The Role of Complement in the Mechanism of Action of Rituximabfor B-Cell Lymphoma: Implications for Therapy

XUHUI ZHOU,a WEIGUO HU,b,c XUEBIN QINb,c

aChangzheng Hospital, Second Military Medical University, Shanghai, China; bDepartment of Medicine,Brigham and Women’s Hospital, Boston, Massachusetts, USA; cHarvard Medical School,

Laboratory for Translational Research, Cambridge, Massachusetts, USA

Disclosure: The content of this article has been reviewed by independent peer reviewers to ensure that it is balanced, objective, andfree from commercial bias. No financial relationships relevant to the content of this article have been disclosed by the authors,planners, independent peer reviewers, or staff managers.

Key Words. Rituximab • CD20 • Mechanism • Resistance • Complement • B-cell non-Hodgkin’s lymphoma

ABSTRACT

Rituximab, a genetically engineered chimeric monoclo-nal antibody specifically binding to CD20, was the firstantibody approved by the U.S. Food and Drug Admin-istration for the treatment of cancer. Rituximab sig-nificantly improves treatment outcome in relapsedor refractory, low-grade or follicular B-cell non-Hodgkin’s lymphoma (NHL). However, there are alsosome challenges for us to overcome: why �50% of pa-tients are unresponsive to rituximab in spite of the ex-pression of CD20, and why some responsive patientsdevelop resistance to further treatment. Although theantitumor mechanisms of rituximab are not completelyunderstood, several distinct antitumor activities of rit-uximab have been suspected, including complement-dependent cytotoxicity (CDC), antibody-dependentcellular cytotoxicity (ADCC), apoptosis, and direct

growth arrest. To counteract resistance to rituximabtherapy, several strategies have been developed to: (a)augment the CDC effect by increasing CD20 expression,heteroconjugating rituximab to cobra venom factor andC3b, and inhibiting membrane complement regulatoryprotein, especially CD59, function; (b) enhance theADCC effect through some immunomodulatory cyto-kines and CR3-binding �-glucan; and (c) reduce the ap-optotic threshold or induce apoptotic signaling on thetumor. Extensive studies indicate that rituximab com-bined with these approaches is more effective than a sin-gle rituximab approach. Herein, the mechanism ofaction of and resistance to rituximab therapy in B-cellNHL, in particular, the involvement of the complementsystem, are extensively reviewed. The Oncologist 2008;13:954–966

INTRODUCTION

Non-Hodgkin’s lymphoma (NHL) is a diverse group oflymphatic malignancies distinct from Hodgkin’s lym-phoma on the basis of their epidemiology, clinical manifes-

tation, therapy, and prognosis, as well as laboratory tests,including pathological features. The incidence of NHL hasbeen steadily rising, currently ranking fifth in cancermortality in the U.S. The annual incidence of NHL in

Correspondence: Xuebin Qin, M.D., Ph.D., Harvard Medical School, One Kendall Square, Building 600, 3rd Floor, Cambridge,Massachusetts 02139, USA. Telephone: 617-621-6102; Fax: 617-621-6148; e-mail: xuebin_qin@hms.harvard.edu ReceivedApril 9, 2008; accepted for publication August 5, 2008; first published online in THE ONCOLOGIST Express on September 8,2008. ©AlphaMed Press 1083-7159/2008/$30.00/0 doi: 10.1634/theoncologist.2008-0089

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Lymphoma

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2000 –2004 was 19.3 cases per 100,000 persons, and ap-proximately 66,120 new cases of NHL were expected to bediagnosed in 2008, representing 5% and 4% of all cancersin males and females, respectively [1]. NHL is classified aseither B-cell or T-cell NHL. B-cell NHL is much morecommon than T-cell NHL. We focus on reviewing the cur-rent treatment of B-cell NHL, but not T-cell NHL.

In the past 10 years or more, significant progress hasbeen made in specifically treating B-cell NHL. While tra-ditional nonspecific cancer therapies such as chemotherapyand radiotherapy mainly focus on killing rapidly dividingcancer cells, they also kill rapidly dividing normal cells,thereby causing obvious multiple side effects. In contrast,specific targeted therapies, including immunotherapy,mainly kill cancer cells, with no or minimal effects on nor-mal cells. Therefore, specific targeted therapies have obvi-ous advantages and are increasingly becoming the preferredtools to conquer cancer. Immunotherapy that targetsreceptors using monoclonal antibodies (mAbs) has anestablished role in the treatment of certain leukemias, lym-phomas, and breast cancers by specifically recognizingcancer cells [2]. Not only can mAbs conjugate with cyto-toxic compounds, radionuclides, and toxins, but unconju-gated mAbs can exert antitumor activity alone. To date, fivemAbs, including three unconjugated mAbs, have been ap-proved for cancer therapy by the U.S. Food and Drug Ad-ministration (FDA). Among the three unconjugated mAbs,the chimeric antibody rituximab, the first mAb approved bythe FDA, has been successfully used in the treatment of B-cell NHL via targeting the membrane molecule of CD20 onB-cell lymphoma, with a great improvement in outcome[3], and is also regarded as the driving force in our currentfocus on immunotherapy of B-cell NHL. Herein, the mech-anism of action of and resistance to rituximab therapy in B-cell NHL, in particular, the involvement of the complementsystem, are described.

RITUXIMAB TARGETING CD20Rituximab (IDEC-C2B8, Rituxan�; IDEC Pharmaceuti-cals, San Diego, CA, and Genentech, Inc, San Francisco,CA) is a genetically engineered chimeric mAb that specif-ically binds to CD20, which contains human �-1 and � con-stant regions with murine variable regions [4]. Rituximabtherapy alone or in combination with chemotherapy has re-markably improved the treatment outcome of patients withB-cell NHL. Its single-agent efficacy with low toxicity wasproven by an overall response rate of 50% with a mediantime to progression in responders of 13.2 months, but someresponsive patients develop resistance to further rituximabtreatment [5, 6]. The mechanism of rituximab unrespon-

siveness remains unclear, and rituximab resistance requiresmore extensive investigation.

The rituximab-targeted molecule, CD20, a 32-kDa non-glycosylated phosphoprotein, provides a universal targetfor immunotherapy, which is especially expressed on thesurface of normal precursor and mature B cells and, impor-tantly, not on early pre-B cells, stem cells, or antigen-presenting dendritic reticulum cells [7]. More than 90% ofB-cell NHLs express this surface protein [8, 9]. The CD20protein has four transmembrane domains and does not mod-ulate from the cell surface in response to antibody binding,thus providing an excellent target for immunotherapeuticstrategies [4]. So far, the natural ligand for CD20 has notbeen identified, and the biological function of CD20 re-mains unclear. CD20 knockout mice show normal B-celldevelopment and function [10]. Via phage display libraries,Binder et al. [11] showed that rituximab binds a discontin-uous conformational epitope on CD20, comprising(170)ANPS(173) and (182)YCYSI(185), with both stringsbrought in steric proximity by a disulfide bridge betweenC(167) and C(183). This structural interaction was furtherproven by crystal structure analysis of the complex of rit-uximab Fab fragment and CD20 fragment (aa163-aa187)[12]. Several distinct antitumor activities of rituximab havebeen suspected in B-cell NHL therapy, including comple-ment-dependent cytotoxicity (CDC), antibody-dependentcellular cytotoxicity (ADCC), apoptosis, and direct growtharrest [13]. In addition, complement-dependent cellular cy-totoxicity (CDCC) has often been included in ADCC [14].

CDC

The Complement SystemThe complement system, a central innate system, is the ef-fector of adaptive immunity. It spontaneously identifies anypotential pathogens and efficiently protects the host fromintruding pathogen attack through a wide range of cellularresponses [15–18]. This system is composed of �30 solu-ble plasma proteins and membrane proteins that can triggerthree distinct protease cascades known as the classical,mannose-binding lectin (MBL), and alternative pathways(Fig. 1). The classical pathway is triggered by antigen-bound antibody molecules and is initiated by the binding ofa specific part of the antibody (Fc) to C1q [15]. The MBLpathway is initiated when plasma MBL in complex MBLand MBL-associated protease (MASP)-1/2 bind to arrays ofcarbohydrates (specifically mannose and fucose residues)on the surface of many pathogens such as bacteria, viruses,fungi, yeasts, and parasites; MASP-1/2 then cleaves C4 andtriggers the subsequent complement cascade [16]. The al-ternative pathway is capable of spontaneous autoactivation,

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termed the “tickover” of C3 at a rate of �1% of total C3 perhour [19, 20], thereby identifying any potential pathogens.All three pathways converge at the C3 level, which allowsC3b to target the pathogens, and at the C5 level, which leadsto polymerization of C9 by C5b-8 binding and to assemblyof a membrane attack complex (MAC) with a diameter of5–10 nm (Fig. 1) [15]. If the MAC inserted into the cellmembrane remains open, it will directly induce targetedcell lysis through subsequent influx of ions and water thatleads to lethal colloid-osmotic swelling, CDC. The CDC ef-fect is characterized by swelling of mitochondria, dilationof the rough endoplasmic reticulum, disruption of the Golgicomplex and of the plasma and nuclear membranes, andheterochromatin disappearance at the ultrastructural levelinvolving the generation of reactive oxygen species [21,22]. For targeted cell lysis, a single MAC is enough forerythrocytes but not for nucleated cells, because nucleatedcells can endocytose MAC and repair the damage unlessmultiple MACs (12- to 16-mers) are assembled [23].

The byproducts produced in complement activation,such as C1q, C3b, iC3b, and C4b, are critical opsonins forhost defense against infection and for disposal of immunecomplexes and dead cell debris by the phagocytosis/lysiseffect of the immune effector cells (macrophages, neutro-phils, natural killer [NK] cells, etc.) through their surfacereceptor binding to these byproducts (Fig. 1 and 2). On the

other hand, the small fragment byproducts such as C3a,C4a, and C5a (Fig. 1), termed anaphylatoxins, also play animportant role in inflammation and especially in host de-fense against parasites. These anaphylatoxins can causemast cell and basophil degranulation, with the release ofhistamine and other substances that increase vascular per-meability, stimulate smooth muscle constriction, and in-duce chemotaxis. The anaphylatoxins can also activatethese immune effector cells by binding to cell surface re-ceptors [24, 25]. Among these anaphylatoxins, C5a has themost potent biological activity.

To prevent the potentially harmful effect of complementactivation on autologous cells, �10 plasma- and mem-brane-bound inhibitory proteins have evolved for restrict-ing complement activation at different stages of activationpathways (Fig. 1 and 2) [26, 27]. The soluble plasma com-plement regulatory proteins include C1 inhibitor, whichregulates C1; factor H and factor I, which regulate thecleavage of C3b and C3/C5 convertases; C4 binding pro-tein, which splits C4 convertase and assists factor I in thecleavage of C4b; and S-protein, clusterin, and serum lipids,which compete with membrane lipids for reacting with nas-cent C5b67 [15]. Membrane complement regulatory pro-teins (mCRPs) include: (a) CD46 (membrane cofactorprotein), which regulates C3 activation by functioning as acofactor protein for factor I–mediated cleavage of C3b and

Figure 1. Three complement activation cascades.Abbreviations: C1 INH, C1 inhibitor; C4BP, C4 binding protein; CR, complement receptor; DAF, decay-accelerating factor;

MAC, membrane attack complex; MBL, mannose-binding lectin; MCP, membrane cofactor protein; MASP, MBL-associatedproteases.

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C4b [28]; (b) CD55 (decay-accelerating factor [DAF]),which inactivates the convertases of C3 (C4b2a andC3bBb) and C5 (C4b2a3b and C3bBb3b) by preventing theformation of new and accelerating the decay of activatedconvertase via binding to C3b and C4b [29]; (c) CD59(membrane inhibitor of reactive lysis), which restrictsMAC assembly on the cell membrane through binding toC8� and C9; (d) CD35 (complement receptor 1), which hasboth CD46 and CD55 functions in humans [26, 27, 30] andis a major immune adherence receptor that plays a role inimmune-complex processing and clearance [31]; and (e)complement-receptor 1-related protein y, present only inrats and mice, which possesses both CD46 and CD55 bio-logical functions [32–35]. CD55 and CD59 attach to thecell surface via a glycosylphosphatidylinositol (GPI) an-chor, whereas CD46 and CD35 associate with the plasmamembrane via their C-terminal transmembrane domains[36]. Structurally, CD55, CD46, and CD35 belong to the

regulators of complement activation (RCA) family andcontain a variable number of short consensus repeat do-mains. CD59 is a much smaller protein with no sequence orstructural resemblance to the RCA family of proteins [27].Several lines of evidence from human and animal studiesindicate that CD59 is more relevant than CD55 and CD46 inprotecting normal cells from MAC formation and MAC-induced phenomena [37–42].

There is a delicate balance between complement activa-tion and regulation on autologous cells that is subject to per-turbation by either increased complement activation ordecreased regulation, which may cause a variety of immunediseases. For example, immune-complex activation of theclassical complement pathway results in autoimmune dis-eases such as systemic lupus erythematosus and glomer-ulonephritis [30, 43]. Conversely, deficiency of GPI-anchored DAF and CD59 in circulating cells—resultingfrom an acquired somatic PIG-A gene mutation—is respon-sible for the hemolytic anemia and thrombosis that charac-terize paroxysmal nocturnal hemoglobinuria [44 – 46].However, the high expression level of mCRPs also confersprotection on cancer cells, invading microorganisms in-cluding HIV for complement attack, and may lead to resis-tance to antibody therapy.

Rituximab-Mediated CDCThe formation of MACs and subsequent cytolysis are usu-ally considered as the activity of CDC via activation of theclassical pathway initiated by C1q binding to antibody Fcfragment in rituximab therapy (Fig. 2). The consequence ofthe formation of MACs generally depends on the copynumbers assembled on the targeted tumor cells. Only athigh copy numbers (12- to 16-mers) per cell (a lytic dose),can MACs induce a loss of membrane integrity and rapidnecrotic-type cell death, possibly by a caspase-independentmechanism [21–23, 47]. At low copy numbers of MACs percell (nonlytic or sublytic dose), MACs exhibit a wide rangeof effects leading to cellular responses such as secretion,adherence, aggregation, chemotaxis, and even cell prolifer-ation activity through various cell-signaling pathways[48 –50].

There is much evidence to highlight the importance ofCDC in rituximab therapy of B-cell NHL. Complement de-pletion by cobra venom factor (CVF) significantly reducedthe antitumor activity of rituximab in severe combined im-munodeficiency (SCID), athymic nude mice [51, 52]. Thisactivity was not affected by depletion of macrophages, NKcells, and/or neutrophils, which are irrelevant to CDC, ineither a disseminated or s.c. tumor mouse model. It was alsocompletely abolished in a C1q-deficient syngeneic mousemodel and in a CVF complement-depleted mouse model

Figure 2. Rituximab-mediated CDC effect and strategies forenhancing the CDC effect. C1q interacts with the rituximab Fcregion exposed after binding to CD20 on the B-cell surface,thus activating the classical complement cascade, and a MACis inserted into the cell membrane, with multiple MACs (12- to16-mers) leading to cytolysis. Strategies to overcome resis-tance to the CDC effect include: (a) inhibitors of the mCRPsDAF and especially CD59, which leads to more complementactivation and more MAC formation on the cell surface; (b)heteroconjugates of rituximab to CVF or C3b, and otherAg-Ab complexes targeting tumor cells, which enhance com-plement activation; and (c) other drugs that can upregulate theexpression of CD20, which include the histone deacetylase in-hibitor trichostatin A and protein kinase C activator bryosta-tin-1.

Abbreviations: Ag-Ab, antigen-antibody; CDC, comple-ment-dependent cytotoxicity; CVF, cobra venom factor; DAF,decay-accelerating factor; MAC, membrane attack complex;MCP, membrane cofactor protein; mCRP, membrane comple-ment regulatory protein.

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[53, 54]. Furthermore, use of inhibitors abrogating mCRP(such as CD55 and CD59) functions can facilitate rituximabtherapy in B-cell NHL, confirming the importance of CDC.However, many groups have claimed that ADCC plays apivotal role in the antiproliferative effect of rituximab. B-cell lymphomas were depleted in vivo mainly by innatemonocytes/macrophages in an Fc�R-dependent manner ofisotype-specific mAb interactions with distinct Fc�Rs. Thisrituximab-mediated ADCC effect on B-cell lymphomas iscompletely effective in C3-, C4-, or C1q-deficient mice[55–57]. The explanation for the discrepancy between rit-uximab-mediated CDC and ADCC is complex and far fromconvincing [3]. A number of factors, including tumor sta-tus, mCRP expression, tumor cell type, tumor-inoculatingmethods, and tumor growth period, may influence the con-tributions of CDC and ADCC differently. However, it iswidely accepted that ADCC and CDC synergistically affectcytotoxicity in cancer cells through the ability of comple-ment to promote inflammation and change the activationstatus of innate effectors [3]. The byproduct of anaphyla-toxin C5a during CDC is pivotal in activating these effectorcells and can upregulate the expression of activating Fc�Rand downregulate the expression of inhibitory Fc�RIIb viainteraction with the C5a receptor, which enhances theADCC effect in tumors [58–61]. Moreover, CDC-resistantcells are sensitive to ADCC and vice versa [62].

The potency of CDC in rituximab therapy is partiallycorrelated with the level of CD20 expression on the B-cellNHL cell membrane [22, 63]. The higher the CD20 level,the more MACs can be assembled. Subsequently, targetedcells are directly lysed. With B-cell NHL cells freshly iso-lated from patients, a level of CD20 expression of �50 �103 CD20 molecules per cell is a prerequisite for the lyticresponse arising from the mechanism of CDC [22]. A sig-moidal or linear correlation between the CD20 expressionlevel and the rituximab-mediated killing effect further dem-onstrates the important role of CDC in the rituximab-medi-ated anticancer effect [22, 62, 63]. Other reports indicatethat the efficacy of CDC in rituximab therapy may be de-termined by the expression levels of CD20 and mCRPs(CD55, and especially CD59) [64], but not of individualCD20 or mCRPs [65, 66].

Overcoming Resistance to Rituximab ThroughEnhancing the CDC Effect

Abrogating mCRP FunctionThe mCRPs CD46, CD55, and CD59 play a critical role intumor resistance to rituximab-mediated CDC (Fig. 2).These mCRPs are expressed widely in almost all cancercells independent of their tissue of origin [13]. The various

expression levels of mCRPs may be regulated by the selec-tive stress of complement attack [67], the stage of differen-tiation [68, 69], and host factors of neighboring tumor orstromal cells. These mCRPs in tumor cells restrict comple-ment activation cascades in distinct stages and reduce MACformation, thereby profoundly protecting the tumor fromrituximab-mediated CDC (Fig. 1 and 2). The rituximab-resistant B-cell NHL cell lines of Raji and Ramos generatedunder the stress of rituximab and normal human serum(complement resource) exhibit upregulation of CD52 andthe complement regulators CD55 and CD59, as well asdownregulation of CD20 [67, 70]. The use of neutralizingantibodies abrogating the function of CD46, CD55, andCD59 markedly enhanced the antitumor activity of ritux-imab in vitro and in vivo [13, 14, 22, 63, 71, 72]. However,these antibodies are not applicable to clinical treatment be-cause they would initiate undesired complement attack bybinding to the host cells expressing mCRPs. To overcomethis problem, Tedesco’s group [73] developed two mini-antibodies (MBs), MB-55 (against CD55) and MB-59(against CD59), containing the human IgG1 hinge-CH2-CH3 domains, two single-chain variable fragments (scFv)without Fc fragment, a region necessary for activating theclassical pathway. Furthermore, they generated two hetero-conjugates of bio-MB55 and bio-MB59 [74] through athree-step biotin–avidin system, which is currently em-ployed in patients to target radionuclides to cancer cells forenhancing the anticancer effect [75–77]. They documentedthat these modified antibodies against CD55 and CD59 sig-nificantly increased the antitumor activity of rituximab invitro [73] and in vivo [74]. Alternatively, enhanced suscep-tibility can be achieved by downregulating the expressionof mCRPs. Knockdown of CD55 expression via siRNAtransfection attenuated the resistance of B-cell lymphomaor breast cancer cells to complement-mediated cytolysis bytreatment with rituximab or trastuzumab [78]. The chemo-therapeutic drug fludarabine showed a synergistic effectwith rituximab treatment in NHL tumors, likely through thedownmodulation of the membrane expression of CD55 [78,79]. Consistent with the critical role of CD59 for restrictingMAC in normal cells, CD59 is also more relevant to protecttumor cells from immunotherapy including rituximab thanCD46 and CD55. Therefore, it is imperative to develop ef-fective and practicable inhibitors against mCRPs, espe-cially CD59, for facilitating rituximab and even otherantibodies in treating tumors. For this reason, we havesearched for an inhibitor for human CD59. Intermedilysin(ILY), a cytolytic pore-forming toxin secreted by Strepto-coccus intermedius, lyses human cells exclusively becausethe toxin binds to human CD59 (hCD59) with high speci-ficity [80–82]. The specificity for hCD59 is derived from

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binding of ILY domain 4 (ILYd4) to amino acids 42–58 inhCD59, which also participate in binding to C8 and C9 [81].Thus, we hypothesize that truncated ILYd4 would abrogatehCD59 function and thereby increase antibody-mediatedCDC in cancer cells. If this is the case, ILYd4 may representan innovative adjuvant for cancer immunotherapy in gen-eral and for antibody-resistant cancers in particular.

Increasing CD20 ExpressionIt is of note that the level of CD20 expression is related tothe number of MACs formed on rituximab-targeted lym-phocytes via binding to CD20, which determines whethertargeted tumor cells can be lysed by CDC or not. Thus, anincreased level of CD20 expression might be a means toovercome resistance to CDC (Fig. 2). However, rituximab–CD20 complex formed by rituximab binding to CD20 onthe targeted cell membrane can be shaved by monocytes/macrophages expressing Fc�RI, a phenomenon called an-tigen modulation [83–85] (Fig. 2). This shaving reactionstarts within 1– 40 hours after rituximab infusion, whichcould substantially compromise the therapeutic efficacy ofrituximab [85]. Interestingly, the histone deacetylase inhib-itor suberoylanilide hydroxamic acid modulated the expres-sion of apoptosis-related genes [86], and another histone,deacetylase inhibitor trichostatin A, could epigeneticallyincrease CD20 mRNA and protein expression in an estab-lished CD20-negative cell line to sensitize rituximab ther-apy [87]. Bryostatin-1, a protein kinase C activator, can alsoenhance expression of CD20 at the level of both mRNA andprotein in human tumor B cells through extracellular sig-nal–related kinase (ERK)-dependent mechanisms, which inturn increases the susceptibility of the tumor to rituximab[88]. Moreover, synthetic CpG oligodeoxynucleotides arecurrently being tested in clinical trials as a vaccine adjuvantand for rituximab immunotherapy of B-cell NHL [89]. Syn-thetic CpG oligodeoxynucleotides resembling sequencesfound in bacterial DNA specifically increase primary ma-lignant B-cell expression of CD20, thereby resulting in en-hanced sensitivity to rituximab treatment [89, 90].

Enhancing Complement ActivationBesides abrogating mCRP function and increasing theCD20 expression level, an alternative strategy that can beemployed to overcome resistance to CDC is to directly en-hance complement activation on tumor cells. A comple-ment-activating protein such as CVF or C3b can beconjugated to the antitumor antibody for the enhancementof complement activation [91] (Fig. 2). This effect wasachieved in vitro by either anti– epithelial cell adhesionmolecule–CVF heteroconjugates targeted to colorectal can-cer cells [92] or anti–GD2-CVF heteroconjugates targeted

to neuroblastoma cells [93, 94]. Additionally, an antibodyagainst iC3b was demonstrated to increase iC3b deposition,which allowed it to interact with effector cells containingboth Fc and complement receptors, and therefore signifi-cantly enhancing rituximab-mediated killing of Raji andDB cells in a cynomologous monkey model [95] (Fig. 2).

ADCC

Effect of ADCC in Rituximab TherapyTogether with CDC, ADCC also plays an important role inrituximab antitumor activity. ADCC triggers tumor cellkilling through interaction between the Fc region of CD20binding rituximab and Fc�Rs, particularly Fc�RI andFc�RIII, activating receptors expressed on immune effec-tor cells such as monocytes/macrophages, granulocytes/neutrophils, and NK cells (Fig. 3). This interaction,mediated by rituximab on activated effector cells, initiates a

Figure 3. Rituximab-mediated ADCC and strategies to en-hance the ADCC effect. CDCC is also classified here, whichnormally has little efficacy in rituximab therapy. This kind ofcellular cytotoxicity can occur after binding of either the ritux-imab Fc region (in ADCC) or C1q, C3b, C4b, and iC3b (inCDCC) to their respective receptors, resulting in either phago-cytosis or cell-mediated lysis of B-lymphocytes, depending onthe effector cell type. The Fc region of cell-bound rituximab isrecognized principally by either the activating receptorsFc�RI/Fc�RIII or the inhibitory receptor Fc�RIIB, whereasthe byproducts of complement activation are recognized byC1qR, CR1, or CR3 on effector cell surfaces. The strategies toovercome resistance to the ADCC/CDCC effect include: (a)anaphylatoxins (C5a, C3a); (b) some cytokines (IL-2, IL-12,M-CSF) that are able to activate effector cells; and (c) CR3-specific polysaccharides such as �-glucan, which primes CR3and therefore triggers cellular cytotoxicity.

Abbreviations: ADCC, antibody-dependent cellular cyto-toxicity; CDCC, complement-dependent cellular cytotoxicity;CR, complement receptor; IL, interleukin.

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series of signaling pathways that lead to the release of in-flammatory and/or cytotoxic immune modulators includingcytokines, chemokines, proteases, and reactive oxygen spe-cies. Eventually, the activated monocytes/macrophages andgranulocytes/neutrophils phagocytose the targeted cancercells, whereas activated NK cells eliminate targeted lym-phoma cells using the granzyme-perforin system. Gener-ally, the resting effector cells where the inhibitory Fc�RIIBis dominantly expressed cannot function unless activated.The inhibitory Fc�RIIB is a potent regulator of ADCC invivo, modulating the activity of Fc�RIII on effector cells[96]. The antitumor activity of rituximab is greatly reducedin Fc�RI/Fc�RIII-deficient mice, whereas disruption of thegene that encodes the inhibitory receptor Fc�RIIB substan-tially enhanced antitumor activity. In addition, rituximabcan induce the �-chemokine CCL3 in vivo, activating in-nate immune cells such as NK cells, macrophages, andpolymorphonuclear cells, which increases the ADCC effect[52]. These results suggest that rituximab-mediated ADCCis important for killing cancer cells [96, 97].

CDCC may also be engaged in destroying invadingpathogens. In CDCC, deposition of C3b, iC3b, C1q, andC4b bound on the surface of targeted cells is able to opso-nize phagocytosis through binding to their receptors onmonocytes/macrophages, NK cells, and polymorphonu-clear leukocytes [95, 98, 99] (Fig. 3). This process can befurther enhanced by chemotactic and cell-activating ana-phylatoxins such as C5a and C3a through their receptors onthe above phagocytes [24, 25], which are pivotal mediatorsof the host defense against infection and of the disposal ofimmune complexes and products of inflammatory injury[16]. To date, there has been little research on the effect ofCDCC on rituximab therapy, perhaps because of its relativeineffectiveness [14].

Enhancing ADCCAntibody-targeted tumor resistance to innate immune cellssuch as macrophages, NK cells, and neutrophils in ADCCmay arise from the suppression of these immune effectorsafter long-term expansion of the tumor. Resistance to ritux-imab in some patients may be linked to a polymorphism inFc�RIIIa affecting isotype preference [100]. Geneticallymodified NK cells carrying a chimeric antigen receptor thatconsists of a CD20-specific scFv antibody fragment con-ferred enhanced cytotoxic activity and could overcomeNK-cell resistance of lymphoma and leukemia cells [101].In addition, the effect of some cytokines on ADCC-relatedresistance was tested. M-CSF in vitro enhanced the cyto-toxicity of monocytes on Daudi B-cell lymphomas throughupregulation of monocyte Fc�RI and Fc�RIII by 1.5-foldand 1.6-fold, respectively, whereas the expression of

Fc�RII remained unchanged [102]. Interleukin (IL)-2 is apleiotropic cytokine that activates selective immune effec-tor cell responses associated with antitumor activity, in-cluding ADCC. Golay et al. [103] demonstrated in vitro thatIL-2–activated NK cells strongly enhanced the therapeuticactivity of rituximab through ADCC in primary B-cell NHLcells freshly isolated from patients. Furthermore, IL-2 syn-ergistically enhanced rituximab efficiency in killing B-cellNHL cells in a xenograft model, in part, through activationand trafficking of monocytes and NK cells to tumors [104];a similar result was obtained with an anti-CD20–IL-2 im-munocytokine [105]. Additionally, IL-12 synergizes the rit-uximab ADCC effect through upregulating �-interferonand interferon-inducible protein 10 expression and increas-ing NK cell lytic activity in vitro [106] (Fig. 3). The con-comitant use of IL-12 and rituximab had only a modesteffect in treating patients with B-cell NHL. The responserate in patients treated with IL-12 in combination with rit-uximab did not seem to be better than that seen with ritux-imab alone. Also, the sequential administration of IL-12after rituximab did not result in additional clinical re-sponses [107].

To enhance the CDCC effect in rituximab treatment,�-glucan can be applied to promote CR3-dependent cellu-lar cytotoxicity. Generally, �-glucan is naturally exposedon the cell wall of yeasts and fungi. Moreover this CR3-mediated cytotoxicity that is responsible for host protectionagainst yeast and fungus infection requires the binding ofCR3 to both iC3b and �-glucan. Normally, this CDCC ef-fect does not happen in rituximab therapy because tumorcells lack �-glucan [108]. However, together with depos-ited iC3b generated by rituximab-mediated complementactivation, the administration of �-glucan can trigger cy-totoxic phagocytosis and degranulation of iC3b-coated tu-mor cells [109, 110], thus regressing tumor growth [110,111] (Fig. 3). The application of �-glucan has consistentlybeen documented to markedly increase the therapeutic ef-ficacy of rituximab in B-cell NHL-inoculated SCID mice[112].

APOPTOSIS

Effect of Apoptosis in Rituximab TherapyDeans et al. [113–116] demonstrated that CD20 is associ-ated with Src family tyrosine kinases, Lyn, Fyn, and Lck,which are involved in rituximab-induced apoptosis. CD20-associated PAG (phosphoprotein associated with GEMs[glycosphingolipid-enriched membrane microdomains]),also known as Csk-binding protein, is a ubiquitous, highlytyrosine-phosphorylated adaptor protein localized exclu-sively in lipid rafts [117, 118]. PAG normally recruits Csk

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to lipid rafts to maintain resident Src family tyrosine ki-nases such as Lyn, Fyn, and Lck in an inactive state. Afterrituximab specifically binds to CD20, it redistributes lipidrafts, subsequently transactivates the above Src family ty-rosine kinase, and initiates downstream signaling pathwaysresulting in apoptosis (Fig. 4).

On the other hand, this redistribution of lipid rafts canalso induce apoptosis by Fas molecule clustering. This clus-tering further induces the formation of the death-inducingsignaling complex (DISC) and downstream activation ofthe death receptor (DR) pathway in Ramos B-cell NHLcells. Upon stimulation with rituximab and subsequentcrosstalking with CD20, Fas-associated death domain pro-tein (FADD) and caspase-8 were recruited into the DISC.This DISC was further confirmed by the identification ofFas, caspase-8, and FADD together with CD20 in sucrose-gradient raft fractions [119]. Therefore, rituximab can sen-sitize lymphoma B cells to Fas-induced apoptosis in acaspase-8–dependent manner (Fig. 4).

Rituximab preferentially inhibits the expression of theantiapoptotic gene products Bcl-2/Bcl-xL, X-linked inhib-itor of apoptosis protein, and myeloid cell leukemia se-quence 1 in B-cell NHL cells through downregulating thep38 mitogen-activated protein kinase (MAPK), nuclearfactor (NF)-�B, ERK-1/2, and Akt survival pathways (Fig.4). The inhibition of these antiapoptosis-related pathwayssensitizes B-cell NHL to undergo apoptosis and even tochemotherapy for killing drug-resistant B-cell NHL celllines [6, 120 –122]. Furthermore, the inhibition of theNF-�B pathway by rituximab treatment can subse-quently block the downstream transcription repressorYin Yang 1, thereby resulting in enhancement of thetranscription of Fas-induced and DR5-induced apoptosis[123, 124] (Fig. 4).

Rituximab also enhances apoptosis by an as yet unclearcaspase-independent mechanism in B-cell NHL cells [125–128]. A broad-spectrum caspase inhibitor, zVAD-fmk, pre-vented processing of caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) as well as DNA fragmentation,but did not block apoptosis as measured by annexin V stain-ing, cell size, and membrane integrity in the rituximab re-sponse, which was also independent of Fas resistance andBcl-2 overexpression. Rituximab-treated cells show leak-age of adenylate kinase, surface expression of phosphati-dylserine, upregulation of the cellular stress protein heatshock protein-70, phosphorylation of the survival proteinAkt, and depolarization of the mitochondrial membrane butno loss of cytochrome c or apoptosis-inducing factor, whichcould not be blocked by caspase inhibitors. Furthermore, incytoplasts that lack mitochondria and in Bcl-2–transfectedcells, phosphatidylserine was still translocated to the cell

surface after CD20 stimulation. Consistently, in support ofthese data, there is no cleavage of caspase-3, caspase-8,caspase-9, PARP, BH3-interacting domain death agonist,or genomic DNA.

Figure 4. Rituximab-induced apoptosis in therapy and strat-egies to enhance apoptosis. PAG normally recruits Csk tomaintain the Src-family kinases Lyn, Fyn, and Lck in an inac-tivated state. CD20 –rituximab crosstalking can redistributelipid rafts and thus transactivate these kinases and initiatedownstream signaling pathways resulting in apoptosis. The re-distribution of lipid rafts also induces apoptosis by Fas mole-cule clustering, which leads to the formation of DISC and thesubsequent recruitment of FADD and caspase-8 into DISC,followed by the activation of the downstream apoptosis path-way. Meanwhile, the redistribution of lipid rafts can also in-hibit the p38 MAPK, ERK-1/2, NF-�B, and Akt signalingpathways and result in the inhibition of both transcription andexpression of many genes, particularly the antiapoptotic genesBcl-2, Bcl-xL, XIAP, and Mcl-1, thereby making B-lympho-cytes susceptible to apoptosis. In addition, inhibition of theNF-�B pathway downregulates the transcription factor YY1,enhancing the transcription of Fas and DR5 and facilitatingFasL- and TRAIL-induced apoptosis. Evidence suggests acaspase-independent apoptosis pathway, whose mechanismneeds more investigation. Strategies to enhance apoptosis afterrituximab treatment include: (a) scFvRit:sFasL; (b) mapatu-mumab, a humanized monoclonal antibody targeting TRAILreceptor 1; (c) a recombinant protein, Apo2L/TRAIL, all threedrugs target DRs to trigger the apoptosis pathway; and (d)other means such as antisense oligonucleotides of related ap-optotic molecules and bortezomib, a proteasome inhibitor thatinduces apoptosis.

Abbreviations: DISC, death-inducing signaling complex;DR, death receptor; ERK, extracellular signal–related kinase;FADD, Fas-associated death domain protein; MAPK, mito-gen-activated protein kinase; Mcl-1, myeloid cell leukemia se-quence 1; NF-�B, nuclear factor �B; PAG, phosphoproteinassociated with GEMs (glycosphingolipid-enriched mem-brane microdomains); TNF, tumor necrosis factor; TRAIL, tu-mor necrosis factor–related apoptosis-inducing ligand; XIAP,X-linked inhibitor of apoptosis protein; YY1, Yin Yang 1.

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Enhancing Apoptosis in Rituximab TherapyRecently, the synergistic effect of apoptosis-inducingdrugs in combination with rituximab in B-cell NHLtreatment has received much attention and interest. Agenetically engineered fusion protein, scFvRit:sFasL,containing a rituximab-derived antibody fragment andFas ligand was successfully used in malignant B cells[129] (Fig. 4). Mapatumumab, a humanized mAb thattargets/activates tumor necrosis factor–related apopto-sis-inducing ligand receptor 1 (TRAIL-R1) was able toaugment the antitumor activity of rituximab via signifi-cant apoptosis [130] (Fig. 4). Another recombinant hu-man mAb, Apo2 ligand (Apo2L)/TRAIL, which was inclinical trials for selectively stimulating apoptosis inother cancer cells through its proapoptotic receptors DR4and/or DR5, was strongly demonstrated to augment rit-uximab antitumor efficacy in mice inoculated with sev-eral different B-cell NHL cell lines [131] (Fig. 4).Alternatively, the downregulation of the apoptosis-sig-naling molecule survivin or Bfl-1 expression by anti-sense oligonucleotides also sensitized rituximab-mediated B-cell NHL cells to apoptosis [132, 133] (Fig.4). Research on patients suffering from relapsed or re-fractory cutaneous B-cell lymphoma disclosed a strongupregulation of the antiapoptotic molecule Bcl-2 com-pared with pretherapeutic levels, which suggests that up-regulation of Bcl-2 plays a major role in therapyresistance [134]. Furthermore, Jazirehi et al. [135] gen-erated rituximab-resistant B-cell NHL cell lines by in-creasing the concentration of rituximab in the culturemedium, and then found lower expression of CD20 inthese resistant clones. Interestingly, these clones exhib-ited hyperactivation of the p38 MAPK, NF-�B, andERK-1/2 pathways and overexpression of Bcl-2/Bcl-xL,indicating that the hyperactivation of these signalingpathways may be related to resistance. Therefore, it isreasonable to assume that the efficacy of rituximab willbe enhanced in combination with antisense Bcl-2 oligonucle-otides in SCID mouse/human lymphoma xenografts [136]. Inaddition, interference with hyperactivated signal pathwayswith various pharmacological and proteasome inhibitors re-versed resistance (Fig. 4). Bortezomib, a proteasome inhibitor,induced apoptosis earlier and sensitized resistant clonesto various cytotoxic drugs and rituximab by preventingthe degradation of proapoptotic factors and then activat-ing caspase-8, caspase-9, caspase-3, and PARP [135,137]. The development/acquisition of rituximab resis-tance may have arisen from perturbations of tumor cellsurvival signaling pathways and failure of rituximab toalter these pathways [6].

DIRECT GROWTH ARREST

The binding of rituximab to CD20 can also induce directgrowth arrest on lymphocytes. Bezombes et al. [138] dem-onstrated in vitro the direct inhibition of tumor growth byrituximab in Daudi and RL B-lymphoma cells. Rituximabinduced moderate accumulation of tumor cells in the G1

phase and significant loss of clonogenic potential withoutapoptosis. They observed that treatment with rituximab re-sulted in a rapid and transient increase in acid-sphingomy-elinase activity and concomitant cellular ceramidegeneration in raft microdomains. This result suggests thatrituximab-induced growth inhibition may be mediatedthrough a ceramide-triggered signaling pathway, leading tothe induction of cell cycle– dependent kinase inhibitorssuch as p27Kip1 through an MAPK-dependent mechanism.Therefore, rituximab mediates direct growth arrest, whichmay also contribute to its anticancer activity.

CONCLUSION

Although rituximab significantly improves the treatmentoutcome of B-cell NHL, it still has two challenges for us:unresponsiveness and resistance. Generally, the antitumoractivity of rituximab is attributed to CDC, ADCC, apopto-sis, and possible direct growth arrest, and CDC and ADCCare considered as the main effects. However, the contribu-tions of CDC and ADCC are very controversial and requirefurther investigation.

On the basis of the mechanisms described above, var-ious strategies may be designed for triumphing over re-sistance to rituximab therapy. For example, complementactivation in CDC can be enhanced by either increasingCD20 expression or heteroconjugating rituximab withCVF or C3b, or inhibiting mCRP (especially CD59)function. In particular, CD59 is a key inhibitor for MACformation to escape the immunoresponse. Sublytic MACis not sufficient to induce cytolysis, but may induce cellresistance by potentially increasing survival of cell-signaling pathways. Thus, it is imperative to develop aninhibitor of CD59 function. To enhance the ADCC ef-fect, some immunomodulatory cytokines such as M-CSF, IL-2, and IL-12, as well as CR3-binding �-glucan,may offer effective strategies to increase the antitumoractivity of rituximab. In addition, reduction of the apop-totic threshold or the induction of apoptotic signalingmay also increase tumor susceptibility to rituximab treat-ment through Bcl-2 inhibitors and bortezomib as well asmolecules targeting the tumor necrosis factor family re-ceptor. In conclusion, although the rational combinationof rituximab treatment with the above strategies towardclinical application requires further evaluation, this ap-

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proach has been extensively shown to be more effectivethan rituximab treatment alone.

ACKNOWLEDGMENTS

This work was supported by the U.S. National Institutes ofHealth grant RO1 AI061174, Harvard Technology Devel-opment Accelerator Fund, and Scientist Development grant0435483N from the American Heart Association. The au-thors take full responsibility for the content of the paper but

thank Jaylyn Olivo from the Brigham and Women’s Hos-pital Editorial Service for her assistance in editing themanuscript.

X.Z. and W.H. contributed equally to the paper.

AUTHOR CONTRIBUTIONSConception/design: Xuhui Zhou, Weiguo Hu, Xuebin QinFinancial support: Xuebin QinManuscript writing: Xuhui Zhou, Weiguo Hu, Xuebin QinFinal approval of manuscript: Xuhui Zhou, Weiguo Hu, Xuebin Qin

REFERENCES

1 Jemal A, Siegel R, Ward E et al. Cancer statistics, 2008. CA Cancer J Clin

2008;58:71–96.

2 Dillman RO. Monoclonal antibodies in the treatment of malignancy: Ba-

sic concepts and recent developments. Cancer Invest 2001;19:833–841.

3 Glennie MJ, French RR, Cragg MS et al. Mechanisms of killing by anti-

CD20 monoclonal antibodies. Mol Immunol 2007;44:3823–3837.

4 Maloney DG, Liles TM, Czerwinski DK et al. Phase I clinical trial using

escalating single-dose infusion of chimeric anti-CD20 monoclonal anti-

body (IDEC-C2B8) in patients with recurrent B-cell lymphoma. Blood

1994;84:2457–2466.

5 White CA, Larocca A, Grillo-Lopez AJ. Anti-CD20 monoclonal antibod-

ies as novel treatments for non-Hodgkin’s lymphoma. Pharm Sci Tech-

nolo Today 1999;2:95–101.

6 Bonavida B. Rituximab-induced inhibition of antiapoptotic cell survival

pathways: Implications in chemo/immunoresistance, rituximab unrespon-

siveness, prognostic and novel therapeutic interventions. Oncogene 2007;

26:3629–3636.

7 Schriever F, Freedman AS, Freeman G et al. Isolated human follicular

dendritic cells display a unique antigenic phenotype. J Exp Med 1989;169:

2043–2058.

8 Anderson KC, Bates MP, Slaughenhoupt BL et al. Expression of human B

cell-associated antigens on leukemias and lymphomas: A model of human

B cell differentiation. Blood 1984;63:1424–1433.

9 Nadler LM, Ritz J, Hardy R et al. A unique cell surface antigen identifying

lymphoid malignancies of B cell origin. J Clin Invest 1981;67:134–140.

10 O’Keefe TL, Williams GT, Davies SL et al. Mice carrying a CD20 gene

disruption. Immunogenetics 1998;48:125–132.

11 Binder M, Otto F, Mertelsmann R et al. The epitope recognized by ritux-

imab. Blood 2006;108:1975–1978.

12 Du J, Wang H, Zhong C et al. Structural basis for recognition of CD20 by

therapeutic antibody rituximab. J Biol Chem 2007;282:15073–15080.

13 Fishelson Z, Donin N, Zell S et al. Obstacles to cancer immunotherapy:

Expression of membrane complement regulatory proteins (mCRPs) in tu-

mors. Mol Immunol 2003;40:109–123.

14 Harjunpää A, Junnikkala S, Meri S. Rituximab (anti-CD20) therapy of B-

cell lymphomas: Direct complement killing is superior to cellular effector

mechanisms. Scand J Immunol 2000;51:634–641.

15 Morgan BP. Regulation of the complement membrane attack pathway.

Crit Rev Immunol 1999;19:173–198.

16 Walport MJ. Complement. First of two parts. N Engl J Med 2001;344:

1058–1066.

17 Qin X, Ferris S, Hu W et al. Analysis of the promoters and 5�-UTR of

mouse Cd59 genes, and of their functional activity in erythrocytes. Genes

Immun 2006;7:287–297.

18 Qin X, Goldfine A, Krumrei N et al. Glycation inactivation of the com-

plement regulatory protein CD59: A possible role in the pathogenesis of

the vascular complications of human diabetes. Diabetes 2004;53:2653–

2661.

19 Muller-Eberhard HJ. Molecular organization and function of the comple-

ment system. Annu Rev Biochem 1988;57:321–347.

20 Thurman JM, Holers VM. The central role of the alternative complement

pathway in human disease. J Immunol 2006;176:1305–1310.

21 Papadimitriou JC, Drachenberg CB, Shin ML et al. Ultrastructural studies

of complement mediated cell death: A biological reaction model to plasma

membrane injury. Virchows Arch 1994;424:677–685.

22 Bellosillo B, Villamor N, Lopez-Guillermo A et al. Complement-medi-

ated cell death induced by rituximab in B-cell lymphoproliferative disor-

ders is mediated in vitro by a caspase-independent mechanism involving

the generation of reactive oxygen species. Blood 2001;98:2771–2777.

23 Ollert MW, Kadlec JV, David K et al. Antibody-mediated complement

activation on nucleated cells. A quantitative analysis of the individual

reaction steps. J Immunol 1994;153:2213–2221.

24 Gerard C, Gerard NP. C5A anaphylatoxin and its seven transmembrane-

segment receptor. Annu Rev Immunol 1994;12:775–808.

25 Chen NJ, Mirtsos C, Suh D et al. C5L2 is critical for the biological activ-

ities of the anaphylatoxins C5a and C3a. Nature 2007;446:203–207.

26 Hourcade D, Holers VM, Atkinson JP. The regulators of complement

activation (RCA) gene cluster. Adv Immunol 1989;45:381–416.

27 Miwa T, Song WC. Membrane complement regulatory proteins: Insight

from animal studies and relevance to human diseases. Int Immunophar-

macol 2001;1:445–459.

28 Liszewski MK, Post TW, Atkinson JP. Membrane cofactor protein (MCP

or CD46): Newest member of the regulators of complement activation

gene cluster. Annu Rev Immunol 1991;9:431–455.

29 Lublin DM, Atkinson JP. Decay-accelerating factor: Biochemistry, mo-

lecular biology, and function. Annu Rev Immunol 1989;7:35–58.

30 Mollnes TE, Song WC, Lambris JD. Complement in inflammatory tissue

damage and disease. Trends Immunol 2002;23:61–64.

31 Ahearn JM, Fearon DT. Structure and function of the complement recep-

tors, CR1 (CD35) and CR2 (CD21). Adv Immunol 1989;46:183–219.

32 Holers VM, Kinoshita T, Molina H. The evolution of mouse and human

complement C3-binding proteins: Divergence of form but conservation of

function. Immunol Today 1992;13:231–236.

33 Li B, Sallee C, Dehoff M et al. Mouse Crry/p65. Characterization of

monoclonal antibodies and the tissue distribution of a functional homo-

logue of human MCP and DAF. J Immunol 1993;151:4295–4305.

963Zhou, Hu, Qin

www.TheOncologist.com

by guest on May 28, 2018

http://theoncologist.alphamedpress.org/

Dow

nloaded from

34 Kurtz CB, Paul MS, Aegerter M et al. Murine complement receptor gene

family. II. Identification and characterization of the murine homolog

(Cr2) to human CR2 and its molecular linkage to Crry. J Immunol 1989;

143:2058–2067.

35 Paul MS, Aegerter M, O’Brien SE et al. The murine complement receptor

gene family. Analysis of mCRY gene products and their homology to

human CR1. J Immunol 1989;142:582–589.

36 Kim DD, Song WC. Membrane complement regulatory proteins. Clin Im-

munol 2006;118:127–136.

37 Telen MJ, Green AM. The Inab phenotype: Characterization of the mem-

brane protein and complement regulatory defect. Blood 1989;74:437–

441.

38 Gossling J. Occurrence and pathogenicity of the Streptococcus milleri

group. Rev Infect Dis 1988;10:257–285.

39 Reid ME, Mallinson G, Sim RB et al. Biochemical studies on red blood

cells from a patient with the Inab phenotype (decay-accelerating factor de-

ficiency). Blood 1991;78:3291–3297.

40 Qian YM, Qin X, Miwa T et al. Identification and functional character-

ization of a new gene encoding the mouse terminal complement inhibitor

CD59. J Immunol 2000;165:2528–2534.

41 Qin X, Krumrei N, Grubissich L et al. Deficiency of the mouse comple-

ment regulatory protein mCd59b results in spontaneous hemolytic anemia

with platelet activation and progressive male infertility. Immunity 2003;

18:217–227.

42 Akatsu H, Yamada T, Okada N et al. Unique expression of HRF20 (CD59)

in human nervous tissue. Microbiol Immunol 1997;41:321–329.

43 Walport MJ. Complement. Second of two parts. N Engl J Med 2001;344:

1140–1144.

44 Takeda J, Miyata T, Kawagoe K et al. Deficiency of the GPI anchor

caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal

hemoglobinuria. Cell 1993;73:703–711.

45 Hillmen P, Richards SJ. Implications of recent insights into the patho-

physiology of paroxysmal nocturnal haemoglobinuria. Br J Haematol

2000;108:470–479.

46 Hillmen P, Young NS, Schubert J et al. The complement inhibitor eculi-

zumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med 2006;355:

1233–1243.

47 Koski CL, Ramm LE, Hammer CH et al. Cytolysis of nucleated cells by

complement: Cell death displays multi-hit characteristics. Proc Natl Acad

Sci U S A 1983;80:3816–3820.

48 Bohana-Kashtan O, Ziporen L, Donin N et al. Cell signals transduced by

complement. Mol Immunol 2004;41:583–597.

49 Shin ML, Carney DF. Cytotoxic action and other metabolic consequences

of terminal complement proteins. Prog Allergy 1988;40:44–81.

50 Morgan BP. Complement membrane attack on nucleated cells: Resis-

tance, recovery and non-lethal effects. Biochem J 1989;264:1–14.

51 Cragg MS, Glennie MJ. Antibody specificity controls in vivo effector

mechanisms of anti-CD20 reagents. Blood 2004;103:2738–2743.

52 Cittera E, Leidi M, Buracchi C et al. The CCL3 family of chemokines and

innate immunity cooperate in vivo in the eradication of an established

lymphoma xenograft by rituximab. J Immunol 2007;178:6616–6623.

53 Golay J, Cittera E, Di Gaetano N et al. The role of complement in the ther-

apeutic activity of rituximab in a murine B lymphoma model homing in

lymph nodes. Haematologica 2006;91:176–183.

54 Di Gaetano N, Cittera E, Nota R et al. Complement activation determines

the therapeutic activity of rituximab in vivo. J Immunol 2003;171:1581–

1587.

55 Uchida J, Hamaguchi Y, Oliver JA et al. The innate mononuclear phago-

cyte network depletes B lymphocytes through Fc receptor-dependent

mechanisms during anti-CD20 antibody immunotherapy. J Exp Med

2004;199:1659–1669.

56 Hamaguchi Y, Xiu Y, Komura K et al. Antibody isotype-specific engage-

ment of Fcgamma receptors regulates B lymphocyte depletion during

CD20 immunotherapy. J Exp Med 2006;203:743–753.

57 Hamaguchi Y, Uchida J, Cain DW et al. The peritoneal cavity provides a

protective niche for B1 and conventional B lymphocytes during anti-

CD20 immunotherapy in mice. J Immunol 2005;174:4389–4399.

58 Shushakova N, Skokowa J, Schulman J et al. C5a anaphylatoxin is a major

regulator of activating versus inhibitory FcgammaRs in immune complex-

induced lung disease. J Clin Invest 2002;110:1823–1830.

59 Schmidt RE, Gessner JE. Fc receptors and their interaction with comple-

ment in autoimmunity. Immunol Lett 2005;100:56–67.

60 Huber-Lang M, Sarma JV, Zetoune FS et al. Generation of C5a in the ab-

sence of C3: A new complement activation pathway. Nat Med 2006;12:

682–687.

61 Kumar V, Ali SR, Konrad S et al. Cell-derived anaphylatoxins as key me-

diators of antibody-dependent type II autoimmunity in mice. J Clin Invest

2006;116:512–520.

62 van Meerten T, van Rijn RS, Hol S et al. Complement-induced cell death

by rituximab depends on CD20 expression level and acts complementary

to antibody-dependent cellular cytotoxicity. Clin Cancer Res 2006;12:

4027–4035.

63 Golay J, Lazzari M, Facchinetti V et al. CD20 levels determine the in vitro

susceptibility to rituximab and complement of B-cell chronic lymphocytic

leukemia: Further regulation by CD55 and CD59. Blood 2001;98:3383–

3389.

64 Manches O, Lui G, Chaperot L et al. In vitro mechanisms of action of rit-

uximab on primary non-Hodgkin lymphomas. Blood 2003;101:949–954.

65 Perz J, Topaly J, Fruehauf S et al. Level of CD 20-expression and efficacy

of rituximab treatment in patients with resistant or relapsing B-cell

prolymphocytic leukemia and B-cell chronic lymphocytic leukemia. Leuk

Lymphoma 2002;43:149–151.

66 Weng WK, Levy R. Expression of complement inhibitors CD46, CD55,

and CD59 on tumor cells does not predict clinical outcome after rituximab

treatment in follicular non-Hodgkin lymphoma. Blood 2001;98:1352–

1357.

67 Takei K, Yamazaki T, Sawada U et al. Analysis of changes in CD20,

CD55, and CD59 expression on established rituximab-resistant B-lym-

phoma cell lines Leuk Res 2006;30:625–631.

68 Koretz K, Bruderlein S, Henne C et al. Expression of CD59, a complement

regulator protein and a second ligand of the CD2 molecule, and CD46 in

normal and neoplastic colorectal epithelium. Br J Cancer 1993;68:926–

931.

69 Inoue H, Mizuno M, Uesu T et al. Distribution of complement regulatory

proteins, decay-accelerating factor, CD59/homologous restriction factor

20 and membrane cofactor protein in human colorectal adenoma and can-

cer Acta Med Okayama 1994;48:271–277.

70 Cruz RI, Hernandez-Ilizaliturri FJ, Olejniczak S et al. CD52 over-expres-

sion affects rituximab-associated complement-mediated cytotoxicity but

not antibody-dependent cellular cytotoxicity: Preclinical evidence that

targeting CD52 with alemtuzumab may reverse acquired resistance to rit-

uximab in non-Hodgkin lymphoma. Leuk Lymphoma 2007;48:2424 –

2436.

71 Treon SP, Mitsiades C, Mitsiades N et al. Tumor cell expression of CD59

964 Mechanisms of Action of and Resistance to Rituximab

by guest on May 28, 2018

http://theoncologist.alphamedpress.org/

Dow

nloaded from

is associated with resistance to CD20 serotherapy in patients with B-cell

malignancies. J Immunother 2001;24:263–271.

72 Golay J, Zaffaroni L, Vaccari T et al. Biologic response of B lymphoma

cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and

CD59 regulate complement-mediated cell lysis. Blood 2000;95:3900 –

3908.

73 Ziller F, Macor P, Bulla R et al. Controlling complement resistance in can-

cer by using human monoclonal antibodies that neutralize complement-

regulatory proteins CD55 and CD59. Eur J Immunol 2005;35:2175–2183.

74 Macor P, Tripodo C, Zorzet S et al. In vivo targeting of human neutralizing

antibodies against CD55 and CD59 to lymphoma cells increases the anti-

tumor activity of rituximab. Cancer Res 2007;67:10556–10563.

75 Goldenberg DM, Sharkey RM, Paganelli G et al. Antibody pretargeting

advances cancer radioimmunodetection and radioimmunotherapy. J Clin

Oncol 2006;24:823–834.

76 Paganelli G, Bartolomei M, Grana C et al. Radioimmunotherapy of brain

tumor. Neurol Res 2006;28:518–522.

77 Paganelli G, Magnani P, Zito F et al. Three-step monoclonal antibody tu-

mor targeting in carcinoembryonic antigen-positive patients. Cancer Res

1991;51:5960–5966.

78 Terui Y, Sakurai T, Mishima Y et al. Blockade of bulky lymphoma-

associated CD55 expression by RNA interference overcomes resistance to

complement-dependent cytotoxicity with rituximab. Cancer Sci 2006;97:

72–79.

79 Di Gaetano N, Xiao Y, Erba E et al. Synergism between fludarabine and

rituximab revealed in a follicular lymphoma cell line resistant to the cy-

totoxic activity of either drug alone. Br J Haematol 2001;114:800–809.

80 Nagamune H, Ohnishi C, Katsuura A et al. Intermedilysin, a novel cyto-

toxin specific for human cells secreted by Streptococcus intermedius

UNS46 isolated from a human liver abscess. Infect Immun 1996;64:3093–

3100.

81 Giddings KS, Zhao J, Sims PJ et al. Human CD59 is a receptor for the

cholesterol-dependent cytolysin intermedilysin. Nat Struct Mol Biol

2004;11:1173–1178.

82 Hu W, Ferris SP, Tweten RK et al. Rapid conditional targeted ablation of

cells expressing human CD59 in transgenic mice by intermedilysin. Nat

Med 2008;14:98–103.

83 Haidar JH, Shamseddine A, Salem Z et al. Loss of CD20 expression in

relapsed lymphomas after rituximab therapy. Eur J Haematol 2003;70:

330–332.

84 Beum PV, Kennedy AD, Williams ME et al. The shaving reaction: Ritux-

imab/CD20 complexes are removed from mantle cell lymphoma and

chronic lymphocytic leukemia cells by THP-1 monocytes. J Immunol

2006;176:2600–2609.

85 Li Y, Williams ME, Cousar JB et al. Rituximab-CD20 complexes are

shaved from Z138 mantle cell lymphoma cells in intravenous and subcu-

taneous SCID mouse models. J Immunol 2007;179:4263–4271.

86 Zhao WL, Wang L, Liu YH et al. Combined effects of histone deacetylase

inhibitor and rituximab on non-Hodgkin’s B-lymphoma cells apoptosis.

Exp Hematol 2007;35:1801–1811.

87 Tomita A, Hiraga J, Kiyoi H et al. Epigenetic regulation of CD20 protein

expression in a novel B-cell lymphoma cell line, RRBL1, established from

a patient treated repeatedly with rituximab-containing chemotherapy. Int

J Hematol 2007;86:49–57.

88 Wojciechowski W, Li H, Marshall S et al. Enhanced expression of CD20

in human tumor B cells is controlled through ERK-dependent mecha-

nisms. J Immunol 2005;174:7859–7868.

89 Jahrsdorfer B, Muhlenhoff L, Blackwell SE et al. B-cell lymphomas differ

in their responsiveness to CpG oligodeoxynucleotides. Clin Cancer Res

2005;11:1490–1499.

90 Jahrsdörfer B, Hartmann G, Racila E et al. CpG DNA increases primary

malignant B cell expression of costimulatory molecules and target anti-

gens. J Leukoc Biol 2001;69:81–88.

91 Reiter Y, Fishelson Z. Targeting of complement to tumor cells by hetero-

conjugates composed of antibodies and of the complement component

C3b. J Immunol 1989;142:2771–2777.

92 Gelderman KA, Kuppen PJ, Bruin W et al. Enhancement of the comple-

ment activating capacity of 17–1A mAb to overcome the effect of mem-

brane-bound complement regulatory proteins on colorectal carcinoma.

Eur J Immunol 2002;32:128–135.

93 Juhl H, Petrella EC, Cheung NK et al. Complement killing of human neu-

roblastoma cells: A cytotoxic monoclonal antibody and its F(ab�)2-cobra

venom factor conjugate are equally cytotoxic. Mol Immunol 1990;27:

957–964.

94 Juhl H, Petrella EC, Cheung NK et al. Additive cytotoxicity of different

monoclonal antibody-cobra venom factor conjugates for human neuro-

blastoma cells. Immunobiology 1997;197:444–459.

95 Kennedy AD, Solga MD, Schuman TA et al. An anti-C3b(i) mAb en-

hances complement activation, C3b(i) deposition, and killing of CD20�

cells by rituximab. Blood 2003;101:1071–1079.

96 Clynes RA, Towers TL, Presta LG et al. Inhibitory Fc receptors modulate

in vivo cytoxicity against tumor targets. Nat Med 2000;6:443–446.

97 Hernandez-Ilizaliturri FJ, Jupudy V, Ostberg J et al. Neutrophils contrib-

ute to the biological antitumor activity of rituximab in a non-Hodgkin’s

lymphoma severe combined immunodeficiency mouse model. Clin Can-

cer Res 2003;9:5866–5873.

98 Kennedy AD, Beum PV, Solga MD et al. Rituximab infusion promotes

rapid complement depletion and acute CD20 loss in chronic lymphocytic

leukemia. J Immunol 2004;172:3280–3288.

99 Gorter A, Meri S. Immune evasion of tumor cells using membrane-bound

complement regulatory proteins. Immunol Today 1999;20:576–582.

100 Cartron G, Dacheux L, Salles G et al. Therapeutic activity of humanized

anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor

FcgammaRIIIa gene. Blood 2002;99:754–758.

101 Mller T, Uherek C, Maki G et al. Expression of a CD20-specific chimeric

antigen receptor enhances cytotoxic activity of NK cells and overcomes

NK-resistance of lymphoma and leukemia cells. Cancer Immunol Immu-

nother 2008;57:411–423.

102 Shimadoi S, Takami A, Kondo Y et al. Macrophage colony-stimulating

factor enhances rituximab-dependent cellular cytotoxicity by monocytes.

Cancer Sci 2007;98:1368–1372.

103 Golay J, Manganini M, Facchinetti V et al. Rituximab-mediated antibody-

dependent cellular cytotoxicity against neoplastic B cells is stimulated

strongly by interleukin-2. Haematologica 2003;88:1002–1012.

104 Lopes de Menezes DE, Denis-Mize K, Tang Y et al. Recombinant inter-

leukin-2 significantly augments activity of rituximab in human tumor

xenograft models of B-cell non-Hodgkin lymphoma. J Immunother 2007;

30:64–74.

105 Gillies SD, Lan Y, Williams S et al. An anti-CD20-IL-2 immunocytokine

is highly efficacious in a SCID mouse model of established human B lym-

phoma. Blood 2005;105:3972–3978.

106 Ansell SM, Witzig TE, Kurtin PJ et al. Phase 1 study of interleukin-12 in

combination with rituximab in patients with B-cell non-Hodgkin lym-

phoma. Blood 2002;99:67–74.

965Zhou, Hu, Qin

www.TheOncologist.com

by guest on May 28, 2018

http://theoncologist.alphamedpress.org/

Dow

nloaded from

107 Ansell SM, Geyer SM, Maurer MJ et al. Randomized phase II study of

interleukin-12 in combination with rituximab in previously treated non-

Hodgkin’s lymphoma patients. Clin Cancer Res 2006;12:6056–6063.

108 Ross GD. Regulation of the adhesion versus cytotoxic functions of the

Mac-1/CR3/alphaMbeta2-integrin glycoprotein. Crit Rev Immunol 2000;

20:197–222.

109 Vetvicka V, Thornton BP, Ross GD. Soluble beta-glucan polysaccharide

binding to the lectin site of neutrophil or natural killer cell complement

receptor type 3 (CD11b/CD18) generates a primed state of the receptor

capable of mediating cytotoxicity of iC3b-opsonized target cells. J Clin

Invest 1996;98:50–61.

110 Xia Y, Vetvicka V, Yan J et al. The beta-glucan-binding lectin site of

mouse CR3 (CD11b/CD18) and its function in generating a primed state

of the receptor that mediates cytotoxic activation in response to iC3b-

opsonized target cells. J Immunol 1999;162:2281–2290.

111 Hong F, Hansen RD, Yan J et al. Beta-glucan functions as an adjuvant for

monoclonal antibody immunotherapy by recruiting tumoricidal granulo-

cytes as killer cells. Cancer Res 2003;63:9023–9031.

112 Modak S, Koehne G, Vickers A et al. Rituximab therapy of lymphoma is

enhanced by orally administered (1--�3),(1--�4)-D-beta-glucan. Leuk

Res 2005;29:679–683.

113 Deans JP, Schieven GL, Shu GL et al. Association of tyrosine and serine

kinases with the B cell surface antigen CD20. Induction via CD20 of ty-

rosine phosphorylation and activation of phospholipase C-gamma 1 and

PLC phospholipase C-gamma 2. J Immunol 1993;151:4494–4504.

114 Deans JP, Robbins SM, Polyak MJ et al. Rapid redistribution of CD20 to

a low density detergent-insoluble membrane compartment. J Biol Chem

1998;273:344–348.

115 Deans JP, Li H, Polyak MJ. CD20-mediated apoptosis: Signalling through

lipid rafts. Immunology 2002;107:176–182.

116 Deans JP, Kalt L, Ledbetter JA et al. Association of 75/80-kDa phospho-

proteins and the tyrosine kinases Lyn, Fyn, and Lck with the B cell mol-

ecule CD20. Evidence against involvement of the cytoplasmic regions of

CD20. J Biol Chem 1995;270:22632–22638.

117 Brdicka T, Pavlistova D, Leo A et al. Phosphoprotein associated with gly-

cosphingolipid-enriched microdomains (PAG), a novel ubiquitously ex-

pressed transmembrane adaptor protein, binds the protein tyrosine kinase

Csk and is involved in regulation of T cell activation. J Exp Med 2000;

191:1591–1604.

118 Kawabuchi M, Satomi Y, Takao T et al. Transmembrane phosphoprotein

Cbp regulates the activities of Src-family tyrosine kinases. Nature 2000;

404:999–1003.

119 Stel AJ, Ten Cate B, Jacobs S et al. Fas receptor clustering and involve-

ment of the death receptor pathway in rituximab-mediated apoptosis with

concomitant sensitization of lymphoma B cells to Fas-induced apoptosis.

J Immunol 2007;178:2287–2295.

120 Byrd JC, Kitada S, Flinn IW et al. The mechanism of tumor cell clearance

by rituximab in vivo in patients with B-cell chronic lymphocytic leuke-

mia: Evidence of caspase activation and apoptosis induction. Blood 2002;

99:1038–1043.

121 Pedersen IM, Buhl AM, Klausen P et al. The chimeric anti-CD20 antibody

rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells

through a p38 mitogen activated protein-kinase-dependent mechanism.

Blood 2002;99:1314–1319.

122 Suzuki E, Umezawa K, Bonavida B. Rituximab inhibits the constitutively

activated PI3K-Akt pathway in B-NHL cell lines: Involvement in chemo-

sensitization to drug-induced apoptosis. Oncogene 2007;26:6184–6193.

123 Vega MI, Huerta-Yepaz S, Garban H et al. Rituximab inhibits p38 MAPK

activity in 2F7 B NHL and decreases IL-10 transcription: Pivotal role of

p38 MAPK in drug resistance. Oncogene 2004;23:3530–3540.

124 Huerta-Yepez S, Vega M, Jazirehi A et al. Nitric oxide sensitizes prostate

carcinoma cell lines to TRAIL-mediated apoptosis via inactivation of NF-

kappa B and inhibition of Bcl-xl expression. Oncogene 2004;23:4993–

5003.

125 van der Kolk LE, Evers LM, Omene C et al. CD20-induced B cell death

can bypass mitochondria and caspase activation. Leukemia 2002;16:

1735–1744.

126 Chan HT, Hughes D, French RR et al. CD20-induced lymphoma cell

death is independent of both caspases and its redistribution into triton

X-100 insoluble membrane rafts. Cancer Res 2003;63:5480–5489.

127 Skvortsova I, Skvortsov S, Popper BA et al. Rituximab enhances radia-

tion-triggered apoptosis in non-Hodgkin’s lymphoma cells via caspase-

dependent and -independent mechanisms. J Radiat Res (Tokyo) 2006;47:

183–196.

128 Daniels I, Abulayha AM, Thomson BJ et al. Caspase-independent killing

of Burkitt lymphoma cell lines by rituximab. Apoptosis 2006;11:1013–

1023.

129 Bremer E, ten Cate B, Samplonius DF et al. Superior activity of fusion

protein scFvRit:sFasL over cotreatment with rituximab and Fas agonists.

Cancer Res 2008;68:597–604.

130 Maddipatla S, Hernandez-Ilizaliturri FJ, Knight J et al. Augmented anti-

tumor activity against B-cell lymphoma by a combination of monoclonal

antibodies targeting TRAIL-R1 and CD20. Clin Cancer Res 2007;13:

4556–4564.

131 Daniel D, Yang B, Lawrence DA et al. Cooperation of the proapoptotic re-

ceptor agonist rhApo2L/TRAIL with the CD20 antibody rituximab against

non-Hodgkin lymphoma xenografts. Blood 2007;110:4037–4046.

132 Ansell SM, Arendt BK, Grote DM et al. Inhibition of survivin expression

suppresses the growth of aggressive non-Hodgkin’s lymphoma. Leuke-

mia 2004;18:616–623.

133 Brien G, Trescol-Biemont MC, Bonnefoy-Bérard N. Downregulation of

Bfl-1 protein expression sensitizes malignant B cells to apoptosis. Onco-

gene 2007;26:5828–5832.

134 Wobser M, Voigt H, Eggert AO et al. Bcl-2 expression in rituximab re-

fractory cutaneous B-cell lymphoma. Br J Cancer 2007;96:1540–1543.

135 Jazirehi AR, Vega MI, Bonavida B. Development of rituximab-resistant

lymphoma clones with altered cell signaling and cross-resistance to che-

motherapy. Cancer Res 2007;67:1270–1281.

136 Smith MR, Jin F, Joshi I. Enhanced efficacy of therapy with antisense

BCL-2 oligonucleotides plus anti-CD20 monoclonal antibody in SCID

mouse/human lymphoma xenografts. Mol Cancer Ther 2004;3:1693–

1699.

137 Wang M, Han XH, Zhang L et al. Bortezomib is synergistic with ritux-

imab and cyclophosphamide in inducing apoptosis of mantle cell lym-

phoma cells in vitro and in vivo. Leukemia 2008;22:179–185.

138 Bezombes C, Grazide S, Garret C et al. Rituximab antiproliferative effect

in B-lymphoma cells is associated with acid-sphingomyelinase activation

in raft microdomains. Blood 2004;104:1166–1173.

966 Mechanisms of Action of and Resistance to Rituximab

by guest on May 28, 2018

http://theoncologist.alphamedpress.org/

Dow

nloaded from