Background Paediatric acute myeloid leukaemia (AML) is characterized by poor outcomes in patients with relapsed/refractory disease, despite the improvements in intensive standard therapy. The ...leukaemic cells of paediatric AML patients show high expression of the CD123 antigen, and this finding provides the biological basis to target CD123 with the chimeric antigen receptor (CAR). However, CAR.CD123 therapy in AML is hampered by on-target off-tumour toxicity and a long "vein-to-vein" time. Methods We developed an off-the-shelf product based on allogeneic natural killer (NK) cells derived from the peripheral blood of healthy donors and engineered them to express a second-generation CAR targeting CD123 (CAR.CD123). Results CAR.CD123-NK cells showed significant anti-leukaemia activity not only in vitro against CD123.sup.+ AML cell lines and CD123.sup.+ primary blasts but also in two animal models of human AML-bearing immune-deficient mice. Data on anti-leukaemia activity were also corroborated by the quantification of inflammatory cytokines, namely granzyme B (Granz B), interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha), both in vitro and in the plasma of mice treated with CAR.CD123-NK cells. To evaluate and compare the on-target off-tumour effects of CAR.CD123-T and NK cells, we engrafted human haematopoietic cells (hHCs) in an immune-deficient mouse model. All mice infused with CAR.CD123-T cells died by Day 5, developing toxicity against primary human bone marrow (BM) cells with a decreased number of total hCD45.sup.+ cells and, in particular, of hCD34.sup.+CD38.sup.- stem cells. In contrast, treatment with CAR.CD123-NK cells was not associated with toxicity, and all mice were alive at the end of the experiments. Finally, in a mouse model engrafted with human endothelial tissues, we demonstrated that CAR.CD123-NK cells were characterized by negligible endothelial toxicity when compared to CAR.CD123-T cells. Conclusions Our data indicate the feasibility of an innovative off-the-shelf therapeutic strategy based on CAR.CD123-NK cells, characterized by remarkable efficacy and an improved safety profile compared to CAR.CD123-T cells. These findings open a novel intriguing scenario not only for the treatment of refractory/resistant AML patients but also to further investigate the use of CAR-NK cells in other cancers characterized by highly difficult targeting with the most conventional T effector cells.
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Despite extensive research and development of new treatments, acute myeloid leukemia (AML)-backbone therapy has remained essentially unchanged over the last decades and is frequently associated with ...poor outcomes. Eradicating the leukemic stem cells (LSCs) is the ultimate challenge in the treatment of AML. Emerging evidence suggests that AML remodels the bone marrow (BM) niche into a leukemia-permissive microenvironment while suppressing normal hematopoiesis. The mechanism of stromal-mediated protection of leukemic cells in the BM is complex and involves many adhesion molecules, chemokines, and cytokines. Targeting these factors may represent a valuable approach to complement existing therapies and overcome microenvironment-mediated drug resistance. Some strategies for dislodging LSCs and leukemic blasts from their protective niche have already been tested in patients and are in different phases of the process of clinical development. Other strategies, such as targeting the stromal cells remodeling processes, remain at pre-clinical stages. Development of humanized xenograft mouse models, which overcome the mismatch between human leukemia cells and the mouse BM niche, is required to generate physiologically relevant, patient-specific human niches in mice that can be used to unravel the role of human AML microenvironment and to carry out preclinical studies for the development of new targeted therapies.
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
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.
Acute myeloid leukemia (AML) is a highly heterogeneous malignancy caused by various genetic alterations and characterized by the accumulation of immature myeloid blasts in the bone marrow (BM). This ...abnormal growth of AML cells disrupts normal hematopoiesis and alters the BM microenvironment components, establishing a niche supportive of leukemogenesis. Bone marrow stromal cells (BMSCs) play a pivotal role in giving rise to essential elements of the BM niche, including adipocytes and osteogenic cells. Animal models have shown that the BM microenvironment is significantly remodeled by AML cells, which skew BMSCs toward an ineffective osteogenic differentiation with an accumulation of osteoprogenitors. However, little is known about the mechanisms by which AML cells affect osteogenesis.
We studied the effect of AML cells on the osteogenic commitment of normal BMSCs, using a 2D co-culture system.
We found that AML cell lines and primary blasts, but not normal hematopoietic CD34
cells, induced in BMSCs an ineffective osteogenic commitment, with an increase of the early-osteogenic marker tissue non-specific alkaline phosphatase (TNAP) in the absence of the late-osteogenic gene up-regulation. Moreover, the direct interaction of AML cells and BMSCs was indispensable in influencing osteogenic differentiation. Mechanistic studies identified a role for AML-mediated Notch activation in BMSCs contributing to their ineffective osteogenic commitment. Inhibition of Notch using a γ-secretase inhibitor strongly influenced Notch signaling in BMSCs and abrogated the AML-induced TNAP up-regulation.
Together, our data support the hypothesis that AML infiltration produces a leukemia-supportive pre-osteoblast-rich niche in the BM, which can be partially ascribed to AML-induced activation of Notch signaling in BMSCs.
CARs are sharpening their weapons Pievani, Alice; Biondi, Marta; Tettamanti, Sarah ...
Journal for immunotherapy of cancer,
01/2024, Volume:
12, Issue:
1
Journal Article
TRPV1, a member of the transient receptor potential (TRP) family, is a nonselective calcium permeable ion channel gated by physical and chemical stimuli. In the skin, TRPV1 plays an important role in ...neurogenic inflammation, pain and pruritus associated to many dermatological diseases. Consequently, TRPV1 modulators could represent pharmacological tools to respond to important patient needs that still represent an unmet medical demand. Previously, we reported the design of capsaicinoid-based molecules that undergo dermal deactivation (soft drugs), thus preventing their long-term dermal accumulation. Here, we investigated the pharmacological properties of the lead antagonist, 2-((4-hydroxy-2-iodo-5-methoxybenzyl) amino)-2-oxoethyl dodecanoate (AG1529), on heterologously expressed human TRPV1 (hTRPV1), on nociceptor excitability and on an in vivo model of acute pruritus. We report that AG1529 competitively blocked capsaicin-evoked activation of hTRPV1 with micromolar potency, moderately affected pH-induced gating, and did not alter voltage- and heat-mediated responses. AG1529 displays modest receptor selectivity as it mildly blocked recombinant hTRPA1 and hTRPM8 channels. In primary cultures of rat dorsal root ganglion (DRG) neurons, AG1529 potently reduced capsaicin-evoked neuronal firing. AG1529 exhibited lower potency on pH-evoked TRPV1 firing, and TRPA1-elicited nociceptor excitability. Furthermore, AG1529 abolished histaminergic and inflammation mediated TRPV1 sensitization in primary cultures of DRG neurons. Noteworthy, dermal wiping of AG1529, either in an acetone-based formulation or in an anhydrous ointment, dose-dependently attenuated acute histaminergic itch in a rodent model. This cutaneous anti-pruritic effect was devoid of the normal nocifensive action evoked by the burning sensation of capsaicin. Taken together, these preclinical results unveil the mode of action of AG1529 on TRPV1 channels and substantiate the tenet that this capsaicinoid-based soft drug is a promising candidate for drug development as a topical anti-pruritic and anti-inflammatory medication.
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Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a ...relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.
Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by a deficiency in lysosomal enzymes catalyzing the stepwise degradation of glycosaminoglycans (GAGs). The current ...therapeutic strategies of enzyme replacement therapy and allogeneic hematopoietic stem cell transplantation have been reported to reduce patient morbidity and to improve their quality of life, but they are associated with persistence of residual disease burden, in particular at the neurocognitive and musculoskeletal levels. This indicates the need for more efficacious treatments capable of effective and rapid enzyme delivery to the affected organs, especially the brain and the skeleton. Gene therapy (GT) strategies aimed at correcting the genetic defect in patient cells could represent a significant improvement for the treatment of MPS when compared with conventional approaches. While in-vivo GT strategies foresee the administration of viral vector particles directly to patients with the aim of providing normal complementary DNA to the affected cells, ex-vivo GT approaches are based on the ex-vivo transduction of patient cells that are subsequently infused back. This review provides insights into the state-of-art accomplishments made with in vivo and ex vivo GT-based approaches in MPS and provide a vision for the future in the medical community.
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Indoleamine 2,3-dioxygenase plays a crucial role in immune tolerance and has emerged as an attractive target for cancer immunotherapy. In this study, the Passerini and Ugi ...multicomponent reactions have been employed to assemble a small library of imidazothiazoles that target IDO1. While the p-bromophenyl and the imidazothiazole moieties have been kept fixed, a full SAR study has been performed on the side-chain, leading to the discovery of nine compounds with sub-micromolar IC50 values in the enzyme-based assay. Compound 7d, displaying a α-acyloxyamide substructure, is the most potent compound, with an IC50 value of 0.20 µM, but a low activity in a cell-based assay. Compound 6o, containing a α-acylaminoamide moiety, shows an IC50 value of 0.81 µM in the IDO1-based assay, a full biocompatibility at 10 µM, together with a modest inhibitory activity in A375 cells. Molecular docking studies show that both 7d and 6o display a unique binding mode in the IDO1 active site, with the side-chain protruding in an additional pocket C, where a crucial hydrogen bond is formed with Lys238. Overall, this work describes an isocyanide based-multicomponent approach as a straightforward and versatile tool to rapidly access IDO1 inhibitors, providing a new direction for their future design and development.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived “mesenchymal stem cells”) can establish the hematopoietic microenvironment within heterotopic ossicles generated by ...transplantation at non-skeletal sites. Here we show that non-mineralized cartilage pellets formed by hBMSCs ex vivo generate complete ossicles upon heterotopic transplantation in the absence of exogenous scaffolds. These ossicles display a remarkable degree of architectural fidelity, showing that an exogenous conductive scaffold is not an absolute requirement for bone formation by transplanted BMSCs. Marrow cavities within the ossicles include erythroid, myeloid and granulopoietic lineages, clonogenic hematopoietic progenitors and phenotypic HSCs, indicating that complete stem cell niches and hematopoiesis are established. hBMSCs (CD146+ adventitial reticular cells) are established in the heterotopic chimeric bone marrow through a unique process of endochondral bone marrow formation, distinct from physiological endochondral bone formation. In this process, chondrocytes remain viable and proliferate within the pellet, are released from cartilage, and convert into bone marrow stromal cells. Once explanted in secondary culture, these cells retain phenotype and properties of skeletal stem cells (“MSCs”), including the ability to form secondary cartilage pellets and secondary ossicles upon serial transplantation. Ex vivo, hBMSCs initially induced to form cartilage pellets can be reestablished in adherent culture and can modulate gene expression between cartilage and stromal cell phenotypes. These data show that so-called “cartilage differentiation” of BMSCs in vitro is a reversible phenomenon, which is actually reverted, in vivo, to the effect of generating stromal cells supporting the homing of hematopoietic stem cells and progenitors.
•Human heterotopic ossicles can be generated in the absence of exogenous scaffolds.•Cartilage pellets convert into ossicles with normal bone–bone marrow structures.•Cartilage pellets develop a marrow cavity through “endochondral myelogenesis”.•The HSC niche can be established in vivo by differentiated chondrocytes.•Chondrocytes in transplanted pellets revert to a stromal stem cell phenotype.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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