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Nicotinamide riboside (NR) is a form of vitamin B

Chimeric antigen receptors (CAR)-modified T cells are an emerging therapeutic tool for chronic lymphocytic leukemia (CLL). However, in patients with CLL, well-known T-cell defects and the inhibitory properties of the tumor microenvironment (TME) hinder the efficacy of CAR T cells. We explored a novel approach combining CARs with lenalidomide, an immunomodulatory drug that tempers the immunosuppressive activity of the CLL TME. T cells from patients with CLL were engineered to express a CAR specific for CD23, a promising target antigen. Lenalidomide maintained the in vitro effector functions of CD23.CAR+ T cells effector functions in terms of antigen-specific cytotoxicity, cytokine release and proliferation. Overall, lenalidomide preserved functional CAR T-CLL cell immune synapses. In a Rag2−/−γc−/−-based xenograft model of CLL, we demonstrated that, when combined with low-dose lenalidomide, CD23.CAR+ T cells efficiently migrated to leukemic sites and delayed disease progression when compared to CD23.CAR+ T cells given with rhIL-2. These observations underline the therapeutic potential of this novel CAR-based combination strategy in CLL.

Chronic inflammation that affects primarily metabolic organs, such as white adipose tissue (WAT), is considered as a major cause of human obesity-associated co-morbidities. However, the molecular mechanisms initiating this inflammation in WAT are poorly understood. By combining transcriptomics, ChIP-seq and modeling approaches, we studied the global early and late responses to a high-fat diet (HFD) in visceral (vWAT) and subcutaneous (scWAT) AT, the first being more prone to obesity-induced inflammation. HFD rapidly triggers proliferation of adipocyte precursors within vWAT. However, concomitant antiadipogenic signals limit vWAT hyperplastic expansion by interfering with the differentiation of proliferating adipocyte precursors. Conversely, in scWAT, residing beige adipocytes lose their oxidizing properties and allow storage of excessive fatty acids. This phase is followed by tissue hyperplastic growth and increased angiogenic signals, which further enable scWAT expansion without generating inflammation. Our data indicate that scWAT and vWAT differential ability to modulate adipocyte number and differentiation in response to obesogenic stimuli has a crucial impact on the different susceptibility to obesity-related inflammation of these adipose tissue depots. Electronic supplementary material The online version of this article (10.1007/s00018-020-03485-z) contains supplementary material, which is available to authorized users.

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T��cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T��cells, enhanced effectiveness of bi-specific T��cell engaging antibodies, and improved CAR T��cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T��cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.

Different complex mechanisms control the morphology of podocyte foot processes and their interactions with the underlying basement membrane. Injuries to this system often cause glomerular dysfunction and albuminuria. The present study aimed at identifying early markers of glomerular damage in diabetic nephropathy. For this purpose, we performed a microarray analysis on kidneys of 3-wk-old peroxisome proliferator-activated receptor-γ (PPARγ)-null and AZIP/F1 mice, which are two models of diabetic nephropathy due to lipodystrophy. This was followed by functional annotation of the enriched clusters of genes. One of the significant changes in the early stages of glomerular damage was the increase of hemicentin 1 (HMCN1). Its expression and distribution were then studied by real-time PCR and immunofluorescence in various models of glomerular damage and on podocyte cell cultures. HMCN1 progressively increased in the glomeruli of diabetic mice, according to disease severity, as well as in puromycin aminonucleoside (PA)-treated rats. Studies on murine and human podocytes showed an increased HMCN1 deposition upon different pathological stimuli, such as hyperglycemia, transforming growth factor-β (TGF-β), and PA. In vitro silencing studies showed that HMCN1 mediated the rearrangements of podocyte cytoskeleton induced by TGF-β. Finally, we demonstrated an increased expression of HMCN1 in the kidneys of patients with proteinuric nephropathies. In summary, our studies identified HMCN1 as a new molecule involved in the dynamic changes of podocyte foot processes. Its increased expression associated with podocyte dysfunction points to HMCN1 as a possible marker for the early glomerular damage occurring in different proteinuric nephropathies.

PPARγ regulates multiple aspects of skin physiology, including sebocyte differentiation, keratinocyte proliferation, epithelial stem cell survival, adipocyte biology, and inflammatory skin responses. However, the effects of its global deletion, namely of nonredundant key functions of PPARγ signaling in mammalian skin, are yet unknown because of embryonic lethality. Here, we describe the skin and hair phenotype of a whole-body PPARγ-null mouse (PpargΔ/Δ), obtained by preserving PPARγ expression in the placenta. PpargΔ/Δ mice exhibited total lipoatrophy and complete absence of sebaceous glands. Right after birth, hair follicle (HF) morphogenesis was transiently delayed, along with reduced expression of HF differentiation markers and of transcriptional regulators necessary for HF development. Later, adult PpargΔ/Δ mice developed scarring alopecia and severe perifollicular inflammation. Skin analyses in other models of lipodystrophy, AZIPtg/+ and Adipoq-Cretg/+Ppargfl/fl mice, coupled with skin graft experiments, showed that the early defects observed in hair morphogenesis were caused by the absence of adipose tissue. In contrast, the late alteration of HF cycle and appearance of inflammation were observed only in PpargΔ/Δ mice and likely were due to the lack sebaceous glands. Our findings underscore the increasing appreciation for the importance of adipose tissue-mediated signals in HF development and function.

// Chiara F. Magnani 1 , Nice Turazzi 1 , Fabrizio Benedicenti 2 , Andrea Calabria 2 , Erika Tenderini 2 , Sarah Tettamanti 1 , Greta M.P. Giordano Attianese 1, 3 , Laurence J.N. Cooper 3 , Alessandro Aiuti 2 , Eugenio Montini 2 , Andrea Biondi 1 , Ettore Biagi 1 1 Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca, San Gerardo Hospital/Fondazione MBBM, Monza, Italy 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy 3 University of Texas MD Anderson Cancer Center, Houston, Texas, USA Correspondence to: Andrea Biondi, email: abiondi.unimib@gmail.com Ettore Biagi, email: e.biagi@hsgerardo.org Keywords: chimeric antigen receptor, Sleeping Beauty transposon, cytokine-induced killer cells, acute lymphoblastic leukemia, acute myelogenous leukemia Received: February 11, 2016 Accepted: May 20, 2016 Published: June 13, 2016 ABSTRACT 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.

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)

CARs are hybrid molecules comprising a tumor antigen-targeting moiety, typically a scFv, followed by a linker, transmembrane (TM) domain, and various endodomains (EDs) involved in T-cell activation. First generation CARs include the ED of CD3ζ only, required for ‘signal 1' of T cell activation, while 2nd and 3rd generation CARs also have one or more co-stimulatory EDs, respectively, such as CD28 and 41BB, to provide ‘signal 2'. CAR T cells have shown robust clinical responses against advanced haematologial malignancies, leading to two recent FDA approvals for CD19-targeting CARs. Solid tumors, however, remain an important challenge to CAR therapy, in part due to the fact there are limited cell surface-expressed antigens (protein, carbohydrate, etc.) that are tumor-restricted, thus running the risk of severe on-target/off-tumor toxicity. Here we describe our progress in the development of a split OFF-switch CAR for which a small molecule is used to stop signaling. We designed a split CAR in which CD3ζ and the co-stimulatory ED are found on separate, co-expressed components. Briefly, Component A includes the scFv, a linker, TM domain, Protein A, and CD28, while the second one, Component B comprises a short extracellular region having no tumor-binding capacity, a TM domain, Protein B, a high affinity binding partner for Protein A, and CD3ζ. Proteins A and B were computationally designed from human proteins that normally do not interact, and upon the design of a novel protein-protein interaction (PPI) they form a heterodimer with an experimentally determined KD of 400 pM. Notably, this novel PPI was rationally designed to be rapidly disrupted by a clinically approved small molecule (KD of 10 pM for Protein B). Lentiviral constructs were built encoding the two components separately, or together on the same vector, and used to engineer both a Jurkat NFAT promoter-mCherry reporter line, and primary human T cells following activation with anti-CD3/anti-CD28 beads. Flow cytometry was used to assess CAR cell-surface expression, and AMNIS imaging to visualize co-localization. Both Jurkat and T cells could co-express Components A and B, but when transduced on their own, neither A nor B could be detected on the cell-surface. However, we observed instability of Component B over time. Finally, we demonstrated co-localization of the chains in Jurkats, as well as functionality of the CAR, as measured by mCherry and IL2 production, in the presence of target cells. Ongoing work includes the titration of the OFF-switch small molecule with target cells in vitro, and in vivo. We have also re-designed Component B to include a larger extracellular domain in an attempt to stabilize its expression. In conclusion, we used computational design to engineer a split-architecture CAR that can specifically activate engineered cells but further optimization is required to enhance its stability. We believe that such OFF-switch CARs hold important promise for increasing patient safety. Citation Format: Elise F. Gray-Gaillard, Greta Giordano Attianese, Pablo Gainza-Cirauqui, Sabrina Vollers, Sailan Shui, Bruno Correia, Melita Irving, George Coukos. OFF-switch CAR T cell for safety-enhanced cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2570.

Gene-manipulation of effector T cells with CARs has recently turned into a powerful tool to redirect antigen specificity for adoptive immunotherapy of tumors. Although promising clinical efficacy has been demonstrated, critical issues concerning the profile of efficacy, safety and feasibility of cell manufacturing and gene therapy still remain partially unsolved. In order to rescue the concerns associated to viral vectors that limit so far their clinical applicability, we have explored here the use of the latest generation Sleeping Beauty Transposon-mediated gene transfer. Since current protocol of nucleofection associated with Transposons impaired subsequent expansion and vitality of modified cells, we generated and propagated CAR+ cytokine-induced killer (CIK) cells with the purpose of optimizing cell expansion. Actually, our experience with CIK cells clearly proved that the production of large numbers of unmanipulated allogeneic cytotoxic effector T cells is feasible under clinical-grade conditions, and repeated infusions in patients are safe and well tolerated (Introna et al., Haematologica 2007). Using an optimized stimulation protocol based on the addition of accessory cells, irradiated PBMCs, after nucleofection, we genetically modified CIK cells to express two distinct 3rd generation CARs (CD28/OX40/TCR zeta) specific for acute myelogenous leukemia (AML) CD123+ or acute lymphoblastic leukemia (ALL) CD19+ blasts. With this system, the average transfection at 24hours was 54.6% (±8.6, n=8) and mean survival percentage was 63.8% (±8.8, n=12). Nucleofection did not affect the phenotype of CIK cells, and, most importantly, the addition of accessory cells was effective in inducing T-cell expansion, with a fold increase of 39.4±9.8 within 3 weeks, sufficient to be translated into adoptive cell therapy clinical protocols. Transposed CIK cells displayed stable expression of CD123-CAR or CD19-CAR with a frequency of modified cells of 48.9%±3.3 (n=11) and 47%±6.4 (n=4), respectively. Efficient lysis of leukemic cell lines and primary blasts was observed and cytotoxic degranulation was associated to CAR expression, indicating a specific target recognition by the CAR. Interestingly, CAR triggering by the encounter with the specific antigen expressed by leukemic cells promoted specific cytokine secretion and proliferation, suggesting activation and selection of modified CIK cells upon encounter with cancer cells. Finally, preliminary insertion-site analysis by LAM-PCR confirmed the polyclonal profile of integrations in the genome of Sleeping Beauty system. These Results provide pre-clinical evidences of efficient transfection of CD123- and CD19- CARs using Sleeping Beauty-mediated gene transfer, specificity of action and improvements in Methods of expansion of cytotoxic effector T cells. The development of an adoptive cell therapy protocol based on a reproducible clinical-grade method of expansion and an innovative gene transfer process will be fundamental to envisage clinical protocols to control relapse in leukemic patients and to improve the range of applications of such novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

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.

Background. Cytokine induced killer cells are ex-vivo expanded cells with potent antitumoral activity. Cytokine induced killer cells infusion in acute myeloid leukemia patients relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses were observed. To improve their effector functions against acute myeloid leukemia, we genetically modified cytokine induced killer cells with chimeric receptors specific for the CD33 myeloid antigen. Design and Methods. SFG-retroviral vectors coding for anti-CD33-ζ and anti-CD33-CD28-OX40-ζ chimeric receptors were used to transduce cytokine induced killer cells. Transduced cells were in vitro characterized for their ability to lyse leukemic targets (4-hour-51Chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate (3H-thymidine-incorporation assay) and to secrete cytokines (Flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34+ hematopoietic progenitors was evaluated by analyzing the colony forming unit capacity after co-incubation. Results. Cytokine induced killer cells were efficiently transduced with the anti-CD33 chimeric receptors, maintaining their native phenotype and functions and acquiring potent cytotoxicity (up to 80% lysis after 4-hour incubation) against different acute myeloid leukemia targets, as also confirmed in long-term killing experiments. Moreover, introduction of the anti-CD33 chimeric receptors was accompanied by a prominent CD33-specific proliferative activity, with a release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of cytokine induced killer cells anti-leukemic activity. Importantly, even though anti-CD33 chimeric receptors-transduced cytokine induced killer cells showed toxicity against normal hematopoietic CD34+ progenitors, a residual clonogenic activity was preserved. Conclusions. Our results indicate that anti-CD33 chimeric receptors strongly enhance anti-leukemic cytokine induced killer functions, suggesting that cytokine induced killer cells transduced with these molecules might represent a promising optimized tool for acute myeloid leukemia immunotherapy.

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) can induce objective clinical responses in patients with malignant diseases. The option of providing a proliferative and survival advantage to adoptively transferred CTLs remains a challenge to improve their efficacy. Host lymphodepletion and administration of recombinant interleukin-2 (IL-2) are currently used to improve CTL survival and expansion after adoptive transfer, but these approaches are frequently associated with significant side effects and may increase proliferation of T regulatory cells. IL-7 is a crucial homeostatic cytokine that has been safely administered as a recombinant protein. However, while IL-7 induces robust expansion of naive and memory T lymphocytes, the lack of expression of the IL-7 receptor alpha chain (IL-7Ralpha) by CTLs precludes their response to this cytokine. We found that CTLs can be genetically modified to re-express IL-7Ralpha, and that this manipulation restores the response of these cells to IL-7 without apparent modification of their antigen specificity or dependency, and without changing their response to other common gamma (gammac) chain cytokines. This approach may allow selective expansion of CTLs without the unwanted effects associated with IL-2.

The antitumor effect of adoptively transferred tumor-specific cytotoxic T lymphocytes (CTLs) is impaired by the limited capacity of these cells to expand within the tumor microenvironment. Administration of interleukin 2 (IL-2) has been used to overcome this limitation, but the systemic toxicity and the expansion of unwanted cells, including regulatory T cells, limit the clinical value of this strategy. To discover whether transgenic expression of lymphokines by the CTLs themselves might overcome these limitations, we evaluated the effects of transgenic expression of IL-2 and IL-15 in our model of Epstein Barr Virus–specific CTLs (EBV-CTLs). We found that transgenic expression of IL-2 or IL-15 increased the expansion of EBV-CTLs both in vitro and in vivo in a severe combined immunodeficiency disease (SCID) mouse model and enhanced antitumor activity. Although the proliferation of these cytokine genes transduced CTLs remained strictly antigen dependent, clinical application of this approach likely requires the inclusion of a suicide gene to deal with the potential development of T-cell mutants with autonomous growth. We found that the incorporation of an inducible caspase-9 suicide gene allowed efficient elimination of transgenic CTLs after exposure to a chemical inducer of dimerization, thereby increasing the safety and feasibility of the approach.

The peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptors that function as transcription factors regulating the expression of genes involved in cellular differentiation, development, metabolism and also tumorigenesis. Three PPAR isotypes (α, β/δ and γ) have been identified, among which PPARβ/δ (PPARD) is the most difficult to functionally examine due to its tissue-specific diversity in cell fate determination, energy metabolism and housekeeping activities. PPARβ/δ acts both in a ligand-dependent and -independent manner. The specific type of regulation, activation or repression, is determined by many factors, among which the type of ligand, the presence/absence of PPARβ/δ-interacting corepressor or coactivator complexes and PPARβ/δ protein post-translational modifications play major roles. Recently, new global approaches to the study of nuclear receptors have made it possible to evaluate their molecular activity in a more systemic fashion, rather than deeply digging into a single pathway/function. This systemic approach is ideally suited for studying PPARβ/δ, due to its ubiquitous expression in various organs and its overlapping and tissue-specific transcriptomic signatures. The aim of the present review is to present in detail the diversity of PPARβ/δ function, focusing on the different information gained at the systemic level, and describing the global and unbiased approaches that combine a systems view with molecular understanding.

Abstract 2446 B-Chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of B-lymphocytes expressing CD19, CD20dim and aberrantly expressing the CD5 T-cell marker. Moreover, they over-express the B-cell activation marker CD23. Chimeric Antigen Receptors (CAR) are engineered molecules able to redirect T-cell killing/effector activity towards a selected target in a non MHC-restricted manner. First trials targeting B-CLL were based on both monoclonal antibodies and anti-CD19/anti-CD20.CAR-transduced T cells. However, this approach causes the elimination of normal B-lymphocytes and B-precursors with consequent impairment of humoral immunity. Selective CD23 expression on B-CLL cells renders this molecule an optimal target to design a specific CAR. We have generated a novel CD23-targeting CAR to redirect T cells against CD23+B-CLL. Transduced T cells were tested for cytotoxicity against different CD23+-targets, using a classic 51Chromium release assay, and for specific cytokine release, by multiplex flow cytomix assay. T cells from B-CLL patients were efficiently transduced with the anti-CD23.CAR (average expression 68%, n=10) and redirected specifically toward autologous blasts (average lysis 58%, n=5). On the contrary, anti-CD23 transduced T-cells did not displayed any relevant killing versus normal B cells (average lysis 13%, n=3), differently from anti-CD19.CAR redirected T-cells, which killed tumor and normal B cells in an indistinct manner. Moreover, anti-CD23.CAR redirected T lymphocytes derived from both healthy donors (HD) and B-CLL patients displayed a specific lytic activity against CD23+EBV-LCLs, even in presence of soluble CD23 enriched plasma without being inhibited (average lysis with no plasma 67%; average lysis with 25% of CD23 enriched plasma 79%; average lysis with 50% of CD23 enriched plasma 88%, n=3). We also demonstrated that the expression of the anti-CD23.CAR caused a significant increase in cytokine release from transduced in vitro activated T cells after a 48h stimulation with CD23+ targets. B-CLL derived CD23.CAR-expressing T cells (n=3) secreted 4-fold more INF-gamma, and 1445-fold more TNF-beta, compared to non transduced T cells. Interleukin-2 was also released (average release 2681 pg/mL, n=3) and sustained the antigen-dependent proliferation of CD23.CAR+T cells. To confirm in vivo the in vitro data, we tested NT and CD23.CAR transduced T cells in a recently published xenograft model of B-CLL (Ref biblio, primo nome, giornale, anno). This model is based on the intravenous or subcutaneous injection of the established human B-CLL cell line MEC1 into Rag2−/− gammac−/− mice, which lack not only B and T cells, but also natural killer (NK) cells, thus presenting a profound immunosuppressive environment leading to an high efficiency of both B-CLL and T-cell engraftment. Moreover, this model reproduces the systemic involvement of the disease and it closely resembles the aggressive form human B-CLL, representing an optimal experimental setting to test the efficacy of new therapeutic agents. In the first set of our experiments, 8 weeks-old male mice were subcutaneously injected with 10*106 MEC1 cells in the left flank. Then, mice bearing an established tumor were treated intravenously with a single dose of NT or engineered CD23.CAR T cells (2*106), without any addition of exogenous IL-2. Animals were monitored twice a week for weight and tumor growth (measuring three perpendicular diameters), and sacrificed when the mean tumor volume reached a dimension of 31000 mm3, before presenting clinical signs and symptoms. Compared with NT-treated mice, the infusion of CD23.CAR+T cells resulted in a significant delay in tumor growth, as measured by tumor volume diameter/day (CD23.CAR+ T cells vs NT T cells: p=0.04 at day 12; n=3). In conclusion, our results suggest that CD23.CAR-redirected T cells provide cytotoxic activity against CD23+ B-CLL cells in vitro and in vivo, while sparing normal B lymphocytes, as compared to other available CARs targeting pan-B-cell antigens, such as CD19 and CD20. These results are encouraging and demonstrate the feasibility of generating CD23.CAR+ T lymphocytes for adoptive T-cell therapy of patients with B-CLL. Disclosures: No relevant conflicts of interest to declare.

B-Chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of mature B-lymphocytes expressing CD19, CD20dim and aberrantly expressing the CD5 T-cell marker. Moreover, they over-express the B-cell activation marker CD23. Chimeric Antigen Receptors (CAR) are engineered molecules able to redirect T-cell killing/effector activity towards a selected target in a non MHC-restricted manner. First trials targeting B-CLL were based on both monoclonal antibodies and anti-CD19/anti-CD20 CAR-transduced T cells. However, this approach causes the elimination of normal B-lymphocytes and B-precursors with consequent impairment of humoral responses. Selective CD23 expression on B-CLL cells renders it an optimal target to design a specific CAR. A new CD23-targeting CAR to redirect T cells against CD23+ B-CLL has been generated. After transduction, modified T cells were tested for cytotoxicity against different CD23+-targets, using a classic chromium release assay and for specific cytokine release by multiplex flow cytomix assay. The anti-CD23 CAR was stably expressed by healthy donor-derived primary T cells after transduction (average expression,20%;range,10%–60%;n=10) and conferred them a strong cytotoxicity against CD23+ tumor cell lines: Epstein Barr Virus transformed lymphoblastoid cell line (EBV-LCL) (average lysis, 50%; range 15%–70%, at 40:1 Effector:Target (E:T) ratio; n=5); Bjab and Jeko cell lines transduced with human CD23 antigen (average lysis, 60%; range, 20%–75%, at 40:1 E:T ratio; n=3). On the contrary, anti-CD23 transduced T-cells displayed no relevant killing versus normal B cells (average lysis, 8%; range, 1%–15% at 40:1 E:T ratio; n=3), differently from anti-CD19 CAR redirected T-cells, which killed tumor and normal B cells in an indistinct manner. T cells from B-CLL patients were also efficiently transduced with the anti-CD23 CAR (average expression, 80%; range, 70%–90%; n=3) and redirected specifically toward autologous blasts (average lysis, 29%; range, 21%–35% at 20:1 E:T ratio; n=3), without being inhibited by soluble CD23-enriched autologous plasma. Moreover, we demonstrated that expression of the anti-CD23 CAR caused a significant increase in cytokine release from transduced in vitro activated T cells after 48h stimulation with irradiated EBV-LCL at 1:1 ratio, both in healthy donors (n=3) and B-CLL patients (n=2). Anti-CD23 CAR expressing T cells from healthy donors secreted 5.5-fold more INF-gamma (3079 pg/ml vs 561pg/mL, p=0.05) and 11-fold more TNF-alpha (187.17 pg/ml vs 16.53 pg/mL, p=0.05), 147-fold more IL-5 (147 pg/ml vs 0 pg/mL, p=0.05) and 13-fold more IL-8 (590 pg/ml vs 43.24pg/mL, p=0.05), compared to non transduced T cells (n=3). In line with these findings, T cells expressing anti-CD23 CAR from B-CLL donors secreted 8.8-fold more INF-gamma (2988 pg/ml vs 337pg/mL, p=0.05) and 17-fold more TNF-gamma (187.17 pg/ml vs 17.34 pg/mL, p=0.05); 25.8-fold more IL-5 (3483.14 pg/ml vs 134.785 pg/mL, p=0.05), 173-fold more IL-8 (2154 pg/ml vs 12.415 pg/mL, p=0.05), compared to non transduced T cells. Altogether these results suggest that for the potentiality to get selective and potent killing of tumor cells, while sparing normal B cells, and for the capability to induce the selective release of immunostimulatory cytokines, CD23-targeting through a specific CAR holds great promises for adoptive immunotherapy of B-CLL.

The PAX5/TEL chimeric gene was cloned from the translocation t(9;12)(q11;p13) in an ALL patient. Recent data indicate that the PAX5/TEL fusion defines the cytogenetic entity dic(9;12)(p13;p13), which accounts for about 1% of childhood ALL, almost exclusively B-progenitor ALL. PAX5/TEL is likely to be an aberrant transcription factor, resulting from joining the 5′ region of PAX5 (a transcription factor essential for B cell development) to the 3′ region of TEL/ETV6, containing the Ets-family DNA binding domain. We have cloned the FLAG-full length chimeric PAX5/TEL cDNA in the retroviral vector pMSCV-IRES-GFP (MigR1) to transduce target cells. We have demonstrated a specific nuclear localization of the chimeric protein in NIH3T3 by immunofluorescence analysis. Moreover, we observed a PAX5/TEL dependent decrease of the cellular growth rate in IL-3 dependent murine proB Ba/F3 cells. We further investigated the function of the PAX5/TEL chimeric protein as a potential oncoprotein in murine preBI cells, as a more physiological model. Murine PAX5 −/− preBI cells and wild type preBI cells were purified as B220+/c-KIT+ cells from mouse fetal liver and they were cultured on OP9 and DL1-OP9 stroma cells in presence of IL-7. The OP9 stroma supports B cell proliferation and survival; the DL1-OP9 stroma expresses Delta-like1, one of the Notch ligands, and it’s important to support T cell development. Both PAX5 −/− preBI cells and wild type preBI cells were transduced with the retroviral construct pMSCV-PAX5/TEL-IRES-GFP to analyze cell proliferation, differentiation and growth-dependence on IL-7. Wild type preBI cells expressing PAX5/TEL showed down modulation of CD19 when cultured on OP9 stroma in presence of IL-7; an inverse correlation was observed between the levels of expression of GFP and of CD19. The down modulation of CD19 can be involved in driving the preBI cell into differentiation block. A possible explanation of CD19 repression can rely on a potential competition between PAX5/TEL and endogenous PAX5 to bind PAX5 consensus region on DNA. On OP9 stroma, PAX5/TEL preBI cells are resistant to TGFbeta anti-proliferative and apoptotic effects, with a three-fold increased growth rate than control cells. Although the specific mechanism of PAX5/TEL disruption of TGFbeta signalling pathway remains to be investigated, we propose the TGFbeta resistance by PAX5/TEL as a way to evade the immunosurveillance. PAX5/TEL-preBI cells cultured on DL1-OP9 showed a different phenotype, with up-regulation of c-KIT and down-regulation of CD44. PAX5−/− preBI cells infected with PAX5TEL and grown on OP9 were CD19 negative even in the presence of PAX5TEL. On DL1-OP9 stroma, PAX5TEL cells were able to differentiate maintaining the developmental plasticity of PAX5 −/− preBI cells. These preliminary results indicate a role of PAX5/TEL as a transcription factor, potentially with a suppressor function, down regulating CD19 expression, thus suggesting a function on B cell differentiation. The chimera is able to interfere with TGFbeta pathway, inducing resistance and conferring an advantage in cell survival, evading the immunosurveillance. PAX5TEL do not replace PAX5 functions in PAX5−/− cells, it cannot activate PAX5 target genes as CD19, important for restoring B cell differentiation. Further analyeis are needed to better evaluate the role of PAX5/TEL protein, both in vivo and in vitro models.

Leukaemia immunotherapy represents a fascinating and promising field of translational research, particularly as an integrative approach of bone marrow transplantation. Adoptive immunotherapy by the use of donor-derived expanded leukaemia-specific T cells has showed some kind of clinical response, but the major advance is nowadays represented by gene manipulation of donor immune cells, so that they acquire strict specificity towards the tumour target and potent lytic activity, followed by significant proliferation, increased survival and possibly anti-tumour memory state. This is achieved by gene insertion of Chimeric T-cell Antigen Receptors (CARs), which are artificial molecules containing antibody-derived fragments (to bind the specific target), joined with potent signalling T-Cell Receptor (TCR)-derived domains that activate the manipulated cells. This review will discuss the main application of this approach particularly focusing on the paediatric setting, raising advantages and disadvantages and discussing relevant perspectives of use in the nearest future.