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Drug-free macromolecular therapeutics (DFMT) utilizes modified monoclonal antibodies (or antibody fragments) to generate antigen-crosslinking-induced apoptosis in target cells. DFMT is a two-component system containing a morpholino oligonucleotide (MORF1) modified antibody (Ab-MORF1) and human serum albumin conjugated with multiple copies of complementary morpholino oligonucleotide (MORF2), (HSA-(MORF2) x ). The two components recognize each other via the Watson-Crick base pairing complementation of their respective MORFs. One HSA-(MORF2) x molecule can hybridize with multiple Ab-MORF1 molecules on the cell surface, thus serving as the therapeutic crosslink-inducing mechanism of action. Herein, various anti-neoplastic agents in combination with the anti-CD20 Obinutuzumab (OBN)-based DFMT system are examined. Three different classes of chemotherapies are examined: DNA alkylating agents; proliferation pathway inhibitors; and DNA replication inhibitors. Chou-Talalay combination index mathematics is utilized to determine which drugs engaged synergistically with OBN-based DFMT. It is determined that OBN-based DFMT synergizes with topoisomerase inhibitors and DNA nucleotide analogs but is antagonistic with proliferation pathway inhibitors. Cell mechanism experiments are performed to analyze points of synergism or antagonism by investigating Ca 2+ influx, mitochondrial health, lysosomal stability, and cell cycle arrest. Finally, the synergistic drug combinatorial effects of OBN-based DFMT with etoposide in vivo are demonstrated using a human xenograft non-Hodgkin's lymphoma mouse model., (© 2023 Wiley-VCH GmbH.)

Abstract: CD20 is an established therapeutic target in B-cell malignancies. The CD20 × CD3 bispecific antibody mosunetuzumab has significant efficacy in B-cell non-Hodgkin lymphomas (NHLs). Because target antigen loss is a recognized mechanism of resistance, we evaluated CD20 expression relative to clinical response in patients with relapsed and/or refractory NHL in the phase 1/2 GO29781 trial investigating mosunetuzumab monotherapy. CD20 was studied using immunohistochemistry (IHC), RNA sequencing, and whole-exome sequencing performed centrally in biopsy specimens collected before treatment at predose, during treatment, or upon progression. Before treatment, most patients exhibited a high proportion of tumor cells expressing CD20; however, in 16 of 293 patients (5.5%) the proportion was <10%. Analyses of paired biopsy specimens from patients on treatment revealed that CD20 levels were maintained in 29 of 30 patients (97%) vs at progression, where CD20 loss was observed in 11 of 32 patients (34%). Reduced transcription or acquisition of truncating mutations explained most but not all cases of CD20 loss. In vitro modeling confirmed the effects of CD20 variants identified in clinical samples on reduction of CD20 expression and missense mutations in the extracellular domain that could block mosunetuzumab binding. This study expands the knowledge about the occurrence of target antigen loss after anti-CD20 therapeutics to include CD20-targeting bispecific antibodies and elucidates mechanisms of reduced CD20 expression at disease progression that may be generalizable to other anti-CD20 targeting agents. These results also confirm the utility of readily available IHC staining for CD20 as a tool to inform clinical decisions. This trial was registered at www.ClinicalTrials.gov as #NCT02500407., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Monoclonal antibody (mAb) therapy directed against CD20 is an important tool in the treatment of B cell disorders. However, variable patient response and acquired resistance remain important clinical challenges. To identify genetic factors that may influence sensitivity to treatment, the cytotoxic activity of three CD20 mAbs: rituximab; ofatumumab; and obinutuzumab, were screened in high-throughput assays using 680 ethnically diverse lymphoblastoid cell lines (LCLs) followed by a pharmacogenomic assessment. GWAS analysis identified several novel gene candidates. The most significant SNP, rs58600101, in the gene MKL1 displayed ethnic stratification, with the variant being significantly more prevalent in the African cohort and resulting in reduced transcript levels as measured by qPCR. Functional validation of MKL1 by shRNA-mediated knockdown of MKL1 resulted in a more resistant phenotype. Gene expression analysis identified the developmentally associated TGFB1I1 as the most significant gene associated with sensitivity. qPCR among a panel of sensitive and resistant LCLs revealed immunoglobulin class-switching as well as differences in the expression of B cell activation markers. Flow cytometry showed heterogeneity within some cell lines relative to surface Ig isotype with a shift to more IgG + cells among the resistant lines. Pretreatment with prednisolone could partly reverse the resistant phenotype. Results suggest that the efficacy of anti-CD20 mAb therapy may be influenced by B cell developmental status as well as polymorphism in the MKL1 gene. A clinical benefit may be achieved by pretreatment with corticosteroids such as prednisolone followed by mAb therapy.

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene ( MS4A1 ) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors., (© 2017 by The American Society of Hematology.)

Treatment response of follicular lymphomas (FL) is highly variable. We, therefore, investigated the role of FL cancer-associated fibroblasts (CAFs) on tumor cell viability, in particular in response to treatment with cytotoxic drugs. Stromal cells outgrown from FL patients were characterized and pure CAF populations were co-cultivated with FL cells. To analyze fibroblast-mediated effects, cells in co-culture were treated with ABT-737 and Bortezomib. The adherent cell population was positive for all fibroblastic markers tested and showed increased mRNA-expression of the activation marker FAP. No effect on FL cell viability was noted when co-cultivating them with CAFs. However, stromal cells protected tumor cells from apoptosis in response to cytotoxic treatment. This might be explained by mRNA-induction of ABCC1 and ABCG2 and up-regulation of BCL2L1 in FL cells. Our finding of protective mechanisms mediated by CAFs is of pivotal impact for further studies of cytotoxic agents in FL.

In spite of newly emerging therapies and the improved survival of patients with non-Hodgkin lymphoma (NHL), relapses or primary refractory disease are commonly observed and associated with dismal prognosis. Although discovery of the anti-CD20 antibody rituximab has markedly improved outcomes in B-cell NHL, rituximab resistance remains an important obstacle to successful treatment of these tumors. To improve the efficacy of CD20-targeted therapy, we fused interleukin 21 (IL-21), which induces direct lymphoma cytotoxicity and activates immune effector cells, to the anti-CD20 antibody (αCD20-IL-21 fusokine). We observed substantially enhanced IL-21R-mediated signaling by the fusokine compared with native IL-21 at equimolar concentrations. Fusokine treatment led to direct apoptosis of lymphoma cell lines and primary tumors that otherwise were resistant to native IL-21 treatment. In addition to direct cytotoxicity, the fusokine enhanced NK cell activation, effector functions, and interferon γ production, resulting in greater antibody-dependent cell-mediated cytotoxicity compared with IL-21 and/or anti-CD20 antibody treatments. Further, the αCD20-IL-21 fusokine stabilizes IL-21 and prolongs its half-life. In vivo αCD20-IL-21 therapy resulted in a significant tumor control in the rituximab-resistant A20-huCD20 tumors. Collectively, the dual functional ability of the αCD20-IL-21 fusokine to induce direct apoptosis and activate immune effector cells may provide benefit over existing treatments for NHL., (© 2017 by The American Society of Hematology.)

Obinutuzumab (GA101) is a type II, glycoengineered anti-CD20 monoclonal antibody for the treatment of hematologic malignancies. Obinutuzumab has mechanisms of action that are distinct from those of rituximab, potentially translating into improved clinical efficacy. We present the pharmacokinetic and clinical data from the phase I/II GAUGUIN and phase I GAUDI studies that were used to identify the obinutuzumab dose and regimen undergoing phase III assessment. In phase I (GAUGUIN and GAUDI), non-Hodgkin lymphoma patients received up to a maximum 9 fixed doses (obinutuzumab 50-2000 mg). In GAUGUIN phase II, patients received obinutuzumab 400/400 mg or 1600/800 mg [first dose day (D)1, D8, cycle (C) 1; second dose D1, C2-C8]. The influence of demographic factors on pharmacokinetics and drug exposure on tumor response and toxicity were analyzed using exploratory graphical analyses. Obinutuzumab serum concentrations with 1600/800 mg were compared with a 1000 mg fixed-dose regimen (D1, D8 and D15, C1; D1, C2-C8) using pharmacokinetic modeling simulations. Factors related to CD20-antigenic mass were more influential on obinutuzumab pharmacokinetics with 400/400 versus 1600/800 mg. Higher serum concentrations were observed with 1600/800 versus 400/400 mg, irrespective of CD20-antigenic mass. Tumor shrinkage was greater with 1600/800 versus 400/400 mg; there was no significant increase in adverse events. Fixed dose 1000 mg with an additional C1 infusion resulted in similar serum concentrations to 1600/800 mg in model-based analyses. The obinutuzumab 1000 mg fixed-dose regimen identified in this exploratory analysis was confirmed in a full covariate analysis of a larger dataset, and is undergoing phase III evaluation. GAUGUIN and GAUDI are registered at www.clinicaltrials.gov (clinicaltrials.gov identifier:00517530 and 00825149, respectively)., (Copyright© Ferrata Storti Foundation.)

CD20 is a differentiation related cell surface phosphoprotein that is expressed during early pre-B cell stages until plasma cell differentiation, and is a suitable molecular target for B-cell malignancies by monoclonal antibodies such as rituximab, ofatumumab, obinutuzumab and others. CD20 expression is confirmed in most B-cell malignancies; however, the protein expression level varies in each patient, even in de novo tumors, and down-modulation of CD20 expression after chemoimmunotherapy with rituximab, resulting in rituximab resistance, has been recognized in the clinical setting. Recent reports suggest that genetic and epigenetic mechanisms are correlated with aberrantly low CD20 expression in de novo tumors and relapsed/refractory disease after using rituximab. Furthermore, some targeting drugs, such as lenalidomide, bortezomib and ibrutinib, directly or indirectly affect CD20 protein expression. CD20-negative phenotypically-changed DLBCL after rituximab use tends to show an aggressive clinical course and poor outcome with resistance to not only rituximab, but also conventional salvage chemo-regimens. Understanding of the mechanisms of CD20-negative phenotype may contribute to establish strategies for overcoming chemo-refractory B-cell malignancies. In this manuscript, recent progress of research on molecular and clinical features of CD20 protein and CD20-negative B-cell malignancies was reviewed.

Purpose: Clinical trials of ibrutinib combined with anti-CD20 monoclonal antibodies (mAb) for chronic lymphocytic leukemia (CLL) report encouraging results. Paradoxically, in preclinical studies, in vitro ibrutinib was reported to decrease CD20 expression and inhibit cellular effector mechanisms. We therefore set out to investigate effects of in vivo ibrutinib treatment that could explain this paradox., Experimental Design: Patients received single-agent ibrutinib (420 mg daily) on an investigator-initiated phase II trial. Serial blood samples were collected pretreatment and during treatment for ex vivo functional assays to examine the effects on CLL cell susceptibility to anti-CD20 mAbs., Results: We demonstrate that CD20 expression on ibrutinib was rapidly and persistently downregulated (median reduction 74%, day 28, P < 0.001) compared with baseline. Concomitantly, CD20 mRNA was decreased concurrent with reduced NF-κB signaling. An NF-κB binding site in the promoter of MS4A1 (encoding CD20) and downregulation of CD20 by NF-κB inhibitors support a direct transcriptional effect. Ex vivo, tumor cells from patients on ibrutinib were less susceptible to anti-CD20 mAb-mediated complement-dependent cytotoxicity than pretreatment cells (median reduction 75%, P < 0.001); however, opsonization by the complement protein C3d, which targets cells for phagocytosis, was relatively maintained. Expression of decay-accelerating factor (CD55) decreased on ibrutinib, providing a likely mechanism for the preserved C3d opsonization. In addition, ibrutinib significantly inhibited trogocytosis, a major contributor to antigen loss and tumor escape during mAb therapy., Conclusions: Our data indicate that ibrutinib promotes both positive and negative interactions with anti-CD20 mAbs, suggesting that successfully harnessing maximal antitumor effects of such combinations requires further investigation., (©2015 American Association for Cancer Research.)

This review is focused on the promises of IgA as a new therapeutic antibody. For more than 30 years IgG molecules have been used in the clinic in the fields of oncology, hematology, auto immune diseases and infections. However, IgA might be a good alternative, since it recruits different effector cells, i.e. polymorphonuclear cells or neutrophils, but can also activate monocytes and macrophages. The present knowledge, but also future direction for IgA- based drugs are discussed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Rituximab is a chimeric monoclonal antibody containing constant kappa-regions of the Fc fragment of IgGI human immunoglobulin and variable fragments of IgG murine immunoglobulin. This modified immunoglobulin structure makes rituximab capable of specifically recognizing CD20 antigen that is mainly expressed on B-lymphocytes. The review considers four main mechanisms of rituximab action leading to reduction of the population of B-lymphocytes in patients with lymphoproliferative processes. All these mechanisms are probably operative under clinical conditions, however, their relative contributions to the clinical effect of rituximab can vary and are yet not defined in full.

Background: The sensitivity of human Burkitt's lymphoma cells to rituximab (Rtx) and tositumomab (Tst) was assessed on cells expressing different levels of CD20 on surface. Cells that harbor low CD20 levels may resists against therapeutics response to CD20-specific antibodies. We postulated that, radiation-induced modulation of CD20 surface levels may play a crucial and central role in determining the relative efficacy of rituximab and tositumomab in treating Burkitt's lymphoma disease. Here, we examined the γ-radiation-induced CD20 expression in the Burkitt lymphoma cell line 'Daudi' and the relation of differential levels of CD20 with anti-CD20 mAbs mediated cell death., Methodology: In this study we examined kinetics of CD20 expression following sub lethal doses ofγ-radiation to Daudi cells and thereafter anti-CD20 mAbs (rituximab and tositumomab) were added in cell suspensions. The correlation of kinetics of CD20 expression and cells treated with anti-CD20 mAbs/or corresponding isotype Abs with special reference to changes in mitochondrial membrane potential and reactive oxygen species generation was also examined. Further, we also investigated the efficacy of anti-CD20 mAbs and possible induction of cell death in relation to levels of CD20 cell surface expression., Conclusion: This report provides evidence that CD20 expression can be induced by exposure of cells to γ-radiation. In addition, these findings demonstrated that the efficacy of anti-CD20 mAbs is dependent on the surface levels of CD20. Based on these findings, we hypothesized (i) irradiation just prior to immunotherapy may provide new treatment options even in aggressive B cell tumors, which are resistant to current therapies in vivo (ii) The efficacy of induction of apoptosis varies with type of monoclonal antibodies in vitro.

CD20 is an attractive therapeutic target given the success of its monoclonal antibody, Rituximab, in the treatment of B-cell malignancies and B-cell-mediated autoimmune diseases. Treatment with Rituximab causes a rapid depletion of B cells and a decrease in disease symptoms. Despite the clinical efficiency of Rituximab, its mechanism of action is not completely understood. In this study, we aimed at further investigating the Rituximab-induced cell death and the factors affecting such responses. Our results indicate that Rituximab-induced cell death depends on the nature of the cells and levels of CD20 expression on the cell surface. Coexpression of CD20 with CD40, a member of the TNF receptor family that is known to be physically associated with CD20 on the cell surface, enhances the apoptotic response induced by Rituximab. Inhibiting the formation of CD40 disulfide-bound-homodimers, a process required for some CD40 signaling, further enhances Rituximab-induced cell death. Cell death induced by anti-CD40 mAb is also upregulated by the presence of CD20, suggesting a bidirectional influence of the CD20/CD40 association. Moreover, treating cells with both anti-CD20 and anti-CD40 antibodies improves the cell death response induced by a single-agent treatment. These results highlight the role of the CD20/CD40 association in triggering B-cell depletion and may pave the way for an alternative more efficient therapeutic strategy in treating B-cell-mediated disorders., (© The Japanese Society for Immunology. 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

CD20 is expressed in most B-cell lymphomas and is a critical molecular target of rituximab. Some B-cell lymphomas show aberrant CD20 expression, and rituximab use in these patients is controversial. Here we show both the molecular mechanisms and the clinical significance of de novo diffuse large B-cell lymphomas (DLBCL) that show a CD20 immunohistochemistry (IHC)-positive and flow cytometry (FCM)- negative (IHC[+]/FCM[-]) phenotype. Both IHC and FCM using anti-CD20 antibodies L26 and B1, respectively, were analyzed in 37 of the 106 cases of de novo DLBCL; 8 (22%) of these cases were CD79a(+)/CD20(+) with IHC and CD19(+)/CD20(-) with FCM. CD20 (MS4A1) mRNA expression was significantly lower in IHC(+)/FCM(-) cells than in IHC(+)/FCM(+) cells (P = 0.0005). No genetic mutations were detected in MS4A1 promoter and coding regions. Rituximab-mediated cytotoxicity in the CDC assay using IHC(+)/FCM(-) primary cells was significantly lower than in IHC(+)/FCM(+) cells (P < 0.05); however, partial effectiveness was confirmed. FCM using rituximab detected CD20 more efficiently than B1. No significant difference was observed between IHC(+)/FCM(-) and IHC(+)/FCM(+) patients in overall survival (P = 0.664). Thus, lower expression of CD20 mRNA is critical for the CD20 IHC(+)/FCM(-) phenotype. Lower CD20 expression with FCM does not rule out rituximab use in these patients if expression is confirmed with IHC. FCM using rituximab may be more informative than B1 for predicting rituximab effectiveness in IHC(+)/FCM(-) cases., (© 2013 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Few studies have statistically investigated reduced CD20 expression in B-cell lymphoma after rituximab therapy and genomic mutation of CD20 associated with reduction. We examined CD20-positive rate in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) by flow cytometry (FCM) and immunohistochemical staining (IHS), comparing 138 cases after rituximab therapy with 360 initial, not yet treated cases. Sequence analysis of exons 3 to 8 of CD20 was performed on 22 cases with low CD20-positive rate after rituximab treatment. The results showed a statistical correlation between CD20-positive rate in FCM and IHS. By FCM, the CD20-positive rate among post-rituximab cases was significantly lower than among initial cases in DLBCL, non-germinal center origin B-cell type (average values [avg] 57.8 and 87.9, respectively) (P < 0.0001), FL2 (avg, 93.9; 103.2) (P = 0.0083), and FL3A (avg, 90.6; 100.7) (P = 0.033). Stratified analyses of post-rituximab cases showed significantly lower CD20-positive rate in cases that were resistant at the start of the treatment and cases with progressive disease during rituximab therapy before biopsy. Sequence analysis showed silent mutation of exon 4 (632 C/T) in seven cases, although this number was not statistically significant. These results suggest the influence of B-lymphoma subtype and a therapeutic effect before biopsy on CD20 expression at relapse and contribute to a better therapeutic approach for relapse cases after rituximab therapy., (© 2012 Japanese Cancer Association.)

Purpose: The aim of this research was to further investigate the contribution of CD20 antigen expression to rituximab activity and define the mechanisms responsible for CD20 downregulation in rituximab-resistant cell lines (RRCL)., Experimental Design: Rituximab-sensitive cell lines, RRCL, and primary neoplastic B cells were evaluated by chromium-51 release assays, ImageStream image analysis, immunohistochemical staining, flow cytometric analysis, CD20 knockdown, promoter activity, chromatin immunoprecipitation (ChIP) analysis of CD20 promoter, and CD20 plasmid transfection experiments to identify mechanisms associated with CD20 regulation in RRCL., Results: RRCL exhibited a gradual loss of CD20 surface expression with repeated exposure to rituximab. We identified a CD20 antigen surface threshold level required for effective rituximab-associated complement-mediated cytotoxicity (CMC). However, a direct correlation between CD20 surface expression and rituximab-CMC was observed only in rituximab-sensitive cell lines. CD20 promoter activity was decreased in RRCL. Detailed analysis of various CD20 promoter fragments suggested a lack of positive regulatory factors in RRCL. ChIP analysis showed reduced binding of several key positive regulatory proteins on CD20 promoter in RRCL. Interleukin-4 (IL-4) induced higher CD20 promoter activity and CD20 expression but modestly improved rituximab activity in RRCL and in primary B-cell lymphoma cells. Forced CD20 expression restored cytoplasmic but not surface CD20, suggesting the existence of a defect in CD20 protein transport in RRCL., Conclusions: We identified several mechanisms that alter CD20 expression in RRCL and showed that, whereas CD20 expression is important for rituximab activity, additional factors likely contribute to rituximab sensitivity in B-cell lymphoma., (©2012 AACR.)

Ofatumumab is a new monoclonal antibody (mAb) targeting a novel membrane-proximal epitope on CD20. To better define ofatumumab's activity, we conducted pre-clinical studies in rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL), ofatumumab-exposed cell lines (OECLs), primary lymphoma cells, and a lymphoma xenograft model. RRCL and OECL were generated by repeated exposure of sensitive cells to escalating doses of rituximab or ofatumumab ± human serum. Antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) assays were performed to assess cellular sensitivity to rituximab or ofatumumab. Ofatumumab elicited a higher rate of CMC in RSCL, RRCL and primary tumour cells. The chronic exposure of lymphoma cells to ofatumumab resulted in rituximab resistance but less ofatumumab resistance. In an in vivo severe combined immunodeficiency mouse model of human lymphoma, ofatumumab prolonged median survival compared to rituximab. While rituximab CMC diminished with CD20 down-regulation in RRCL passages, ofatumumab activity in vitro diminished to a lesser degree. Our data suggest that ofatumumab is more potent than rituximab in rituximab-sensitive or rituximab-resistant models and has the potential to decrease the development of biological resistance in patients with repeated exposure to anti-CD20 mAbs., (© 2011 Blackwell Publishing Ltd.)

Plitidepsin (Aplidin), an antitumor agent of marine origin, presently is undergoing phase II/III clinical trials, and has shown promise for the treatment of lymphoma. Here, we describe the antitumor effects of plitidepsin alone and in combination with rituximab and investigated the effects of each drug and the combination on the cell cycle and mechanism of cell death. Several Diffuse Large Cell Lymphoma (DLCL) lines and Burkitt cell lines were tested for sensitivity to plitidepsin and rituximab. All DLCL and Burkitt lymphoma cell lines were inhibited by plitidepsin in nanomolar concentrations, while rituximab sensitivity varied among different cell lines. Ramos and the RL cell lines proved sensitive to rituximab and were used to test the effects of each of the two drugs. The two agents exhibited synergism at all tested concentrations. For in vivo studies, irradiated athymic nude mice were engrafted with the Ramos lymphoma. Treatment was initiated when the tumors were ~0.5 cm in diameter, and toxic and therapeutic effects were monitored. In the in vivo study, additive effects of the combined two drugs, was demonstrated without an increase in host toxicity. The in vitro synergy and the in vivo additive antitumor effects without an increase in host toxicity with two relatively non-marrow suppressive agents encourages further development of this combination for treatment of aggressive B-cell lymphomas.

Unresponsiveness to rituximab treatment develops in many patients prompting elucidation of underlying molecular pathways. It was recently observed that rituximab-resistant lymphoma cells exhibit up-regulation of components of the ubiquitin-proteasome system (UPS). Therefore, we investigated in more detail the role of this system in the regulation of CD20 levels and the influence of proteasome inhibitors on rituximab-mediated complement-dependent cytotoxicity (R-CDC). We observed that incubation of Raji cells with rituximab leads to increased levels of ubiquitinated CD20. However, inhibition of the UPS was not associated with up-regulation of surface CD20 levels, although it significantly increased its ubiquitination. Short-term (24 hours) incubation of Raji cells with 10 or 20 nM bortezomib did not change surface CD20 levels, but sensitized CD20(+) lymphoma cells to R-CDC. Prolonged (48 hours) incubation with 20 nM bortezomib, or incubation with 50 nM bortezomib for 24 hours led to a significant decrease in surface CD20 levels as well as R-CDC. These effects were partly reversed by bafilomycin A1, an inhibitor of lysosomal/autophagosomal pathway of protein degradation. These studies indicate that CD20 levels are regulated by 2 proteolytic systems and that the use of proteasome inhibitors may be associated with unexpected negative influence on R-CDC.

A pilot study previously demonstrated that thrice-weekly, fractionated-dose intravenous rituximab (RTX) limits CD20 loss from chronic lymphocytic leukemia (CLL) B cells, thereby enhancing immunotherapeutic targeting. Here, we investigated the feasibility of giving 20 mg rituximab subcutaneously thrice weekly for up to 12 weeks in 4 previously treated CLL patients. Subcutaneous rituximab was well-tolerated with minimal injection site reactions; a variable degree of efficacy was observed, likely influenced by the size of the patients' B cell/CD20 burden. Subcutaneous RTX largely preserved CD20 expression on leukemic cells but the most effective therapeutic dosing regimen needs to be established (ClinicalTrials.gov Identifier: NCT00366418).

Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st position (Kabat numbering) in CDR3 of the variable heavy chain (V(H)), having aspartic acid (Asp) instead of asparagine (Asn), with framework regions of epratuzumab, a humanized anti-CD22 antibody. When compared with rituximab, veltuzumab has significantly reduced off-rates in 3 human lymphoma cell lines tested, as well as increased complement-dependent cytotoxicity in 1 of 3 cell lines, but no other in vitro differences. Mutation studies confirmed that the differentiation of the off-rate between veltuzumab and rituximab is related to the single amino acid change in CDR3-V(H). Studies of intraperitoneal and subcutaneous doses in mouse models of human lymphoma and in normal cynomolgus monkeys disclosed that low doses of veltuzumab control tumor growth or deplete circulating or sessile B cells. Low- and high-dose veltuzumab were significantly more effective in vivo than rituximab in 3 lymphoma models. These findings are consistent with activity in patients with non-Hodgkin lymphoma given low intravenous or subcutaneous doses of veltuzumab. Thus, changing Asn(101) to Asp(101) in CDR3-V(H) of rituximab is responsible for veltuzumab's lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients.

The anti-CD20 monoclonal antibody rituximab has been less successful in treating chronic lymphocytic leukemia (CLL) than lymphoma, possibly due to the lower density of CD20 on B lymphocytes from CLL patients than on those from lymphoma patients. This lowering may result from insufficiency of one of the transcription factors of cd20. Of these, purine-rich box-1 (PU.1) is poorly expressed in CLL. To estimate its weight in CD20 expression, pu.1 cDNA was transfected into CLL B cells and shown to raise the membrane expression of CD20 and to improve the rituximab-induced lysis of transfected cells. Granulocyte macrophage colony-stimulating factor and all-trans-retinoic acids were not involved in the defective expression of PU.1 or the excessive methylation of the pu.1 gene, because 6 of 14 CLL samples tested were normally methylated. This was confirmed by the failure of DNA methyltransferase inhibitors to restore pu.1 transcription in hypermethylated CLL, and, in fact, the expression of PU.1 was down-regulated by excessive expression of the FMS proto-oncogene-like tyrosine kinase 3 (Flt3) receptor. This abnormality is consistent with our finding of elevated levels of Flt3 ligand (FL) in 20 of 23 CLL sera tested. We propose that FL-dependent increased Flt3 signaling prevents the expression of PU.1, which down-regulates that of CD20, and accounts for resistance of leukemic B cells to rituximab-induced lysis.

Acquirement of resistance to rituximab has been observed in lymphoma patients. To define mechanisms associated with rituximab resistance, we developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 expression/structure, lipid raft domain (LRD) reorganization, calcium mobilization, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity (CMC) between parental and RRCL. Significant changes in surface CD20 antigen expression were shown in RRCL. Decreased calcium mobilization and redistribution of CD20 into LRD were found in RRCL. Western blotting identified a unique 35 kDa protein band in RRCL, which was not seen in parental cells and was secondary to an increase in surface and cytoplasmic expression of IgM light chains. CD20 gene expression was decreased in RRCL. In vitro exposure to PS341 increased CD20 expression in RRCL and minimally improved the sensitivity to rituximab-associated CMC. Our data strongly suggest that the acquisition of rituximab resistance is associated with global gene and protein down-regulation of the CD20 antigen affecting LRD organization and downstream signaling. CD20 expression seems to be regulated at the pretranscriptional and posttranscriptional levels. Proteasome inhibition partially reversed rituximab resistance, suggesting the existence of additional mediators of rituximab resistance. Future research is geared to identify drugs and/or biological agents that are effective against RRCL.

With the introduction of rituximab to chemotherapy in lymphoma, CHOP changed to R-CHOP in elderly, intermediate risk DLBCL (diffuse large B-cell lymphoma) patients. Although the treatment is not yet standard, due to insufficient evidence, in clinical practice it is an R-containing regimen, for example, in mantle cell lymphoma, such as HyperCVAD/MA to R-HyperCVAD/MA. Recently, another group and ours reported the presence of rituximab resistance during R-containing chemotherapy. If the lymphoma is bulky,the overexpression of CD 55 (complement regulatory molecule) leads to resistance to rituximab. When the patients evidenced the loss of CD 20 antigen in refractory/relapsed lymphoma after R-containing therapy, some patients showed the presence of CD 20 point mutation. In the cases of refractory/relapsed cases, radioimmunotherapy or other monoclonal antibodies are prepared, including Zevalin and CD 22, CD 40, CD 74, and HLA-DR targeting antibodies. Not only monoclonal antibodies but also HDACI or bortezomib (NF-kappaB) and other signal inhibitors (for Akt, ERK/MAPK) have been developed. In Japan, we must consider the higher speed of infusion rituximab and we must prepare standard therapy for lymphoma because of recruiting phase I/II clinical trials after use of rituximab for easy entry.

The clinical application of rituximab (chimeric mouse anti-human CD20 mAb, Rituxan, IDEC-C2B8), alone and/or combined with chemotherapy, has significantly ameliorated the treatment outcome of patients with relapsed and refractory low-grade or follicular non-Hodgkin's lymphoma (NHL). The exact in vivo mechanisms of action of rituximab are not fully understood, although antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis have been suggested. We have proposed that modifications of the cellular signaling pathways by rituximab may be crucial for its clinical response. The B-cell restricted cell surface phosphoprotein CD20 is involved in many cellular signaling events including proliferation, activation, differentiation, and apoptosis upon crosslinking. Monomeric rituximab chemosensitizes drug-resistant NHL cells via selective downregulation of antiapoptotic factors through the type II mitochondrial apoptotic pathway. Several signaling pathways are affected by rituximab which are implicated in the underlying molecular mechanisms of chemosensitization. ARL (acquired immunodeficiency syndrome (AIDS)-related lymphoma) and non-ARL cell lines have been examined as in vitro model systems. In ARL, rituximab diminishes the activity of the p38MAPK signaling pathway resulting in inhibition of the interleukin (IL)-10/IL-10R autocrine/paracrine cytokine autoregulatory loop leading to the inhibition of constitutive STAT-3 activity and subsequent downregulation of Bcl-2 expression leading to chemosensitization. Rituximab upregulates Raf-1 kinase inhibitor protein (RKIP) expression in non-ARL cells. Through physical association with Raf-1 and nuclear factor kappaB (NF-kappa B)-inducing kinase (NIK), RKIP negatively regulates two major survival pathways, namely, the extracellular signal-regulated kinase1/2 (ERK1/2) and the NF-kappa B pathways, respectively. Downmodulation of the ERK1/2 and NF-kappa B pathways inhibits the transcriptional activity of AP-1 and NF-kappa B transcription factors, respectively, both of which lead to the downregulation of Bcl-(xL) (Bcl-2 related gene (long alternatively spliced variant of Bcl-x gene)) transcription and expression and sensitization to drug-induced apoptosis. Bcl-(xL)-overexpressing cells corroborated the pivotal role of Bcl-(xL) in chemosensitization. The specificity of rituximab-mediated signaling and functional effects were corroborated by the use of specific pharmacological inhibitors. Many patients do not respond and/or relapse and the mechanisms of unresponsiveness are unknown. Rituximab-resistant B-NHL clones were generated to investigate the acquired resistance to rituximab-mediated signaling, and chemosensitization. Resistant clones display different phenotypic, genetic and functional properties compared to wild-type cells. This review summarizes the data highlighting a novel role of rituximab as a signal-inducing antibody and as a chemosensitizing agent through negative regulation of major survival pathways. Studies presented herein also reveal several intracellular targets modified by rituximab, which can be exploited for therapeutic and prognostic purposes in the treatment of patients with rituximab- and drug-refractory NHL.

Imatinib mesylate and rituximab are molecularly targeted drugs against the BCR-ABL fusion protein and the CD20 antigen, respectively. Although these drugs have excellent anticancer effects, a major concern is drug resistance. We have investigated the case of a patient with Philadelphia chromosome-positive and CD20+ acute lymphocytic leukemia who acquired resistance to imatinib and rituximab. Imatinib therapy resulted in prompt cytogenetic remission, but resistance developed shortly thereafter. Sequencing of the kinase domain of the ABL gene and allele-specific polymerase chain reaction analysis revealed a point mutation resulting in an E255V substitution that was present before the therapy. After the patient received mild chemotherapy followed by rituximab administration, hematologic and cytogenetic remission was sustained for 5.5 months. The recurrent leukemic cells after the rituximab therapy showed not only the E255V mutation in the ABL gene but also loss of the CD20 antigen due to impaired transcription of the CD20 gene. The results of 2-color flow cytometry analysis showed that a small population of CD20(-) leukemic cells existed before the imatinib therapy. These results suggest that leukemic subclones carrying a genetic perturbation of the targeted molecules for both imatinib and rituximab were present before the therapies. The preexistence of primary resistant clones suggests the inability of combination therapy with 2 molecularly targeted drugs to overcome drug resistance in leukemia.

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.

Objective: Anti-CD20 chimeric monoclonal antibody rituximab (Mabthera; IDEC-C2B8) is currently tested in several clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). In the present study, we investigated whether rituximab therapy may select for CD20(-) subclones., Materials and Methods: Leukemic B-CLL cells were isolated from patients with B-CLL and sensitivity to rituximab-induced cell death was examined. Levels of CD20 protein and mRNA were determined using flow cytometry and real-time PCR, respectively. Clonality analyses of leukemic cells throughout rituximab therapy were performed by GeneScan analysis of patient clone specific rearrangements of the complementarity determining region III of the heavy chain immunoglobulin., Results: Cytotoxicity of rituximab in vitro did not depend on the protein levels of CD20. During therapy with rituximab CD20(+) B-CLL cells were depleted and CD20(-) leukemic cells emerged. After treatment, the initial CD20(+) B-CLL cell clone reexpanded. CD20(-) B-CLL cells retained their capacity to synthesize the CD20 molecule., Conclusions: These data support the concept that in B-CLL rituximab treatment may not lead to the emergence of CD20(-) leukemic variants. Our findings support clinical studies investigating the benefit of prolonged period of rituximab therapy in B-CLL disease.

CD20 is a B-cell-restricted antigen that, for the most part, is expressed from the pre-B-cell to the mature B-cell stage of B-cell differentiation. Several transcription factors regulate CD20 expression during B-cell differentiation, the most important of which appear to be PU.1 and Pip (PU.1 interacting protein). As B cells differentiate to plasma cells, CD20 expression is down-regulated, which coincides with PU.1 downregulation in plasma cells. Analogous to their normal B-cell counterparts, CD20 is expressed on malignant lymphoplasmacytic cells from most patients with Waldenstrom's macroglobulinemia and on malignant plasma cells from a fraction (20%) of multiple myeloma patients. CD20 also is expressed on subpopulations of normal donor plasma cells, which may include autoantibody-secreting plasmacytes. In view of these findings, the anti-CD20 chimeric monoclonal antibody, rituximab (Rituxan; Genentech, Inc, South San Francisco, CA and IDEC Pharmaceutical Corporation, San Diego, CA), has been evaluated in the treatment of Waldenstrom's macroglobulinemia and multiple myeloma, as well as in nonmalignant plasma cell disorders including IgM polyneuropathies, immune thrombocytopenias, and autoimmune hemolytic anemias, with reported activity in these entities. An update of these clinical efforts is presented in this report.

Rituximab is a chimeric antibody with human gamma-1 and kappa constant regions and murine variable regions. It recognizes the CD20 antigen, a pan B-cell marker. Therapeutic trials in patients with B-cell non-Hodgkin's lymphoma (NHL) have shown significant efficacy with a primary response rate of 50%, and a secondary response rate of 44% after repeat treatments in prior responders. The selection for proliferating tumor cells that no longer express CD20 may compromise repeated treatment. We have identified a patient who developed a transformed NHL that lost CD20 protein expression after two courses of therapy with rituximab. In a pretreatment lymph node biopsy, 83% of B cells (as defined by CD19 and surface immunoglobulin) expressed surface CD20. A biopsy from the recurrent tumor after two courses of rituximab revealed a diffuse large cell NHL where 0% of B cells expressed CD20 with no evidence of bound rituximab. Cytoplasmic staining showed no CD20 protein. Sequencing of immunoglobulin heavy chain cDNA identified identical variable sequences in the initial and recurrent lymphomas, confirming the association between the two tumors. Literature and database review suggests that approximately 98% of diffuse large cell lymphomas express CD20, which suggests that these tumors rarely survive without CD20. This is the first identified case of loss of CD20 expression in a lymphoma that has relapsed after rituximab therapy, although several other cases have since been identified. Considering the significant number of patients treated with anti-CD20 antibodies, this may occur only rarely and is unlikely to preclude recurrent therapy with anti-CD20 antibodies in the majority of patients. However, because many patients have relapsed after anti-CD20 antibody therapy and have not been biopsied to identify clones with down-regulated CD20 antigen, we do not currently know the true frequency of this phenomenon. When possible, patients should undergo evaluation for CD20 expression before repeated courses of anti-CD20 therapy.