CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies

Antibody-dependent cell-mediated cytotoxicity (ADCC), which is largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells after activation, including NK cells. In the present study, we show that an anti-CD137 agonistic mAb enhances the antilymphoma activity of rituximab by enhancing ADCC. Human NK cells up-regulate CD137 after encountering rituximab-coated tumor B cells, and subsequent stimulation of these NK cells with anti-CD137 mAb enhances rituximab-dependent cytotoxicity against the lymphoma cells. In a syngeneic murine lymphoma model and in a xenotransplanted human lymphoma model, sequential administration of anti-CD20 mAb followed by anti-CD137 mAb had potent antilymphoma activity in vivo. These results support a novel, sequential antibody approach against B-cell malignancies by targeting first the tumor and then the host immune system.     

Glucocorticoids and rituximab in vitro: synergistic direct antiproliferative and apoptotic effects

Rituximab, a chimeric human immunoglobulin G(1) (IgG(1)) anti-CD20 monoclonal antibody has been shown to mediate cytotoxicity in malignant B cells via several mechanisms in vitro. These include direct antiproliferative and apoptotic effects, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). Glucocorticoids (GCs) are often administered in conjunction with rituximab in chemotherapeutic regimens or as premedication to reduce infusion-related symptoms. The effects of GCs on CDC and ADCC, and the direct apoptotic and antiproliferative effects of rituximab are unknown. Therefore, we evaluated these mechanisms in 9 B-cell non-Hodgkin lymphoma (B-NHL) cell lines using rituximab and GCs. Rituximab and dexamethasone induced synergistic growth inhibition in 6 B-NHL cell lines. Dexamethasone and rituximab induced significant G(1) arrest in 9 of 9 cell lines. The combination of rituximab and dexamethasone resulted in supra-additive increases in phosphatidylserine exposure and hypodiploid DNA content in 5 and 3 B-NHL cell lines, respectively. CDC and ADCC were neither impaired nor enhanced when dexamethasone and rituximab were administered concurrently. However, preincubation of both effector and tumor cells with dexamethasone reduced specific lysis in ADCC assays in 4 B-NHL cell lines. Preincubation of tumor cell lines with dexamethasone significantly increased cell sensitivity to CDC in 3 B-NHL cell lines. We conclude that the addition of dexamethasone to rituximab results in supra-additive cytotoxicity with respect to its direct antiproliferative and apoptotic effects, induces a cell-dependent increased sensitivity to rituximab-induced CDC, and has minimal negative impact on ADCC when used simultaneously with rituximab.     

Acquired immunodeficiency syndrome-associated lymphomas are efficiently lysed through complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity by rituximab

Rituximab (Mabthera) and alemtuzumab (Campath(R), Mabcampath(R)) are non-conjugated IgG1 therapeutic monoclonal antibodies directed against the CD20 and CD52 surface antigens respectively. They are presently used in the therapy of indolent B-cell non-Hodgkin's lymphoma (B-NHL) and of B-cell chronic lymphocytic leukaemia, and are thought to act mainly through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Here we have analysed the capacity of these two monoclonal antibodies to lyse cell lines of acquired immunodeficiency syndrome (AIDS)-related B-NHL through either complement activation or antibody-dependent cytotoxicity. Rituximab strongly activated both CDC and ADCC against CD20-positive AIDS-NHL cells lines, inducing up to 60-98% and 20% specific lysis respectively. In contrast, alemtuzumab was a poor activator of CDC, even in the AIDS-NHL cell lines expressing high amounts of CD52, leading to a lysis of only 1-30%, whereas it was at least as strong as rituximab in inducing ADCC of the same lines (up to 30% specific lysis). Altogether, these data offer a first in vitro rationale supporting the therapeutic use of rituximab for CD20-positive AIDS-NHL.     

4. Antibody therapy for malignant lymphoma

Rituximab, a genetically engineered, chimeric anti-CD20 monoclonal antibody, induces the apoptosis of B-lymphoma cells, in addition to the lyses by complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), as shown in Fig. 1. A Japanese phase I study of rituximab in relapsed or refractory patients with B-cell non-Hodgkin's lymphoma (B-NHL) showed an overall response rate (ORR) of 64% (7/11) with minimal toxicities. Elimination half-life (T(1/2)) of serum rituximab was 445+/-361 hours, and the serum rituximab was detectable at three months. In the subsequent phase II study, 90 relapsed or refractory patients with indolent B-NHL or mantle cell lymphoma (MCL) were treated with rituximab at 375 mg/m2x4 weekly infusions. ORRs in indolent B-NHL and MCL were 61% (37/61) and 46% (6/13), respectively. In this presentation, the results of clinical trials of antibody therapy of malignant lymphoma are summarized, focusing on the two recent Japanese multicenter trials of rituximab and a Japanese feasibility study of anti-CD20 radioimmunotherapy with yttrium-90-lableled ibritumomab tiuxetan.     

Anti-CD20 monoclonal antibody with enhanced affinity for CD16 activates NK cells at lower concentrations and more effectively than rituximab

Growing evidence indicates that the affinity of monoclonal antibodies (mAbs) for CD16 (FcgammaRIII) plays a central role in the ability of the mAb to mediate antitumor activity. We evaluated how CD16 polymorphisms, and mAb with modified affinity for target antigen and CD16, affect natural killer (NK) cell phenotype when CD20(+) malignant B cells were also present. The mAb consisted of rituximab (R), anti-CD20 with enhanced affinity for CD20 (AME-B), and anti-CD20 with enhanced affinity for both CD20 and CD16 (AME-D). Higher concentrations of mAb were needed to induce CD16 modulation, CD54 up-regulation, and antibody-dependent cellular cytotoxicity (ADCC) on NK cells from subjects with the lower affinity CD16 polymorphism. The dose of mAb needed to induce NK activation was lower with AME-D irrespective of CD16 polymorphism. At saturating mAb concentrations, peak NK activation was greater for AME-D. Similar results were found with measurement of CD16 modulation, CD54 up-regulation, and ADCC. These data demonstrate that cells coated with mAb with enhanced affinity for CD16 are more effective at activating NK cells at both low and saturating mAb concentrations irrespective of CD16 polymorphism, and they provide further evidence for the clinical development of such mAbs with the goal of improving clinical response to mAb.     

The anti-lymphoma effect of antibody-mediated immunotherapy is based on an increased degranulation of peripheral blood natural killer (NK) cells

BACKGROUND: In patients treated with rituximab and alemtuzumab for lymphomas or CLL, antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of action. Therefore, assessment of ADCC is mandatory to understand the complex mechanisms leading to the anti-lymphoma effects of monoclonal antibodies (mAb). Due to methodical difficulties, little is yet known about the relevant cell subpopulations and effector mechanisms leading to tumor lysis in ADCC. METHODS: We used a novel flow cytometric assay that detects CD107a as a marker for NK-cell degranulation to characterize and quantify peripheral blood natural killer (NK) cells mediating ADCC in vitro and in vivo. RESULTS: We observed specific and dose-dependent NK-cell activation after administration of rituximab and alemtuzumab. The number of degranulating NK cells was closely related to the concentration of mAb and the effector:target ratio. We were able to quantify and characterize the peripheral blood NK cells mediating ADCC. The majority of degranulating NK cells had the phenotype: CD56(dim), CD69(+), NKG2D(+), NKp30(-), NKp46(-), and CD94(-). Furthermore, we found that the CD107a assay can also visualize ADCC under clinical conditions as we observed increased numbers of NK cells degranulating in response to CD20(+) lymphoma cell lines in patients with non-Hodgkin's lymphoma treated with rituximab. CONCLUSIONS: We were able to quantify and characterize NK cells mediating ADCC with a new and feasible method. The CD107a assay may be useful for predicting treatment responses of individual patients and may help find the optimal dosage and timing for treatment with mAb.     

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.     

A glycoengineered anti-CD19 antibody with potent antibody-dependent cellular cytotoxicity activity in vitro and lymphoma growth inhibition in vivo

Human cluster of differentiation (CD) antigen 19 is a B cell-specific surface antigen and an attractive target for therapeutic monoclonal antibody (mAb) approaches to treat malignancies of B cell origin. MEDI-551 is an affinity-optimized and afucosylated CD19 mAb with enhanced antibody-dependent cellular cytotoxicity (ADCC). The results from in vitro ADCC assays with Natural Killer cells as effector cells, demonstrate that MEDI-551 is effective at lower mAb doses than rituximab with multiple cell lines as well as primary chronic lymphocytic leukaemia and acute lymphoblastic leukaemia samples. Targeting CD19 with MEDI-551 was also effective in several severe combined immunodeficiency lymphoma models. Furthermore, the combination of MEDI-551 with rituximab resulted in prolonged suppression of tumour growth, demonstrating that therapeutic mAbs with overlapping effector function can be combined for greater tumour growth inhibition. Together, the data demonstrate that MEDI-551 has potent antitumour activity in preclinical models of B cell malignancies. The results also suggest that the combination of the ADCC-enhanced CD19 mAb with an anti-CD20 mAb could be a novel approach for the treatment of B cell lymphomas.     

Characterization of a humanized IgG4 anti-HLA-DR monoclonal antibody that lacks effector cell functions but retains direct antilymphoma activity and increases the potency of rituximab

HLA-DR is under investigation as a target for monoclonal antibody (mAb) therapy of malignancies. Here we describe a humanized IgG4 form of the anti-HLA-DR mAb L243, hL243gamma4P (IMMU-114), generated to provide an agent with selectivity toward neoplastic cells that can kill without complement-dependent cytotoxicity (CDC) or antibody-dependent cellular-cytotoxicity (ADCC), so as to reduce reliance on intact immunologic systems in the patient and effector mechanism-related toxicity. In vitro studies show that replacing the Fc region of hL243gamma1, a humanized IgG1 anti-HLA-DR mAb, with the IgG4 isotype abrogates the effector cell functions of the antibody (ADCC and CDC) while retaining its antigen-binding properties, antiproliferative capacity (in vitro and in vivo), and the ability to induce apoptosis concurrent with activation of the AKT survival pathway. Growth inhibition was evaluated compared with and in combination with the anti-CD20 mAb rituximab, with the combination being more effective than rituximab alone in inhibiting proliferation. Thus, hL243gamma4P is indistinguishable from hL243gamma1 and the parental murine mAb in assays dependent on antigen recognition. The abrogation of ADCC and CDC, which are believed to play a major role in side effects of mAb therapy, may make this antibody an attractive clinical agent. In addition, combination of hL243gamma4P with rituximab offers the prospect for improved patient outcome.     

Two pathways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+ CD56+ killer cells

Cytokine-induced killer (CIK) cells are non-major histocompatibility complex-restricted cytotoxic cells generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody (MoAb), interleukin-2 (IL-2), IL-1, and interferon-gamma. Cells with the greatest effector function in CIK cultures coexpress CD3 and CD56 surface molecules. CIK cell cytotoxicity can be blocked by MoAbs directed against the cell surface protein leukocyte function associated antigen-1 but not by anti-CD3 MoAbs. CIK cells undergo release of cytoplasmic cytotoxic granule contents to the extracellular space upon stimulation with anti-CD3 MoAbs or susceptible target cells. Maximal granule release was observed from the CD3+ CD56+ subset of effector cells. The cytoplasmic granule contents are lytic to target cells. Treatment of the effector cells with a cell-permeable analog of cyclic adenosine monophosphate (cAMP) inhibited anti-CD3 MoAb and target cell- induced degranulation and cytotoxicity of CIK cells. The immunosuppressive drugs cyclosporin (CsA) and FK506 inhibited anti-CD3- mediated degranulation, but did not affect cytotoxicity of CIK cells against tumor target cells. In addition, degranulation induced by target cells was unaffected by CsA and FK506. Our results indicate that two mechanisms of cytoplasmic granule release are operative in the CD3+ CD56+ killer cells; however, cytotoxicity proceeds through a cAMP- sensitive, CsA- and FK506-insensitive pathway triggered by yet-to-be- identified target cell surface molecules.     

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)     

Comparison of the in vitro effects of the anti-CD20 antibodies rituximab and GA101 on chronic lymphocytic leukaemia cells

The effects of two CD20 antibodies, namely rituximab, the current standard for treatment of chronic lymphocytic leukaemia (CLL) in combination with chemotherapy, and GA101, a glyco-engineered type II antibody were compared on CLL cells ex vivo. Antibody-induced phosphatidylserine exposure was examined in isolated CLL cells. For a more comprehensive assessment of antibody-mediated cell killing including Fc-mediated mechanisms, B cell depletion from whole blood samples was monitored. Treatment with rituximab or GA101 reduced the average viability of isolated CLL cells by 6% or 11%, and the ratio of B to T cells in whole blood samples by 12% or 33%, respectively. Combination with GA101 enhanced the cytotoxicity of the chemotherapeutic agent chlorambucil on isolated CLL cells. CD20 surface expression on CLL cells correlated with GA101-induced B cell depletion, but not with direct cell death induction. Treatment of whole blood samples from CLL patients with a CpG-containing oligonucleotide increased CD20 expression on CLL cells and GA101-dependent B cell depletion. Despite the variable responses of individual CLL samples, the CLL cell depletion from whole blood by GA101 was consistently much stronger than by rituximab, which argues for clinical investigation of GA101 in CLL patients.     

Rituximab blocks binding of radiolabeled anti-CD20 antibodies (Ab) but not radiolabeled anti-CD45 Ab

Rituximab therapy is associated with a long in vivo persistence, yet little is known about the effect of circulating rituximab on B-cell non-Hodgkin lymphoma (B-NHL) targeting by the other available anti-CD20 monoclonal antibodies (MoAbs) ¹³¹iodine-tositumomab and ⁹⁰yttrium-ibritumomab tiuxetan. Therefore we assessed the impact of preexisting rituximab on the binding and efficacy of second anti-CD20 MoAbs to B-NHL and determined whether targeting an alternative lymphoma-associated antigen, CD45, could circumvent this effect. We demonstrated that rituximab concentrations as low as 5 µg/mL nearly completely blocked the binding of a second anti-CD20 MoAbs (P < .001), but had no impact on CD45 targeting (P = .89). Serum from patients with distant exposures to rituximab also blocked binding of anti-CD20 MoAbs to patient-derived rituximab-naive B-NHL at concentrations at low as 7 µg/mL, but did not affect CD45 ligation. A mouse xenograft model (Granta, FL-18, Ramos cell lines) showed that rituximab pretreatment significantly reduced B-NHL targeting and tumor control by CD20-directed radioimmunotherapy (RIT), but had no impact on targeting CD45. These findings suggest that circulating rituximab impairs the clinical efficacy of CD20-directed RIT, imply that novel anti-CD20 MoAbs could also face this same limitation, and indicate that CD45 may represent an alternative target for RIT in B-NHL.     

Characterization of in vitro migratory properties of anti-CD19 chimeric receptor-redirected CIK cells for their potential use in B-ALL immunotherapy

OBJECTIVE: Cytokine-induced killer (CIK) cells are ex vivo expanded cells enriched in CD3(+)CD56(+) natural killer T (NKT) cells with major histocompatibility-unrestricted cytotoxicity against several tumoral targets, except B-lineage acute lymphoblastic leukemia (B-ALL). We redirected CIK cells cytotoxicity toward B-ALL with a chimeric receptor specific for the CD19 antigen and then explored if modified-CIK cells maintain the same chemotactic properties of freshly isolated NKT cells, whose trafficking machinery reflects their preferential localization into the sites of B-ALL infiltration. MATERIAL AND METHODS: CIK cells were expanded ex vivo for 21 days and analyzed for expression of adhesion molecules and chemokine receptors regulating adhesion and homing toward leukemia-infiltrated tissues. CIK cells were then transduced with the anti-CD19-zeta-internal ribosomal entry site-green fluorescent protein retroviral vector and characterized for their cytotoxicity against B-ALL cells in a (51)Cr-release assay and for their trafficking properties, including chemotactic activity, adhesion and transendothelial migration, and metalloproteases-dependent invasion of Matrigel. RESULTS: Similarly to freshly isolated NKT cells, CD49d and CD11a were highly expressed on CIK cells. Moreover, CIK cells expressed CXCR4, CCR6, and CCR7 (mean expression 72%, 60%, and 32%, respectively), presenting chemotactic activity toward their respective ligands. Anti-CD19 chimeric receptor-modified CIK cells became cytotoxic against B-ALL cells (mean lysis, 60%) and showed, after exposure to a CXCL12 gradient, high capacity to adhere and transmigrate through endothelial cells and to invade Matrigel. CONCLUSION: The potential capacity to localize into leukemia-infiltrated tissues of anti-CD19 chimeric receptor-redirected CIK cells, together with their ability to efficiently kill B-ALL cells, suggests that modified-CIK cells represent a valuable tool for leukemia immunotherapy.     

Requirements for the promotion of allogeneic engraftment by anti-CD154 (anti-CD40L) monoclonal antibody under nonmyeloablative conditions.

The promotion of alloengraftment in the absence of global immune suppression and multiorgan toxicity is a major goal of transplantation. It is demonstrated that the infusion of a single modest bone marrow dosage in 200 cGy-irradiated recipients treated with anti-CD154 (anti-CD40L) monoclonal antibody (mAb) resulted in chimerism levels of 48%. Reducing irradiation to 100 or 50 cGy permitted 24% and 10% chimerism, respectively. In contrast, pan-T-cell depletion resulted in only transient engraftment in 200 cGy-irradiated recipients. Host CD4(+) cells were essential for alloengraftment as depletion of CD4(+) cells abrogated engraftment in anti-CD154-treated recipients. Strikingly, the depletion of CD8(+) cells did not further enhance engraftment in anti-CD154 mAb-treated recipients in a model in which rejection is mediated by both CD4(+) and CD8(+) T cells. However, anti-CD154 mAb did facilitate engraftment in a model in which only CD8(+) T cells mediate rejection. Furthermore, CD154 deletional mice irradiated with 200 cGy irradiation were not tolerant of grafts, suggesting that engraftment promotion by anti-CD154 mAb may not simply be the result of CD154:CD40 blockade. Together, these data suggest that a CD4(+) regulatory T cell may be induced by anti-CD154 mAb. In contrast to anti-CD154 mAb, anti-B7 mAb did not promote donor engraftment. Additionally, the administration of either anti-CD28 mAb or anti-CD152 (anti-CTLA-4) mAb or the use of CD28 deletional recipients abrogated engraftment in anti-CD154 mAb-treated mice, suggesting that balanced CD28/CD152:B7 interactions are required for the engraftment-promoting capacity of anti-CD154 mAb. These data have important ramifications for the design of clinical nonmyeloablative regimens based on anti-CD154 mAb administration.     

CD48 as a novel molecular target for antibody therapy in multiple myeloma

Monoclonal antibody (mAb) drugs are desirable for the improvement of multiple myeloma (MM) treatment. In this study, we found for the first time that CD48 was highly expressed on MM plasma cells. In 22 out of 24 MM patients, CD48 was expressed on more than 90% of MM plasma cells at significantly higher levels than it was on normal lymphocytes and monocytes. CD48 was only weakly expressed on some CD34(+) haematopoietic stem/progenitor cells, and not expressed on erythrocytes or platelets. We next examined whether CD48 could serve as a target antigen for mAb therapy against MM. A newly generated in-house anti-CD48 mAb induced mild antibody-dependent cell-mediated cytotoxicity and marked complement-dependent cytotoxicity against not only MM cell lines but also primary MM plasma cells in vitro. Administration of the anti-CD48 mAb significantly inhibited tumour growth in severe combined immunodeficient mice inoculated subcutaneously with MM cells. Furthermore, anti-CD48 mAb treatment inhibited growth of MM cells transplanted directly into murine bone marrow. Finally and importantly, we demonstrated that the anti-CD48 mAb did not damage normal CD34(+) haematopoietic stem/progenitor cells. These results suggest that the anti-CD48 mAb has the potential to become an effective therapeutic mAb against MM.     

The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism

Antibodies against CD20 can activate complement and induce antibody-dependent cellular cytotoxicity (ADCC) in B lymphocytes. In B-cell lines, such antibodies also induce apoptosis. In this study, the expression and function of CD20 on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed. Flow cytometric analysis demonstrated that B-CLL cells express CD20 with a fluorescence intensity that is significantly weaker than that of normal CD5(+) and CD5(-) B cells and that of malignant CD5(-) low-grade non-Hodgkin lymphoma cells. A small population of cells from healthy donors that have an expression pattern of CD5 and CD20 identical to that of B-CLL cells were identified, and this population was confirmed to be of T lineage, not B lineage. Culture of freshly isolated B-CLL cells in the presence of the chimeric anti-CD20 antibody rituximab and a cross-linking F(ab)(2) fragment, resulted in dose- and time-dependent induction of apoptosis. The induction of apoptosis occurred under conditions in which the influence of complement activation and ADCC was negligible. Cross-linking of rituximab induced strong and sustained phosphorylation of the 3 mitogen activated protein (MAP) kinases c-Jun NH2-terminal protein kinase, extracellular signal-regulated kinase, and p38. Introduction of the p38 inhibitor SB203580 into the system completely blocked signaling downstream of p38, as evidenced by the absence of MAPKAP K2 activity, and significantly reduced the degree of anti-CD20-induced apoptosis. These results demonstrate that cross-linking of rituximab bound to CD20 on freshly isolated B-CLL cells induces apoptosis through a signaling pathway that is dependent on p38 MAP-kinase activation.     

γδ T-cell killing of primary follicular lymphoma cells is dramatically potentiated by GA101, a type II glycoengineered anti-CD20 monoclonal antibody

Background

Anti-CD20 monoclonal antibodies are major therapeutic agents for patients with follicular lymphoma and work through complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. Optimization of antibody-dependent cellular cytotoxicity, in particular by amplifying its effectors, could further increase the efficacy of anti-CD20 monoclonal antibodies.

Design and Methods

We investigated the cytotoxic activity of Vγ9Vδ2 T cells against follicular lymphoma cells and whether this killing could be increased by promoting antibody-dependent cellular cytotoxicity with anti-CD20 monoclonal antibodies, in particular a type-II glycoengineered anti-CD20. Vγ9Vδ2 T cells were expanded in vitro in the presence of bromohydrin pyrophosphate (Phosphostim) and interleukin-2 and their ability to kill follicular lymphoma primary cells or cell lines was evaluated by flow cytometry cytotoxic T-lymphocyte assays in the presence or absence of three anti-CD20 monoclonal antibodies: the afucosylated GA101, the chimeric rituximab or the humanized ofatumumab. The ability of these cells to release perforin/granzyme and secrete interferon-γ when co-cultured with follicular lymphoma primary cells or cell lines in the presence or not of the three anti-CD20 monoclonal antibodies was also evaluated by CD107a staining and Elispot assays.

Results

Phosphostim and interleukin-2 expanded Vγ9Vδ2 T cells were cytotoxic to primary follicular lymphoma cells and their cytotoxic potential was dramatically increased by GA101, a type II glycoengineered anti-CD20 monoclonal antibody, and to a lesser extent, by rituximab and ofatumumab. The increased cytotoxicity was associated with increased secretion of perforin/granzyme and interferon-γ.

Conclusions

In-vitro expanded Vγ9Vδ2 T cells efficiently kill primary follicular lymphoma cells and express CD16; anti-CD20 monoclonal antibodies, in particular GA101, dramatically increase the cytotoxic activity of expanded Vγ9Vδ2 T cells. These preclinical results prompt the development of clinical trials using this antibody dependent cellular cytotoxicity property of Vγ9Vδ2 T cells and anti-CD20 monoclonal antibodies.     

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.