Adoptive transfer of cytolytic natural killer (NK) cells is limited by the short-term persistence of NK cells and their impaired effector function after infusion. We have previously shown that ...ex-vivo expansion of NK cells with nicotinamide (NAM) resulted in potent cytotoxicity against multiple tumor cell lines, and robust homing, proliferation and retention in vivo (Blood 2017 130:657). We now report the first-in-human experience of NAM-NK in patients with relapsed/refractory non-Hodgkin lymphoma (NHL) and multiple myeloma (MM).
Following donor apheresis, CD3-depleted mononuclear cells were cultured for 2 weeks in the presence of NAM (5mM) and IL-15 (20ng/ml). Patients received lymphodepleting chemotherapy followed by two doses of NAM-NK (Days 0 and 2) and low-dose IL-2. Rituximab or elotuzumab was administered to patients with NHL or MM, respectively, to facilitate tumor targeting and antibody-dependent cellular cytotoxicity.
As of Oct 2018, 9 patients were enrolled. Of 7 evaluable patients, 5 had refractory NHL (3 follicular and 2 diffuse large cell lymphoma) and 2 had MM. Final NAM-NK product (n=7) contained a median of 98% NK cells. In vitro culture with NAM and IL15 resulted in a 3.8-fold increase in TNC and 40-fold increase in NK cells after 14-16 day culture; expression of homing receptor CD62L increased from 2.9% in apheresis to 21% in final product. CD3 content was kept <0.5% (<5 × 105/kg/dose). NAM-NK was infused in 3 patients at the initial dose (2 × 107/kg) without dose-limiting toxicity (DLT). Transient neutropenia was observed in all 3 with neutropenic fever in 1 patient. The 2nd dose level of NAM-NK (1 × 108/kg) was well-tolerated in 4 patients without DLT or grade 3 or 4 adverse events; no cytokine release syndrome or neurotoxicity were observed. Response assessment at 2 months showed that 3 patients achieved complete metabolic remission (follicular lymphoma 2 patients (Figure 1b), transformed lymphoma 1 patient). One patient with MM had stable disease at 2 months and the second with MM experienced progressive disease. Flow cytometry analysis of peripheral blood showed proliferation of NAM-NK in blood between days 2 and 7 in all tested patients (range 2-55% donor NK cells; Figure 1a). Compared to host NK cells, donor NAM-NK cells in blood demonstrated higher CD16 expression (median 68% vs 82%) and enhanced proliferation (median Ki67 81% vs 99%).
NAM-NK cells have been safely administered, were well tolerated and proliferated and persisted in vivo. Promising early evidence of clinical activity was observed in patients with advanced disease. Dose escalation will be followed by an expansion cohort at the MTD.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
To examine whether ex-vivo expanded human bone marrow (BM)-derived AC133+ cells may participate in post myocardial infarction (MI) healing, we have examined the effect of the copper chelator ...tetraethylenepentamine (TEPA) on the ex-vivo expansion potential of BM-derived stem cell populations and the effect of the resulting ex-vivo expanded AC133+ cells in a MI animal model. AC133+ cells isolated from human BM, using the CliniMACS device, at purities greater than 90% were expanded in Teflon bags, in the presence of IL-6, TPO, Flt-3 ligand, and SCF with or without TEPA for three weeks. The progenitor cell composition and potential were examined at the end of the treatment time and after long-term incubation in culture. After 3 weeks in TEPA-treated cultures the total nuclear cell expanded more than 200±20 fold. The increase in the CD34+, AC133+ and AC133+/CD38-cell populations was 17±13, 16±1 and 270±110 - fold, respectively, and the CFU content was 84±28 fold higher than at the initiation of the cultures. Contrary to TEPA treated cultures, the cultures treated in the absence of the chelator lost their progenitor populations by week 5-7 in culture. Ex-vivo expanded AC133+ cells for 3 weeks expressed VEGF and VEGF receptor RNA as examined by RT-PCR.
An MI model was established in athymic nude rats by permanent ligation of the left anterior descending coronary artery. Ex-vivo expanded AC133+ cells (6x106 cells/rat) or saline (control) were injected at the scar tissue 6 days post MI. Four weeks after cell therapy, the hearts were harvested and examined. Staining for smooth-muscle alpha-actin detected a 1.6-fold increase in capillary and arteriole density in the expanded cell-treated vs. control hearts. Preliminary echocardiographic studies compared 4 weeks post-treatment with those observed prior to treatment. Expanded BM-derived AC133+ injection into the infarcted myocardium improved left ventricular (LV) remodeling as demonstrated by increasing LV systolic dimensions only by 11%±4 while increasing by 53%±17 in control animals (p=0.02). Similarly, AC133+ cell injection improved LV contractility as demonstrated by increasing fractional shortening (FS) by 58%±44 whereas FS decreased by 20%± 5 in control animals (p=0.14). In addition, AC133+ cell injection prevented scar thinning as demonstrated by a decrease in anterior wall thickness of 10%±4 whereas the decrease in anterior wall thickness was 30%±7.5 in control saline-treated animals (p<0.05). Clinical trials examining the safety and feasibility of intra-corronary injection of ex-vivo expanded autologous BM-derived AC133+ to patients with ischemic heart disease, are currently in preparation.
Conclusion: A. TEPA allows BM-derived AC133+ cells to self renew by permitting proliferation ex-vivo while hindering their differentiation. B. Our preliminary results suggest that injection of ex-vivo expanded BM-derived AC133+ cells into infarcted myocardium results in new vessel formation and improves left ventricular function.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
This study aimed to assess the effect of the M20 interleukin-1 (IL-1) inhibitor on normal and leukemic hematopoietic cells. The M20-derived IL-1 inhibitor was found to inhibit the growth of various ...hematopoietic cells. The in vitro proliferation of myeloid cell lines in serum-containing medium or proliferation of these cells induced by IL-1 in serum-free medium (measured by 3H-TdR) were inhibited by the M20 IL-1 inhibitor. In addition, growth of normal progenitors and fresh leukemic cells stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) (as measured by colony and liquid systems) was also inhibited by this factor. After the removal of the IL-1 inhibitor at the peak of growth inhibition, leukemic and normal progenitor cells retain their ability to grow and develop into GM-CSF colonies. These results show that the growth inhibition phenomena were reversible and did not result from a cytotoxic effect. Our data suggest that the M20-derived IL-1 inhibitor might function as a true negative growth regulator of normal and leukemic hematopoietic cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The effect of an inhibitor of IL-1, purified from a human myelomonocytic cell line (M20) on the development of human erythroid cell development was studied. The inhibitor, is a protein of 52 kD ...molecular weight that is distinct immunologically and functionally from other reported IL-1 inhibitors. The experiments were performed in a two-phase culture system that allows separation of the erythroid cell development into an erythropoietin (EPO)-independent phase, where early erythroid-committed BFUe proliferate and differentiate into the more mature progenitors, CFUe, and EPO-dependent phase, where CFUe further proliferate and mature into hemoglobin-containing orthochromatic normoblasts. The results indicated that in both developmental stages the M20-derived inhibitor reversibly blocked cell proliferation without interfering with cell differentiation.
More than 80% of cells from a human promyelocytic leukemic cell line (HL-60) possess the capacity for self-renewal as evidenced by their ability to form large primary colonies in semisolid medium and ...the presence within these colonies of cells capable of subsequent colony formation. Colony development is independent of the normal regulator--the myeloid colony stimulating factor. The observed autostimulation suggests the production of specific growth promoters by the cells. Differentiation either to mature granulocytes or macrophages, induced by various agents, was associated with reduced cloning potential. Nevertheless, colonies containing differentiated cells could be developed either by cloning cells in the presence of suboptimal concentrations of inducer or by adding inducers over colonies developed in its absence. Upon differentiation, there was a morphological change from compact to diffused colony morphology due to cell mobility in the semisolid medium. Even at suboptimal concentrations of inducer more than 95% of the colonies became diffused, indicating clonal homogeneity of the population with respect to differentiation capacity. The loss of self-renewal was found to be one of the early properties which changed following the initiation of differentiation. The loss preceded not only the overt expression of maturation-specific functions but also cellular commitment to terminal differentiation; shorter contact with the inducer was required to cause loss of self-renewal than to induce an irreversible transition to differentiation. This resulted in cells that lost their self-renewal potential without being able to complete their program of differentiation.
The relationship between the effects of two types of inducers on the maturation of a line of human promyelocytic cells (HL-60) was studied. The dual potentiality of these promyelocytes was ...demonstrated by the ability of isolated colonies to differentiate into granulocytes, following induction by dimethylsulphoxide (DMSO) or express properties specific to macrophages following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). The differentiation process involved an irreversible step which occurred 48 h after exposure to DMSO, and a few h after exposure to TPA. This implies that the presence of the inducer in the culture medium is no more required for completion of the differentiation programme. Removal of the inducer prior to this step resulted in reversal of all the inducer-mediated effects. During the period of non-commitment the specific pathway of maturation was still undetermined; cells in which differentiation was initiated by exposure to DMSO were able to develop into macrophages after substitution of DMSO by TPA. Moreover, pre-exposure to DMSO and other inducers of granulocyte differentiation resulted in higher sensitivity to TPA, as indicated by the cell response to low TPA concentrations and more rapid expression of macrophage specific properties. These results suggest that the early stages in HL-60 differentiation are probably common to the granulocyte and the macrophage pathways.
The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Regulator-, and ...vacuolar adenosine triphosphatase–dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Regulator in an amino acid–sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Regulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.
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