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In acute myeloid leukemia (AML), malignant stem cells hijack the normal bone marrow niche where they are largely protected from the current therapeutic approaches. Thus, eradicating these progenitors is the ultimate challenge in the treatment of this disease. Specifically, the development of chimeric antigen receptors (CARs) against distinct mesenchymal stromal cell subpopulations involved in the maintenance of leukemic stem cells within the malignant bone marrow microenvironment could represent a new strategy to improve CAR T-cell therapy efficacy, which is still unsuccessful in AML. As a proof of concept, we generated a novel prototype of Tandem CAR, with one specificity directed against the leukemic cell marker CD33 and the other against the mesenchymal stromal cell marker CD146, demonstrating its capability of simultaneously targeting two different cell types in a 2D co-culture system. Interestingly, we could also observe an in vitro inhibition of CAR T cell functionality mediated by stromal cells, particularly in later effector functions, such as reduction of interferon-gamma and interleukin-2 release and impaired proliferation of the CAR+ effector Cytokine-Induced Killer (CIK) cells. Taken together, these data demonstrate the feasibility of a dual targeting model against two molecules, which are expressed on two different target cells, but also highlight the immunomodulatory effect on CAR CIK cells exerted by stromal cells, confirming that the niche could be an obstacle to the efficacy of CAR T cells. This aspect should be considered in the development of novel CAR T cell approaches directed against the AML bone marrow niche.

Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 106 CD3+ cells/cm2 of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph+ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.

Acute myeloid leukemia (AML) is a hematological malignancy derived from neoplastic myeloid progenitor cells characterized by abnormal clonal proliferation and differentiation. Although novel therapeutic strategies have recently been introduced, the prognosis of AML remains unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)-T therapy in AML has been hampered by several factors including the poor accumulation of the blood-injected cells in the leukemia bone marrow (BM) niche, where chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within the niche, could improve T cell homing to the BM and consequently enhance their intimate contact with BM-resident AML cells facilitating disease eradication. Specifically, we engineered conventional CD33.CAR-cytokine induced killer cells (CIKs) with the wild-type CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of WHIM syndrome patients. Overexpression of both CXCR4wt and CXCR4mut in CD33.CAR-CIKs resulted in significant improvement of chemotaxis toward recombinant CXCL12 or BM stromal cell conditioned medium with no observed impairment of cytotoxic potential in vitro. Moreover, CXCR4-overexpressing CD33.CAR-CIKs showed enhanced in vivo BM homing, associated with a prolonged retention for the CXCR4R334X variant. However, only CD33.CAR-CIKs co-expressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a non-canonical role for CXCR4 in modulating CAR-CIK functions independent of BM homing. Taken together, these data suggest that arming CAR-CIKs with CXCR4 may represent a promising strategy for increasing their therapeutic potential for AML.

Acute Myeloid Leukemia (AML) still represents an unmet clinical need for adult and pediatric patients. Adoptive cell therapy by chimeric antigen receptor (CAR)-engineered T cells demonstrated a high therapeutic potential, but further development is required to ensure a safe and durable disease remission in AML, especially in elderly patients. To date, translation of CAR T cell therapy in AML is limited by the absence of an ideal tumor-specific antigen. CD123 and CD33 are the two most widely overexpressed LSCs biomarkers but their shared expression with endothelial and hematopoietic stem and progenitor cells (HSPCs) increases the risk of undesired vascular and hematologic toxicities. To counteract this issue, we established a balanced Dual CAR strategy aimed at reducing off-target toxicities while retaining full functionality against AML. Cytokine-Induced Killer (CIK) cells, co-expressing a first-generation low affinity anti-CD123 IL3-zetakine and an anti-CD33 as costimulatory receptor (CCR) without activation signaling domains, demonstrated a powerful antitumor efficacy against AML targets without any relevant toxicity on HSPCs and endothelial cells. The proposed optimized Dual CAR CIK strategy could offer the opportunity to unleash the potential of specifically target CD123+/CD33+ leukemic cells while minimizing toxicity against healthy cells.

In spite of the remarkable progress in basic and preclinical studies of acute myeloid leukemia (AML), the five-year survival rate of AML patients remains poor, highlighting the urgent need for novel and synergistic therapies. Over the past decade, increased attention has been focused on identifying suitable immunotherapeutic strategies for AML, and in particular on targeting leukemic cells and their progenitors. However, recent studies have also underlined the important contribution of the leukemic microenvironment in facilitating tumor escape mechanisms leading to disease recurrence. Here, we describe the immunological features of the AML niche, with particular attention to the crosstalk between the AML blasts and the cellular components of the altered tumor microenvironment (TME) and the mechanisms of immune escape that hamper the therapeutic effects of the most advanced treatments. Considering the AML complexity, immunotherapy approaches may benefit from a rational combination of complementary strategies aimed at preventing escape mechanisms without increasing toxicity.

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. TRIAL REGISTRATION: ClinicalTrials.gov NCT03389035. FUNDING: This study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundación Científica de la Asociación Española Contra el Cáncer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB).

INTRODUCTION An accurate diagnostic classification of thyroid lesions remains an important clinical aspect that needs to be addressed in order to avoid 'diagnostic' thyroidectomies. Among the several 'omics' techniques, proteomics is playing a pivotal role in the search for diagnostic markers. In recent years, different approaches have been used, taking advantage of the technical improvements related to mass spectrometry that have occurred. Areas covered: The review provides an update of the recent findings in diagnostic classification, in genetic definition and in the investigation of thyroid lesions based on different proteomics approaches and on different type of specimens: cytological, surgical and biofluid samples. A brief section will discuss how these findings can be integrated with those obtained by metabolomics investigations. Expert commentary: Among the several proteomics approaches able to deepen our knowledge of the molecular alterations of the different thyroid lesions, MALDI-MSI is strongly emerging above all. In fact, MS-imaging has also been demonstrated to be capable of distinguishing thyroid lesions, based on their different molecular signatures, using cytological specimens. The possibility to use the material obtained by the fine needle aspiration makes MALDI-MSI a highly promising technology that could be implemented into the clinical and pathological units.

Chimeric Antigen Receptor (CAR) T cell therapy is a promising treatment for acute myeloid leukemia (AML), but a limited efficacy was reported from ongoing clinical trials. The capacity of engineered T cells to infiltrate into the bone marrow (BM) niche, where leukemic stem cells (LSC) reside, strongly impacts the success of the treatment. Ex vivo manipulation of CAR T cells affects the expression of several chemokine receptors and may alter the capacity of infused cells to migrate to BM. The chemokine ligand 12 (CXCL12), released by mesenchymal stromal cells (MSCs) within the medullary niche, and its chemokine receptor 4 (CXCR4) regulate leukocytes trafficking to the BM. In AML, CXCL12 binds CXCR4 over-expressed on blasts, promoting their homing in the niche. CXCR4 expression is drastically downregulated during the culture of cytokine induced killer (CIK) cells, an interesting effector T cell population with acquired NK-like cytotoxicity along with minimal alloreactivity. Therefore, combining the expression of CD33.CAR with the over-expression of CXCR4 might facilitate CAR-CIKs homing to the BM and subsequent leukemia eradication. We designed two bicistronic Sleeping Beauty transposon vectors: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. The monocistronic CD33.CAR was used as control. We observed that both CD33.CAR(2A)CXCR4-CIKs (n=22, P Disclosures Dotti: Tessa Therapeutics: Consultancy; Bellicum Pharmaceuticals: Consultancy; Catamaran: Consultancy. Biondi: Bluebird: Other: Advisory Board; Amgen: Honoraria; Incyte: Consultancy, Other: Advisory Board; Novartis: Honoraria; Colmmune: Honoraria.

Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.

Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients, thus demanding advanced and personalized therapies. In this regard, different targeted immunotherapeutic approaches are available, ranging from naked monoclonal antibodies (mAb) to conjugated and multifunctional mAbs (i.e., BiTEs and DARTs). Recently, researchers have focused their attention on novel techniques of genetic manipulation specifically to redirect cytotoxic T cells endowed with chimeric antigen receptors (CARs) toward selected tumor associated antigens. So far, CAR T cells targeting the CD19 antigen expressed by B-cell origin hematological cancers have gained impressive clinical results, leading to the possibility of translating the CAR platform to treat other hematological malignancies such as AML. However, one of the main concerns in the field of AML CAR immunotherapy is the identification of an ideal target cell surface antigen, being highly expressed on tumor cells but minimally present on healthy tissues, together with the design of an anti-AML CAR appropriately balancing efficacy and safety profiles. The current review focuses mainly on AML target antigens and the related immunotherapeutic approaches developed so far, deeply dissecting methods of CAR T cell safety improvements, when designing novel CARs approaching human studies.

MALDI-MSI represents an ideal tool to explore the spatial distribution of proteins directly in situ, integrating molecular and cytomorphological information, enabling the discovery of potential diagnostic markers in thyroid cytopathology. However, red cells present in the fine needle aspiration biopsy (FNAB) specimens caused ion suppression of other proteins during the MALDI-MSI analysis due to large amount of haemoglobin. Aim of this study was to set up a sample preparation workflow able to manage this haemoglobin interference. Three protocols were compared using ex vivo cytological samples collected from fresh thyroid nodules of 9 patients who underwent thyroidectomy: (A) conventional air-dried smears, (B) cytological smears immediately fixed in ethanol, and (C) ThinPrep liquid-based preparation. Protocols C and A were also evaluated using real FNABs. Results show that protocol C markedly decreased the amount of haemoglobin, with respect to protocols A and B. Protein profiles obtained with protocols A and B were characterised by high inter-patient variability, probably related to the abundance of the haemoglobin, whereas similar spectra were observed for protocol C, where haemoglobin contents were lower. Our findings suggest protocol C as the sample preparation method for MALDI-MSI analysis. Graphical abstract.

Purpose Membranous nephropathy (MN) is the most frequent cause of nephrotic syndrome in adults and the disease course is characterized by the "rule of third", with one-third of patients experiencing complete remission and the remaining experiencing relapses or progression of the disease. Additionally, the therapeutic approach is not standardized, leading to further heterogeneity in terms of response and outcome. Experimental design In this pilot study, MALDI-MSI analysis is performed on renal biopsies (n = 13) obtained from two homogeneous groups of patients, which differentially responded to the immunosuppressive treatments (Ponticelli regimen). Results A signal at m/z 1303 displays the greatest discriminatory power when comparing the two groups and is observed to be of higher intensity in the glomeruli of the non-responding patients. The corresponding tryptic peptide is identified as macrophage migration inhibitory factor (MIF). Conclusions and clinical relevance Despite much effort being made in recent years to understand the pathogenesis of MN, a biomarker able to predict the outcome of these patients following therapeutic treatment is still lacking. Here, a protein (MIF), verified by immunohistochemistry, that can differentiate between these MN patients and could be a valuable starting point for a further study focused on verifying its predictive role in therapy response is highlighted.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Nowadays the survival rate is around 85%. Nevertheless, an urgent clinical need is still represented by primary refractory and relapsed patients who do not significantly benefit from standard approaches, including chemo-radiotherapy and hematopoietic stem cell transplantation (HSCT). For this reason, immunotherapy has so far represented a challenging novel treatment opportunity, including, as the most validated therapeutic options, cancer vaccines, donor-lymphocyte infusions and tumor-specific immune effector cells. More recently, unexpected positive clinical results in ALL have been achieved by application of gene-engineered chimeric antigen expressing (CAR) T cells. Several CAR designs across different trials have generated similar response rates, with Complete Response (CR) of 60-90% at 1 month and an Event-Free Survival (EFS) of 70% at 6 months. Relevant challenges anyway remain to be addressed, such as amelioration of technical, cost and feasibility aspects of cell and gene manipulation and the necessity to face the occurrence of relapse mechanisms. This review describes the state of the art of ALL immunotherapies, the novelties in terms of gene manipulation approaches and the problems emerged from early clinical studies. We describe and discuss the process of clinical translation, including the design of a cell manufacturing protocol, vector production and regulatory issues. Multiple antigen targeting and combination of CAR T cells with molecular targeted drugs have also been evaluated as latest strategies to prevail over immune-evasion.

As significant numbers of acute myeloid leukemia (AML) patients are still refractory to conventional therapies or experience relapse, immunotherapy using T-cells expressing chimeric antigen receptors (CARs) might represent a valid treatment option. AML cells frequently overexpress the myeloid antigens CD33 and CD123, for which specific CARs can be generated. However, CD33 is also expressed on normal hematopoietic stem/progenitors cells (HSPCs), and its targeting could potentially impair normal hematopoiesis. In contrast, CD123 is widely expressed by AML, while low expression is detected on HSPCs, making it a much more attractive target. In this study we describe the in vivo efficacy and safety of using cytokine-induced-killer (CIK) cells genetically modified to express anti-CD33 or anti-CD123 CAR to target AML. We show that both these modified T-cells are very efficient in reducing leukemia burden in vivo, but only the anti-CD123 CAR has limited killing on normal HSPCs, thus making it a very attractive immunotherapeutic tool for AML treatment.Leukemia accepted article preview online, 7 February 2014; doi:10.1038/leu.2014.62.

Use of adoptive T-cell therapy (ACT) is increasing; however, T-cell therapy can result in severe toxicity. Consequently, several suicide-gene strategies that allow selective destruction of the infused T cells have been described. We compared effectiveness of four such strategies in vitro in Epstein Barr virus (EBV)-cytotoxic T lymphocytes (CTLs). Herpes simplex virus thymidine kinase (HSV-TK), human inducible caspase 9 (iCasp9), mutant human thymidylate kinase (mTMPK), and human CD20 codon optimized genes were cloned in frame with 2A-truncated codon optimized CD34 (dCD34) in a retroviral vector. Codon-optimization considerably improved CD20 expression. EBV-CTLs could be efficiently transduced in all constructs, with transgene expression similar to the control vector containing dCD34 alone. Expression was maintained for prolonged cultures. Expression of the suicide genes was not associated with alterations in immunophenotype, proliferation, or function of CTLs. Activation of HSV-TK, iCasp9, and CD20 ultimately resulted in equally effective destruction of transduced T cells. However, while iCasp9 and CD20 effected immediate cell-death induction, HSV-TK-expressing T cells required 3 days of exposure to ganciclovir to reach full effect. mTMPK-transduced cells showed lower T-cell killing all time points. Our results suggest that the faster activity of iCasp9 might be advantageous in treating certain types of acutely life-threatening toxicity. Codon-optimized CD20 has potential as a suicide gene.

Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19(+)CD5(+)CD20(dim) B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23. CAR). CD23.CAR(+) T cells showed specific cytotoxic activity against CD23(+) tumor cell lines (average lysis 42%) and primary CD23(+) CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR(+) T cells released inflammatory cytokines (1445-fold more TNF-beta, 20-fold more TNF-alpha, and 4-fold more IFN-gamma). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR(+) T cells. Redirected T cells were also effective in vivo in a CLL Rag2(-/-)gamma(-/-)(c) xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23. CAR(+) T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23. CAR(+) T cells represent a selective immunotherapy for the elimination of CD23(+) leukemic cells in patients with CLL. (Blood. 2011; 117(18): 4736-4745) Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19(+)CD5(+)CD20(dim) B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23. CAR). CD23.CAR(+) T cells showed specific cytotoxic activity against CD23(+) tumor cell lines (average lysis 42%) and primary CD23(+) CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR(+) T cells released inflammatory cytokines (1445-fold more TNF-beta, 20-fold more TNF-alpha, and 4-fold more IFN-gamma). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR(+) T cells. Redirected T cells were also effective in vivo in a CLL Rag2(-/-)gamma(-/-)(c) xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23. CAR(+) T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23. CAR(+) T cells represent a selective immunotherapy for the elimination of CD23(+) leukemic cells in patients with CLL. (Blood. 2011; 117(18): 4736-4745)

Purpose MALDI-MS imaging (MALDI-MSI) is an emerging technology that enables the spatial distribution of biomolecules within tissue to be combined with the traditional morphological information familiar to clinicians. Thus, for diagnostic or prognostic purposes, along with predicting response to therapeutic treatment, it is important to properly collect and handle biological specimens in order to avoid degradation or the formation of artifacts in the morphological structure and proteomic profile. Experimental design In this work, the morphological and proteomic stability of thyroid fine needle aspiration biopsies in PreservCyt (up to 14 days) and CytoLyt (up to 7 days) solutions at 4 °C has been verified, by MALDI-MSI analysis. Moreover, a new measure has been introduced in order to assess the similarity of the obtained MALDI-MSI spectra, by equally taking into account the number of signals (fit and retrofit), and their intensities (Spearman's correlation and spectra overlap). Results Results show no degradation of the cellular morphology and a good stability of the samples up to 14 days in PreservCyt solution. Conclusions and clinical relevance Moreover, this protocol can be easily implemented in pathological units, allowing simple sample collection and shipment to be used not only for the proteomic MALDI-MSI analysis of thyroid FNABs but also for other biological liquid based specimens.

In the context of acute myeloid leukemia (AML) treatment, the interface between chemotherapy and immunotherapy is at present getting closer as never before. Scientific research is oriented in overcoming the main limits of actual chemotherapeutic regimens against AML, which still accounts for a considerable number of relapsed or resistant forms.A lot of investments have been done in the use of monoclonal antibodies (mAbs) and recently gene-modified immune cells have been considered as an alternative approach whenever chemotherapy fails to eradicate the disease. In this sense, AML is a potential suitable target for immunotherapeutic approaches, due to overexpression of several tumor antigens.Here we describe the state of the art of mAbs and cellular therapies employing engineered immune effectors, developed against specific AML antigens, in a window embracing preclinical research and translational studies to the clinical setting. © 2013 Elsevier B.V.

Despite the survival of pediatric patients affected by hematological malignancies being improved in the last 20 years by chemotherapy and hematopoietic stem cell transplantation (HSCT), a significant amount of patients still relapses. Treatment intensification is limited by toxic side effects and is constrained by the plateau of efficacy, while the pipeline of new chemotherapeutic drugs is running short. Therefore, novel therapeutic strategies are essential and researchers around the world are testing in clinical trials immune and gene therapy approaches as second-line treatments. The aim of this review is to give a glance at these novel promising strategies of advanced medicine in the field of pediatric leukemias. Results from clinical protocols using new targeted smart drugs, immunotherapy and gene therapy are summarized, and important considerations regarding the combination of these novel approaches with standard treatments to promote safe and long-term cure are discussed.

Summary Current therapeutic regimens for acute myeloid leukaemia (AML) are still associated with high rates of relapse. Immunotherapy with T-cells genetically modified to express chimeric antigen receptors (CARs) represents an innovative approach. Here we investigated the targeting of the interleukin three receptor alpha (IL3RA; CD123) molecule, which is overexpressed on AML bulk population, CD34+ leukaemia progenitors, and leukaemia stem cells (LSC) compared to normal haematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine-induced killer (CIK) cells were transduced with SFG-retroviral-vector encoding an anti-CD123 CAR. Transduced cells were able to strongly kill CD123+ cell lines, as well as primary AML blasts. Interestingly, secondary colony experiments demonstrated that anti-CD123.CAR preserved in vitro HSPCs, in contrast to a previously generated anti-CD33.CAR, while keeping an identical cytotoxicity profile towards AML. Furthermore, limited killing of normal monocytes and CD123-low-expressing endothelial cells was noted, thus indicating a low toxicity profile of the anti-CD123.CAR. Taken together, our results indicate that CD123-specific CARs strongly enhance anti-AML CIK functions, while sparing HSPCs and normal low-expressing antigen cells, paving the way to develop novel immunotherapy approaches for AML treatment.

Abstract 4225 Despite the progress in the treatment of acute myeloid leukemia (AML) achieved in the last decades, a significant number of patients are still refractory to or relapse after conventional chemotherapy regimens. Therefore it is necessary to develop novel alternative approaches. Immunotherapy with T cells genetically modified to express chimeric antigen receptors (CARs) represent a valid option in this sense. CARs are artificial T-cell receptors constituted by a specific antigen-binding domain, and a signaling region, that, upon antigen recognition, leads to T-cell activation, and lysis of the target cells. AML is a potential optimal target for CAR strategy because of the over-expression of a number of surface antigens like CD33, CD123. Since CD33 is also expressed on normal hematopoietic stem/progenitors cells (HSPCs) resulting in a potential severe impairment of normal myelopoiesis, CD123 has recently emerged as new potential attractive molecules based on its differential expression pattern, being still wildly overexpressed by AML population, and at the same time less expressed on HSPCs. Here we describe the in vivo efficacy and the safety of this approach based on Cytokine-Induced-Killers (CIK) cells genetically modified to express CAR molecules specific for the CD33 or CD123 antigen. Once injected into low-level AML engrafted NSG mice (median of hCD45+CD33+ 0.6% before treatment), genetically modify T cells had a potent antitumor effect. Indeed, the bone marrow of control untreated animals or mice treated with un-manipulated CIK cells, was infiltrated by leukemic cells (86% and 81% leukemic engraftment), while in 7/8 anti-CD33-CD28-OX40-ζ and 8/10 anti-CD123-CD28-OX40-ζ treated mice we couldn't detect any AML cells. Similar results have been obtained when the treatment via T cell injection start when high AML burden has been obtained (median of hCD45+CD33+ 70% before treatment). One week after the last CIK's injection the level of AML engraftment was 96%, 87%, 0.35% and 0.34% for untreated mice, mice treated with un-manipulated CIK cells and with anti-CD33-CD28-OX40-ζ and anti-CD123-CD28-OX40-ζ transduced CIK-cells respectively. We performed secondary transplantation on the residual AML cells present in these animals and mice were treated again with transduced CIK cells. Residual AML cells were still sensitive to CARs approach, leading once again to an almost complete eradication of the disease (median level of hCD45+CD33+ engraftment was 98%, 0.02% and 0.04% respectively for untreated mice, anti-CD33-CD28-OX40-ζ and anti-CD123-CD28-OX40-ζ transduced CIK-cells). Furthermore, a fundamental issue was to determine the safety profile of such approach against normal hematopoietic precursors. In untreated mice injected with primary cord blood derived CD34+ cells the level of engraftment of hCD45 compartment was 42% whilst in mice treated with un-manipulated, anti-CD33-CD28-OX40-ζ or anti-CD123-CD28-OX40-ζ transduced CIK-cells the levels of human compartment was 40%, 11.7% and 26.3% respectively. Moreover when we consider specifically the CD34+CD38- compartment, enriched in HSC, the level of engraftment was 1.92%, 1.02%, 0.55% and 0.83%. Secondary transplantations are now ongoing to give a more complete profile about the remaining HSC repopulating capability after treatment. To more closely mimic a physiological context, similar experiments are ongoing using mice engrafted with normal adult bone marrow instead of umbilical cord blood. These experiments should offer relevant information concerning the efficacy and safety of the proposed strategy particularly in the context of minimal residual disease in high-risk transplanted AML patients. Moreover CAR approach could be potentially used to treat patients resistant to conventional chemotherapeutic approaches or for whom high dose chemotherapy treatment could not be proposed. Disclosures: No relevant conflicts of interest to declare.