/Cas9, abbiamo eliminato dai linfociti il TCR endogeno, e abbiamo re-direzionato la loro specificità tramite trasduzione lentivirale, ottenendo linfociti T specifici per MUC1 e funzionali nell’eliminare le cellule bersaglio. Inoltre, abbiamo effettuato la disruption di PD1 e CD39, allo scopo di rendere i linfociti T selettivamente resistenti al microambiente del CRC. Lo scopo ultimo, mettendo insieme i risultati finora ottenuti, è quello di generare un prodotto cellulare da usare come strategia di ACT per il trattamento del CRC, capace di riconoscere il tumore in maniera specifica e di contrastare il TME immunosoppressivo.

(2021). Effector T cells co-expressing PD1 and CD39 are enriched in colorectal tumors: implications for cancer immunotherapy. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2021).

Effector T cells co-expressing PD1 and CD39 are enriched in colorectal tumors: implications for cancer immunotherapy

POTENZA, ALESSIA
2021

Abstract

Colorectal cancer (CRC) is the 2nd cause of cancer-related death. Despite standard therapies, more than 50% of patients experience relapse, eventually with metastatic disease. The CRC microenvironment is densely infiltrated by T cells, which have a role in immune surveillance and modulation of tumor progression, and their presence correlates with improved overall survival. To counteract immune exhaustion, inhibitory receptors (IRs) blockade has been exploited, but efficacy was limited to a small group of CRC patients characterized by high mutational burden. Adoptive T cell therapy (ACT) with genetically engineered T cells could represent an innovative strategy to harness T cell function and specificity. Initially relying on isolation, ex vivo expansion and re-infusion of tumor-infiltrating T cells (TILs), the development of ACT cellular products has now been prompted by the newest gene transfer and genome editing techniques. However, the widening of ACT with genetically engineered T cells is still limited from by paucity of anti-tumoral T cell receptors (TCRs) and by the need to counteract the immune-suppressive tumor microenvironment (TME). This work aims at setting the basis for the development of effective T cell products for the ACT of CRC, endowed with the capacity to specifically recognize cancer cells and counteract the immune-suppressive CRC TME. We employed high-dimensional flow cytometry coupled with an advanced pipeline of data handling by dimensionality reduction and clustering algorithms to describe the phenotype and the exhaustion features of TILs retrieved from the healthy, peritumoral and neoplastic tissue of treatment-naïve primary CRC patients and from the peritumoral and tumoral tissue of CRC patients undergoing surgery for liver metastasis. Unsupervised analyses highlighted the co-expression of multiple IRs and activations markers in T cells within the tumors. Populations of TILs described by a peculiar IRs signature were enriched both in primary CRC and liver metastasis. Of note, the signatures retrieved from primary and metastatic CRC overlapped for the upregulation of PD1 and CD39 thus underlining these molecules as relevant targets for T cells engineering. With the aim of exploiting this exhaustion signature to retrieve new anti-tumor specificities, we performed TCRαβ sequencing on PD1+CD39+ T cells isolated from primary CRC samples, obtaining different results from MSS and MSI tumors, where the repertoire was more oligoclonal. Of note, a small but consistent subpopulation of PD1+CD39+ T cells was also enriched in the peripheral blood of CRC patients compared to healthy donors (HDs), suggesting that exhausted tumor-specific T cells might circulate although at low frequencies. This signature could thus be used to isolate CRC-specific T cells and TCRs. As an alternative approach, we exploited a strategy to isolate CRC-specific T cells starting from HD peripheral blood mononuclear cells (PBMCs). We repetitively stimulated HD PBMCs with autologous antigen-presenting cells loaded with a pool of peptides selected to be immunogenic and expressed by CRC. We efficiently expanded T cells specific for tumor-associated antigens and neoantigen epitopes and we sequenced their TCR. To set up a T cell genetic modification pipeline, we employed a published MUC-1 TCR. We used CRISPR/Cas9 to render T cells completely devoid of the endogenous TCR, and we redirected T cells specificity by lentiviral transduction, obtaining MUC-1 specific T cells functionally able to kill target cells. Moreover, we set up the efficient disruption of PD1 and CD39 by CRISPR/Cas9 with the aim of rendering T cells selectively resistant to CRC TME. Overall, by coupling these findings we aim at generating a library of T cell products able to specifically recognize tumor antigens and to counteract the immune-suppressive TME, to be tested in adoptive T cell therapy trials for the treatment of CRC.
BONINI, CHIARA
Tumore del colon; Immunoterapia; Linfociti T; PD1; CD39
Colorectal cancer; Immunotherapy; T cells; PD1; CD39
MED/06 - ONCOLOGIA MEDICA
Italian
23-mar-2021
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET
33
2019/2020
open
(2021). Effector T cells co-expressing PD1 and CD39 are enriched in colorectal tumors: implications for cancer immunotherapy. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2021).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/309596
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