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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 risultat, 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

Le neoplasie mieloidi sono un gruppo di malattie caratterizzate da un'origine clonale mieloide comune. In ogni singolo gruppo di queste malattie il decorso clinico è eterogeneo e difficile da prevedere, in particolare per la Leucemia Mieloide Acuta (LMA) e per la Mielofibrosi (MF). Recentemente, l'identificazione di mutazioni molecolari mediante sequenziamento ad alto rendimento (NGS) ha notevolmente migliorato la nostra conoscenza della genetica molecolare di LAM e MF e ha messo in evidenza le mutazioni geniche e il loro significato prognostico. La mia attività di dottorato mirava a definire meglio il ruolo delle alterazioni geniche nel predire il decorso clinico delle malattie e guidare le strategie terapeutiche. In questo contesto, abbiamo definito il profilo mutazionale in una coorte di 221 LAM a cariotipo normale (NK) arruolate in uno studio clinico prospettico randomizzato. In questa coorte di NK-LAM, mediante analisi multivariata, il raggiungimento della remissione completa è stato influenzato negativamente dalla mutazione SRSF2. Le alterazioni di FLT3 (FLT3-ITD) e U2AF1 erano associate a un peggioramento della sopravvivenza globale (OS) e della sopravvivenza libera da malattia (DFS) (p <0,05). I pazienti positivi per FLT3-ITD sottoposti a alloHSCT avevano una probabilità di sopravvivenza simile a quella negativa per FLT3-ITD. Inoltre, con l'obiettivo di implementare la definizione di AML secondaria, abbiamo analizzato una coorte prospettica di 413 pazienti con AML concentrandoci su una signature mutazionale cromatina-spliceosoma (CS). Le mutazioni CS sono state identificate nel 17,6% dei casi de novo clinicamente definiti, mostrando caratteristiche cliniche più vicine alla sAML. Gli esiti clinici in questo gruppo erano avversi e paragonabili a quelli dei pazienti con sAML. AlloHSCT ha migliorato i risultati sia nei pazienti sAML che CS-AML. Questi dati supportano una definizione molecolare di sAML, con implicazioni per un trattamento ottimizzato. Inoltr, Myeloid malignancies are a group of diseases characterized by a common myeloid clonal origin. In every single group of these diseases the clinical course is heterogeneous difficult to predict, particularly for Acute Myeloid Leukemia (AML) and for Myelofibrosis (MF). Recently, the identification of molecular mutations by high throughput sequencing (NGS) has dramatically improved our knowledge of AML and MF molecular genetics and shed new light on the prognostic significance gene mutations. My PhD activity aimed to better define the role of genes alterations to predict the clinical course of the diseases and guide therapeutic strategies. In this context, we defined the mutational profile in a cohort of 221 normal karyotype (NK) AML enrolled into a prospective randomized clinical trial. In this cohort ok NK-AML, by multivariate analysis, the achievement of complete remission was negatively affected by the SRSF2 mutation. Alterations of FLT3 (FLT3-ITD) and U2AF1 were associated with a worse overall (OS) and disease-free survival (DFS) (p < 0.05). FLT3-ITD positive patients who underwent alloHSCT had a survival probability similar to FLT3-ITD negative. Moreover, aiming to implement the definition of secondary AML, we analyzed a prospective cohort of 413 AML patients focusing on a chromatin-spliceosome (CS) mutational signature. CS mutations were identified in 17.6% of clinically defined de novo cases, showing clinical characteristics closer to sAML. Clinical outcomes in this group were adverse and comparable to those of sAML patients. AlloHSCT improved outcomes in both sAML and CS-AML patients. These data support a molecular definition of sAML, with implications for optimized treatment. Furthermore, we evaluated the post alloHSCT outcome of MF patients enrolled into a multicentric perspective clinical trial. NGS allowed us to classify patients by MIPSS scoring systems, which also consider the prognostic value of HMR mutations, obtaining that the great part of them result

Background aims: Advanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area. Methods: We have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy “Plagencell network” for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years. Results: The cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at <–150°C and did not show any tendency for diminished viability or efficacy for up to 13.5 years. Conclusions: Our data indicate that new guidelines for stability studies, specific for ATMPs and based on risk analyses, should be drafted to harmonize practices, significantly reduce the costs of stability studies without diminishing safety. Some specific suggestions are presented in the discussion.

Breast cancer is the most common malignancy among women worldwide, and HER2-positive breast cancer accounts for approximately 15% of all breast cancer diagnoses. The advent of HER2-targeting therapies has dramatically improved the survival of these patients, significantly reducing their risk of recurrence and death. However, as a significant proportion of patients ultimately develop resistance to these therapies, it is extremely important to identify new treatments to further improve their clinical outcomes. Immunotherapy has revolutionized the treatment and history of several cancer types, and it has already been approved as a standard of care for patients with triple-negative breast cancer. Based on a strong preclinical rationale, immunotherapy in HER2-positive breast cancer represents an intriguing field that is currently under clinical investigation. There is a close interplay between HER2-targeting therapies (both approved and under investigation) and the immune system, and several new immunotherapeutic strategies, including immune checkpoint inhibitors, CAR-T cells and therapeutic vaccines, are being studied in this disease. In this narrative review, we discuss the clinical evidence and the future perspectives of immunotherapy for patients with HER2-positive breast cancer., SCOPUS: re.j, info:eu-repo/semantics/published

BACKGROUND. Chimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. METHODS. We report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells. RESULTS. The cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3+ lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD–). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion. CONCLUSION. SB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.