Concetta Quintarelli

SHP-1 expression accounts for resistance to imatinib treatment in Philadelphia chromosome-positive cells derived from patients with chronic myeloid leukemia

We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL-independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P < .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

Selective strong synergism of Ruxolitinib and second generation tyrosine kinase inhibitors to overcome bone marrow stroma related drug resistance in chronic myelogenous leukemia.

The IC50 of TKIs is significantly increased when BCR-ABL+ K562 cell line is cultured in stroma conditioned media produced by BM mesenchymal cells. In particular, while the Imatinib IC50 in the stromal co-cultures was well above the in vivo through levels of the drug, the IC50s of second generation TKIs were still below their through levels. Moreover, we provide a formal comparison of the synergy between first and second generation TKIs with the JAK inhibitor Ruxolitinib to overcome BM stroma related TKI resistance. Taken together, our data provide a rationale for the therapeutic combination of TKIs and Ruxolitinib with the aim to eradicate primary BCR-ABL+ cells homed in BM niches.

Choice of costimulatory domains and of cytokines determines CAR T-cell activity in neuroblastoma

ABSTRACT Chimeric antigen receptor (CAR) T-cell therapy has been shown to be dramatically effective in the treatment of B-cell malignancies. However, there are still substantial obstacles to overcome, before similar responses can be achieved in patients with solid tumors. We evaluated both in vitro and in a preclinical murine model the efficacy of different 2nd and 3rd generation CAR constructs targeting GD2, a disial-ganglioside expressed on the surface of neuroblastoma (NB) tumor cells. In order to address potential safety concerns regarding clinical application, an inducible safety switch, namely inducible Caspase-9 (iC9), was also included in the vector constructs. Our data indicate that a 3rd generation CAR incorporating CD28.4-1BB costimulatory domains is associated with improved anti-tumor efficacy as compared with a CAR incorporating the combination of CD28.OX40 domains. We demonstrate that the choice of 4-1BB signaling results into significant amelioration of several CAR T-cell characteristics, including: 1) T-cell exhaustion, 2) basal T-cell activation, 3) in vivo tumor control and 4) T-cell persistence. The fine-tuning of T-cell culture conditions obtained using IL7 and IL15 was found to be synergic with the CAR.GD2 design in increasing the anti-tumor activity of CAR T cells. We also demonstrate that activation of the suicide gene iC9, included in our construct without significantly impairing neither CAR expression nor anti-tumor activity, leads to a prompt induction of apoptosis of GD2.CAR T cells. Altogether, these findings are instrumental in optimizing the function of CAR T-cell products to be employed in the treatment of children with NB.

Adoptive Immunotherapy Using PRAME-Specific T Cells in Medulloblastoma

Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity. Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated the expression of the tumor-associated antigen PRAME in biopsies from 60 patients with medulloblastoma. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A*02+ DAOY cells as well as primary HLA-A*02+ medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T-cell-related toxicity, an inducible caspase-9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating patients with HLA-A*02+ medulloblastoma.Significance: These findings identify PRAME as a medulloblastoma tumor-associated antigen that can be targeted using genetically modified T cells. Cancer Res; 78(12); 3337-49. ©2018 AACR.

Antigen-specificity and DTIC before peptide-vaccination differently shape immune-checkpoint expression pattern, anti-tumor functionality and TCR repertoire in melanoma patients

ABSTRACT We have recently described that DNA-damage inducing drug DTIC, administered before peptide (Melan-A and gp100)-vaccination, improves anti-tumor CD8+ Melan-A-specific T-cell functionality, enlarges the Melan-A+ TCR repertoire and impacts the overall survival of melanoma patients. To identify whether the two Ags employed in the vaccination differently shape the anti-tumor response, herein we have carried out a detailed analysis of phenotype, anti-tumor functionality and TCR repertoire in treatment-driven gp100-specific CD8+ T cells, in the same patients previously analyzed for Melan-A. We found that T-cell clones isolated from patients treated with vaccination alone possessed an Early/intermediate differentiated phenotype, whereas T cells isolated after DTIC plus vaccination were late-differentiated. Sequencing analysis of the TCRBV chains of 29 treatment-driven gp100-specific CD8+ T-cell clones revealed an oligoclonal TCR repertoire irrespective of the treatment schedule. The high anti-tumor activity observed in T cells isolated after chemo-immunotherapy was associated with low PD-1 expression. Differently, T-cell clones isolated after peptide-vaccination alone expressed a high level of PD-1, along with LAG-3 and TIM-3, and were neither tumor-reactive nor polyfunctional. Blockade of PD-1 reversed gp100-specific CD8+ T-cell dysfunctionality, confirming the direct role of this co-inhibitory molecule in suppressing anti-tumor activity, differently from what we have previously observed for Melan-A+CD8+ T cells, expressing PD-1 but highly functional. These findings indicate that the functional advantage induced by combined chemo-immunotherapy is determined by the tumor antigen nature, T-cell immune-checkpoints phenotype, TCR repertoire diversity and anti-tumor T-cell quality and highlights the importance of integrating these parameters to develop effective immunotherapeutic strategies.

The EURE-CART project as a prototype model for CAR T-cell immunotherapy in Europe

Immunotherapy approaches have revolutionized the clinical management of patients suffering from either hematological or solid neoplasia [1, 2]. This notion is particularly true in the context of adoptive cellular immunotherapy for patients with hematological diseases, for which cells derived from the patient’s immune system are genetically modified, using chimeric antigen receptors (CARs), in order to be redirected against leukemia or lymphoma cells [2–5]. CAR T-cell therapies have been successfully established, especially, but not only, for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) [3–7] or B-cell aggressive non-Hodgkin lymphoma (B-NHL) [8, 9]. Such remarkable successes, reported even in patients with advanced disease, have been obtained also thanks to the close interaction between highly specialized academic research groups in US and pharmaceutical companies, as exemplified by the collaboration between the University of Pennsylvania (UPenn) and Novartis (CTL019 product), or by that between the National Cancer Institute and Kite Pharma (KTE-C19 product), or by the joint venture developed between the Fred Hutchinson Cancer Research Center (FHCRC), the Memorial Sloan Kettering Cancer Center (MSKCC), and the Seattle Children’s Research with Juno Therapeutics (JCAR014, JCAR015, JCAR017, JCAR021 clinical trials). Different types of gene therapy platforms have been developed, including viral and non-viral ones, to induce a stable expression of a specific CAR in T lymphocytes. Irrespectively of the differences in CAR design, T-cell manufacturing process and lymphodepleting therapy used for promoting CAR T-cell expansion, impressive rates of sustained remission have been achieved in patients with both BCP-ALL and B-NHL, especially with the infusion of CTL019. Indeed, complete remission (CR) was recorded in 93% of children and adults with relapsed/refractory ALL given CTL019 CAR T cells [6] and in 54%–57% of patients with diffuse large B-cell or transformed follicular lymphoma [7, 8]. In light of the complexity in manufacturing the cell drug product, it has been unclear whether the approach could be used to treat a large number of patients in clinical settings other than the highly specialized academic centers pioneering these techniques. Although the initial studies were conducted in single institutions with dedicated CAR T-cell programs [6, 7, 10], more recently, trials conducted in multicenter settings substantially confirmed that a personalized geneengineered T-cell product can be generated at a centralized cell-manufacturing facility and safely administered to patients at transplantation-capable medical centers [8, 11]. The exciting results obtained in patients with B-cell lymphoproliferative disorders resulted in the very recent marketing authorization by the Food and Drug Administration (FDA) of two different cellular CAR T-cell products (namely CTL019 and KTE-C19). At the same time, several EU Hospitals have started to enroll patients in phase I/II clinical trials based on CAR Tcell products. Further confirmatory clinical trials will certainly be conducted by pharmaceutical companies in both the US and EU, but this does not mean that academic initiatives, involving networks of centers of excellence, will not have a role in the future development of CAR T cells. In particular, academia is called to contribute to the search of strategies able to optimize the approach and to enlarge the population of patients that can benefit from this novel form of immunotherapy. Indeed, there are several problems related to the use of CAR T cells that are still unsolved, including: (i) standardization of manufacturing process; (ii) reduction of relapse rates after initial response; iii) reduction of the toxicity associated with the treatment (e.g. cytokine release syndrome or neuro-toxicity); (iv) standardization of the cell dose to be infused; and (v) identification of the key factors associated with CAR T-cell persistence. These open questions are even more compelling in the context of CAR T-cell approaches targeting hematological malignancies other than ALL, such as acute myeloid leukemia (AML), which represents the most frequent type of acute leukemia in adults with an annual incidence of 4–5 new cases per 100,000 subjects and with a median age at diagnosis of 65 years [12]. Currently, although conventional therapies based on the use of cytotoxic drugs are able to induce CR in most AML patients, around 30% of patients with variants other than acute promyelocytic leukemia still experience disease relapse. The cumulative incidence of relapse is even higher in elderly patients, who often have also primary resistant disease [12]. Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective curative approach for patients with relapsed/refractory AML, but it is still associated with a high rate of lethal complications, such as infections and acute graftversus-host-disease (GvHD). Thus, given the observation that AML mainly occurs in elderly people, who are less able to tolerate aggressive conventional therapies, including HSCT, developing new and safer treatment strategies is of paramount importance for improving the outcome of these patients. For these reasons, along with the emerging clinical success of anti-CD19 CAR T-cell therapies in ALL, many studies have attempted to also translate CAR T-cell use to AML.