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Despite recent advances in immunotherapies targeting single tumor-associated antigens,patients with multiple myeloma eventually relapse. ISB 2001 is a CD3+ T cell engager (TCE) co-targeting BCMA and CD38 designed to improve cytotoxicity against multiple myeloma. Targeting of two tumor-associated antigens by a single TCE resulted in superior cytotoxic potency across a variable range of BCMA and CD38 tumor expression profiles mimicking natural tumor heterogeneity,improved resistance to competing soluble factors and exhibited superior cytotoxic potency on patient-derived samples and in mouse models. Despite the broad expression of CD38 across human tissues,ISB 2001 demonstrated a reduced T cell activation profile in the absence of tumor cells when compared to TCEs targeting CD38 only. To determine an optimal first-in-human dose for the ongoing clinical trial (NCT05862012),we developed an innovative quantitative systems pharmacology model leveraging preclinical data,using a minimum pharmacologically active dose approach,therefore reducing patient exposure to subefficacious doses of therapies. Perro and colleagues develop a CD3+ T cell engager co-targeting BCMA and CD38 to improve immunotherapy for multiple myeloma,demonstrate cytotoxicity in patient-derived samples and murine models and develop a quantitative systems pharmacology model. View Publication

IntroductionCD8+ T cells are vital in the immune control of cancer and a key player in cell-based cancer immunotherapy. Recent studies have shown that microbial short-chain fatty acids (SCFA) can promote both effector and memory phenotypes in CD8+ T cells and may thereby enhance protection against cancer.MethodsIn this study,we determined the effect of SCFA butyrate on mouse CD8+ T cell function in vitro and in vivo,using the OT-I model.ResultsButyrate co-culture with anti-CD3 + anti-CD28 activated T cells in vitro enhanced the frequency of effector CD8+ IFN-γ-producing cells,and the amount of cytokine produced per cell. Culture with butyrate also enhanced the activation,TCR expression,and levels of phosphorylated mTOR proteins within CD8+ T cells but reduced proliferation rate and increased apoptosis. Butyrate-treated activated cells conferred tumor protection after adoptive transfer. Butyrate-treated cells were present at higher frequencies within the tumor compared to non-butyrate treated cells,and expressed IFN-γ. When analyzed using high dimensional cytometry,the tumors of mice that received butyrate-treated cells were enriched in clusters displaying an effector memory phenotype with high expression of IL-15Rβ and T-bet.DiscussionOur findings show that butyrate promotes the effector activity of CD8+ T cells in culture,which can persist in vivo while also stimulating memory phenotypes. Consequently,butyrate treatment may have strong application in T cell-based immunotherapies to improve protective cell functions and patient outcomes. View Publication

OBJECTIVE: Multipotent mesenchymal stromal cells (MSCs) are endowed with multilineage differentiative potential and immunomodulatory properties. It is still a matter of debate whether donor MSCs have sustained engraftment potential in host bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to analyze the donor/recipient origin of MSCs in children receiving allogeneic either BM or cord blood (CB) transplantation. METHODS: Thirty-seven pediatric patients undergoing allo-HSCT for either a malignant or a nonmalignant disorder were enrolled in the study; 19 received CB and 18 BM transplantation. Results were compared with those obtained in 14 adults given BM transplantation for either malignant or nonmalignant disorders. MSCs were grown from BM aspirates obtained 1-17 and 2-192 months after allo-HSCT in pediatric and adult patients,respectively. MSC samples at the third-fourth passage were phenotypically characterized. Donor/recipient origin of MSCs was assessed by amelogenin assay and microsatellite analysis. RESULTS: MSCs could be grown from 30 of 37 children; at the third-fourth passage MSCs resulted positive (textgreater or = 98%) for CD73,CD105,CD106,CD29,CD13,CD44 and negative (textless or = 1%) for CD34,CD45,CD14. Mixed chimerism with donor cells was observed in 4 BM and 5 CB transplantation recipients,respectively; full recipient chimerism was detected in the remaining children. Full recipient MSC chimerism was observed also in all assessable (12/14) adult patients. CONCLUSIONS: BM of pediatric patients might be a more favorable milieu than that of adults for sustained engraftment of transplanted MSCs. MSCs able to engraft in the host can be transferred with cryopreserved CB units. View Publication

Histone deacetylase inhibitors (HdI) could potentially improve the differentiation of leukemic dendritic cells (DC). Therefore,bone marrow samples from 100 children with acute lymphoblastic leukemia (ALL) were cultured in the cytokines TNF-alpha,GM-CSF,c-kit ligand,and fetal liver tyrosine kinase 3 ligand,with or without IL-3 and -4 and after administration of HdI valproic acid (VAL),suberoylanilide hydroxamic acid (SAHA),isobutyramid,or trichostatin A. Among the tested samples,25 were positive for the chromosomal translocation t(12;21),encoding the fusion gene translocation ETS-like leukemia/acute myeloid leukemia 1 (TEL/AML1). SAHA increased CD83 expression of TEL/AML1-positive blasts in conditions without ILs,and SAHA and VAL increased the number of CD86(+)80(-) cells in the presence of ILs. VAL and isobutyramid supported the allostimulatory capacities of TEL/AML1-positive,leukemic DC; VAL and SAHA reduced those of TEL/AML1-negative DC. Cytotoxic T cells sensitized with leukemic DC produced more IFN-gamma and TNF-alpha upon presentation of the TEL/AML1 peptide. They also induced the cytotoxic lysis of nondifferentiated blasts,which was enhanced when TEL/AML1-positive DC had developed after addition of VAL or SAHA. Therefore,the use of HdI in the differentiation of leukemic DC from patients with TEL/AML1-positive ALL is recommended. View Publication

Protein tyrosine kinases of the Janus kinase (JAK) family are associated with many cytokine receptors,which,on ligand binding,regulate important cellular functions such as proliferation,survival,and differentiation. In multiple myeloma,JAKs may be persistently activated due to a constant stimulation by interleukin (IL)-6,which is produced in the bone marrow environment. INCB20 is a synthetic molecule that potently inhibits all members of the JAK family with a 100- to 1,000-fold selectivity for JAKs over textgreater70 other kinases. Treatment of multiple myeloma cell lines and patient tumor cells with INCB20 resulted in a significant and dose-dependent inhibition of spontaneous as well as IL-6-induced cell growth. Importantly,multiple myeloma cell growth was inhibited in the presence of bone marrow stromal cells. The IL-6 dependent cell line INA-6 was particularly sensitive to the drug (IC50textless1 micromol/L). Growth suppression of INA-6 correlated with an increase in the percentage of apoptotic cells and inhibition of signal transducer and activator of transcription 3 phosphorylation. INCB20 also abrogated the protective effect of IL-6 against dexamethasone by blocking phosphorylation of SHP-2 and AKT. In contrast,AKT phosphorylation induced by insulin-like growth factor-I remained unchanged,showing selectivity of the compound. In a s.c. severe combined immunodeficient mouse model with INA-6,INCB20 significantly delayed INA-6 tumor growth. Our studies show that disruption of JAKs and downstream signaling pathways may both inhibit multiple myeloma cell growth and survival and overcome cytokine-mediated drug resistance,thereby providing the preclinical rationale for the use of JAK inhibitors as a novel therapeutic approach in multiple myeloma. View Publication

Off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication. To identify such compounds,we conducted 2 independent cell-based chemical screens and identified the antimicrobial ciclopirox olamine (CPX) in both screens. CPX decreased cell growth and viability of malignant leukemia,myeloma,and solid tumor cell lines as well as primary AML patient samples at low-micromolar concentrations that appear pharmacologically achievable. Furthermore,oral CPX decreased tumor weight and volume in 3 mouse models of leukemia by up to 65% compared with control without evidence of weight loss or gross organ toxicity. In addition,oral CPX prevented the engraftment of primary AML cells in nonobese diabetic/severe combined immunodeficiency mouse models,thereby establishing its ability to target leukemia stem cells. Mechanistically,CPX bound intracellular iron,and this intracellular iron chelation was functionally important for its cytotoxicity. By electron paramagnetic resonance,CPX inhibited the iron-dependent enzyme ribonucleotide reductase at concentrations associated with cell death. Thus,in summary,CPX has previously unrecognized anticancer activity at concentrations that are pharmacologically achievable. Therefore,CPX could be rapidly repurposed for the treatment of malignancies,including leukemia and myeloma. View Publication

Aldehyde dehydrogenase 1 A1 (ALDH1A1) has recently been suggested as a marker for cancer stem or stem-like cancer cells of some human malignancies. The purpose of this study was to investigate the stem cell-related function and clinical significance of the ALDH1A1 in bladder urothelial cell carcinoma. Aldefluor assay was used to isolate ALDH1A1+ cells from bladder cancer cells. Stem cell characteristics of the ALDH1A1+ cells were then investigated by in vitro and in vivo approaches. Immunohistochemistry was done for evaluating ALDH1A1 expression on 22 normal bladder tissues and 216 bladder tumor specimens of different stage and grade. The ALDH1A1+ cancer cells displayed higher in vitro tumorigenicity compared with isogenic ALDH1A1- cells. The ALDH1A1+ cancer cells could generate xenograft tumors that resembled the histopathologic characteristics and heterogeneity of the parental cells. High ALDH1A1 expression was found in 26% (56 of 216) of human bladder tumor specimens and significantly related to advanced pathologic stage,high histologic grade,recurrence and progression,and metastasis of bladder urothelial cell carcinomas (all P textless 0.05). Furthermore,ALDH1A1 expression was inversely associated with cancer-specific and overall survivals of the patients (P = 0.027 and 0.030,respectively). Therefore,ALDH1A1+ cell population could be enriched in tumor-initiating cells. ALDH1A1 may serve as a useful marker for monitoring the progression of bladder tumor and identifying bladder cancer patients with poor prognosis who might benefit from adjuvant and effective treatments. View Publication

Almost 25 centuries ago,Hippocrates,the father of medicine,proclaimed Let food be thy medicine and medicine be thy food." Exploring the association between diet and health continues today. For example View Publication

Infection with human immunodeficiency virus (HIV)-1 induces a progressive deterioration of the immune system that ultimately leads to acquired immune deficiency syndrome (AIDS). Murine models indicate that the common γ-chain (γ(c))-sharing cytokine interleukin (IL)-21 and its receptor (IL-21R) play a crucial role in maintaining polyfunctional T cell responses during chronic viral infections. Therefore,we analyzed the ability of this cytokine to modulate the properties of human CD8 T cells in comparison with other γ(c)-sharing cytokines (IL-2,IL-7,and IL-15). CD8 T cells from healthy volunteers were stimulated in vitro via T cell receptor signals to mimic the heightened status of immune activation of HIV-infected patients. The administration of IL-21 upregulated cytotoxic effector function and the expression of the costimulatory molecule CD28. Notably,this outcome was not accompanied by increased cellular proliferation or activation. Moreover,IL-21 promoted antiviral activity while not inducing HIV-1 replication in vitro. Thus,IL-21 may be a favorable molecule for immunotherapy and a suitable vaccine adjuvant in HIV-infected individuals. View Publication

Use of animal feeder layers and serum containing media in the derivation and propagation of induced pluripotent stem cells (iPSCs) can hinder clinical translation,because of the presence of xeno-material/pathogens. A defined and standardized system would be ideal for generating a homogenous population of iPSCs,which closely resembles human embryonic stem cells (hESCs). This article presents a novel and extensive comparison between in-house produced iPSCs and hESCs under feeder" and "feeder-free" conditions View Publication

Xenotransplantation of acute myeloid leukemia (AML) into immunodeficient mice has been critical for understanding leukemogenesis in vivo and defining self-renewing leukemia-initiating cell subfractions (LICs). Although AML-engraftment capacity is considered an inherent property of LICs,substrains of NOD/SCID mice that possess additional deletions such as the IL2Rγc(null) (NSG) have been described as a more sensitive recipient to assay human LIC function. Using 23 AML-patient samples,39% demonstrated no detectable engraftment in NOD/SCID and were categorized as AMLs devoid of LICs. However,33% of AML patients lacking AML-LICs were capable of engrafting NSG recipients,but produced a monoclonal T-cell proliferative disorder similar to T-ALL. These grafts demonstrated self-renewal capacity as measured by in vivo serial passage and were restricted to CD34-positive fraction,and were defined as LICs. Molecular analysis for translocations in MLL genes indicated that these AML patient-derived LICs all expressed the MLL-AFX1 fusion product. Our results reveal that the in vivo human versus xenograft host microenvironment dictates the developmental capacity of human LICs residing in a small subset of patients diagnosed with AML harboring MLL mutations. These findings have implications both for the basic biology of CSC function,and for the use of in vivo models of the leukemogenic process in preclinical or diagnostic studies. View Publication