ALBERT

Description: Leukemia is a type of hematopoietic stem/progenitor cell malignancy characterized by the accumulation of immature cells in the blood and bone marrow. Treatment strategies mainly rely on the administration of chemotherapeutic agents, which, unfortunately, are known for their high toxicity and side effects. The concept of targeted therapy as magic bullet was introduced by Paul Erlich about 100 years ago, to inspire new therapies able to tackle the disadvantages of chemotherapeutic agents. Currently, nanoparticles are considered viable options in the treatment of different types of cancer, including leukemia. The main advantages associated with the use of these nanocarriers summarized as follows: i) they may be designed to target leukemic cells selectively; ii) they invariably enhance bioavailability and blood circulation half-life; iii) their mode of action is expected to reduce side effects. FDA approval of many nanocarriers for treatment of relapsed or refractory leukemia and the desired results extend their application in clinics. In the present review, different types of nanocarriers, their capability in targeting leukemic cells, and the latest preclinical and clinical data are discussed.

Description: In B-cell chronic lymphocytic leukemia (B-CLL), defective apoptosis causes the accumulation of mature CD5+ B cells in lymphoid organs, bone marrow (BM), and peripheral blood (PB). These cells are the progeny of a proliferating pool that feeds the accumulating compartment. The authors sought to determine which molecular mechanisms govern the proliferating pool, how they relate to apoptosis, and what the role is of the microenvironment. To begin to resolve these problems, the expression and modulation of the family of inhibitor of apoptosis proteins (IAPs) were investigated, with consideration given to the possibility that physiological stimuli, such as CD40 ligand (CD40L), available to B cells in the microenvironment, might modulate IAP expression. The in vitro data on mononuclear cells from PB or BM of 30 patients demonstrate that B-CLL cells on CD40 stimulation express Survivin and that Survivin is the only IAP whose expression is induced by CD40L. Through immunohistochemistry, in vivo Survivin expression in lymph node (LN) and BM biopsies was evaluated. In reactive LN, Survivin was detected only in highly proliferating germinal center cells. In LN from patients with B-CLL, Survivin was detected only in pseudofollicles. Pseudofollicle Survivin+ cells were actively proliferating and, in contrast to Survivin+ B cells found in normal GC, were Bcl-2+. In B-CLL BM biopsies, CD5+, Survivin+ cells were observed in clusters interspersed with T cells. These findings establish that Survivin controls the B-CLL proliferative pool interfacing apoptosis and that its expression may be modulated by microenvironmental stimuli.

Description: Abstract 4289 The aim of this study was to generate cytotoxic T-lymphocytes (CTL) clones directed against AML cells and to identify new immunogenic antigens with the following properties: i) to be overexpressed by leukemic cells and not by normal tissues; ii) to be shared among different AML subtypes; iii) to play a role in leukemic growth/survival; iv) to be expressed by leukemic stem cells. To this end, we loaded normal dendritic cells (DC) from a healthy donor with apoptotic bodies from primary AML cells and used loaded DC to stimulate autologous lymphocytes (i.e. lymphocytes from the donor). Donor was selected to be partially matched with the leukemic patient for MHC-class I, in that he shared two MHC-class I alleles at the HLA-supertype level (HLA-B7 and HLA-B44). With this strategy, a CD8+ T-cell line was generated that recognized both loaded DC and AML cells used for loading, but not DC loaded with normal cells derived from the patient (PHA-blasts). This CTL line was cloned by limiting dilutions and 180 clones were screened by IFN-g elispot against 2 different AML samples that were expressing both HLA-B7 and HLA-B44; one additional mismatched AML sample was used as negative control. Two clones were selected that recognized HLA-B7+/HLA-B44+ AML cells but not the negative control (namely, clone 31D3 and 8E12). To determine the HLA-restriction element and to verify shared antigen expression among AML subtypes, we tested both clones against a panel of 18 HLA-typed primary AML samples. We found that clone 31D3 was restricted by HLA-B7 and clone 8E12 by HLA-B44. Each clone recognized 5/5 HLA-matched AML samples of different subtype. In addition, the clones did not recognize both resting and activated normal myeloid, nor lymphoid and CD34+ cells expressing the proper HLA-restriction allele. In addition to elispot activity, both clones showed killing of AML cells in a CFSE-based cytotoxic assay. To confirm the lack of reactivity against normal tissues, we analyzed the activity of both clones against HLA matched or mismatched fibroblasts and we found that clone 8E12 displayed a low reactivity activity against normal matched fibroblasts, while clone 31D3 was not reactive. We then focused the analysis on clone 31D3 and tested whether its activity was restricted to leukemia or also directed against a panel of HLA-B7 positive (N=3) or negative (N=5) solid tumors. Interestingly, we found that the clone 31D3 could recognize one colon carcinoma and one melanoma cell line expressing the HLA-B7 supertype. In particular, the melanoma cell line (G4-mel) was HLA-B35+ (HLA-B35 belongs to the HLA-B7 supertype) and was recognized at very high levels. The availability of a tumor cell line expressing adequate amounts of antigen will make the generation of a cDNA library for antigen identification more feasible than with primary leukemic cells. Finally, to further confirm that HLA-B35 is the proper MHC restriction element and to determine to what extent the antigen is shared among different tumors, we transduced 8 tumor cell lines, 3 normal B-lymphoblastoid and 1 fibroblast cell line with the HLA-B35 allele. Clone 31D3 efficiently recognized 6/8 HLA-B35 transduced tumor cell lines (1 AML, 2 melanomas, 2 colon and 1 breast carcinoma) but none of the control cell lines. In conclusion, clone 31D3 is restricted by the HLA-B7 supertype, it recognizes an antigen that is expressed by leukemic cells and not by normal tissues and, most importantly, is shared among tumors of different histology. Since the vast majority of malignant cells tested (both primary cells and in vitro established cell lines) were targeted by this clone, the putative antigen might be a protein playing an important role in tumor growth or survival. We are now testing the activity of the clone against purified leukemic stem cells and attempting to identify this shared tumor antigen. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1051 Churg Strauss Syndrome (CSS) is a systemic small-vessel necrotizing vasculitis characterized by asthma, chronic rhinosinusitis, pulmonary infiltrations, eosinophil-rich inflammation, vascular and extra-vascular granulomas. We recently documented in the peripheral blood of CSS patients the presence of one or two numerically expanded V-Beta (V-B) families of CD8+ lymphocytes with an effector memory phenotype and with a monoclonal or oligoclonal T-cell receptor (TCR), as shown by TCR-γ rearrangement analysis. To define the characteristics of numerically expanded families at a molecular level, we purified the expanded V-B family of two CSS patients and cloned purified T-cells by limiting dilution. Ten clones from each patient were randomly selected for molecular analysis and the Complementarity-Determining Region 3 (CDR3) of their TCR alpha e beta chain was sequenced. In each patient, 9/10 clones had an identical CDR3 sequence. We also amplified by PCR the cDNA obtained from whole PBMC with specific primers for the expanded V-B family of the same patients (i.e. V-B1 and V-B8) and sequenced the PCR product. The sequence obtained from the entire V-B family (as detected by PCR amplification from whole PBMC) was identical to that obtained from each T-cell clone. These data indicate the presence of a dominant clonotype within a numerically expanded V-B family, which is detectable by molecular analysis not only of single cells but also of whole PBMC. To compare the TCR V-B diversity of CSS patients with healthy donors (HD), we performed TCR-CDR3 length analysis by spectratyping of purified PBMC, CD4 and CD8 cells in CSS patients and in age-matched HD. Analysis was performed by determining the complexity score of 22 V-B families in 8 patients and 8 HD. The TCR repertoire of CSS circulating T-cells was skewed (i.e. with a significantly lower complexity score than HD) and this skewing was found exclusively within the CD8+ subset. Therefore, the CD8+ repertoire of CSS patients is composed by one or two TCR V-B families that are numerically expanded and by several other V-B families that have a skewed spectrum, but are not numerically expanded. In one patient, we also analyzed the cDNA of 11 different V-B families with a low complexity score on a polyacrylamide gel. Five discrete bands were detected, that were purified and sequenced. Sequences from 2/5 PCR-amplified V-B families were readable and representative of a productive and clonal VDJ rearrangement of the CDR3 region. This confirmed that a skewed V-B family at spectratyping is very often the result of an antigen-driven proliferation of a single cell with a distinct TCR. These molecular data revealed that alterations of the T-cell repertoire in CSS patients are more profound than initially expected, involving many CD8+ V-B families. We hypothesized that functional analysis of the whole CD8+ fraction could reveal the main functional characteristics of the several clonal expansions observed within this subset. We therefore analyzed cytokines and chemokine receptor expression of CD8+ and CD4+ cells from the same CSS patients and HD. A significantly higher percentage of both CD8+ and CD4+ cells producing INFγ and TNFα was observed in CCS compared to HS. IL1β, IL2, IL4, IL6, IL13, IL17, GM-CSF, and TGF-β in CD4+ and CD8+ cells were not different between CSS patients and controls, even if a trend toward a higher expression of IL-13 was observed. CD4+ cells, but not CD8+ cells, from CSS patients had a significantly higher production of IL5 and IL10 than HS. These data suggested that patients' CD8+ cells have a Th1/proinflammatory profile, while the functional profile of CD4+ T-cells is less clearly polarized. Expression of CCR5 and, to a lesser extent, of CXCR3 by total and Vβ-expanded CD8+ cells was significantly higher in CSS than in HS. Expression of CRTH2, but not of CCR4, was significantly higher in CD4+ cells of CSS patients than in controls. In conclusion, the molecular profiling of T-cells revealed that monoclonal expansion of CD8+ lymphocytes in CSS patients are frequent and involve several V-B families. CD8+ cells of patients have a Th1/proinflammatory profile, potentially involved in vasculitic damage with the cooperation of CD4+/Th1 cells. CD4+/Th2 cells (but not CD8+ cells) are functionally apt to mediate eosinophil-rich inflammation and asthma. Disclosures: No relevant conflicts of interest to declare.

Description: In acute myeloid leukemia (AML), blasts lose their ability to differentiate into mature cells and to undergo apoptosis. Accordingly, a proapoptotic and differentiating therapy (arsenic and retinoic acid) has dramatically improved survival in acute promyelocytic leukemia; however, a similar combination therapy is not available for other AML subtypes. In 2016, inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme of the pyrimidine biosynthesis, was found to induce differentiation in several AML models; for in vivo studies, brequinar (BRQ) was utilized. Starting from BRQ and applying a scaffold-hopping replacement, we have recently developed a new DHODH inhibitor, Meds433, which could induce differentiation at a 1-log lower concentration compared to BRQ (Sainas, J Med Chem 2018). Here we characterize Meds433 with in vitro and in vivo experiments, showing that it has a significant pro-apoptotic effect in several AML cell lines, which is at least partially independent from the differentiating effect. Analyzing the kinetic of differentiation induced by Meds433 on U937 and THP1 cell lines, and comparing the data with the number of viable cells, we noticed that cells started to die before the differentiation effect could be significant. Hence, we decided to investigate the proapoptotic effect of Meds433, treating several AML (U937, THP1, OCI-AML3, NB4) and non-AML (CEM, P3j, peripheral blood mononuclear cells-PBMC) cell lines with Meds433, and analyzing the expression of Annexin V and propidium in flow cytometry. Experiments demonstrated that Meds433 had a significant pro-apoptotic effect on several AML cell lines (Fig.1), but not on non-AML cell lines. The apoptotic rate increased with the time of exposure (3 vs 6 days), allowing to obtain a good apoptotic rate also in OCI-AML3, the most resistant cell line in our hands. As for the differentiation experiments, Meds433 could induce apoptosis at a 1-log inferior concentration compared to BRQ. More interestingly, in NB4 cells, a strong apoptotic effect was not associated with any differentiating feature, indicating that DHODH inhibition can induce apoptosis directly. As NB4 is a promyelocytic cell line, we also compared the effects of ATRA (all-trans retinoic acid), Meds433 and their combination. ATRA alone was found to induce strong differentiation and mild apoptosis on NB4 cells, while Meds433 alone could induce exclusively apoptosis; finally, the combination of ATRA and Meds433 increased both the rate of differentiating and apoptotic cells compared to ATRA only. We next tried to further characterize the apoptotic effect. When experiments were performed in the presence of uridine, a downstream product of DHODH in the pyrimidine biosynthesis, the apoptotic effect was totally abrogated. This phenomenon was already observed in the differentiation experiments (Sainas, J Med Chem 2018), and it confirms that both differentiation and apoptosis are indeed caused by the pyrimidine depletion rather than off-target mechanisms. Moreover, when experiments were performed in hypoxic conditions, the rate of apoptosis did not change, suggesting that Meds433 could work in the bone marrow hypoxic niche of (leukemic) stem cells. Further analyzing the effect of Meds433 on non-AML cells, we evaluated the maturation of T-lymphocytes, from naïve to TEMRA (T effector memory RA), finding no influence at all. Finally, preliminary results from in vivo experiments show that i) Meds433 is not toxic on Balb/c mice after 5 weeks of intraperitoneal administration; ii) the half-life is limited to 4-6 hours and iii) Meds433 has a good antileukemic activity (approximately 50% reduction of the tumor volume compared with control, after an 18-day treatment of THP1-xenograft in NSG mice). In conclusion, our work demonstrates that: i) DHODH inhibition can induce apoptosis in AML cells both directly and as a result of differentiation, partially depending on the cell line; ii) Meds433 is a novel, promising antileukemic drug, with limited toxicity, which could be active on a wide variety of AML. Since continuous exposure to the drug is fundamental in the pyrimidine starvation strategy, we are currently optimizing Meds433 pharmacokinetic profile in order to maximize the in vivo antileukemic activity. Disclosures Saglio: BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy.

Description: Growth and survival of chronic B-cell tumors are favored by the malignant cell's capacity to respond to selected microenvironmental stimuli provided by nontumoral bystander cells. To investigate which mechanisms operate in these crosstalks and whether they are malignancy-related or reproduce the mechanisms used by normal B cells we have studied the expression and functional role of semaphorin CD100 (now called Sema4D) in chronic lymphocytic leukemia (CLL) cells and normal CD5+ B cells. We demonstrate here that (1) leukemic and normal CD5+ B lymphocytes uniformly express CD100; (2) the CD100 high-affinity receptor Plexin-B1 is expressed by bone marrow stromal cells, follicular dendritic cells, and activated T lymphocytes, and is thus available to CD100+ lymphocytes in different specific microenvironments; and (3) upon interaction between CD100 and Plexin-B1 both CLL and normal CD5+ B cells increase their proliferative activity and extend their life span. These findings establish that Plexin-B1 is an easily accessible receptor for CD100 within the immune system. The encounter of CD100+ leukemic cells with Plexin-B1 may promote the proliferation and survival of malignant cells. The crosstalk operated by the CD100/Plexin-B1 interaction is not malignancy related but reproduces a mechanism used by normal CD5+ B cells.

Description: Abstract 1917 Cytokine Induced Killer (CIK) cells are a heterogeneous population of T lymphocytes sharing NK phenotype and functional properties: they are CD3+/CD56+ and have a potent MHC-unrestricted antitumor activity. We hypothesized that the therapeutic potential of CIK cells might be increased if they acquired the ability to recognize MHC-restricted tumor associated antigens. To this end, we transduced CIK cells with an HLA-A2 restricted T-Cell Receptor (TCR) directed against the melanocyte associated antigen Mart-1. PBMC were incubated with IFN-γ on day 0 and supplemented with anti-CD3 and IL-2 on day +1 to generate CIK cells. Cultures were transduced at day 4 with concentrated lentiviral particles and successfully expanded over a 4 week period. This allowed to generate CIK cells that contained 11±9% Mart-1 TCR positive cells, as detected by staining with a Mart-1 specific tetramer. Transduced CIK cultures contained 61±19% CD3+/CD56+ cells. Tetramer positive cells were both CD3+/CD56- and CD3+/CD56+ (31±8% and 59±9%, respectively), indicating that both MHC-restricted T-cells and MHC-unrestricted CIK cells could be targeted by lentiviral transduction. TCR-transduced CIK cells specifically recognized tumor cells presenting the relevant peptide and maintained their MHC-unrestricted tumor activity at the same time. The cytotoxic activity of Mart-1 redirected CIK against HLA-A2+ melanoma cell lines was 2.8 fold higher than the untransduced counterparts (62%±9 vs 22±6% lysis at an effector/target ratio of 20:1), while the cytotoxic activity against a Mart-1+/HLA-A2- melanoma cell line was similar in transduced and untransduced CIK cells (24%±8 vs 22±6% lysis), indicating that the increased activity was due to HLA-restricted recognition. This was confirmed by blocking experiments with an HLA-Class I antibody. At the end of the culture, the majority of both unmodified and transduced CIK cells expressed an effector memory phenotype, with few residual central memory cells. In TCR redirected cells there was a slight increase of cells with a naive phenotype compared to unmodified cells (19±5% vs 9±4%). These data suggest that the naive and central memory pool of redirected CIK cells might efficiently expand in vivo and support a long lived memory response, whereas the terminally differentiated pool might mediate short lived but potent MHC-restricted and unrestricted activity. To demonstrate that TCR transduced CIK cells display an increased antitumor activity also in vivo, we have conducted preparative experiments in humanized immunocompromised mice (NOD/scid/γ(c)(−/−), NSG). Results obtained so far have shown that: i) When 5×106 or 10×106 CIK cells (both TCR-transduced and unmodified) were injected intravenously, they stably engrafted NSG mice, homing predominantly to spleen and liver and also, to a lesser extent, to bone marrow and kidney (36±9%, 39±12%, 4±3%, 1.6±3% of human CD3+/CD45+ cells at 3 months in the spleen, liver, bone marrow and kidney, respectively); circulating cells were also detected in the peripheral blood. Engrafted CIK cells maintained high expression of CD8 but progressively downregulated and finally lost CD56 expression. When 10×106 CFSE-marked CIK cells where injected intravenously, they similarly engrafted and proliferated in NSG mice, reaching a peak of proliferation 2–3 weeks after injection; at 4 weeks, the CFSE dye was already completely diluted out. ii) Differently from normal PBMC, CIK cells did not induce any appreciable clinical sign of acute or chronic Graft versus Host Disease (GVHD), as determined by the general appearance of the fur, mobility and weight loss. Mice were observed up to 5 months, and both unmodified and TCR transduced CIK cells displayed the same behavior. iii) NSG accepted the graft of as few as 50.000 matrigel-resuspended melanoma cells that were injected subcutaneously, with the appearance of a measurable mass (〉3mm) ten days after inoculation. From these in vivo experiments we conclude that: i) human CIK cells engraft and proliferate in NSG mice; ii) CIK cells do not cause GVHD; iii) the human melanoma cell lines used in vitro can grow in NSG mice. We are now testing whether TCR transduced CIK cells have superior antitumor activity than unmodified CIK in the NSG mice model. Taken together, our data suggest that TCR transfer into CIK cells is feasible and greatly improves their antitumor potential in vitro and possibly in vivo. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Endogenous intronic long terminal repeats promote the ectopic expression of truncated ERBB4 transcripts in 24% of ALK-negative ALCL. The expression of ERBB4-aberrant transcripts defines a new subclass of ALK-negative ALCL and may contribute to ALCL transformation.

Description: Adoptive immunotherapy strategies enrolling T regulatory cells (Tregs) might have a great potential in modulating donor T cells alloreactivity after Hematopoetic Cell Transplant (HCT). In murine models of HCT Tregs were shown to promote engraftment and contribute controlling graft versus host disease (GVHD) while still not conclusive data are available on humans. Ex-vivo engineering conventional CD4+ T cells to over-express the transcription factor FOXP3 is an intriguing approach to overcome the main difficulty of obtaining large amount of Tregs for experimental studies. Reports of retrovirus-mediated expression of FOXP3 not consistently resulted in functional Tregs while, recently, a lentivirus-mediated strategy was successfully reported to result in homogeneous and stable expression of FOXP3. Lentiviral transduced Tregs were able to suppress a polyclonal proliferation of CD4 purified lymphocytes stimulated with soluble Ab anti-CD3. In our study we generated lentiviral engineered Tregs (eng-Tregs) and investigated their inhibitory effect on unselected lymphocytes alloreactivity across major HLA barriers. Within the bulk lymphocytes population we separately tracked the suppressive influence of eng-Tregs on both CD4+ and CD8+ T cells. To obtain eng-Tregs, CD4+ T cells were purified from healthy donors and transduced with a bidirectional lentiviral vector encoding for FOXP3 and the truncated Nerve Growth Factor Receptor (ΔNGFR). Prior to transduction CD4+ cells were activated for 72 hours with IL2 (100U/ml), IL7 (20ng/ml) and soluble Ab anti-CD3 (200 ng/ml, only IL2 was added to the culture medium after transduction. The lentiviral transduction efficiency ranged from 8 to 25%, ΔNGFR+ T cells were positively selected and tested for their ability to suppress a mixed lymphocyte reaction across major HLA barriers. Effector peripheral blood mononuclear cells (PBMC) were collected from the same donors from whom eng-Tregs were generated. Effector PBMC were stained with CFSE in oder to separately track the alloreactive proliferation of both CD8+ and CD4+ subsets of T cells. Eng-Tregs were added on day 0 and HLA-mismatched irradiated PBMC were used as stimulators; both eng-Tregs and irradiated stimulators were used in a 1:1 ratio with the effectors. No cytokines or additional soluble stimulators were added to the MLR culture medium. The alloreactive proliferation of T cell subsets was determined by evaluating the logarithmic decrease of CFSE fluorescence intensity. The flow cytometry analysis on day +7 showed that alloreactive proliferation of both CD4+ and CD8+ effector cells was significantly inhibited (〉75%) by the addition of eng-Tregs compared to controls. In order to rule out a possible role played by the naturally present Tregs (nat-Tregs), the effectors were depleted of the CD4+CD25high subpopulation before the MLR started. The observed alloreactive proliferation was higher after the depletion of nat-Tregs but still it could be significantly inhibited by the addition of eng-Tregs. Eng-Tregs did not significantly expanded when cultured in vitro (up to 2 weeks) with IL2 (100U/ml) but maintained a stable expression of the transgene and retained their suppressive capacity. Our data show that lentiviral engineered Tregs can efficiently down-modulate both CD4+ and CD8+ T cell alloreactivity across major HLA barriers. The observed independence from the presence of nat-Tregs might be important in future experimental HCT settings where the adoptive infusion of eng-Tregs might encounter a great variability in the number and activity of recipient’s nat-Tregs. The possibility of transducing a potentially unlimited number of CD4+ cells makes this strategy appealing for future pre-clinical studies to control GVHD in HCT settings.

Description: The introduction of different generations of tyrosine kinase inhibitors (TKIs) significantly improved outcome and survival rate in chronic myeloid leukemia (CML) patients. However, long-term use of TKIs is concomitant with many side effects that affect the quality of life in patients. Approximately half of CML patients achieve deep molecular response (DMR), this makes them suitable candidates to discontinue the TKI therapy in a controlled condition, and about half of them will remain in treatment free remission (TFR) after discontinuation. It has been shown that a small population of leukemia stem cells (LSCs) as the residual disease burden is present at diagnosis, during the treatment, and in patients who are in TFR. While CML LSCs have many features in common with HSCs, they express specific markers such as CD25, CD26, IL1-RAP, etc., which can be used for the diagnosis and targeting. Protection by the bone marrow microenvironment and activity of signaling pathways such as WNT/β catenin, Hedgehog, PI3K, JAK/STAT in CML LSCs in a BCR-ABL dependent and independent manner guarantee their survival and elimination of these cells solely using TKIs seems ineffective. Herein we designed a pegylated liposomal nanocarrier conjugated with a specific antibody against CD26 (Begelomab, ADIENNE, Lugano, Switzerland). Then we loaded this immunoliposome with venetoclax, a BCL2 inhibitor, to eliminate CML LSCs selectively and to spare normal HSCs. First, we measured the expression of CD26 in the bone marrow and peripheral blood samples of newly diagnosed patients. We had a high expression of CD26 in CD34+/CD38- of both PB and BM, and a low expression on CD34+/CD38+ (progenitors) cells. Also, the expression of this marker in resistant patients to TKIs was visible while it was absent in normal stem cells. After the synthesis of the liposome, we conjugated Begelomab to the liposome. Then, we tested the selectivity of the designed system in different positive and negative cells. Our designed immunoliposome showed a strong selectivity toward CD26 positive cells. We also tested the selectivity on CML primary cells; in particular, we sorted newly diagnosed CML samples based on CD34+/CD38-/CD26- for HSCs and CD34+/CD38-/CD26+ for LSCs. Based on the confocal and flow cytometry analysis, our designed immunoliposome selectively targets LSCs and spares HSCs. Then we loaded this immunoliposome with venetoclax, and we treated CD26 positive and negative cells with this system. Based on our preliminary results, this immunoliposome loaded venetoclax specifically induced apoptosis in CD26+ cells, with higher activity compared to free venetoclax at the same dose. However, more analysis will be performed to confirm the selectivity of this system. Based on the obtained results, CD26 in newly diagnosed CML patients is expressed by CML LSCs and is a suitable option for diagnosis and targeting. Our preliminary data strongly suggest that we can selectively target CML LSCs. The main advantage of this system is its precision to hit the target. So we expect that after the drug release, the LSCs will be eliminated without any side effects on normal cells. Liposomes are suitable carriers because of their biocompatibility, self-assembly, large drug payload, and minimal toxicity. This strategy may help us to increase the number of patients attaining and maintaining TFR without relapsing. Disclosures Saglio: Ariad: Research Funding; Pfizer: Research Funding; Incyte: Research Funding; Roche: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Research Funding.