Josée Golay

Complement Activation Determines the Therapeutic Activity of Rituximab In Vivo

Rituximab is an anti-CD20 chimeric mAb effective for the treatment of B-NHL. It can lyse lymphoma cells in vitro through both C- and Ab-dependent cellular cytotoxicity. The mechanism of action of rituximab in vivo is however still unclear. We have set up a new in vivo model in nonimmunodeficient mice by stable transduction of the human CD20 cDNA in the murine lymphoma line EL4. Animals injected i.v. with the EL4-CD20+ lymphoma cells died within 30 days with evident liver, spleen, and bone marrow involvement, confirmed by immunohistochemistry and PCR analysis. A single injection of rituximab or the murine anti-CD20 Ab 1F5, given i.p. 1 day after the tumor, cured 100% of the animals. Indeed, at week 4 after tumor cell inoculation, CD20+ cells were undetectable in all organs analyzed in rituximab-treated animals, as determined by immunohistochemistry and PCR. Rituximab had no direct effect on tumor growth in vitro. Depletion of either NK cells or neutrophils or both in tumor-injected animals did not affect the therapeutic activity of the drug. Similarly, rituximab was able to eradicate tumor cells in athymic nude mice, suggesting that its activity is T cell independent. In contrast, the protective activity of rituximab or the 1F5 Ab was completely abolished in syngeneic knockout animals lacking C1q, the first component of the classical pathway of C (C1qa−/−). These data demonstrate that C activation is fundamental for rituximab therapeutic activity in vivo.

Synergism between fludarabine and rituximab revealed in a follicular lymphoma cell line resistant to the cytotoxic activity of either drug alone.

We have shown previously that the anti‐CD20 chimaeric monoclonal antibody rituximab exerts its effects on neoplastic B‐lymphoma cell lines in part via complement‐dependent cytotoxicity. In addition, membrane expression levels of complement inhibitory proteins CD55 and CD59 play a role in determining susceptibility to lysis. We have identified one t(14;18)‐positive human B‐cell non Hodgkins lymphoma cell line (Karpas 422) that is resistant to rituximab and complement and used it for subsequent studies on the possible interaction between this novel therapeutic agent and established antineoplastic drugs. We have exposed Karpas to several chemotherapeutic agents (doxorubicin, idarubicin, cisplatin, taxol) for different time periods and subsequently exposed the cells to rituximab and human complement. The combination of these drugs with rituximab induced an additive cytotoxic effect. In contrast, exposure to fludarabine (1 µg/ml for 48–72 h) showed a synergistic effect, with cell lysis increasing from 10% to 20% using fludarabine or rituximab and complement alone to about 70% with both cytotoxic agents. Analysis of the mechanism for this synergistic effect showed that fludarabine downmodulates the membrane expression of CD55 (from 96% to 55% positive cells) without significantly altering CD20 levels. Northern analysis demonstrated that fludarabine induced a general downmodulation of steady state mRNA levels with no change in transcription rate detected in run‐off assays. The study of the effect of fludarabine and rituximab in six freshly isolated B‐cell chronic lymphocytic leukaemia (B‐CLL) samples showed that, in most cases, fludarabine has an additive cytotoxic activity with rituximab and complement. This report gives a rational support for clinical studies with combinations of drugs, including monoclonal antibodies and fludarabine.

Autologous Mesenchymal Stromal Cells and Kidney Transplantation: A Pilot Study of Safety and Clinical Feasibility

BACKGROUND AND OBJECTIVES Mesenchymal stromal cells (MSCs) abrogate alloimmune response in vitro, suggesting a novel cell-based approach in transplantation. Moving this concept toward clinical application in organ transplantation should be critically assessed. DESIGN, SETTING, PARTICIPANTS & MEASUREMENTS A safety and clinical feasibility study (ClinicalTrials.gov, NCT00752479) of autologous MSC infusion was conducted in two recipients of kidneys from living-related donors. Patients were given T cell-depleting induction therapy and maintenance immunosuppression with cyclosporine and mycophenolate mofetil. On day 7 posttransplant, MSCs were administered intravenously. Clinical and immunomonitoring of MSC-treated patients was performed up to day 360 postsurgery. RESULTS Serum creatinine levels increased 7 to 14 days after cell infusion in both MSC-treated patients. A graft biopsy in patient 2 excluded acute graft rejection, but showed a focal inflammatory infiltrate, mostly granulocytes. In patient 1 protocol biopsy at 1-year posttransplant showed a normal graft. Both MSC-treated patients are in good health with stable graft function. A progressive increase of the percentage of CD4+CD25highFoxP3+CD127- Treg and a marked inhibition of memory CD45RO+RA-CD8+ T cell expansion were observed posttransplant. Patient T cells showed a profound reduction of CD8+ T cell activity. CONCLUSIONS Findings from this study in the two patients show that MSC infusion in kidney transplant recipients is feasible, allows enlargement of Treg in the peripheral blood, and controls memory CD8+ T cell function. Future clinical trials with MSCs to look with the greatest care for unwanted side effects is advised.

M2 Macrophages Phagocytose Rituximab-Opsonized Leukemic Targets More Efficiently than M1 Cells In Vitro

Because macrophages have been implicated as major players in the mechanism of action of rituximab, we have investigated the factors that modulate their tumor cell killing potential. Human macrophages, differentiated in vitro from peripheral blood monocytes, were used in binding and phagocytosis assays using B-chronic lymphocytic leukemia or lymphoma target cells opsonized with rituximab. Phagocytosis was maximal at 0.1 μg/ml rituximab and was not significantly affected by CD20 expression levels or by CD16A polymorphism at position 158 (Val/Phe). The role of FcγRs was demonstrated by complete inhibition of phagocytosis by excess human Igs. Because macrophages can be differentiated to M1- or M2-type cells with either GM-CSF or M-CSF, respectively, and can be classically activated by proinflammatory stimuli (IFN-γ/LPS) or undergo alternative activation with cytokines such as IL-4 or IL-10, we have analyzed the effect of these different polarization programs on the phagocytosis mediated by rituximab. Macrophages differentiated in presence of M-CSF showed a 2- to 3-fold greater phagocytic capacity compared with GM-CSF-induced cells. Furthermore, addition of IL-10 significantly increased, whereas IL-4 decreased phagocytosis by both M-CSF- and GM-CSF-differentiated macrophages. LPS/IFN-γ had little effect. Expression of CD16, CD32, and CD64 in different macrophage populations correlated with phagocytic activity. Interestingly, several B lymphoma cell lines were observed to secrete 400-1300 pg/ml IL-10 in vitro, and coculture of human macrophages with lymphoma conditioned medium increased significantly their phagocytic capacity. Our data suggest that cytokines secreted by lymphoma cells can favor alternate activation of macrophages with a high phagocytic capacity toward rituximab-opsonized targets.

Mechanism of Action of Type II, Glycoengineered, Anti-CD20 Monoclonal Antibody GA101 in B-Chronic Lymphocytic Leukemia Whole Blood Assays in Comparison with Rituximab and Alemtuzumab

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of CD19+ B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1–4 h (62%). In contrast, rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5% cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r2 = 0.88 and 0.85, respectively). Interestingly, lysis by all three Abs at high concentrations was mostly complement dependent, because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion, it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays, but phagocytosis was not significant in whole blood due to excess Igs. Finally, GA101 at 1–100 μg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not rituximab, also mediated significant NK cell degranulation in whole blood samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays.

Dual-functional capability of CD3+CD56+ CIK cells, a T-cell subset that acquires NK function and retains TCR-mediated specific cytotoxicity.

CD3(+)CD56(+) cytokine-induced killer (CIK) cells display a potent cytolytic activity. The adhesion molecule lymphocyte function-associated antigen-1 plays a crucial role in binding as well as in cytolytic activity of CIK cells against tumor target cells expressing the corresponding ligands. CIK cells express activating natural killer (NK) receptors, including NKG2D, DNAX accessory molecule-1 (DNAM-1), and low levels of NKp30. Cell signaling not only through TCR/CD3 but also through NKG2D, DNAM-1, and NKp30 leads to CIK cell activation resulting in granule exocytosis, cytokine secretion, and cytotoxicity. Antibody blocking experiments showed that DNAM-1, NKG2D, and NKp30 are involved in the TCR-independent tumor cell recognition and killing. Anti-CMV-specific CIK cells could be expanded in standard CIK cultures and mediate both specific, MHC-restricted recognition and TCR-independent NK-like cytolytic activity against leukemic cell lines or fresh leukemic blasts. Antibody blocking of lymphocyte function-associated antigen-1 and DNAM-1 led to significant reduction of both CTL and NK-cell functions, whereas blocking of NKG2D and NKp30 only inhibited NK-like cytotoxicity. Their dual-effector function suggests that CIK cells, when used in a clinical setting, may control both neoplastic relapses and viral infections, 2 frequently associated complications in patients who received a transplant.

Mutations in v-myb alter the differentiation of myelomonocytic cells transformed by the oncogene

Chick myelomonocytic cells transformed by the v-myb oncogene-containing viruses E26 and AMV differ in that the former resemble myeloblasts and express the v-myb-regulated granulocyte-specific mim-1 gene, while the latter resemble monoblasts and are mim-1 negative. We constructed a series of AMV-E26 chimeras and localized the critical differences between these viruses to three point mutations within the second repeat of the v-myb DNA binding domain. These three positions are altered in the v-myb protein of AMV relative to the proteins encoded by c-myb or E26 v-myb. Back mutating AMV v-myb at any of these three sites restored the oncogenes ability to activate the mim-1 gene. Surprisingly, two of these changes led to the transformation, in vitro and in vivo, of cells having a promyelocyte-like phenotype. These results indicate that different forms of v-myb impose alternate phenotypes of differentiation on transformed myeloid cells, probably by regulating unique sets of differentiation-specific genes.

Treatment of Graft versus Host Disease with Mesenchymal Stromal Cells: A Phase I Study on 40 Adult and Pediatric Patients

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10(6)/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.

Cytokine-induced killer cells are terminallydifferentiated activated CD8 cytotoxic T-EMRA lymphocytes

OBJECTIVE Cytokine-induced killer cells (CIK) are CD3(+)CD56(+) T cells with natural killer (NK)-like cytotoxic activity used for the immunotherapy of tumors. We aimed to fully characterize CIK cells and define their ontogeny. MATERIALS AND METHODS CIK were generated in vitro by stimulation of peripheral blood mononuclear cells or T-cell subsets with interferon-gamma, anti-CD3 and interleukin-2. They were fully characterized in terms of phenotype, cytotoxic activity, and gene expression with respect to circulating CD3(+)CD56(+) cells, NK cells, and CD56(-) T cells present in CIK cultures. RESULTS We demonstrate that CIK are terminally differentiated CD8 T cells that derive from proliferating CD3(+)CD56(-)CD8(+) T cells. They express polyclonal T-cell receptor Vbeta chains and have acquired CD56, NKG2D, and large granular lymphocyte morphology, but lack expression of most NK-specific activating (NKp30, NKp44, NKp46) and inhibitory (KIR2DL1, KIR2DL2, KIR3DL1, NKG2A, CD94) receptors, and can kill K562 targets. Circulating CD3(+)CD56(+) cells are also CD8(+)CD16(-), but are oligoclonal, poorly cytotoxic for K562, and express lower levels of CD56 and NKG2D. Gene profiling of CIK, CD56(-) T and NK cells present at the end of culture shows that differences are much more limited between CIK and CD56(-) T compared to CIK and NK cells. Most of the genes upregulated in CIK cells compared to CD56(-) T cells are part of the tumor necrosis factor gene network. CONCLUSIONS The CIK phenotype, that is CD45RA(+), CCR7(-), CD62L-weakly positive, CD11a(+), CD27(+), CD28(-), macrophage inflammatory protein 1alpha(+), perforin(+), Fas ligand(+) coincides almost exactly with that described for the T RA(+) effector memory CD27 single positive subset of terminally differentiated human memory T cells.

Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3–3 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs.