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The tumor microenvironment hinders CAR T-cell access, activation, and persistence at the tumor site, thereby impacting on the therapeutic efficacy. To tackle these obstacles, ongoing efforts are focusing on further engineering CAR T-cells to enhance their homing, fitness, long-term persistence, and antitumor activity. Advances in genetic modification have prompted the development of armored CAR T-cells equipped with a combination of synergistic elements strengthening their function. These include cytokine release, chemokine receptor expression, immune checkpoint inhibition, gene-editing of inhibitory molecules, or metabolic reprogramming, among others. Multiarmored CAR T-cells may allow addressing the unmet clinical needs of patients with solid tumors or hard-to-treat hematological malignancies who do not benefit from conventional CAR T-cell therapy. Accordingly, several clinical trials are currently assessing the safety and efficacy of these novel CAR constructs.

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.

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.

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 2 most widely overexpressed leukemic stem cell biomarkers but their shared expression with endothelial and hematopoietic stem and progenitor cells 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, coexpressing a first-generation low affinity anti-CD123 interleukin-3–zetakine (IL-3z) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR), demonstrated a powerful antitumor efficacy against AML targets without any relevant toxicity on hematopoietic stem and progenitor cells and endothelial cells. The proposed optimized dual-CAR cytokine-induced killer cell strategy could offer the opportunity to unleash the potential of specifically targeting CD123+/CD33+ leukemic cells while minimizing toxicity against healthy cells.

Chimeric Antigen Receptor (CAR) T-cell therapy has become a new therapeutic reality for refractory and relapsed leukemia patients and is also emerging as a potential therapeutic option in solid tumors. Viral vector-based CAR T-cells initially drove these successful efforts; however, high costs and cumbersome manufacturing processes have limited the widespread clinical implementation of CAR T-cell therapy. Here we will discuss the state of the art of the transposon-based gene transfer and its application in CAR T immunotherapy, specifically focusing on the Sleeping Beauty (SB) transposon system, as a valid cost-effective and safe option as compared to the viral vector-based systems. A general overview of SB transposon system applications will be provided, with an update of major developments, current clinical trials achievements and future perspectives exploiting SB for CAR T-cell engineering. After the first clinical successes achieved in the context of B-cell neoplasms, we are now facing a new era and it is paramount to advance gene transfer technology to fully exploit the potential of CAR T-cells towards next-generation immunotherapy.

Membranous Nephropathy (MN) is the most frequent cause of nephrotic syndrome in adults and the disease course is characterised 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 standardised, leading to further heterogeneity in terms of response and outcome. In this pilot study, MALDI-MSI analysis was performed on renal biopsies (n = 13) obtained from two homogeneous groups of patients which differentially responded to the immunosuppressive treatments (Ponticelli regimen). A signal at m/z 1303 displayed the greatest discriminatory power when comparing the two groups and was observed to be of higher intensity in the glomeruli of the non-responding patients. The corresponding tryptic peptide was identified as macrophage migration inhibitory factor (MIF). 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, we highlight a protein (MIF), verified by immunohistochemistry, which 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. This article is protected by copyright. All rights reserved

Chimeric Antigen Receptor (CAR) cytokine-induced killer (CIK) cell therapy is a promising treatment for acute myeloid leukemia (AML). Specifically, it is crucial to improve CAR CIK-cells infiltration ability into the bone marrow (BM) niche to eradicate leukemia stem cells (LSCs) at their location. Actually, BM mesenchymal stromal cells (MSCs) interact with LSCs, residing in the niche, releasing different chemokines and soluble factors. The chemokine ligand 12 (CXCL12), produced by MSCs, and its chemokine receptor 4 (CXCR4) regulate leukocytes trafficking to the BM. In AML, CXCL12 binds CXCR4 overexpressed on blasts, promoting their homing in the niche. On the contrary, CXCR4 expression is drastically downregulated on CIKs during culture. Combining the expression of CD33.CAR and 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 both CD33.CAR(2A)CXCR4-CIKs (n=22, P<0.0001) and CXCR4(IRES)CD33.CAR-CIKs (n=9, P<0.0001) maintained CXCR4 overexpression during culture, whereas in CD33.CAR-CIKs was drastically downregulated (n=22). However, CD33.CAR expression was lower in CXCR4(IRES)CD33.CAR-CIKs (n=8, P<0.0001) compared with CD33.CAR-CIKs, while CD33.CAR(2A)CXCR4-CIKs (n=11) exhibit a significant co-expression of both proteins against control (P=0.001). Chemotaxis assays toward recombinant CXCL12 confirmed both CXCR4(IRES)CD33.CAR-CIKs (n=7, P=0.01) and CD33.CAR(2A)CXCR4-CIKs (n=8, P=0.0006) displayed a migration advantage over CD33.CAR-CIKs (n=12) with a mean percentage of migration of 58.5% and 67.2% respectively, compared to 40.1%. Interestingly, CD33.CAR(2A)CXCR4-CIKs (n=2) showed an increased specific chemotactic response toward HD- (n=3) and AML-MSCs (n=2) supernatants, as demonstrated by the use of CXCR4 antagonist Plerixafor. Moreover, CXCR4(IRES)CD33.CAR

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

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.

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

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.

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.

Infusion of patient-derived CD19-specific chimeric antigen receptor (CAR) T cells engineered by viral vectors achieved complete remission and durable response in relapsed and refractory (r/r) B-lineage neoplasms. Here, we expand on those findings by providing a preclinical evaluation of allogeneic non-viral cytokine-induced killer (CIK) cells transfected with the Sleeping Beauty (SB) transposon CD19CAR (CARCIK-CD19). Specifically, thanks to a large-scale 18-day manufacturing process, it was possible to achieve stable CD19CAR expression (62.425 ± 6.399%) and efficient T-cell expansion (23.36 ± 3.00-fold). Frozen/thawed CARCIK-CD19 remained fully functional both in vitro and in an established patient-derived xenograft (PDX) of MLL-ENL rearranged acute lymphoblastic leukemia (ALL). CARCIK-CD19 showed a dose-dependent antitumor response and prolonged persistence in a PDX, bearing the feature of a Philadelphia-like ALL with PAX5/AUTS2 translocation, and in a survival model of lymphoma, achieving complete eradication of disseminated tumors. Finally, the infusion of CARCIK-CD19 proved to be safe and well tolerated in a biodistribution and toxicity model. The infused cells persisted in the hematopoietic and post-injection perfused organs until the end of the study and consisted of CD8+, CD56+, and CAR+ T cells. Overall, these findings provide important implications for non-viral technology and the proof-of-concept that donor-derived CARCIK-CD19 are indeed effective against relapsed ALL, a possibility that will be tested in Phase I/II clinical trials after allogeneic hematopoietic stem-cell transplantation

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.

Chimeric antigen receptor (CAR)-modified T-cell adoptive immunotherapy is a remarkable therapeutic option proven effective in the treatment of hematological malignancies. In order to optimize cell manufacturing, we sought to develop a novel clinical-grade protocol to obtain CAR-modified cytokine-induced killer cells (CIKs) using the Sleeping Beauty (SB) transposon system. Administration of irradiated PBMCs overcame cell death of stimulating cells induced by non-viral transfection, enabling robust gene transfer together with efficient T-cell expansion. Upon single stimulation, we reached an average of 60% expression of CD123- and CD19- specific 3rd generation CARs (CD28/OX40/TCRzeta). Furthermore, modified cells displayed persistence of cell subsets with memory phenotype, specific and effective lytic activity against leukemic cell lines and primary blasts, cytokine secretion, and proliferation. Adoptive transfer of CD123.CAR or CD19.CAR lymphocytes led to a significant anti-tumor response against acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) disseminated diseases in NSG mice. Notably, we found no evidence of integration enrichment near cancer genes and transposase expression at the end of the differentiation. Taken all together, our findings describe a novel donor-derived non-viral CAR approach that may widen the repertoire of available methods for T cell-based immunotherapy.

Despite the survival of pediatric patients affected by hematological malignancies being improved in the last 20 years by chemotherapy and hematopoietic stem cell transplantation, 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

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.

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.

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