Zhao-Yang Lu

Interleukin-6 as a Therapeutic Target

Human IL6 is a cytokine produced by many cell types that has pleiotropic effects. In agreement, anti-IL6 therapy reduces inflammation, hepatic acute phase proteins, and anemia and has antiangiogenic effects. Blocking IL6 has demonstrated therapeutic efficacy with drug registration in Castleman disease and inflammatory diseases (rheumatoid arthritis) without major toxicity. Interestingly, the inhibition of C-reactive protein (CRP) production is a trustworthy surrogate marker of anti-IL6 therapy efficacy. Clinically registered IL6 inhibitors include siltuximab, an anti-IL6 mAb, and tocilizumab, an anti-IL6R mAb. In various cancers, in particular plasma cell cancers, large randomized trials showed no efficacy of IL6 inhibitors, despite a full inhibition of CRP production in treated patients in vivo, the numerous data showing an involvement of IL6 in these diseases, and initial short-term treatments demonstrating a dramatic inhibition of cancer cell proliferation in vivo. A likely explanation is the plasticity of cancer cells, with the presence of various subclones, making the outgrowth of cancer subclones possible using growth factors other than IL6. In addition, current therapeutic strategies used in these cancers already target IL6 activity. Thus, anti-IL6 therapeutics are able to neutralize IL6 production in vivo and are safe and useful in inflammatory diseases and Castleman disease. Clin Cancer Res; 21(6); 1248–57. ©2015 AACR.

Interleukin‐1 in multiple myeloma: producer cells and their role in the control of IL‐6 production

We studied the role of interleukin (IL)‐1β in patients with multiple myeloma. By in situ hybridization and immunochemistry, myeloid and megakaryocytic cells expressed high levels of the IL‐1β gene and produced IL‐1β. Myeloma cells less potently expressed the IL‐1β gene and IL‐1β protein. IL‐1β gene expression was not constitutive since it was detected in the bone marrow myeloma cells of two patients, unlike circulating tumoural cells. In addition, nine myeloma cell lines failed to express the IL‐1β gene and this expression could not be induced by 12 different cytokines. We demonstrated that IL‐1 was mainly responsible for IL‐6 production in the tumoural environment through a PGE2 loop. In fact, an IL‐1 receptor antagonist (IL‐1RA) blocked PGE2 synthesis and IL‐6 production by 80%; this blockage could be reversed by adding synthetic PGE2. Similar findings were found with indomethacin, an inhibitor of cyclooxygenase that blocks PGE2 synthesis. Taken together, these data emphasize the possibility of blocking IL‐1 by using IL‐1RA or other antagonists in order to block IL‐6 production, which is a major tumoural survival and proliferation factor.

Cooperation between heparin-binding EGF-like growth factor and interleukin-6 in promoting the growth of human myeloma cells

Interleukin-6 (IL-6) is a major survival and proliferation factor of human malignant plasma cells and IL-6 dependent myeloma cell lines can be obtained from patients with terminal disease. We show here that mutated diphtheria toxin, a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF), blocked the IL-6-induced growth of two myeloma cell lines (XG-1 and XG-14) and did not significantly affect that of two other cell lines (XG-6 and XG-13). The IL-6 mediated growth of myeloma cells was also inhibited by antibodies to ErbB1, a receptor for HB-EGF. The XG-1 and XG-14 cell lines that are sensitive to HB-EGF inhibitors overexpressed HB-EGF and EGF receptor (ErbB1) genes. They also overexpressed CD9, a tetraspanin that binds to the heparin-binding domain of HB-EGF and is critical for promoting ErbB1 activation by HB-EGF. The XG-6 and XG-13 myeloma cells that were not significantly sensitive to HB-EGF antagonists, poorly expressed HB-EGF, ErbB1 and CD9 genes or proteins. We demonstrated that recombinant HB-EGF supported the long-term growth of myeloma cells, as did IL-6. The myeloma cell growth factor activity of HB-EGF was completely inhited by antibodies to ErbB1, but also by antibodies to gp130 IL-6 transducer or to IL-6. These data indicate that in the XG-1 and XG-14 IL-6-dependent myeloma cell lines, the CD9/HB-EGF/erbB1 and the IL-6/IL-6R/gp130 pathways cooperate synergistically to trigger myeloma cell growth. They suggest that inhibitors of the EGF receptor or HB-EGF may be useful for inducing myeloma cell apoptosis in patients with multiple myeloma.

Functional regulatory T cells are collected in stem cell autografts by mobilization with high-dose cyclophosphamide and granulocyte colony-stimulating factor.

High-dose cyclophosphamide (Cy) and G-CSF are widely used to mobilize hemopoietic stem cells for treating patients with high-dose chemotherapy and autologous stem cell transplantation (ASCT). Because lymphocyte count in the graft collected after Cy-G-CSF treatment is an independent survival factor after ASCT for patients with multiple myeloma, our purpose was to study how Cy-G-CSF treatment affects the phenotype and function of T cells in patients with multiple myeloma. Cy induced a 3-fold decrease of T cell counts with a slow and partial T cell recovery of one-third at the time of hemopoietic stem cell collection. Cy-G-CSF treatment did not affect the relative ratios of central memory, effector memory, and late effector CD4+ or CD8+ T cells, but a decrease in the percentage of naive CD4+ cells was observed. The percentages of CD25+ cells increased 2- to 3-fold in CD4+ and CD8+ T cells, the former including both activated CD25low and CD25high cells. CD4+CD25high cells were regulatory T cells (Treg) that expressed high levels of FOXP3, CTLA-4, and GITR and displayed in vitro suppressive properties. The recovery of Treg absolute counts after Cy-G-CSF treatment was higher than the recovery of other lymphocyte subpopulations. In conclusion, Cy-G-CSF treatment induces a severe T cell count decrease without deleting Treg, which are potent inhibitors of antitumor response. The present data encourage novel therapeutic strategies to improve T cell recovery following ASCT while limiting Treg expansion.

Enhanced graft-versus-tumor effect following dose-reduced conditioning and allogeneic transplantation for refractory lymphoid malignancies after high-dose therapy

Non-myeloablative regimens have been proven to allow engraftment following allogeneic stem cells transplantation (allo-SCT) with minimal procedure-related toxicity. Conventional allo-SCT may produce remissions in patients with relapsed and refractory lymphoid malignancies (LM) but these good results may be achieved at the cost of high treatment-related morbidity and mortality. Application of allo-SCT using less intensive regimens may temper the frequency of these complications, allowing a potent graft-versus-tumor effect (GVT). We present our data on 11 patients with LM receiving allo-SCT with a reduced regimen. Ten patients had received previous high-dose therapy, and were at high risk for toxicity, thus precluding the use of allo-SCT. A fludarabine and low-dose busulfan combination facilitated engraftment while exerting GVT. Hematological recovery was quick, and full donor T cell chimerism preceded acute GVHD. GVHD and infections were the major problems. Spontaneous acute GVHD occurred in eight patients (72%). Five patients (45%) achieved complete remission, and the GVT effect was closely associated with GVHD. These results support the concept that GVT is effective against LM in patients who have been heavily pretreated. Further studies are needed to investigate strategies to generate more specific alloreactive effects providing optimal GVT and an acceptable risk of GVHD and infections. Bone Marrow Transplantation (2001) 28, 335–339.

Large scale and clinical grade purification of syndecan-1+ malignant plasma cells.

For cancer immunotherapy, it is usually necessary to obtain a large number of tumor cells from patients. We have previously reported that syndecan-I is present only on malignant plasma cells in samples from patients with multiple myelomatosis. We report here that this antigen is cleaved by chymopapain. This makes it possible to develop a rapid and clinical grade procedure to purify large numbers of myeloma cells using anti-syndecan-1 mAb, magnetic beads and chymopapain.

All-trans retinoic acid (ATRA) induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity.

NK cell is an innate immune system lymphocyte lineage with natural cytotoxicity. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and encounter with target cells activate NK cells and induce proliferation, and this could depend on the presence of other immune cells. Here we activated PBMCs during 5 days with IL-2, with IL-2 plus the tumor cell line K562 and with the lymphoblastoid cell line R69 and perform integrated analyses of microRNA and mRNA expression profiles of purified NK cells. The samples cluster depending on the stimuli and not on the donor, indicating that the pattern of NK cell stimulation is acutely well conserved between individuals. Regulation of mRNA expression is tighter than that of miRNA expression. All stimuli induce a common preserved genetic remodeling. In addition, encounter with target cells mainly activates pathways related to metabolism. Different target cells induce different NK cell remodeling which affects cytokine response and cytotoxicity, supporting the notion that encounter with different target cells significantly changing the activation pattern. We validate our analysis by showing that activation down regulates miR-23a, which is a negative regulator of cathepsin C (CTSC) mRNA, a gene up regulated by all stimuli. The peptidase CTSC activates the granzymes, the main effector proteases involved in NK cell cytotoxicity. All-trans retinoic acid (ATRA), which induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity in an in vivo mouse model.

Overall interleukin-6 production exceeds 7 mg/day in multiple myeloma complicated by sepsis

We previously reported that injection of anti-IL-6 monoclonal antibody (mAb) in a patient with multiple myeloma (MM) induced the circulation of high amounts of IL-6 in the form of IL-6/anti-IL-6 monomeric complexes. This made it possible to estimate overall daily IL-6 production in patients in vivo, which had not been achieved in animals or humans before. In this study, estimations are given for a patient with MM who developed Escherichia coli sepsis during anti-IL-6 mAb. During the first 12 days, the overall IL-6 production was estimated at 1.5 to 2.0 micrograms/day. On day 13, serum IL-6 concentration, in the form of IL-6/anti-IL-6 complexes, increased 1000-fold and was 1.7 x 10(6) pg/ml, in relation with the development of E. coli sepsis. Overall IL-6 production was estimated to be greater than 7 mg/day, i.e. 3500 times higher than before sepsis. Serum IL-6 levels in the form of monomeric immune complexes remained very high for 20 days after sepsis indicating the persistence of very high overall IL-6 production (100 to 3500-fold greater than pre-sepsis production). This study demonstrates a considerable and persistent potential for IL-6 production in this patient during and after sepsis.

Low doses of GM-CSF (molgramostim) and G-CSF (filgrastim) after cyclophosphamide (4 g/m2) enhance the peripheral blood progenitor cell harvest: results of two randomized studies including 120 patients.

The use of a combination of G-CSF and GM-CSF versus G-CSF alone, after cyclophosphamide (4 g/m2) was compared in two randomized phase III studies, including 120 patients. In study A, 60 patients received 5 × 2 μg/kg/day of G-CSF and GM-CSF compared to 5 μg/kg/day of G-CSF. In study B, 60 patients received 2.5 × 2 μg/kg/day G-CSF and GM-CSF compared to G-CSF alone (5 μg/kg/day). With the aim to collect at least 5 × 106/kg CD34 cells in a maximum of three large volume leukapherises (LK), 123 LK were performed in study A, showing a significantly higher number of patients reaching 10 × 106/kg CD34 cells (21/29 in G+GM-CSF arm vs 11/27 in G-CSF arm, P=0.00006). In study B, 109 LK were performed, with similar results (10/27 vs 15/26, P=0.003). In both the study, the total harvest of CD34 cells/kg was twofold higher in G-CSF plus GM-CSF group (18.3 × 106 in study A and 15.85 × 106 in study B) than in G-CSF group (9 × 106 in study A and 8.1 × 106 in study B), a significant difference only seen in multiple myeloma, with no significant difference in terms of mobilized myeloma cells between G-CSF and GM-CSF groups.

Mobilization of plasma cells in healthy individuals treated with granulocyte colony-stimulating factor for haematopoietic stem cell collection

In mice, the plasma cell (PC) niche in the bone marrow is close to the haematopoietic stem cell (HSC) niche. We investigated whether PCs can be mobilized into the peripheral blood (PB) in healthy donors receiving granulocyte colony‐stimulating factor (G‐CSF) for the induction of HSC mobilization into the PB. G‐CSF increased the count of circulating PCs 6‐fold, that of circulating B lymphocytes 4‐fold and that of circulating HSCs 44‐fold. Mobilized circulating PCs comprised CD138− (62·2%) and CD138+ (37·8%) PCs, the latter being more mature based on increased CD27, CD38 and cytoplasmic immunoglobulin expression. Mobilized PCs had a phenotype close to that of steady‐state PB PCs or in vitro generated PCs, but they expressed L‐selectin only weakly. Finally, a median value of 0·4 × 106/kg donor PCs – one‐thirtieth of the overall PC count in a healthy adult – was grafted into patients, which could contribute to immune memory recovery.