Search

•CAR T-cell therapy in AML is hampered by the poor accumulation of effector cells in the BM niche in which leukemic stem cells are nested.•Engineering CAR-CIKs with CXCR4 promotes their homing in the BM and improves their antileukemic efficacy in AML.

The use of small molecules to induce targeted protein degradation is increasingly growing in the drug discovery landscape, and protein degraders have progressed rapidly through the pipelines. Despite the advances made so far, their synthesis still represents a significant burden and new approaches are highly demanded. Herein we report an unprecedented platform that leverages the modular nature of both multicomponent reactions and degraders to enable the preparation of highly decorated PROTACs. As a proof of principle, our platform has been applied to the preparation of potential BRD4-degrading PROTACs, resulting in the discovery of a set of degraders endowed with high degradation efficiency. Compared to the existing methods, our approach offers a versatile and cost-effective means to access libraries of protein degraders and increase the chance of identifying successful candidates.

Store-operated Ca2+-entry is a cellular mechanism that governs the replenishment of intracellular stores of Ca2+upon depletion caused by the opening of intracellular Ca2+-channels. Gain-of-function mutations of the 2 key proteins of store-operated Ca2+-entry, STIM1 and ORAI1, are associated with several ultra-rare diseases clustered as tubular aggregate myopathies. Our group has previously demonstrated that a mouse model bearing the STIM1 p.I115F mutation recapitulates the main features of the STIM1 gain-of-function disorders: muscle weakness and thrombocytopenia. Similar findings have been found in other mice bearing different mutations on STIM1. At present, no valid treatment is available for these patients. In the present contribution, we report that CIC-39Na, a store-operated Ca2+-entry inhibitor, restores platelet number and counteracts the abnormal bleeding that characterizes these mice. Subtle differences in thrombopoiesis were observed in STIM1 p.I115F mice, but the main difference between wild-type and STIM1 p.I115F mice was in platelet clearance and in the levels of platelet cytosolic basal Ca2+. Both were restored on treatment of animals with CIC-39Na. This finding paves the way to a pharmacological treatment strategy for thrombocytopenia in tubular aggregate myopathy patients.

Store-operated Ca2+-entry is a cellular mechanism that governs the replenishment of intracellular stores of Ca2+ upon depletion caused by the opening of intracellular Ca2+-channels. Gain-of-function mutations of the 2 key proteins of store-operated Ca2+-entry, STIM1 and ORAI1, are associated with several ultra-rare diseases clustered as tubular aggregate myopathies. Our group has previously demonstrated that a mouse model bearing the STIM1 p.I115F mutation recapitulates the main features of the STIM1 gain-of-function disorders: muscle weakness and thrombocytopenia. Similar findings have been found in other mice bearing different mutations on STIM1. At present, no valid treatment is available for these patients. In the present contribution, we report that CIC-39Na, a store-operated Ca2+-entry inhibitor, restores platelet number and counteracts the abnormal bleeding that characterizes these mice. Subtle differences in thrombopoiesis were observed in STIM1 p.I115F mice, but the main difference between wild-type and STIM1 p.I115F mice was in platelet clearance and in the levels of platelet cytosolic basal Ca2+. Both were restored on treatment of animals with CIC-39Na. This finding paves the way to a pharmacological treatment strategy for thrombocytopenia in tubular aggregate myopathy patients.

The improvement of hematopoietic stem and progenitor cell (HSPC) engraftment remains a high-priority goal when limited cell doses are available, such as in cord blood (CB) transplantation and HSC gene therapy. We observed that monocytes are highly effective at improving the engraftment of both CB-CD34+and lentivirus-transfected CD34+cells in a xenogeneic model of HSC transplantation. Moreover, monocytes, in particular the CD14+CD16−classical subset, in co-culture systems increase survival and stemness of CB-CD34+cells. Both soluble factors and direct-cell contact interactions, such as JAG/NOTCH and COX-2/PGE2 pathways, are critically involved in the HSC-monocyte crosstalk. Our results indicate that the infusion of monocytes improves engraftment when cell dose is a limiting factor.

Starting from a wide range of α-acylamino amide substructures synthesized using tritylamine as an ammonia surrogate in the Ugi reaction, Burgess-type reagents enable cyclodehydration and afford unprecedented oxazole scaffolds with four points of diversity, including a sulfamide moiety in the 5-position. The synthetic procedure employs readily available starting materials and proceeds smoothly under mild reaction conditions with good tolerance for a variety of functional groups, coming to fill a gap in the field of oxazole compounds.

The World Health Organization assigns international nonproprietary names (INN), also known as common names, to compounds upon request from drug developers. Structures of INNs are publicly available and represent a source, albeit underused, to understand trends in drug research and development. Here, we explain how a common drug name is composed and analyze chemical entities from 2000 to 2021. In the analysis, we describe some changes that intertwine chemical structure, newer therapeutic targets (e.g., kinases), including a significant increase in the use of fluorine and of heterocycles, and some other evolutionary modifications, such as the progressive increase in molecular weight. Alongside these, small signs of change can be spotted, such as the rise in spirocyclic scaffolds and small rings and the emergence of unconventional structural moieties that might forecast the future to come.

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.

Store-operated calcium entry (SOCE) is important in the maintenance of calcium homeostasis and alterations in this mechanism are responsible for several pathological conditions, including acute pancreatitis. Since the discovery of SOCE, many inhibitors have been identified and extensively used as chemical probes to better elucidate the role played by this cellular mechanism. Nevertheless, only a few have demonstrated drug-like properties so far. Here, we report a class of biphenyl triazoles among which stands out a lead compound, 34, that is endowed with an inhibitory activity at nanomolar concentrations, suitable pharmacokinetic properties, and in vivoefficacy in a mouse model of acute pancreatitis.

Since 1977, the World Health Organization publishes a list of essential medicines, i.e., those that satisfy the priority health care needs of the population and are selected with regard to disease prevalence and public health relevance, evidence of clinical efficacy, and safety, as well as comparative costs and cost-effectiveness. The Essential Medicines List (EML) is an invaluable tool for all countries to select those medicines that have an excellent risk/benefit ratio and that are reputed to be of pivotal importance to health. In the present perspective, we describe the chemical composition and the main features of the small molecules that are included in the EML, spanning from their origin, to their stereochemistry and measure of drug-likeness. Most and foremost, we wish to disseminate the importance of the EML, which can be both a helpful teaching tool in an ever-expanding world of medicines and an inspiration for those involved in pharmaceutical R&D.

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 vitroand in vivoin 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.

In this study, a successful medicinal chemistry campaign that exploited virtual, biophysical, and biological investigations led to the identification of a novel class of IDO1 inhibitors based on a benzimidazole substructure. This family of compounds is endowed with an extensive bonding network in the protein active site, including the interaction with pocket C, a region not commonly exploited by previously reported IDO1 inhibitors. The tight packing of selected compounds within the enzyme contributes to the strong binding interaction with IDO1, to the inhibitory potency at the low nanomolar level in several tumoral settings, and to the selectivity toward IDO1 over TDO and CYPs. Notably, a significant reduction of L-Kyn levels in plasma, together with a potent effect on abrogating immunosuppressive properties of MDSC-like cells isolated from patients affected by pancreatic ductal adenocarcinoma, was observed, pointing to this class of molecules as a valuable template for boosting the antitumor immune system.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1in HSPCs from patients with hypomorphic RAG1mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1–/–mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

•SETBP1mutations, acting as a first-hit/early event, drive an aggressive myelofibrosis-like myeloproliferative disorder.•SETBP1mutations discriminate between 2 subtypes of triple-negative myelofibrosis, with different genetic landscape and aggressiveness.

The use of deuteration in medicinal chemistry has exploded in the past years, and the FDA has recently approved the first deuterium-labeled drug. Precision deuteration goes beyond the pure and simple amelioration of the pharmacokinetic parameters of a drug and might provide an opportunity when facing problems in terms of metabolism-mediated toxicity, drug interactions, and low bioactivation. The use of deuterium is even broader, offering the opportunity to lower the degree of epimerization, reduce the dose of coadministered boosters, and discover compounds where deuterium is the basis for the mechanism of action. Nevertheless, designing, synthesizing, and developing a successful deuterated drug is far from straightforward, and the translation from concept to practice is often unpredictable. This Perspective provides an overview of the recent developments of deuteration, with a focus on deuterated clinical candidates, and highlights both opportunities and challenges of this strategy.

Somatic SETBP1mutations are found in various myeloid disorders covering both myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS).

Chimeric Antigen Receptor (CAR)-T cells have proven highly successful in the treatment of some haemopoietic neoplasms. However, in several cases the treatment fails and disease does not respond and/or relapses. Amongst the different factors impacting on a negative clinical outcome, an unfavourable immunosuppressive microenvironment has been proposed as playing a critical role, particularly in the context of acute myeloid leukemia. We hypothesised that bone marrow mesenchymal stromal cells (BM-MSC) acquire immunosuppressive properties after exposure to the by-stander effects of activated CAR-T cells against leukemic cells. We used a CAR-T cell consisting of a CD33 binder and CD28z-OX40 costimulatory moiety. BM-MSC were exposed to the CAR following in vitro activation on CD33-coated plates for 24 hours. To model the different ways by which stromal cells can license their immunosuppressive activity, BM-MSC were exposed to the supernatant harvested from the cultures of activated CARs or in direct contact with the activated CARs themselves. In the first case, after incubation with the CAR-conditioned medium (CAR-cm), BM-MSCs were investigated for their transcriptome by RNASeq. Gene ontology analysis of the transcriptome revealed a strong NF-κB and STAT1 signature in BM-MSC exposed to the CAR-cm compared to those which were not exposed. Amongst the most upregulated molecules, we identified several chemokines (CXCL10, CCL8, CXCL8, and CXCL9), PDL1 and CECAM1, IL-6, indoleamine-2,3 dyoxigenase (IDO), and most prominently PTGES and PTGS2. We then interrogated the immune functional profile by incubating by CAR-cm licensed BM-MSC with fresh CD33-activated CAR-T cells. We observed that CAR proliferation, cytotoxicity and cytokine production was significantly inhibited. CAR function was restored if Rel-A - a REL-associated protein critically involved in NF-κB heterodimer formation, nuclear translocation and activation - was inhibited in BM-MSC by shRNA before exposure to CAR-cm. We previously described that BM-MSC undergo apoptosis when in contact with activated cytotoxic T cells and that this also triggers their immunosuppressive capability. Therefore, we evaluated the impact of cell contact dependent mechanisms on BM-MSC licensing. CD33-activated CAR-T cells were incubated with BM-MSC and the proportion of annexin-V positive BM-MSC were assessed after 4 hours. We observed that a large fraction of BM-MSC underwent apoptosis and that this was dependent on caspase activation, but not RIP kinase 3, and the release of cytolytic granules in a contact-dependent fashion. The supernatant of apoptotic BM-MSC but not the terminally apoptotic and neither the live cells exhibited a strong inhibitory activity on CAR-T cell proliferation, cytotoxicity and effector cytokine production. Similarly to what described with the CAR-cm induced profile, BM-MSC immunosuppression required NF-κB and was effected via the COX2 pathway. We finally tested the impact of apoMSC in vivo in a xenograft model of AML (Fig. 1). CD33-positive KG1 cells were injected in NSG mice and 2 weeks later, CD33-specific CAR-T were administered with or without (controls) apoMSC. Peripheral blood samples were collected 7, 14, and 21 days after. We observed an inhibitory effect of apoMSc on the in vivo expansion of CAR-T cells which was accompanied by disease progression. Accordingly, the survival of the mice treated with apoMSC was markedly reduced compared to those receiving CAR-T cells only.

In recent years, channels that mediate store-operated calcium entry (SOCE, i.e., the ability of cells to sense a decrease in endoplasmic reticulum luminal calcium and induce calcium entry across the plasma membrane) have been associated with a number of disorders, spanning from immune disorders to acute pancreatitis and have been suggested to be druggable targets. In the present contribution, we exploited the click chemistry approach to synthesize a class of SOCE modulators where the arylamide substructure that characterizes most inhibitors so far described is substituted by a 1,4-disubstituted 1,2,3-triazole ring. Within this series, inhibitors of SOCE were identified and the best compound proved effective in an animal model of acute pancreatitis, a disease characterized by a hyperactivation of SOCE. Strikingly, two enhancers of the process were discovered, affording invaluable research tools to further explore the (patho)physiological role of capacitative calcium entry.

Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WTor BRD4(1)L94C, to determine their selectivity for BRD4(1)L94Cover other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94Cover all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.

•Dual CAR strategy by low affinity interleukin-3–zetakine and CD33 costimulatory receptor minimizes on-target/off-tumor toxicity in CD123 and/or CD33 healthy cells.•Dual interleukin-3–zetakine /CD33 costimulatory-receptor cytokine-induced killer cells demonstrate high efficacy and specificity for CD123+/CD33+leukemic cells in preclinical models.

The Gsα/cAMP signaling pathway mediates the effect of a variety of hormones and factors that regulate the homeostasis of the post-natal skeleton. Hence, the dysregulated activity of Gsα due to gain-of-function mutations (R201C/R201H) results in severe architectural and functional derangements of the entire bone/bone marrow organ. While the consequences of gain-of-function mutations of Gsα have been extensively investigated in osteoblasts and in bone marrow osteoprogenitor cells at various differentiation stages, their effect in adipogenically-committed bone marrow stromal cells has remained unaddressed. We generated a mouse model with expression of GsαR201Cdriven by the Adiponectin(Adq) promoter. Adq-GsαR201Cmice developed a complex combination of metaphyseal, diaphyseal and cortical bone changes. In the metaphysis, GsαR201Ccaused an early phase of bone resorption followed by bone deposition. Metaphyseal bone formation was sustained by cells that were traced by Adq-Creand eventually resulted in a high trabecular bone mass phenotype. In the diaphysis, GsαR201C, in combination with estrogen, triggered the osteogenic activity of Adq-Cre-targeted perivascular bone marrow stromal cells leading to intramedullary bone formation. Finally, consistent with the previously unnoticed presence of Adq-Cre-marked pericytes in intraosseous blood vessels, GsαR201Ccaused the development of a lytic phenotype that affected both cortical (increased porosity) and trabecular (tunneling resorption) bone. These results provide the first evidence that the Adq-cell network in the skeleton not only regulates bone resorption but also contributes to bone formation, and that the Gsα/cAMP pathway is a major modulator of both functions.

Neonatal bone marrow transplantation (BMT) could offer a novel therapeutic opportunity for genetic disorders by providing sustainable levels of the missing protein at birth, thus preventing tissue damage. We tested this concept in mucopolysaccharidosis type I (MPS IH; Hurler syndrome), a lysosomal storage disorder caused by deficiency of α-l-iduronidase. MPS IH is characterized by a broad spectrum of clinical manifestations, including severe progressive skeletal abnormalities. Although BMT increases the life span of patients with MPS IH, musculoskeletal manifestations are only minimally responsive if the timing of BMT delays, suggesting already irreversible bone damage. In this study, we tested the hypothesis that transplanting normal BM into newborn MPS I mice soon after birth can prevent skeletal dysplasia. We observed that neonatal BMT was effective at restoring α-l-iduronidase activity and clearing elevated glycosaminoglycans in blood and multiple organs. At 37 weeks of age, we observed an almost complete normalization of all bone tissue parameters, using radiographic, microcomputed tomography, biochemical, and histological analyses. Overall, the magnitude of improvements correlated with the extent of hematopoietic engraftment. We conclude that BMT at a very early stage in life markedly reduces signs and symptoms of MPS I before they appear.

Neonatal bone marrow transplantation (BMT) could offer a novel therapeutic opportunity for genetic disorders by providing sustainable levels of the missing protein at birth, thus preventing tissue damage. We tested this concept in mucopolysaccharidosis type I (MPS IH; Hurler syndrome), a lysosomal storage disorder caused by deficiency of α-l-iduronidase. MPS IH is characterized by a broad spectrum of clinical manifestations, including severe progressive skeletal abnormalities. Although BMT increases the life span of patients with MPS IH, musculoskeletal manifestations are only minimally responsive if the timing of BMT delays, suggesting already irreversible bone damage. In this study, we tested the hypothesis that transplanting normal BM into newborn MPS I mice soon after birth can prevent skeletal dysplasia. We observed that neonatal BMT was effective at restoring α-l-iduronidase activity and clearing elevated glycosaminoglycans in blood and multiple organs. At 37 weeks of age, we observed an almost complete normalization of all bone tissue parameters, using radiographic, microcomputed tomography, biochemical, and histological analyses. Overall, the magnitude of improvements correlated with the extent of hematopoietic engraftment. We conclude that BMT at a very early stage in life markedly reduces signs and symptoms of MPS I before they appear.

Mesenchymal stromal cells (MSCs), seeded onto a scaffold and associated with platelet-gel, may represent an innovative treatment to improve bone repair. The preparation of MSCs for clinical use requires the fulfillment of Good Manufacturing Practice indications. The aim of this study was to validate a Good Manufacturing Practice–grade protocol of tissue engineering for bone regeneration, seeding platelet lysate (PL)–cultured MSCs onto an hydroxyapatite clinical-grade scaffold. Six large-scale experiments were performed. MSC expansions were performed comparing fetal bovine serum 10% and PL 5%. We demonstrated that PL lots contain high levels of growth factors possibly responsible of accelerated growth rate, since the number of colony-forming unit–fibroblast and population doublings were always significantly higher in PL cultures. MSCs were characterized for their phenotype and multilineage differentiation capacity, demonstrating appropriate features for both conditions. Gene expression analysis revealed higher expression of typical osteogenic genes of PL-cultured MSCs, when compared to fetal bovine serum MSCs. Cell transformation was excluded by analysis of karyotype, absence of growth without anchorage, and p53/c-mycgene expression. Scaffolds were precoated with retronectin before MSC seeding. MSC adhesion, distribution, and proliferation were demonstrated through the whole surface of the scaffold by scanning electron microscopy analysis or by immunofluorescence and MSC osteogenic differentiation through quantitative reverse transcriptase–polymerase chain reaction of typical osteogenic genes. The present report offers a model of an MSC-based bioengineered device, using an hydroxyapatite clinical-grade scaffold, and supports its potential use in tissue engineering to repair bone defects.

The recent introduction of new technologies such as Luminex has provided alternative methods to the Complement Dependent Cytotoxicity (CDC) test for HLA specific antibody detection. In this study we compared the results obtained with CDC to those obtained using a Luminex method with the aim of evaluating the impact of this new technology on antibody screening policies in our transplant setting.A total of 1,421 sera, acquired from patients on the waiting list for a kidney transplant or following transplantation, were tested by both methodologies. CDC was performed using a whole lymphocyte population comprising a panel of 52 cells. The percentage panel reactive antibodies (PRA) and antibody specificity were evaluated using Lambda Scan Analysis software. For the Luminex method sera screening and identification of antibody specificity were carried out using the LABScreen Mixed and LABScreen PRA respectively.The overall concordance between the results obtained using the CDC and the Luminex methods was 85%. HLA antibody specificity was confirmed in 96% of the sera which tested positive using the Luminex system and serum positivity corresponded with a previous sensitisation event in these individuals. Using the Luminex method 18% of patients on the waiting list were considered and managed as sensitised as compared to 7% when testing with CDC alone. The Luminex method was able to detect a number of antibody specificities significantly more frequently than the CDC method and in addition the CDC method failed to detect some of the antibody specificities detected by the Luminex system.Based on this comparison study we have incorporated the Luminex methodology into our screening strategy. © 2007 Clinical Cytometry Society

We have investigated the capacity of two human immunodeficiency virus type 1-derived lentivectors, differing in the presence of a 118-bp pol fragment containing the cPPT/CTS element, to transduce human normal primary cells of different hematopoietic lineages. Infection of resting monocytes with a high multiplicity of infection (MOI > 10) revealed that the lentivirus carrying the pol fragment (cPPT) is effective, transducing 75% of cells compared with 36% for the no-cPPT vector. Even at low MOIs (≤1) the cPPT vector still shows a better transduction efficiency than the no-cPPT vector. Moreover, transduction does not require dendritic cell differentiation. In contrast, infection of nonactivated T lymphocytes showed that both vectors, tested at high MOIs, can transduce a small, although measurable, percentage of cells (up to 10%), which may correspond to G1a "activated" cells as detected by simultaneous staining of DNA and RNA, in our cultures in the presence of medium alone. Furthermore, we show that the sole addition of interleukin 2 or interleukin 15 represents a full proliferative signal under our conditions and permits high transduction efficiency (up to 30% with the cPPT vector and 15% with the no-cPPT vector). Still higher transduction of T lymphocytes can be achieved after stimulation with phytohemagglutinin and interleukin 2 (up to 78% with the cPPT vector vs. 42% with the no-cPPT vector). Finally, both viruses do not transduce either resting or proliferating tonsillar B lymphocytes.

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 vivomanipulation 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<0.0001) and CXCR4(IRES)CD33.CAR-CIKs (n=9, P<0.0001) maintained CXCR4 over-expression 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 to CD33.CAR-CIKs, while CD33.CAR(2A)CXCR4-CIKs (n=11) exhibited a significant co-expression of both proteins against control (P=0.001). CXCR4(IRES)CD33.CAR-CIKs and CD33.CAR(2A)CXCR4-CIKs maintained all CAR-associated in vitroeffector functions, eliminating CD33+ KG1 target cell line, releasing cytokines (IL-2 and IFN-γ) and proliferating in an antigen-specific manner. However, CXCR4(IRES)CD33.CAR-CIKs exhibited lower effector responses against control, due to lower CAR expression. 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 healthy (n=3) and AML-derived MSC (n=2) supernatants, which could be inhibited by the use of the CXCR4 antagonist Plerixafor. Moreover, when infused intravenously into NSG mice, significantly higher proportions of CD33.CAR(2A)CXCR4-CIKs were recovered in the femur BM compared to controls (P=0.0068). In conclusion, CD33.CAR(2A)CXCR4-CIKs, reaching the medullary niche more effectively, have the potential to more efficiently target the residing LSC responsible for the high relapse rates in AML.

Acute Myeloid Leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients. Adoptive cell therapy by chimeric antigen receptor (CAR) T cells demonstrated a high therapeutic potential in B-acute lymphoblastic leukemia, but translation in AML is limited by the absence of an ideal tumor-specific antigen. CD123 (known as IL-3 receptor alfa) and CD33 satisfy several features of ideal target antigens as they are expressed in almost all AML patients (mainly overexpressed in NPM1 and FLT-3 mutated AML), and conserved at disease relapse .Despite that, CD33 expression on hemopoietic stem/progenitor cells (HSPC) induced prolonged myelotoxicity after anti-CD33 CAR-T cell therapy and CD123 expression on endothelium was responsible for severe capillary leak syndrome after anti-CD123 CAR therapy. With the aim to improve selectivity for CD123+/CD33+ AML cells while minimizing toxicity against healthy cells, we probe a dual targeting model by Cytokine Induced Killer (CIK) cells co-expressing a first generation low affinity anti-CD123 IL-3 zetakine (IL3z.CAR) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR). This trans-signaling strategy could allow: 1)low toxicity profile against CD123+ endothelial cells and HSPC, due to a reduced cell activation given by the suboptimal first-generation CAR signal; 2)no or low myelotoxicity against CD33+ HSPC cells, due to absence of CIK cell activation upon the sole costimulatory signal engagement; 3)full CAR-CIK activation only against double expressing CD123 + / CD33 + leukemic cells.

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.