Leukemia Research 29 (2005) 679–683

Case report

Rituximab therapy of lymphoma is enhanced by orallyadministered (1→ 3),(1→ 4)-d-�-glucan

Shakeel Modaka,∗, Guenther Koehneb, Andrew Vickersb,c,Richard J. O’Reillya, Nai-Kong V. Cheunga

a Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY, USAb Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY, USA

c Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center,1275 York Avenue, New York, NY, USA

Received 20 August 2004; accepted 27 October 2004Available online 2 February 2005

Abstract

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By activating complement, antitumor monoclonal antibodies coat tumor cells with iC3b.�-glucans, naturally occurring glucose polymeind to the lectin domain of the leukocyte receptor CR3, prime it for binding to iC3b, and trigger cytotoxicity of iC3b-coated tume studied the combination of the complement-activating antibody rituximab with barley-derived (1→ 3),(1→ 4)-�-d-glucan (BG) againsD-20 positive lymphoma xenografts in SCID mice. Growth of established subcutaneous non-Hodgkin’s lymphoma (NHL) (Daudi aerived B-NHL) or Hodgkin’s disease (Hs445 and RPMI6666) was significantly suppressed in mice treated with a combination of inituximab and oral BG, when compared to mice treated with rituximab or BG alone. Survival of mice with disseminated lymphignificantly increased in the combination group as compared to other treatment groups. No clinical toxicity was observed. The tfficacy and lack of toxicity of this combination supports further investigation into its clinical utility.2004 Elsevier Ltd. All rights reserved.

eywords:Rituximab;�-Glucan; iC3b-mediated cytotoxicity

. Introduction

The chimeric anti-CD20 antibody rituximab is beingvaluated in an increasing number of disorders. After clinicalfficacy was initially demonstrated against relapsed andefractory follicular/low grade non-Hodgkin’s lymphoma,esponses to rituximab have been reported in other malignantnd non-malignant B-cell disorders. Several mechanismsf action have been proposed including activation ofpoptotic pathways, elaboration of cytokines and elicitationf host complement-dependent cytotoxicity (CDC) and

∗ Corresponding author. Tel.: +1 212 6397623; fax: +1 212 7442245.E-mail addresses:modaks@mskcc.org (S. Modak),

uentherkoehne@rush.edu (G. Koehne), vickersa@mskcc.orgA. Vickers), oreillyr@mskcc.org (R.J. O’Reilly), cheungn@mskcc.orgN.-K.V. Cheung).

antibody-dependent cell-mediated cytotoxicity (ADCAlthough many patients with B-cell disorders respondrituximab, remissions are often transient. More than 5of lymphomas recurrent after rituximab treatment fato respond the second time. Mechanisms of resistanrituximab are as yet unclear, and may include paucitloss of target antigen, pharmacokinetic variations amindividual patients, FcR polymorphism, resistancecomplement activity or inherent gene expression oflymphoma[1].

The iC3b receptor, CR3 is found on monocymacrophages, neutrophils, NK cells and cytotoxiclymphocytes. CR3 activation requires the engagemetwo sites on its�-subunit (CD11b): the iC3b binding swithin the I-domain at the N-terminus and a lectin sitethe C-terminus.�-glucans, complex polymers of glucosetractable from barley, mushrooms, seaweed and yeasts

145-2126/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2004.10.008

680 S. Modak et al. / Leukemia Research 29 (2005) 679–683

specifically to the lectin site, as demonstrated initially by Rosset al.[2]. With bound�-glucan, CR3 is primed to engage iC3bfragments deposited on cells by complement-activating an-tibodies such as rituximab. CR3 mediates the diapedesis ofleukocytes through the endothelium and stimulates phago-cytosis, degranulation and tumor cytotoxicity. Many fungipresent�-glucan or�-glucan-like CR3 binding ligands ontheir cell surface. Hence, when iC3b deposition occurs, bothCD11b and lectin sites become engaged, and phagocytosisand respiratory burst is triggered. In contrast, tumor cellslack such molecules, and even when coated with iC3b do notgenerally activate CR3 and cannot activate leucocytes. Sol-uble forms of�-glucan bind to lectin sites and prime bothphagocytic and NK cells to kill iC3b-coated tumor targets[3,4].

(1→ 3),(1→ 4)-d-�-glucan (BG), a soluble, barley-derived �-glucan has advantages over previously studied(1→ 3),(1→ 6)-�-glucans, particularly efficacy when ad-ministered orally and a good safety profile[2]. We re-cently demonstrated in vivo synergism between BG and thecomplement-activating antibody 3F8 against human neurob-lastoma xenografts[3,4]. We now report on the synergismbetween BG and rituximab against lymphoma.

2. Materials and methods

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2.2. Tumor models

Subcutaneous tumors were established for all cell linesby injecting 5× 106 cells suspended in 0.1 ml of Matrigel(Becton-Dickinson, Franklin Lakes, NJ) into mice flanks. Tu-mor dimensions were measured two to three times a weekand tumor size was calculated as product of the two largestdiameters. Mice were sacrificed when maximum tumor di-mension exceeded 20 mm. A disseminated tumor model wasestablished in SCID mice as previously described[6]. Briefly,5× 106 Daudi or Hs445 cells suspended in 100�l normalsaline were injected intravenously (i.v.) into SCID mice. Tu-mors grew systemically and mice became paralyzed whentumor cells infiltrated the spinal cord, resulting in hind-legparalysis. Mice were sacrificed at onset of paralysis or whenanimals lost 10% of their body weight.

2.3. Treatment regimens

For mice with subcutaneous tumors, therapy was initiatedafter tumors were established (7–8 mm diameter). For thedisseminated tumor model, therapy was initiated ten daysafter injection of tumor cells. Groups of four or five mice pertreatment regimen received either rituximab, BG, neither orboth. Rituximab (Genentech, San Francisco, CA) at a dose of4 l)w nsa mln vaged erea eklya

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) l (days)

23

25

S Hs445

.1. Cell lines and mice

Human Burkitt’s lymphoma cell line, Daudi, aodgkin’s disease (HD) cell lines Hs445 and RPMI 6ere purchased from American Type Culture Collec

Rockville, MD). Human Epstein–Barr virus-derivedymphoblastoid (EBV-BLCL) cell lines were establishedng previously described methods[5]. All cell lines were

aintained in RPMI 1640 supplemented with 10% bovalf serum, 2 mM glutamine, 100 IU/ml of penicillin, a00�g/ml of streptomycin. Female Fox Chase ICR SCice (Taconic, White Plains, NY) were maintained un

nstitutionally approved guidelines and protocols.

able 1

Disseminated model

Number treated Median survival (days

DaudiNo treatment 18 48Rituximab alone 47 77BG alone 15 43Rituximab + BG 61 124*

Hs445No treatment 4 12BG alone 4 16Rituximab alone 8 31Rituximab + BG 8 243*

urvival data in mice with disseminated and subcutaneous Daudi and∗ p< 0.05 for combination group compared to all other groups.

0�g (disseminated model) or 200�g (subcutaneous modeas injected i.v. twice weekly for a total of eight injectiond 400�g BG (Sigma, St. Louis, MO) dissolved in 0.1ormal saline was administered orally via intragastric gaaily for 29 days. The first doses of rituximab and BG wdministered concurrently. Animals were weighed wend observed clinically at least once daily.

.4. Statistical analysis

Tumor growth was calculated by fitting a regression sor each individual mouse to log transformed values oor size.p-Values for the difference between groups w

alculated usingt-tests for the area-under-the-curve for

Subcutaneous model

% surviving >365 days Number treated Median surviva

0 5 270 8 390 5 34

9 102*

0 4 2000 5 28

5 45*

lymphoma xenografts.

S. Modak et al. / Leukemia Research 29 (2005) 679–683 681

mor size, using a previously described method for censoredobservations[7]. Survival in mice with disseminated diseasewas compared using Kaplan–Meier analysis and proportionof deaths was compared by Fisher’s exact test. Analyses wereconducted using STATA 7 (Stata Corporation, College Sta-tion, TX).

3. Results

3.1. Subcutaneous xenografts

Numbers of mice treated for Daudi and Hs445 xenograftsare represented inTable 1. For EBV-BLCL and RPMI6666xenografts, at least nine mice each were included in rituximabalone and combination groups. In all subcutaneous xenograftmodels, significant reduction in tumor growth was noted inmice treated with a combination of rituximab and BG. Micetreated with rituximab alone showed a modest reduction intumor growth, while those treated with BG alone or left un-treated had unabated tumor growth. In a multivariable linearmodel of tumor growth rate, using dummy variables for treat-ment, the interaction between BG and rituximab was positiveand significant, demonstrating synergism. The reduction intumor growth per day in the group receiving BG in addi-tion to rituximab compared to rituximab alone was 2.0%( -B %C %C esf BV-B rt mms tiont treat-m odel( intoa henc rD

3

abh o alloff eaa s445t tux-i ntin-u trast,0 tivegw t ad-

Fig. 1. Subcutaneous xenograft growth in SCID mice. SCID mice with es-tablished subcutaneous Daudi (A), EBV-BLCL (B), Hs445 (C), xenograftswere treated either with intravenous rituximab, BG or a combination of rit-uximab and BG, or left untreated. Percentage tumor growth is plotted ony-axis and days after treatment was commenced onx-axis. Error bars repre-sent standard errors of mean and have been shown only for rituximab aloneand combination groups. For all xenografts, only combination treatment wasassociated with reduction in tumor growth.

95% CI 1.3–2.7%;p< 0.001) for Daudi, 0.8% for EBVLCL (95% CI 0.4–1.2%;p< 0.001), 2.2% for Hs445 (95.I. 1.3%–3.0%;p= 0.001), and 1.8% for RPMI6666 (95I 1.0–2.7 %;p< 0.0002) xenografts. Tumor growth curv

rom representative experiments for Daudi, Hs445 and ELCL xenografts are shown inFig. 1. All tumors except fo

hose treated with combination therapy grew beyond 20ize and mice had to be sacrificed. Mice on combinareatment had persistent tumor suppression even afterent was stopped, particularly for the Daudi xenograft m

Table 1). The reduction in tumor growth rate translatedsurvival advantage for mice on the combination group wompared to all other treatment groups (p< 0.0001 both foaudi and Hs445 groups) (Fig. 1).

.2. Disseminated xenografts

Mice treated with a combination of BG and rituximad a significantly increased survival when compared tther treatment groups (p< 0.0005 for Daudi andp= 0.001

or Hs445) or when compared to rituximab alone (p< 0.0005or Daudi andp= 0.01 for Hs445) (Fig. 2). Numbers of micnd survival data for each group are shown inTable 1. 10/44nd 2/8 evaluable mice with disseminated Daudi and H

umors, respectively, treated with combination BG and rimab were surviving >12 months after therapy was discoed suggesting complete eradication of disease. In con/47 and 0/8 mice receiving rituximab alone in respecroups survived (23% versus 0% survival;χ2 = 0.01). Thereas no significant weight loss or other clinically apparen

682 S. Modak et al. / Leukemia Research 29 (2005) 679–683

Fig. 2. Survival in SCID mice with disseminated lymphoma xenografts. Kaplan–Maier survival curves for mice with disseminated (A: Daudi and B: Hs445)and subcutaneous (C: Daudi and D: Hs445) are shown. Therapeutic regimens utilized were: combination of rituximab and BG (—), rituximab alone (- - -), BGalone (. . .) or no treatment ( ). Mice treated with a combination of BG and rituximab had a significantly increased survival when compared to all othertreatment groups.

verse effects. That BG is absorbed can be inferred from thefact that it could be detected intracellularly within fixed andpermeabilized peripheral blood leucocytes by immunofluo-rescence (data not shown).

4. Discussion

In our preclinical studies, synergism between BG andrituximab was highly significant irrespective of the typeof CD20-positive lymphoma. Improved responses in Daudixenografts as compared to Hs445 may be attributable to

higher CD20 expression in the former (Mean geometric fluo-rescence channel for Daudi 241 compared to 184 for Hs445).When tumors that progressed were examined for CD20 ex-pression by immunofluorescence studies of single cell sus-pensions or indirect immunohistochemistry of frozen sec-tions, no significant difference was noted between groupstreated with rituximab, BG alone or rituximab + BG (data notshown), indicating that treatment with rituximab + BG wasnot associated with loss of CD20. We and others have pre-viously demonstrated synergism between other complement-activating monoclonal antibodies and BG[3,4,8]. The currentdata extend this observation to rituximab.

S. Modak et al. / Leukemia Research 29 (2005) 679–683 683

In other SCID models of CD20-positive lymphoma, neu-trophils were demonstrated to be significant contributors tothe anti-tumor effect of rituximab[9]. The addition of BG,the action of which is mediated primarily by neutrophils, canaugment their anti-tumor effect in rituximab-treated mice.The mechanism of glucan processing and anti-tumor activityhas recently been elucidated. Orally administered�-glucansare taken up by macrophages from the gut and transportedto spleen, lymph nodes and bone marrow. Within the bonemarrow, smaller, soluble�-(1→ 3) glucan fragments are pro-duced by macrophage degradation. These fragments are thentaken up by the CR3 receptor of neutrophils which then killiC3b-opsonized tumor cells following their recruitment to asite of complement activation. The absolute requirement foriC3b deposition on tumors and CR3 on granulocytes wasdemonstrated by the lack of therapeutic effect in C3 or CR3deficient mice[10].

Complement plays a key role in the mechanism of action ofrituximab. Rodent complement is not inhibited efficiently byhuman complement regulatory proteins (mCRPs). Therefore,CDC can be an effective anti-tumor mechanism in xenograftmodels. However, in our study, at sub-therapeutic doses ofantibody, rituximab-mediated ADCC and CDC were not suf-ficient to effect tumor cell killing. Since BG has no directeffect on ADCC, this synergy is most likely a result of iC3b-mediated tumor cytotoxicity. iC3b was found to co-localizew es-e anp m-p ento ofl dingC oxi-c mentr C-m anb mon-s nliket tu-m

bes icitym ancei r Bc avea map isor-d oxico ofc ea iner is ap areu s aren evel-o ent

of B-cell disorders and in antibody-based therapies of othercancers.

Acknowledgments

Supported in part by the Leukemia Lymphoma Society,Hope Street Kids, Robert Steel Foundation, and Katie-Find-a-Cure Fund.

Contributions.S. Modak contributed to the concept anddesign, interpreted and analysed the data, provided draftingof the article, provided study materials/patients, obtained afunding source, provided administrative support, collectedand assembled the data. G. Koehne interpreted and analysedthe data, provided study material study materials/patients,provided administrative support, collected and assembled thedata. A. Vickers supplied statistical expertise. R. O’Reillyprovided critical revisions and important intellectual content.N.-K.V. Cheung contributed to the concept and design, pro-vided critical revisions and important intellectual content, andgave final approval.

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