Cytokine Induced Killer (CIK) cells are CD3+CD56+ double positive T cells that have acquired phenotypic markers of natural killer (NK) cells and show non-restricted cytotoxicity against different antigens. Clinical trials have demonstrated CIK cells therapeutic activity only in low tumor burden context, so strategies to increase the therapeutic efficacy of CIK cells are required. In the first part of my project, I compared the activity of two different methods to improve CIK cells anti-tumor efficacy and specificity against CD19. One method was to genetically engineer CIK cells to express a chimeric antigen receptor (CAR), using stable transfection of a Sleeping Beauty transposon plasmid. Using this transposon system, two anti-CD19 CARs, recapitulating the structure of the clinical anti-CD19 CAR Tisagenlecleucel (Novartis), originally inserted in a lentivirus, and the CAR-CD19 used in current clinical CARCIK-CD19 protocol (Fondazione Matilde Tettamanti Menotti De Marchi Onlus) were compared. The second strategy investigated was to combine CIK cells with Blinatumomab, an anti-CD3xCD19 bispecific antibody, which can bridge CD19+ targets with CD3+ CIK, perhaps in a similar way to CD19-CAR, but without genetic modification. The activity of the different CARs or Blinatumomab were compared in vitro in terms of CIK cells proliferation, cytokines release, cytotoxicity and NFAT and NF-kB signaling against two CD19+ target cell lines. The data obtained suggest that the addition of Blinatumomab to CIK cultures may be as efficacious, if not more, as the CAR gene transduction in vitro. Comparisons in vivo in a mouse model are in progress. Future results will complete this work and verify whether specific in vitro and in vivo activities correlate, and if so in what manner. The second part of the project is based on the knowledge gained from the anti-CD19 CARs, but is focused on a novel tumor target antigen for CAR-CIK therapy, a tumor microenvironment protein, refered to here as TMA (Tumor Microenvironment Antigen). TMA is a known constituent of the Extra-Cellular Matrix (ECM), and its real identity is not revealed here for patenting reasons. TMA can be a good target for solid tumors thanks to its specific localization, it is secreted in response to tissue damage and in growing tumors, reducing the probability of off-tumor toxicity. Different anti-TMA mAbs have been developed by different groups and some of them have reached early phase clinical testing. Firstly, TMA expression was investigated by flow cytometry and immunofluorescence microscopy on putative targets, primary cells and a panel of tumor cell lines. High expression of TMA was observed on mesenchymal stromal cells (MSCs) and monocytes differentiated to M1 and M2 macrophages. In parallel, 2 different anti-TMA CAR constructs were designed and generated. The CARs differed in their CAR backbone, recapitulating one the signaling domains present in the anti-CD19 CAR Tisagenlecleucel (Novartis) and one the CAR-CD19 domains used in the context of CARCIK-CD19 trial. Both structures were used with the transposon platform Sleeping Beauty and were shown to be expressed on the surface of transfected mononuclear cells expanded in vitro to CIK. In vitro functional assays demonstrated the ability of anti-TMA CARCIK cells to be cytotoxic against TMA+ targets, to proliferate in response to antigen binding and to secrete the inflammatory cytokines IFN-γ and IL-2. In vivo animal models have been set up with the TMA+ cell lines HT-29, MDA-MB-231 and the B cell lymphoma BJAB. Work is in progress to analyze the anti-tumor activity of CARCIK-TMA cells in these models.

Le cellule CIK (Cytokine Induced Killer) sono cellule T citotossiche indotte da citochine che esprimono marcatori fenotipici delle cellule Natural Killer (NK) ed esibiscono una elevata capacità citotossica. Diversi studi clinici hanno dimostrato che le cellule CIK hanno un minimo livello di alloreattività e hanno attività terapeutica, quest’ultima solo in contesti con bassi livelli di malattia. Per questo motivo, nuove strategie che aumentino l’attività terapeutica delle cellule CIK sono necessarie. Nella prima parte del mio progetto ho confrontato l’attività in vitro di due strategie in grado di aumentare la specificità delle cellule CIK. La prima strategia è modificare geneticamente le cellule CIK per esprimere un recettore chimerico CAR, usando il sistema trasposonico Sleeping Beauty. Abbiamo confrontato due costrutti CAR anti-CD19, ricalcando la struttura di prodotti già utilizzati nella pratica clinica, uno il CAR anti-CD19 Tisagenlecleucel (Novartis) e il secondo il CAR del protocollo clinico CARCIK-CD19 (Fondazione Matilde Tettamanti Menotti De Marchi Onlus). La seconda strategia è la combinazione di cellule CIK con l’anticorpo bispecifico Blinatumomab, CD3xCD19, il quale agisce da “ponte” tra le cellule CIK CD3+ e le cellule tumorali CD19+. Abbiamo studiato l’attività in vitro delle due tipologie di cellule CARCIK e delle cellule CIK+Blinatumomab in termini di citotossicità, proliferazione indotta dal legame con l’antigene, rilascio di citochine e attivazione intracellulare di NFAT e NF-kB. I dati ottenuti suggeriscono che tutte e tre le strategie per migliorare la specificità delle cellule CIK sono funzionali e che l’aggiunta dell’anticorpo bispecifico Blinatumomab è comparabile, se non in alcuni casi più efficiente, delle CARCIK in vitro. Il confronto in vivo in modelli murini dell’attività di queste cellule è in corso e sarà fondamentale per completare la valutazione della loro attività. La seconda parte del progetto si basa sui risultati dello studio delle CARCIK anti-CD19, ma diretto contro un nuovo antigene di interesse. Abbiamo studiato un antigene del microambiente tumorale che abbiamo chiamato TMA (Tumor Microenvironment Antigen), la cui vera identità non può essere rivelata al momento per ragioni brevettuali. TMA è una proteina della matrice extracellulare altamente espressa a livello embrionale e nell’adulto in contesti infiammatori e nei tumori. La specificità della sua espressione rende la proteina TMA un buon target immunoterapico. Abbiamo studiato l’espressione di TMA su diverse cellule primarie e linee cellulari, in modo da individuare dei target contro cui testare le nostre cellule CARCIK-TMA. Le linee cellulari di colon adenocarcinoma HT-29 e carcinoma mammario MDA-MB-231 hanno dimostrato un’alta espressione di TMA, assieme ad alcune linee di linfoma di Burkitt e linfoma mantellare. In parallelo abbiamo generato cellule CARCIK-TMA esprimenti due diversi CAR, ricalcanti la struttura dei due costrutti anti-CD19 studiati in precedenza. Entrambi i CAR sono stati trasdotti nelle cellule CIK utilizzando il sistema a trasposoni Sleeping Beauty e abbiamo confermato l’espressione sulla superficie delle cellule CIK. I test funzionali in vitro hanno dimostrato l’attività delle cellule CARCIK anti-TMA nei confronti di linee cellulari TMA+ in termini di citotossicità, rilascio di citochine e proliferazione indotta dal legame con l’antigene. Per lo studio dell’attività in vivo delle cellule CARCIK anti-TMA abbiamo messo a punto tre modelli di tumori umani inoculati in modelli murini, utilizzando le linee cellulari HT-29, MDA-MB-231 e la linea cellulare di linfoma a cellule B BJAB. Un primo esperimento in vivo ha dimostrato una parziale attività delle cellule CARCIK anti-TMA e l’assenza di tossicità per l’animale, altri esperimenti saranno necessari per valutare più in dettaglio l’attività di queste cellule.

(2023). Development of innovative CAR molecules to be transduced in Cytokine Induced Killer cells for the treatment of different neoplasia. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2023).

Development of innovative CAR molecules to be transduced in Cytokine Induced Killer cells for the treatment of different neoplasia

ZANINELLI, SILVIA
2023

Abstract

Cytokine Induced Killer (CIK) cells are CD3+CD56+ double positive T cells that have acquired phenotypic markers of natural killer (NK) cells and show non-restricted cytotoxicity against different antigens. Clinical trials have demonstrated CIK cells therapeutic activity only in low tumor burden context, so strategies to increase the therapeutic efficacy of CIK cells are required. In the first part of my project, I compared the activity of two different methods to improve CIK cells anti-tumor efficacy and specificity against CD19. One method was to genetically engineer CIK cells to express a chimeric antigen receptor (CAR), using stable transfection of a Sleeping Beauty transposon plasmid. Using this transposon system, two anti-CD19 CARs, recapitulating the structure of the clinical anti-CD19 CAR Tisagenlecleucel (Novartis), originally inserted in a lentivirus, and the CAR-CD19 used in current clinical CARCIK-CD19 protocol (Fondazione Matilde Tettamanti Menotti De Marchi Onlus) were compared. The second strategy investigated was to combine CIK cells with Blinatumomab, an anti-CD3xCD19 bispecific antibody, which can bridge CD19+ targets with CD3+ CIK, perhaps in a similar way to CD19-CAR, but without genetic modification. The activity of the different CARs or Blinatumomab were compared in vitro in terms of CIK cells proliferation, cytokines release, cytotoxicity and NFAT and NF-kB signaling against two CD19+ target cell lines. The data obtained suggest that the addition of Blinatumomab to CIK cultures may be as efficacious, if not more, as the CAR gene transduction in vitro. Comparisons in vivo in a mouse model are in progress. Future results will complete this work and verify whether specific in vitro and in vivo activities correlate, and if so in what manner. The second part of the project is based on the knowledge gained from the anti-CD19 CARs, but is focused on a novel tumor target antigen for CAR-CIK therapy, a tumor microenvironment protein, refered to here as TMA (Tumor Microenvironment Antigen). TMA is a known constituent of the Extra-Cellular Matrix (ECM), and its real identity is not revealed here for patenting reasons. TMA can be a good target for solid tumors thanks to its specific localization, it is secreted in response to tissue damage and in growing tumors, reducing the probability of off-tumor toxicity. Different anti-TMA mAbs have been developed by different groups and some of them have reached early phase clinical testing. Firstly, TMA expression was investigated by flow cytometry and immunofluorescence microscopy on putative targets, primary cells and a panel of tumor cell lines. High expression of TMA was observed on mesenchymal stromal cells (MSCs) and monocytes differentiated to M1 and M2 macrophages. In parallel, 2 different anti-TMA CAR constructs were designed and generated. The CARs differed in their CAR backbone, recapitulating one the signaling domains present in the anti-CD19 CAR Tisagenlecleucel (Novartis) and one the CAR-CD19 domains used in the context of CARCIK-CD19 trial. Both structures were used with the transposon platform Sleeping Beauty and were shown to be expressed on the surface of transfected mononuclear cells expanded in vitro to CIK. In vitro functional assays demonstrated the ability of anti-TMA CARCIK cells to be cytotoxic against TMA+ targets, to proliferate in response to antigen binding and to secrete the inflammatory cytokines IFN-γ and IL-2. In vivo animal models have been set up with the TMA+ cell lines HT-29, MDA-MB-231 and the B cell lymphoma BJAB. Work is in progress to analyze the anti-tumor activity of CARCIK-TMA cells in these models.
INTRONA, MARTINO
cellule CAR T; CIK; immunoterapia; microambiente; tumori solidi
CAR T cells; CIK cells; immunotherapy; microenvironment; solid tumors
MED/04 - PATOLOGIA GENERALE
English
27-feb-2023
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET
35
2021/2022
open
(2023). Development of innovative CAR molecules to be transduced in Cytokine Induced Killer cells for the treatment of different neoplasia. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2023).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/405201
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