A randomized,open‐label,multicentre, phase 2/3 study to evaluate the safety and efficacy of lumiliximab in combination with fludarabine,cyclophosphamide and rituximab versus fludarabine,cyclophosphamide and rituximab alone in subjects with relapsed chronic lymphocytic leukaemia

Lumiliximab is a chimeric monoclonal antibody that targets CD23 on the surface of chronic lymphocytic leukaemia (CLL) B‐cells. Early phase clinical studies with lumiliximab alone and in combination with fludarabine, cyclophosphamide and rituximab (FCR) established its potential efficacy and tolerability. The 152CL201 trial [Lumiliximab with fludarabine, cyclophosphamide and rituximab (FCR) versus FCR alone in subjects with relapsed CLL; LUCID] was a phase 2/3, randomized (1:1), open‐label, multicentre study of lumiliximab in combination with FCR versus FCR alone in patients with relapsed CLL. Six hundred and twenty‐seven patients were randomized to either arm. Overall the combination of lumiliximab with FCR was not significantly better than FCR alone (overall response rate 71% vs. 72%, complete response rate 16% vs. 15%, median progression‐free survival 24.6 vs. 23.9 months respectively, for FCR with and without lumiliximab). There was a slightly increased incidence of adverse events with lumiliximab but these increases did not appear to lead to differences in eventual outcomes. An interim analysis failed to show sufficient efficacy of the combination of lumiliximab with FCR. The study was therefore stopped early for lack of efficacy. Despite the eventual outcome, the LUCID trial is one of the largest studies that provides valuable insight into the efficacy and tolerability of FCR as a therapeutic option for patients with relapsed CLL.     

The mechanism of tumor cell clearance by rituximab in vivo in patients with B-cell chronic lymphocytic leukemia: evidence of caspase activation and apoptosis induction.

Rituximab is a chimeric monoclonal antibody directed at CD20 with significant activity in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). A variety of pathways of tumor cytotoxicity different from cytotoxic chemotherapy have been proposed for this therapeutic antibody including antibody-dependent cellular cytotoxicity and complement-mediated cell lysis. This report describes that a proportion of patients with CLL receiving rituximab treatment have in vivo activation of caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) cleavage in blood leukemia cells immediately following infusion of rituximab. This suggests that apoptosis using a pathway similar to fludarabine and other chemotherapeutic agents is intricately involved in the blood elimination of tumor cells after rituximab treatment. Patients having caspase-3 activation and PARP cleavage in vivo had a significantly lower blood leukemia cell count after treatment as compared to those without caspase activation. Significant down-modulation of the antiapoptotic proteins XIAP and Mcl-1 was also noted, possibly explaining in part how rituximab sensitizes CLL cells to the cytotoxic effect of chemotherapy in vivo. These findings suggest that the therapeutic benefit of antibody-based therapy in vivo for patients with CLL depends in part on induction of apoptosis and provides another area of focus for studying mechanisms of antibody-resistance in neoplastic cells.     

Chronic lymphocytic leukaemia cells are efficiently killed by an anti-CD20 monoclonal antibody selected for improved engagement of FcgammaRIIIA/CD16

Patients with chronic lymphocytic leukaemia (CLL) treated with a combination of fludarabine, cyclophosphamide and rituximab show a high response rate. However, only a poor response is observed following rituximab monotherapy. The use of chemo-immunotherapy is often associated with haematological and infectious complications. Thus, an antibody with an enhanced ability to kill CLL cells could lead to better clinical responses to antibody monotherapy and the possibility of lowering drug doses during chemo-immunotherapy. We generated a chimeric anti-CD20 monoclonal antibody (mAb), EMAB-6, which has a low fucose content. Apoptosis and complement activities for EMAB-6 were similar to those seen for rituximab. By contrast, EMAB-6 mAb showed improved Fcγ receptor IIIA (FcγRIIIA)/CD16 binding and FcγRIIIA-dependent effector functions. It induced a higher in vitro antibody-dependent cellular cytotoxicity against CLL cells and a higher FcγRIIIA-mediated interleukin-2 production by FcγRIIIA⁺ Jurkat cells in the presence of CLL cells at both low and maximally saturating concentrations. Comparative studies between CLL and lymphoma cells coated with EMAB-6 or rituximab indicated that the difference of efficacy was more pronounced at low doses and when target cells expressed fewer CD20 molecules. Thus, EMAB-6 mAb represents a promising drug candidate for the treatment of CLL by inducing a strong cytotoxicity against tumour cells that express low CD20 levels.     

IL-21 mediates apoptosis through up-regulation of the BH3 family member BIM and enhances both direct and antibody-dependent cellular cytotoxicity in primary chronic lymphocytic leukemia cells in vitro

Interleukin-21 (IL-21) is a recently identified -chain receptor cytokine family member that promotes B-cell apoptosis as well as activation of innate immune system. Based on this, we hypothesized that IL-21 might enhance the apoptosis induced by fludarabine and rituximab and also play a role in augmenting immune-mediated clearance of the chronic lymphocytic leukemia (CLL) cells. Our studies demonstrate that the majority of CLL patients have surface IL-21 receptor-, and its expression correlates with apoptosis, tyrosine phosphorylation of STAT1, and up-regulation of the proapoptotic BH3 domain protein BIM. IL-21–induced BIM up-regulation is critical for apoptosis because inhibition of BIM expression using small interfering RNA prevented IL-21–induced apoptosis. IL-21 treatment of CLL cells but not normal T cells with fludarabine or rituximab additively enhanced the direct cytotoxic effect of these therapies. In addition to its proapoptotic effect, IL-21 promoted STAT1 and STAT5 phosphorylation in natural killer cells with concurrent enhanced antibody-dependent cellular cytotoxicity against rituximab-coated CLL cells in vitro. These data provide justification for combination studies of IL-21 with fludarabine and rituximab in CLL and suggest that BIM up-regulation might serve as relevant pharmacodynamic end point to measure biologic effect of this cytokine in vivo.     

Anti-CD20 antibody (IDEC-C2B8, rituximab) enhances efficacy of cytotoxic drugs on neoplastic lymphocytes in vitro: role of cytokines, complement, and caspases.

BACKGROUND AND OBJECTIVES: Monoclonal antibody IDEC-C2B8 (rituximab) has been shown to be highly effective in the treatment of non-Hodgkin's lymphomas (NHL). The present study was designed to investigate relationships between the efficacy of IDEC-C2B8 and expression of CD20, presence of complement, and effects of differently acting chemotherapeutic agents used in lymphoma treatment (doxorubicin, mitoxantrone, cladribine, bendamustine). DESIGN AND METHODS: DOHH-2, WSU-NHL and Raji lymphoma cell lines and ex vivo cells from patients with chronic lymphocytic leukemia (CLL) (n=17) and leukemic B-cell lymphomas (n=9) were studied. Additionally, the effect of interleukin (IL)-2, IL-4, IL-6, IL-13, granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)alpha on expression of CD20 molecules per cell was determined. RESULTS: We demonstrate that 10 mg/mL rituximab saturated 80-95% of CD20 molecules per cell in all tested lymphoma samples. Although rituximab induced only a minor increase of apoptosis, combinations of rituximab with different cytotoxic drugs significantly decreased the IC(30)- and IC(50) dosages of the chemotherapeutic agents necessary for induction of apoptosis irrespective of addition of complement, demonstrating a chemosensitizing effect of rituximab in combination with cytotoxic drugs in the neoplastic lymphocytes. This effect seemed to be independent of the percentage of saturated CD20 molecules. After addition of caspase inhibitors to the cell lines incubated with rituximab and cytotoxic agents, caspase-7 and -8 were found, by Western blotting, to be the executioner caspases, possibly explaining the rituximab-sensitized apoptosis. Preincubation of lymphoma cells with cytokines did not alter the expression of CD20; IL-2 and IL-4 even decreased the rate of apoptosis. INTERPRETATION AND CONCLUSIONS: We conclude that rituximab sensitizes lymphoma cells to the effect of differently acting cytotoxic drugs used in lymphoma treatment, that this effect does not require complement, and that caspase-7 and -8 may represent the main executioner caspases in chemosensitization by rituximab.     

CD40 triggering enhances fludarabine-induced apoptosis of chronic lymphocytic leukemia B-cells through autocrine release of tumor necrosis factor-alpha and interferon-gama and tumor necrosis factor receptor-I-II upregulation

BACKGROUND AND OBJECTIVES: In chronic lymphocytic leukemia (CLL) B-cells are refractory to activation signals and to apoptosis. CD40 triggering, however, rescues CLL B-cells from their anergic state and upregulates the FAS receptor. We therefore studied whether CD40 triggering enhances CLL B-cell sensitivity to fludarabine, and receptors or cytokines potentially involved in apoptosis. DESIGN AND METHODS: CD40-activation of CLL B-cells was carried out by co-culture with CD40L-transfected cells. After fludarabine treatment, apoptosis was evaluated by propidium iodide (PI), annexin-V/PI or DiOC6 staining and flow cytometry analysis. Modulation of Bcl-2, of tumor necrosis factor receptor (TNFRI/II) and release of tumor necrosis factor (TNF)alpha/interferon (IFN)gamma was also analyzed. Furthermore, addition of caspase-inhibitors or anti-TNFalpha/-IFNgamma monoclonal antibodies to fludarabine-treated cells allowed us to determine the mediators of apoptosis. Student's t tests or ANOVA variance statistical analysis were performed to evaluate whether any differences observed might be considered significant. RESULTS: CD40 triggering enhanced fludarabine sensitivity of CLL B-cells, downmodulated Bcl-2 and upregulated TNFRI/II. Caspases 1 and 6 were the major caspases involved in fludarabine apoptosis induction in resting B cells, while only anti-TNFalpha/-IFNgamma monoclonal antibodies reduced apoptosis in activated cells. In agreement with this observation, autocrine production of TNFalpha and IFNgamma by CD40-activated CLL B cells was found. INTERPRETATION AND CONCLUSIONS: B-cells from a considerable proportion of CLL cases studied (11/20) are more prone to fludarabine-induced apoptosis after CD40 triggering; accordingly Bcl-2 expression was lower in activated cells. Moreover, upregulation of TNFRI/II, release of TNFalpha and IFNgamma, and inhibition of apoptosis by anti-TNFalpha/-IFNgamma monoclonal antibodies in CD40-activated cells strongly suggest that these cytokines may play a role in sensitizing B-cells to fludarabine treatment.     

Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis

The chimeric anti-CD20 MAb rituximab has recently become a treatment of choice for low-grade or follicular non-Hodgkin's lymphomas (FL) with a response rate of about 50%. In this report, we have investigated the mechanism of action of rituximab on 4 FL and 1 Burkitt's lymphoma (BL) cell lines, 3 fresh FL samples and normal B cells in vitro. Rituximab efficiently blocks the proliferation of normal B cells, but not that of the lymphoma lines. We did not detect significant apoptosis of the cell lines in response to rituximab alone. All cell lines were targets of antibody-dependent cellular cytotoxicity (ADCC). On the other hand, human complement-mediated lysis was highly variable between cell lines, ranging from 100% lysis to complete resistance. Investigation of the role of the complement inhibitors CD35, CD46, CD55, and CD59 showed that CD55, and to a lesser extent CD59, are important regulators of complement-mediated cytotoxicity (CDC) in FL cell lines as well as in fresh cases of FL: Blocking CD55 and/or CD59 function with specific antibodies significantly increased CDC in FL cells. We conclude that CDC and ADCC are major mechanisms of action of rituximab on B-cell lymphomas and that a heterogeneous susceptibility of different lymphoma cells to complement may be at least in part responsible for the heterogeneity of the response of different patients to rituximab in vivo. Furthermore, we suggest that the relative levels of CD55 and CD59 may become useful markers to predict the clinical response. (Blood. 2000;95:3900-3908)     

Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression

Chemoimmunotherapy with anti-CD20 monoclonal antibody rituximab is increasingly used for the treatment of patients with chronic lymphocytic leukemia (CLL). Antibody-dependent cytotoxicity (ADCC) is one of the most important mechanisms of action of rituximab against B-cell malignancies. We studied ways to increase the cytotoxic effect of rituximab on CLL cells by enhancing ADCC. Peripheral blood mononuclear cell (PBMC) or purified natural killer (NK) cells from healthy donors were activated with interleukin-15 (IL-15) and cultured with rituximab-coated CLL cells, and ADCC was evaluated using a (51)chromium release assay. The IL-15 significantly enhanced in?vitro ADCC against CLL cells, and this effect was mainly mediated by NK cells. The IL-15 treated effector cells with the low affinity FcγRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcγRIIIA form (158VF). In?addition, IL-15 enhanced rituximab-mediated ADCC of CLL cells in the presence of transforming growth factor-beta. The IL-15 increases rituximab-mediated ADCC against CLL, and supports the use of such agents with the goal of improving clinical response to chemoimmunotherapy in patients with CLL.     

Fludarabine treatment of patients with chronic lymphocytic leukemia induces a p53-dependent gene expression response

Fludarabine, the current standard treatment for B-cell chronic lymphocytic leukemia (CLL), can induce apoptosis in CLL cells in vitro, and a number of molecular mechanisms contribute to its cytotoxicity. Using gene expression profiling, we investigated the molecular consequences of fludarabine treatment of patients with CLL in vivo. In 7 patients with CLL, a consistent gene expression signature of in vivo fludarabine exposure was identified. Many of the fludarabine signature genes were known p53 target genes and genes involved in DNA repair. In vitro treatment of CLL cells with fludarabine induced the same set of genes as observed in vivo, and many of these genes were also induced by in vitro exposure of CLL cells to ionizing radiation. Using isogenic p53 wild-type and null lymphoblastoid cell lines, we confirmed that many of the fludarabine signature genes were also p53 target genes. Because in vivo treatment with fludarabine induces a p53-dependent gene expression response, fludarabine treatment has the potential to select p53-mutant CLL cells, which are more drug resistant and associated with an aggressive clinical course. These considerations suggest that fludarabine treatment should be given in strict accordance to the current National Cancer Institute (NCI) guidelines that have established criteria of disease activity that warrant treatment.     

T cells armed with anti-CD3 x anti-CD20 bispecific antibody enhance killing of CD20+ malignant B cells and bypass complement-mediated rituximab resistance in vitro

OBJECTIVE: Resistance to rituximab, a chimeric monoclonal antibody that binds to CD20, is a major limitation for the successful treatment of patients with non-Hodgkin lymphoma and other CD20+ B-cell malignancies. To circumvent rituximab resistance in these patient populations, we have constructed a bispecific antibody (BiAb), anti-CD3 x anti-CD20 (CD20Bi), that combines rituximab targeting with non-major histocompatibility complex (non-MHC)-restricted cytotoxicity mediated by activated T cells (ATC). MATERIALS AND METHODS: Activated T cells were obtained from anti-CD3 activated peripheral blood mononuclear cells (PBMC) of normal donors or the leukapheresis products of patients by culturing in the presence of interleukin-2 for 6-14 days. After ATC expansion, the cells were armed with CD20Bi. Killing activity was evaluated by 51Cr-release assay. RESULTS: Arming ATC with as little as 5 ng CD20Bi/10(6) cells significantly increased cytotoxicity above unarmed ATC. CD20Bi-armed ATC (50 ng/10(6) cells) efficiently lysed CD20+ cell lines at E:T of 6.25-50, but not the nonhematologic, CD20- SK-BR-3 cell line. High levels of cytotoxicity mediated by CD20Bi-armed ATC (p < 0.05) could not be blocked by an 8000-fold excess of soluble rituximab. CD20Bi-armed ATC in the presence of complement killed ARH-77 cells, a rituximab-complement pathway-resistant multiple myeloma, significantly (p < 0.05) better than rituximab or unarmed ATC, suggesting that CD20Bi-armed ATC may be clinically effective for treatment of rituximab-resistant CD20+ hematologic malignancies. CONCLUSIONS: Our findings demonstrate that CD20Bi-armed ATC enhance cytotoxicity against CD20+ B-cell lines and circumvent complement-mediated rituximab resistance, providing a strong rationale for this immune-based strategy for the treatment of rituximab-refractory CD20+ B-cell malignancies.     

Targeting Bcl-2 family proteins modulates the sensitivity of B-cell lymphoma to rituximab-induced apoptosis

The chimeric monoclonal antibody rituximab is the standard of care for patients with B-cell non-Hodgkin lymphoma (B-NHL). Rituximab mediates complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity of CD20-positive human B cells. In addition, rituximab sensitizes B-NHL cells to cytotoxic chemotherapy and has direct apoptotic and antiproliferative effects. Whereas expression of the CD20 antigen is a natural prerequisite for rituximab sensitivity, cell-autonomous factors determining the response of B-NHL to rituximab are less defined. To this end, we have studied rituximab-induced apoptosis in human B-NHL models. We find that rituximab directly triggers apoptosis via the mitochondrial pathway of caspase activation. Expression of antiapoptotic Bcl-xL confers resistance against rituximab-induced apoptosis in vitro and rituximab treatment of xenografted B-NHL in vivo. B-NHL cells insensitive to rituximab-induced apoptosis exhibit increased endogenous expression of multiple antiapoptotic Bcl-2 family proteins, or activation of phosphatidylinositol-3-kinase signaling resulting in up-regulation of Mcl-1. The former resistance pattern is overcome by treatment with the BH3-mimetic ABT-737, the latter by combining rituximab with pharmacologic phosphatidylinositol-3-kinase inhibitors. In conclusion, sensitivity of B-NHL cells to rituximab-induced apoptosis is determined at the level of mitochondria. Pharmacologic modulation of Bcl-2 family proteins or their upstream regulators is a promising strategy to overcome rituximab resistance.     

Apoptosis induced by molecular targeting therapy in hematological malignancies

Molecular targeting therapies for hematological malignant diseases such as monoclonal antibodies and small molecules have been reviewed. Imatinib mesylate (STI571) targets the tyrosine kinase activity of the BCR-ABL fusion protein in CML, and was superior to IFN-alpha plus low-dose cytarabine in newly diagnosed chronic-phase CML in a phase III randomized study. Imatinib induced apoptosis in BCR-ABL-positive cells in vitro, and activates several signaling pathways such as PI3K/Akt, STAT5 and Ras/MAPK. Combination therapies with imatinib and new strategies for downregulation of intracellular BCR-ABL protein levels have also been investigated from the phenomenon of resistance to imatinib. Anti-CD20 (rituximab) became the first monoclonal antibody approved for the treatment of a relapsed/refractory follicular/low-grade NHL and promising results were obtained from a phase III randomized study. Although antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity are likely to be the major effectors of B-cell depletion in vivo, direct cytotoxicity by CD20 monoclonal antibody on B-cell lines in vitro has been reported. Anti-CD33 (Mylotarg) and FLT3 inhibitors for AML have also been used in clinical trials and signaling pathways induced by these agents are under intensive investigation. Arsenic trioxide, like all-TRANS-retinoic acid (ATRA), downregulates promyelocytic leukemia protein/retinoic acid receptor-alpha (PML/RARalpha) fusion protein and induced apoptosis in APL cells, and promising results were obtained from ATRA-resistant APL patients. Finally we show our promising in vitro and in vivo data of R-etodolac (a non-steroidal anti-inflammatory drug lacking cyclooxygenase inhibitor activity) against chronic lymphocytic leukemia (CLL) cells.     

Antiproliferative activity of a humanized anti-CD74 monoclonal antibody, hLL1, on B-cell malignancies

The humanized anti-CD74 monoclonal antibody (mAb) hLL1 is under evaluation as a therapeutic agent. The effects of hLL1-at times in comparison with the CD20 mAb rituximab-were assessed on non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) cell lines and in tumor-bearing SCID mice. In vitro, hLL1 caused growth inhibition and induction of apoptosis in B-cell lines when cross-linked with an antihuman immunoglobulin G (IgG) second antibody. The sensitivity profile of the cell lines was different for hLL1 and rituximab, and antiproliferative activity was augmented when the 2 mAbs were combined. Unlike rituximab, hLL1 did not induce antibody-dependent cellular cytotoxicity or complement-mediated cytotoxicity. In xenograft models of NHL and MM, treatment with hLL1 yielded significant survival benefits without cross-linking agents. Efficacy was greater in the MM model, in which median survival time was increased more than 4.5-fold. Thus, hLL1 has therapeutic potential as a naked mAb for B-cell malignancies because of high antigen expression on malignant cells, specifically MM, with limited expression on normal tissue, and because of its antiproliferative activity. Further, hLL1 may be a therapeutic candidate for rituximab-resistant disease because the 2 antibodies apparently act through distinct mechanisms and exhibit different expression and sensitivity profiles, and activity can be augmented when the mAbs are combined.     

Rituximab reduces the number of peripheral blood B-cells in vitro mainly by effector cell-mediated mechanisms

BACKGROUND AND OBJECTIVES: The humanized CD20 mono- clonal antibody, rituximab, has significant anti-tumor activity in patients with B-cell non-Hodgkin's lymphoma and induces depletion of B-cells in vivo. It was the objective of this study to define the contribution of the different mechanisms of action of rituximab on primary normal and malignant B-cells. DESIGN AND METHODS: Primary human B-lymphocytes and effector cell fractions were isolated from peripheral blood of normal donors using an immunomagnetic separation technique. Blood samples from 20 patients with chronic lymphocytic leukemia (CLL) were studied and the B-lymphoblastoid Daudi cell line was used as a control. B-cells were cultured in the presence or absence of rituximab adding a secondary hyper-crosslinking antibody, serum as source of complement or different effector cell fractions. The cells were analyzed by immunofluorescence staining and flow cytometry. RESULTS: In contrast to the B-lymphoblastoid Daudi cell line, the number of highly purified normal peripheral blood CD19+ cells was only minimally affected by rituximab in the presence of autologous serum. A significant reduction in the number of B-cells was observed when mononuclear cells from peripheral blood were added back. To identify the cell type which mediates this effect, CD3+ T-cells, CD56+ cells, and CD14+ monocytes were added to selected CD22+ B-cells. A marked B-cell decrease was only observed in the presence of CD56+ and CD14+ cells in an effector to target ratio of 10:1. The experiments with mononuclear cells from patients with CLL showed a B-cell reduction by rituximab, which was significantly enhanced following addition of granulocyte-macrophage colony-stimulating factor (GM-CSF). INTERPRETATION AND CONCLUSIONS: These data support the important role of cell-mediated mechanisms in the B-cell-depleting action of rituximab and suggest that pre-treatment with GM-CSF could improve the response to rituximab in patients with CLL.     

Rituximab and 17-allylamino-17-demethoxygeldanamycin induce synergistic apoptosis in B-cell chronic lymphocytic leukaemia

Treatment options for chronic lymphocytic leukaemia (CLL) are limited and eventually fail because of the development of toxicities or drug resistance. Thus, identification of new therapeutic strategies and targets is a high priority. The semisynthetic geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits heat shock protein 90 (Hsp90) binding to client proteins, thereby leading to their degradation. We demonstrate that at biologically active and clinically attainable levels (1 mumol/l), 17-AAG treatment of CLL B cells in vitro causes modest apoptosis as well as decreased AKT protein levels. Given the potential activation of AKT following antibody therapy in CLL, we evaluated the combination of 17-AAG and rituximab. These agents produced synergistic cytotoxicity of CLL cells in vitro. However, rituximab-mediated antibody-dependent cellular cytotoxicity was modestly reduced with 17-AAG, and complement-dependent cytotoxicity was not altered. We conclude that the combination of Hsp90 inhibitors with therapeutic antibodies, such as rituximab may represent a novel strategy to enhance therapeutic response in CLL. Furthermore, our data indicates that AKT and Hsp70 protein levels are relevant pharmacodynamic endpoints to monitor the in vivo effect of 17-AAG therapy.     

Combination of two anti‐CD5 monoclonal antibodies synergistically induces complement‐dependent cytotoxicity of chronic lymphocytic leukaemia cells

The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti‐CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement‐dependent cytotoxicity (CDC) and antibody‐dependent cell‐mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non‐overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti‐CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab‐induced CD5 internalization and degradation. Importantly, an anti‐CD5 mAb combination enhanced CDC of CLL cells when combined with the anti‐CD20 mAbs rituximab and ofatumumab as well as with the anti‐CD52 mAb alemtuzumab. These results suggest that an anti‐CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5‐expressing haematological or lymphoid malignancies.     

Future prospects for fludarabine-containing regimens in the treatment of hematological cancers

Developments in the understanding of disease biology and in therapeutic approaches during the last decade have failed to alter the natural history of many human hematological malignancies, including chronic lymphocytic leukemia (CLL). Despite better appreciation of the molecular foundations and phenotypic characteristics of these cancers, improvements to disease classification and prognosis, and the discovery of more effective cytotoxic and biological agents, these disorders remain largely incurable. The development of a new class of cytotoxic agents in the 1980 s, namely the purine analogs, heralded a period of renewed optimism in the treatment of indolent lymphoid leukemias and lymphomas. More recently, monoclonal antibodies that selectively target cell-surface proteins, and agents that target cell-cycle or apoptotic pathways, have been developed and their use alone or in combination promise to have a major impact on the treatment of these conditions. Of these, the anti-CD52 antibody alemtuzumab and the anti-CD20 antibody rituximab have demonstrated the most potential for the treatment of CLL. Further, because of their different mode of action in comparison to conventional cytotoxic agents, combination regimens involving fludarabine and these antibodies are showing particular promise in early trials. The encouraging findings with these combination therapies are moving the intent of therapy from palliation towards cure. This paper reviews the therapeutic application of present and future fludarabine-containing approaches. The strategies that are discussed include fludarabine given with alemtuzumab, rituximab, or other monoclonal antibodies, and the potential benefits of combining fludarabine with more experimental agents such as antisense oligonucleotides, immunotoxins, or radioimmunoconjugates. The successful application of fludarabine plus alemtuzumab, or fludarabine in combination with other cytotoxic agents, as preparative regimens for stem cell transplantation techniques is also covered.