krn-7000 and Multiple-Myeloma

Multiple myeloma is a hematologic malignancy characterized by growth of malignant plasma cells in the bone marrow. Tumor cells in myeloma express CD1d and are sensitive to lysis by CD1d restricted natural killer T (NKT) cells. Here we discuss recent studies to harness the properties of these cells in the context of human myeloma. In spite of large body of preclinical data, attempts to fully harness the properties of these cells in the clinic are in early stages. Early phase clinical studies document the capacity of human monocyte-derived dendritic cells to expand NKT cells in vivo in myeloma patients. These results have set the stage for ongoing studies combining NKT activation with immune-modulatory drugs. Lessons learnt from these studies may inform the optimal application of human NKT based therapies in other settings as well.

Topics: Adjuvants, Immunologic; Antigens, CD1d; Dendritic Cells; Galactosylceramides; Humans; Immunotherapy, Adoptive; Lymphocyte Activation; Multiple Myeloma; Natural Killer T-Cells; T-Lymphocyte Subsets

Natural killer T (iNKT) cells can help mediate immune surveillance against tumors in mice. Prior studies targeting human iNKT cells were limited to therapy of advanced cancer and led to only modest activation of innate immunity. Clinical myeloma is preceded by an asymptomatic precursor phase. Lenalidomide was shown to mediate antigen-specific costimulation of human iNKT cells. We treated 6 patients with asymptomatic myeloma with 3 cycles of combination of α-galactosylceramide-loaded monocyte-derived dendritic cells and low-dose lenalidomide. Therapy was well tolerated and led to reduction in tumor-associated monoclonal immunoglobulin in 3 of 4 patients with measurable disease. Combination therapy led to activation-induced decline in measurable iNKT cells and activation of NK cells with an increase in NKG2D and CD56 expression. Treatment also led to activation of monocytes with an increase in CD16 expression. Each cycle of therapy was associated with induction of eosinophilia as well as an increase in serum soluble IL2 receptor. Clinical responses correlated with pre-existing or treatment-induced antitumor T-cell immunity. These data demonstrate synergistic activation of several innate immune cells by this combination and the capacity to mediate tumor regression. Combination therapies targeting iNKT cells may be of benefit toward prevention of cancer in humans.

Topics: Aged; Antineoplastic Agents; Combined Modality Therapy; Dendritic Cells; Drug Synergism; Female; Galactosylceramides; Humans; Killer Cells, Natural; Lenalidomide; Lymphocyte Activation; Male; Middle Aged; Monocytes; Multiple Myeloma; Thalidomide; Treatment Outcome

Due to the CD1d restricted recognition of altered glycolipids, Vα24-invariant natural killer T (iNKT) cells are excellent tools for cancer immunotherapy with a significantly reduced risk for graft-versus-host disease when applied as off-the shelf-therapeutics across Human Leukocyte Antigen (HLA) barriers. To maximally harness their therapeutic potential for multiple myeloma (MM) treatment, we here armed iNKT cells with chimeric antigen receptors (CAR) directed against the MM-associated antigen CD38 and the plasma cell specific B cell maturation antigen (BCMA). We demonstrate that both CD38- and BCMA-CAR iNKT cells effectively eliminated MM cells in a CAR-dependent manner, without losing their T cell receptor (TCR)-mediated cytotoxic activity. Importantly, iNKT cells expressing either BCMA-CARs or affinity-optimized CD38-CARs spared normal hematopoietic cells and displayed a Th1-like cytokine profile, indicating their therapeutic utility. While the costimulatory domain of CD38-CARs had no influence on the cytotoxic functions of iNKT cells, CARs containing the 4-1BB domain showed a better expansion capacity. Interestingly, when stimulated only via CD1d

Topics: ADP-ribosyl Cyclase 1; B-Cell Maturation Antigen; Bone Marrow Cells; Cytokines; Cytotoxicity, Immunologic; Dendritic Cells; Galactosylceramides; Hematopoietic Stem Cells; HLA Antigens; Humans; Immunotherapy, Adoptive; Killer Cells, Natural; Leukocytes, Mononuclear; Membrane Glycoproteins; Multiple Myeloma; Natural Killer T-Cells; Protein Domains; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; Risk; Th1 Cells; Tumor Necrosis Factor Receptor Superfamily, Member 9

NK cells represent a first line of immune defense, but are progressively dysregulated in multiple myeloma (MM) patients. To restore and facilitate their antitumor effect, NK cells are required in sufficient quantities and must be stimulated. We initially assessed the proportions of NKT and NK cells in 34 MM patients. The frequencies of both in PBMC populations correlated with those in BMMNCs irrespective of low BMMNC numbers. We then assessed the adjunctive effect of stimulating NKT cells with CD1d and α-GalCer complexes on the NK cells. The expression of NKG2D on CD56

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD1d; Antigens, Differentiation, T-Lymphocyte; Antineoplastic Agents; Female; Galactosylceramides; Glucocorticoids; Humans; Killer Cells, Natural; Lymphocyte Activation; Male; Middle Aged; Multiple Myeloma; Natural Killer T-Cells; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Antigen, T-Cell

Recent research showed that invariant natural killer T (iNKT) cells take part in the regulation of osteoclastogenesis. While the role of iNKT cells in myeloma bone disease (MBD) remains unclear. In our study, the quantity of iNKT cells and the levels of cytokines produced by them were measured by flow cytometry. iNKT cells and osteoclasts were induced from peripheral blood mononuclear cells after activation by α-GalCer or RANKL in vitro. Then, gene expressions and the levels of cytokines were determined by RT-PCR and ELISA, respectively. The results showed that the quantity of iNKT and production of IFN-γ by iNKT cells were significantly decreased in newly diagnosed MM (NDMM), and both negatively related with severity of bone disease. Then, the osteoclasts from healthy controls were cultured in vitro and were found to be down-regulated after α-GalCer-stimulated, while there was no significant change with or without α-GalCer in NDMM patients, indicating that the regulation of osteoclastogenesis by iNKT cells was impaired. Furthermore, the inhibition of osteoclastogenesis by iNKT cells was regulated by IFN-γ production, which down-regulated osteoclast-associated genes. In conclusion, the role of α-GalCer-stimulated iNKT cells in regulation of osteoclastogenesis was impaired in MBD, as a result of iNKT cell dysfunction.

Topics: Aged; Bone Diseases; Case-Control Studies; Cell Proliferation; Female; Galactosylceramides; Humans; Interferon-gamma; Lymphocyte Subsets; Male; Middle Aged; Multiple Myeloma; Natural Killer T-Cells; Osteoclasts; Osteogenesis; Remission Induction

A hallmark of bone marrow changes with aging is the increase in adipocyte composition, but how this impacts development of multiple myeloma (MM) is unknown. Here, we report the role of the adipokine leptin as master regulator of anti-myeloma tumor immunity by modulating the invariant natural killer T (iNKT) cell function. A marked increase in serum leptin levels and leptin receptor (LR) expression on iNKT cells in MM patients and the 5T33 murine MM model was observed. MM cells and leptin synergistically counteracted anti-tumor functionality of both murine and human iNKT cells. In vivo blockade of LR signaling combined with iNKT stimulation resulted in superior anti-tumor protection. This was linked to persistent IFN-γ secretion upon repeated iNKT cell stimulation and a restoration of the dynamic antigen-induced motility arrest as observed by intravital microscopy, thereby showing alleviation of iNKT cell anergy. Overall our data reveal the LR axis as novel therapeutic target for checkpoint inhibition to treat MM.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cytokines; Disease Models, Animal; Galactosylceramides; Humans; Leptin; Lymphocyte Activation; Mice; Mice, Knockout; Molecular Targeted Therapy; Multiple Myeloma; Natural Killer T-Cells; Receptors, Leptin; Xenograft Model Antitumor Assays

Tumour pathogenesis in multiple myeloma (MM) correlates with a high vascular index. Therefore, targeting angiogenesis is an important therapeutic tool to reduce MM progression. This study aimed to investigate the role of invariant natural killer T (iNKT) cells in angiogenesis and the mechanisms behind the stimulation by α-Galactosylceramide (α-GalCer). We have previously found that α-GalCer could increase the survival of 5T33MM mice and here we demonstrate that α-GalCer reduces the microvessel density. We performed both in vivo and in vitro angiogenic assays to confirm this observation. We found that conditioned medium of α-GalCer stimulated iNKT cells reduced neovascularization in the chick chorioallantoic membrane and in matrigel plug assays. Moreover, we observed a reduction in proliferation, migration and network formation and an induction of apoptosis upon exposure of murine endothelial cell lines to this conditioned medium. We furthermore observed that the JAK-STAT signaling pathway was highly activated in endothelial cells in response to stimulated iNKT cells, indicating the possible role of IFN-γ in the anti-angiogenic process. In conclusion, these results highlight the possibility of recruiting iNKT cells to target MM and angiogenesis. This gives a rationale for combining immunotherapy with conventional anti-tumour treatments in view of increasing their therapeutic potential.

Topics: Animals; Cell Line; Galactosylceramides; Immunotherapy; Interferon-gamma; Janus Kinases; Mice; Multiple Myeloma; Natural Killer T-Cells; Neoplasms, Experimental; Signal Transduction; STAT Transcription Factors

Immunomodulators have been used in recent years to reactivate host anti-tumor immunity in several hematological malignancies. This report describes the effect of activating natural killer T (NKT) cells by α-Galactosylceramide (α-GalCer) in the 5T33MM model of multiple myeloma (MM). NKT cells are T lymphocytes, co-expressing T and NK receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain. We followed iNKT numbers during the development of the disease in both 5T33MM mice and MM patients and found that their numbers dropped dramatically at the end stage of the disease, leading to a loss of total IFN-γ secretion. We furthermore observed that α-GalCer treatment significantly increased the survival of 5T33MM diseased mice. Taken together, our data demonstrate for the first time the possibility of using a preclinical murine MM model to study the effects of α-GalCer and show promising results of α-GalCer treatment in a low tumor burden setting.

Topics: Animals; Antigens, CD1d; Disease Models, Animal; Galactosylceramides; Humans; Interferon-gamma; Lymphocyte Count; Mice; Multiple Myeloma; Natural Killer T-Cells

One of the greatest challenges in all of medicine is to improve on the life of an asymptomatic patient; however, in this issue of Blood, Richter and colleagues share provocative new data taking steps toward accomplishing this goal.

Topics: Dendritic Cells; Female; Galactosylceramides; Humans; Killer Cells, Natural; Lenalidomide; Male; Multiple Myeloma; Thalidomide

Natural killer T (NKT) cells are CD1d-restricted glycolipid reactive innate lymphocytes that play an important role in protection from pathogens and tumors. Pharmacologic approaches to enhance NKT cell function will facilitate specific NKT targeting in the clinic. Here we show that lenalidomide (LEN), a novel thalidomide (Thal) analog, enhances antigen-specific expansion of NKT cells in response to the NKT ligand alpha-galactosylceramide (alpha-GalCer) in both healthy donors and patients with myeloma. NKT cells activated in the presence of LEN have greater ability to secrete interferon-gamma. Antigen-dependent activation of NKT cells was greater in the presence of dexamethasone (DEX) plus LEN than with DEX alone. Therapy with LEN/Thal also led to an increase in NKT cells in vivo in patients with myeloma and del5q myelodysplastic syndrome. Together these data demonstrate that LEN and its analogues enhance CD1d-mediated presentation of glycolipid antigens and support combining these agents with NKT targeted approaches for protection against tumors.

Topics: Antigen Presentation; Cells, Cultured; Galactosylceramides; Humans; Killer Cells, Natural; Lenalidomide; Ligands; Lymphocyte Activation; Multiple Myeloma; Thalidomide

Most multiple myeloma (MM) patients could not be cured by high-dose chemotherapy and bone marrow transplantation. This study was designed to investigate in vitro killing effect of tumor-specific cytotoxic T lymphocytes (CTLs) stimulated by idiotype protein (Id)-pulsed dendritic cells (DCs) on autologous MM cells.. DCs were generated from peripheral blood monocytes of 6 MM patients using interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). After cultured for 5 days, immature DCs were pulsed with Idû tumor necrosis factor-alpha (TNF-alpha) was added at the 7th day. Id-pulsed DCs were cocultured with autologous T cells for 3 days to induce tumor-specific CTLs. MTT assay was used to detect proliferation of autologous T cells, and evaluate killing effect of CTLs on autologous MM cells.. Mature DCs were successfully induced. Id-pulsed DCs markedly increased proliferation of autologous T cells in a dose-dependent manner; stimulation index (SI) of Id-pulsed DCs was the highest [(39.1+/-6.0)%] when the radio of DCs to T cells was 10:1, which was significantly higher than those of unpulsed mature DCs [(19.3+/-7.7)%], Id-pulsed immature DCs [(15.9+/-6.1)%], and unpulsed immature DCs [(11.4+/-4.9)%] (P < 0.01). Id-pulsed DCs induced anti-MM activity of CTLs in a dose-dependent manner. Unpulsed mature DCs also induced cytotoxicity of CTLs against autologous MM cellsû however, when DC:T was 30:1, killing rate of MM cells was significantly higher in Id-pulsed mature DCs group than in unpulsed mature DCs group [(70.1+/-7.9)% vs. (40.8+/-7.8)%,P < 0.05]. KRN7000-pulsed mature DCs stimulated proliferation of allogeneic T cells in a dose-dependent manner; when DC:T was 1:10, SI was significantly higher in KRN7000-pulsed mature DCs group than in unpulsed mature DCs group [(38.5+/-5.7)% vs. (20.2+/-5.7)%, P < 0.05].. Mature DCs could be induced and Id with biological activity could be extracted from peripheral blood of MM patients. Id-pulsed DCs could induce antitumor immune response. KRN7000 could improve the immune function of in vitro cultured DCs.

Topics: Adult; Aged; Cell Proliferation; Cytotoxicity, Immunologic; Dendritic Cells; Dose-Response Relationship, Drug; Female; Galactosylceramides; Humans; Immunoglobulin Idiotypes; Male; Middle Aged; Multiple Myeloma; T-Lymphocytes; T-Lymphocytes, Cytotoxic