orally administered b-glucans enhance anti-tumor effects of monoclonal antibodies

7/30/2019 Orally Administered B-glucans Enhance Anti-tumor Effects of Monoclonal Antibodies

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O R IGINA L A R T IC L E

Nai-Kong V. Cheung Shakeel ModakAndrew Vickers Benny Knuckles

Orally administered b-glucans enhance anti-tumor effectsof monoclonal antibodies

Received: 21 May 2002 / Accepted: 25 July 2002 / Published online: 20 September 2002 Springer-Verlag 2002

Abstract b-Glucan primes leukocyte CR3 for enhancedcytotoxicity and synergizes with anti-tumor monoclonalantibodies (mAb). We studied readily available (1fi3)-b-D-glucan using the immune deficient xenograft tumor

models, and examined the relationship of its anti-tumoreffect and physico-chemical properties. Establishedsubcutaneous (s.c.) human xenografts were treated for29 days orally with daily b-glucan by intragastric injec-tion and mAb intravenously (i.v.) twice weekly. Controlmice received either mAb alone or b-glucan alone. Tu-mor sizes were monitored over time. b-Glucans werestudied by carbohydrate linkage analysis, and highperformance size-exclusion chromatography with mul-tiple angle laser scattering detection. Orally administeredb-D-glucan greatly enhanced the anti-tumor effects ofmAb against established tumors in mice. We observedthis b-glucan effect irrespective of antigen (GD2, GD3,

CD20, epidermal growth factor-receptor, HER-2), hu-man tumor type (neuroblastoma, melanoma, lympho-ma, epidermoid carcinoma and breast carcinoma) ortumor sites (s.c. versus systemic). This effect correlatedwith the molecular size of the (1fi3),(1fi4)-b-D-glucan.Orally administered (1fi3),(1fi6)-b-D-glucans also syn-ergized with mAb, although the effect was generally lessmarked. Given the favorable efficacy and toxicity profileof oral b-D-glucan treatment, the role of natural prod-

ucts that contain b-glucan in cancer treatment as anenhancer of the effect of mAb therapy deserves furtherstudy.

Keywords ADCC

CMC

CR3

(1fi3),(1fi4)-b-D-glucan

Introduction

Evidence of the efficacy of monoclonal antibodies (mAb)against human cancer in clinical trials is increasinglyevident. However, induced or administered antibodies tohuman tumors have not realized their fullest therapeuticpotential, even when they can activate complement-mediated cytotoxicity (CMC) and antibody-dependentcell-mediated cytotoxicity (ADCC). The deposition of

C3b and iC3b on tumor cells fails to stimulate phago-cytosis or extracellular cytotoxicity by C3-receptor-bearing neutrophils, macrophages, and natural killer(NK) cells, even though these same effector cells canefficiently kill C3b and iC3b opsonized microorganisms.The receptor for iC3b, CR3 (also called CD11b/CD18,Mac-1, or aMb2-integrin), is found in monocytes/mac-rophages, NK cells, and immune cytotoxic T lympho-cytes (CTL). CR3 activation requires the engagement oftwo sites on its a-subunit (CD11b): the iC3b-binding sitewithin the I-domain at the N-terminus and a lectin siteat the C-terminus [41, 42]. b-Glucans are specific for thelectin site. When coated with iC3b, yeast cells (with their

b-glucan-containing cell wall), engage both iC3b andlectin binding sites on leukocytes, triggering phagocy-tosis and respiratory burst [6, 42]. In contrast, tumorcells coated with iC3b cannot activate leukocytes be-cause they lack the CR3-binding b-glucan [20, 29, 44,54]. Soluble forms ofb-glucans can bind to the lectin site[50, 57] and prime both phagocytic and NK cells tokill iC3b-coated tumor targets [53, 54, 57]. Whenthis strategy was tested in mice carrying mammarytumors, where natural IgM anti-tumor antibodiesdeposited iC3b on tumor cell surface, intravenous (i.v.)

Cancer Immunol Immunother (2002) 51: 557564DOI 10.1007/s00262-002-0321-3

N.-K.V. Cheung (&) S. ModakDepartment of Pediatrics,

Memorial Sloan-Kettering Cancer Center,1275 York Avenue, New York, NY 10021, USAE-mail: [email protected].: +212-639-8401Fax: 212-744-2245

A. VickersDepartment of Integrative Medicine and Biostatistics Service,Memorial Sloan-Kettering Cancer Center,New York, NY 10021, USA

B. KnucklesUSDAARS, Western Regional Research Center,Albany, CA 94710, USA


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administration of b-glucan alone induced impressivetumor shrinkage [58]. In the absence of such naturalantibodies (SCID mice), or of C3 (C3 knockout mice),or of leukocyte CR3 (CR3 knockout mice), these tumorswere resistant to b-glucan therapy [58].

b-Glucans are polymers of glucose that are extract-able from cereals, mushrooms, seaweed and yeasts [4].They are (1fi3)-b-D-glucopyranosyl polymers withrandomly dispersed single b-D-glucopyranosyl unitsattached by (1fi6)-b linkages, giving a comb-likestructure. The (1fi3)-b-backbone and the (1fi6)-linkedbranches were thought to be important for their immuneeffects. Lentinan (b-glucan extracted from from themushroom Lentinus edodes, Basidiomycete family) is ahigh molecular weight (m.w.) b-glucan with (1fi6)branches off every three (1fi3)-b-D-glucopyranosyl res-idues, and has been licensed in Japan for cancer treat-ment. Schizophyllan (from Schizophyllum commune,Basidiomycete family) and b-glucan from Bakers yeast(Saccharomyces cerevisiae) have similar structures.Laminarin (from seaweed), a small m.w. b-glucan, has(1fi6)-b branches occurring at every ten (1fi3)-b-D-

glucopyranosyl units. On the other hand, b-glucan frombarley, oat or wheat has mixed (1fi3),(1fi4)-b linkagein the backbone, but no (1fi6)-b branches and is gen-erally of high m.w.

More recently, the human b-glucan receptor, DEC-TIN-1, was cloned and found to be expressed onmonocytes, macrophages, dendritic cells (DC), and NKcells [55]. This is a type II transmembrane receptor witha single extracellular carbohydrate recognition domainand an immunoreceptor tyrosine activation motif in itscytoplasmic tail. It functions as a pattern recognitionreceptor, recognizing a variety of (1fi3)-b- and/or(1fi6)-b-linked glucans, including mixed linkage

(1fi3),(1fi4)-b-glucans. This receptor also binds Tlymphocytes using a domain distinct from the b-glucanbinding site. DECTIN-1 gene has been mapped tochromosome 12p13 within the NK gene cluster complex[46].

Oral administration of botanical and yeast extractscontaining (1fi3),(1fi6)-b-glucans has shown anti-tu-mor effects in animal studies [13, 16, 33, 34, 37, 48], andin patients with colorectal cancer [52] and gastric cancer[32], although results have been less encouraging forbreast cancer [18, 51] and leukemia [39]. However, noneof these previous reports recognized the critical contri-bution from anti-tumor antibodies in orally adminis-

tered b-glucan activity. Furthermore, oral b-glucanshave not been rigorously tested in immune-deficientmodels, a mimic of real-life situation among cancer pa-tients undergoing chemotherapy. In addition, theformulations previously explored were not chemicallypure glucans, a major limitation for quality control andfurther clinical development. Although (1fi3),(1fi6)-b-glucans were the primary focus of most previousinvestigations, (1fi3),(1fi4)-b-glucans, despite theirwide distribution in the daily diet, have not beenrigorously tested in their purified forms.

(1fi3),(1fi4)-b-Glucans derived from barley havebeen shown to bind to CR3 in vitro [57], to activateADCC mediated by NK cells [11, 53, 54], monocyte [10,50], and neutrophils [50], as well as to stimulate tumornecrosis factor (TNF) production by monocytes [43].However, their in vivo immunomodulatory effects, es-pecially when administered by oral route, have not beentested. We have recently reported on an unusually strongsynergism between anti-GD2 antibodies and intragastricinjection of barley b-D-glucan against human neuro-blastoma xenografts [7]. NK cells were only partlyresponsible for the anti-tumor effect, and the IgManti-GD2 antibody 3G6 (class switch variant of 3F8with no ADCC activity) was equally effective. Here weextend this observation to a diverse panel of clinicallyimportant mAb in a broad spectrum of common humancancers. We further examine the relationship betweenmolecular size (1fi3)-b-D-glucan and its synergy withmAb.

Materials and methods

Cell lines

The cell lines Daudi, SKMel-28 and A431 were obtained from theAmerican Type Culture Collection (ATCC; Rockville, Md.).LAN-1 was provided by R. Seeger (Childrens Hospital of LosAngeles, Los Angeles, Calif.); NMB7 by S.K. Liao (McMasterUniversity, Ontario, Canada); human breast carcinoma cell lineBT474 was kindly provided by D. Solit (MSKCC; MemorialSloan-Kettering Cancer Center, N.Y.); and SKNJD and SKNERwere established at MSKCC. BT474 was cultured in Dulbeccosmodified Eagles medium (DMEM) with nutrient mixture F12(DMEM/F-12; Life Technologies, Gaithersburg, Md.) in a 1:1mixture fortified with 10% newborn calf serum (Hyclone, Logan,Utah), MEM non-essential amino acids (Life Technologies),

100 U/ml of penicillin (Sigma, St. Louis, Mo.), and 100 lg/ml ofstreptomycin (Sigma). All other cell lines were cultured in RPMI1640 (Life Technologies) containing 10% defined calf serum(Hyclone) and 100 U/ml of penicillin, 100 lg/ml of streptomycinand 2 mM L-glutamine (Sigma).

Antibodies

mAb 3F8 (mouse IgG3) and 3G6 (mouse IgM) reactive againstGD2 ganglioside expressed on neuroectodermal tumors, and mAb8H9 (mouse IgG1) reactive with a glycoprotein expressed on thesesame tumors, have been previously described [8, 31]. They werepurified to >90% purity by affinity chromatography: protein A(Pharmacia, Piscataway, N.J.) for 3F8, and protein G (Pharmacia)for 8H9. Anti-GD3 antibody (R24) [17] was provided by P.Chapman (MSKCC). Hybridomas producing the anti-epidermalgrowth factor receptor (EGF-R) antibodies 528 (IgG2a) and 455(IgG1) were obtained from ATCC [28]. Rituximab (anti-CD20)and Herceptin (anti-HER2) were purchased from Genentech (SanFrancisco, Calif.).

b-Glucan

Barley, oat and lichenan b-D-glucans were purchased from Sigmaand Megazyme International Ireland (Wicklow, Ireland). Althoughendotoxin cannot prime CR3 in the same way as b-glucan [53],barley b-glucans were tested and found negative for bacterial ormycoplasma contamination and


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kinetic-chromogenic limulus amebocyte lysate (LAL) test. In ad-dition, they were all found to contain


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for individual tumors in each treatment group and ex-pressed as a percent of that in the saline control. Whennormalized to the range between minimally suppressed(45 kDa) and maximally suppressed (404 kDa), sup-pression of tumor growth was highly correlated withmolecular size for both 40 lg and 400 lg b-glucan doses(P


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mice. We observed this effect irrespective of the route ofb-glucan administration (intragastric or intraperitoneal),antigen (GD2, GD3, CD20, EGFR, HER2), humantumor type (NB, melanoma, epidermoid carcinoma,

lymphoma, breast cancer), mouse strain (athymic nu/nu,severe combined immune deficiency mice), or tumor site(s.c. versus systemic). b-Glucan was heat-stable, its anti-tumor effect was dose- and schedule-dependent, requir-ing antibody-Fc, but not cytophilicity of the antibody.Neither antibody nor b-glucan alone was effective. Thissynergy of (1fi3),(1fi4)-b-D-glucan with mAb increasedwith b-glucan m.w. This is the first report demonstratingthe importance of molecular size in orally administered(1fi3),(1fi4)-b-D-glucan. These findings were not en-tirely intuitive, since small molecules (often thought tobe easier to process or absorb) were less effective in ourtumor model. More importantly, we extended our initial

observation of the effect of oral administration of barleyb-glucan on antibody function in neuroblastoma [7] to abroad variety of clinically relevant mAb in well estab-lished, clinically relevant xenograft models. These find-ings laid to rest our concern that the anti-tumor effectwhen barley b-glucan was combined with anti-disialo-ganglioside antibody was a consequence of direct orindirect interaction of b-glucan with carbohydrate tu-mor antigen or epitopes (e.g. GD2), or a unique inter-action on neuroblastoma tumors.

b-Glucans have been tested for tumor therapy in micefor nearly 40 years [12, 49]. Several forms of mushroom-derived b-glucans are used clinically in Japan to treat

cancer, including polysaccharide Kureha (PSK; fromCoriolus versicolor), Lentinan and Schizophyllan. InJapan, PSK and Schizophyllan were shown to improvesurvival rates in some cancer trials [14, 30, 35, 36, 4040],but with less encouraging results in others [38, 51]. Whileb-glucans are not used by western oncologists, b-glucancontaining botanical medicines such as Ling-Zhi, mai-take and green barley are widely used to treat cancerpatients in the US as alternative/complementary cancertherapies, often with insufficient clinical validation orquality control.

Given the biology of iC3b targeted cytotoxicity, b-glucan should have clinical potential. However, there areseveral limitations to existing b-glucan strategies. Theyare generally expensive and inconvenient to administer:

e.g. Lentinan and Schizophyllan are given i.v. daily overlong periods of time. Besides being insoluble, they con-tain proteins and non-b-glucan carbohydrates, whichconfound mechanistic studies and complicate the man-ufacturing and control process. Because of proteincontaminants they are potentially allergenic. The spon-taneous cross-linking of CR3 by b-glucan of high m.w.can cause neutrophil degranulation and cytokine releasefrom macrophages, resulting in undesirable clinicaltoxicities. Regarding low m.w. b-glucans, besides theirlow affinity for CR3, they have rapid renal clearance.Without anti-tumor antibodies to activate human com-plement, b-glucan is largely ineffective. In this report we

addressed these limitations by: (1) using b-glucan fromreadily available sources, e.g. (1fi3),(1fi4)-b-D-glucanfrom barley; (2) administering b-glucan orally instead ofintravenously; and (3) including the coadministration oftumor-specific antibodies to ensure complement activa-tion.

Previous studies have demonstrated that orally ad-ministered b-glucans activate splenic and peritonealmacrophages for tumor cytotoxicity. In a study of 14C-labeled orally administered b-glucan, sequestration inthe liver was observed [19], suggesting that it entered theblood and behaved pharmacokinetically in a similarmanner to i.v. administered low m.w. b-glucan [45, 47,

59]. These studies also suggested that processing by thegastrointestinal tract produced b-glucan with high ac-tivity for CR3. Besides this model of intravasation ofprocessed barley b-glucan, leukocytes could also be ac-tivated directly in the gut before homing to the tumor.Despite the abundance of b-glucan (3% of the dryweight) in grains, its bioavailability from cereals is lim-ited since high m.w. b-glucans require high temperature(>60oC) for solubilization and gelling. It is of interestthat unpurified b-glucan (Betratrim) has low solubilityand poor gelling properties, and has low biologic activity

Table 1. Anti-tumor effect oforally administered b-glucanswhen used in combination withi.v. 3F8

b-Glucans Description Molecular weight (kDa) A nti-tumor effect (%)b

(1fi3),(1fi4)-b-D-glucana

Standard barley glucan 206 100Barley High viscosity 321 40

Medium viscosity 178 92Low viscosity 102


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in our model. Recent studies using high-fiber (13.5 g/day) wheat-bran supplement [1] did not show anti-tumoreffect in human trials. Besides the issue of bioavailabil-ity, the effectiveness of glucans or other agents such aswheat bran cannot be assessed in the absence of mAbadministration, since our studies clearly showed thateven the most effective b-glucan required the simulta-neous administration of mAb. Previous studies thatinvestigated the therapeutic effect ofb-glucan administ-ration did not incorporate co-administration of mAb aspart of the protocol. It is likely that when b-glucanfunctions without co-administration of mAb, its tumorcytotoxic effect requires the presence of naturally-oc-curring antitumor antibodies that target the tumor withiC3b.

Our findings using (1fi3),(1fi4)-b-D-glucan frombarley were unexpected. In previous studies, (1fi3),(1fi6)-b linkage (e.g. Lentinan, Schizophyllan, Lami-narin, and b-glucan from Bakers yeast) was deemedrequisite for its anti-tumor effect [4]. In those models,however, T cells were essential. This T cell requirementwas later shown to involve helper T cell function needed

for the generation of naturally-occurring antitumorantibodies. Thus, the therapy was shown to be effectivein T cell deficient SCID or nude mice given naturalantibodies isolated from normal mouse serum [58]. Inour studies, while (1fi3),(1fi4)-b-D-glucan alone wascompletely inactive, it enhanced the anti-tumor effect ofmAb in immunodeficient mice, supporting the model inwhich b-glucans function in tumor therapy by binding toCR3 in vitro to mediate leukocyte CR3-dependentcytotoxicity, and that neither T nor B cells are needed.Since most cancers express mCRP (CD46, CD55, CD59)on their cell surface [3, 5, 15, 21, 22, 23, 26, 27],complement-mediated tumor lysis is typically inefficient.

Nevertheless, despite these inhibitory proteins, iC3b hasbeen detected on tumor cells isolated from fresh humanbreast tumors, and enough levels could be deposited byantibodies in vitro to opsonize tumor cells for phago-cytes and NK cells in vitro [24]. It is possible thatsublethal levels of complement activation depositedenough iC3b to optimize tumor killing, a strategy thatcan be enhanced by oral administration ofb-glucans.

Acknowledgements This study was supported in part by grantsfrom the NIH (CA 096321) and the Robert Steel Foundation,Hope Street Kids, Katie-Find-a-Cure Fund, and the Catie HochFoundation.

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