ORAL LACTOFERRIN INHIBITS GROWTH OF ESTABLISHED TUMORS AND POTENTIATES CONVENTIONAL CHEMOTHERAPY Atul VARADHACHARY 1 , * Jeffrey S. WOLF 2 , Karel PETRAK 1 , Bert W. O’MALLEY, Jr. 3 , Michela SPADARO 4 , Claudia CURCIO 4 , Guido FORNI 4 and Federica PERICLE 1 1 Agennix, Inc., Houston, TX, USA 2 Department of Otolaryngology-Head and Neck Surgery, University of Maryland School of Medicine, Baltimore, MD, USA 3 Department of Otorhinolaryngology—Head and Neck Surgery, University of Pennsylvania Health System, Philadelphia, PA, USA 4 Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy In this work, we investigated the anticancer activity of orally administered recombinant human lactoferrin (rhLF) alone and in combination with chemotherapy in tumor-bear- ing mice. rhLF inhibited the growth of squamous cell carci- noma (O12) tumors in T cell–immunocompromised nu/nu mice by 80% when administered at 1,000 mg/kg (2.9 g/m 2 ) by oral gavage twice daily for 8 days (p < 0.001). Similar activity was observed in syngeneic, immunocompetent BALB/c mice, where orally administered rhLF (1,000 mg/kg, 2.9 g/m 2 once daily) halted the growth of mammary adenocarcinoma TUBO. Oral rhLF (200 mg/kg, 0.57 g/m 2 ) was also used alone and in combination with cis-platinum (5 mg/kg) to treat head-and-neck squamous cell carcinoma in a syngeneic mu- rine model. Monotherapy with oral rhLF or cis-platinum caused 61% or 66% tumor growth inhibition over placebo, respectively. Mice receiving both therapies showed 79% growth inhibition, a statistically significant improvement over each drug alone. We then demonstrated that adminis- tration of oral rhLF (300 mg/kg, 0.86 g/m 2 ) to tumor-bearing or naive mice resulted in (i) significantly increased production of IL-18 in the intestinal tract, (ii) systemic NK cell activation and (iii) circulating CD8  T-cell expansion. These data sug- gest that oral rhLF is an immunomodulatory agent active against cancer as a single agent and in combination chemo- therapy, exerting its systemic effect through stimulation of IL-18 and other cytokines in the gut enterocytes. rhLF has been administered orally to 211 people without a single se- rious drug-related adverse event. Thus, rhLF shows promise as a safe and well-tolerated novel immunomodulatory anti- cancer agent. © 2004 Wiley-Liss, Inc. Key words: lactoferrin; immunotherapy; IL-18 LF is an 80 kDa iron-binding glycoprotein, first identified in breast milk as a protein product of mammary epithelial cells. 1 The protein is present in exocrine secretions that are commonly ex- posed to normal flora: milk, tears, nasal exudates, saliva, bronchial mucus, gastrointestinal fluids, cervicovaginal mucus and seminal fluid. Additionally, LF is a major constituent of the secondary specific granules of circulating PMNCs. It is secreted by neutro- phils and present at high levels at sites of bacterial infection. 2 rhLF is overexpressed in, and isolated from, Aspergillus niger var. awamori. rhLF is structurally and functionally equivalent to native human LF in all material respects, differing only in the nature of its glycosylation. 3 The 3D structure of rhLF appears identical to that of human milk LF as shown by X-ray crystallography. 4 Pharmaceutical-grade rhLF is manufactured at large scale, to cGMP specifications. LF scavenges free iron in fluids and inflamed areas, suppressing free radical–mediated damage and decreasing the availability of the metal to invading microbial and neoplastic cells. 5 LF binds 2 Fe 3+ atoms through a conformational change that opens up the molecule along a hinge behind each iron-binding site, providing access to the iron-binding sites. 6 A wide array of LF functions related to host primary defense and unrelated to its iron-binding ability have been shown, 7,8 with anti-inflammatory and antimicro- bial effects thought to occur via activation of NK and lymphokine- activated killer cells 9 and enhancement of PMNCs and macro- phage cytotoxicity. 10 LF is a multifunctional protein having several distinct biologic activities that derive from different sur- face properties. These include the ability to bind anionic molecules such as DNA, heparin and glycosaminoglycans and to bind cells of several histotypes and modulate their activity. 11 Administration of exogenous bLF to infected hosts with in- flamed sites has resulted in prophylactic or therapeutic effects. 12 LF contributes to enhanced Th1 response, which may explain the lower rate of allergies in breast-fed children. 13 Although LF was originally identified as a mucosal host defense mediator and inflammatory immune modulator, it also displays antineoplastic activity. Marked inhibition of tumor growth and reduced lung colonization by B16-F10 melanoma experimental metastasis were found in mice treated with hLF injected i.p. 14 As LF receptors have been found in the gut epithelium, oral LF appears possible as a route of administration. bLF added to drink- ing water inhibited 4-nitroquinoline 1-oxide carcinogenesis in rats. 15 Moreover, rats fed a diet supplemented with bLF showed reduced diethylnitrosamine-, dihydroxy-di-N-propylnitrosamine- and N-nitrosomethylbenzylamine-induced esophageal and lung cancers. 16 In another study, oral bLF reduced lung colonization by colon carcinoma cells in mice. 17 Despite the evidence that LF possesses chemopreventive activ- ity, little is known about (i) the anticancer activity of oral hLF against established tumors; (ii) its ability to potentiate chemother- apy, as described with other immunotherapeutics; or (iii) the immune mechanisms by which its antitumor activity is mediated. Here, we show that oral rhLF inhibits the growth of established tumors at distal sites and potentiates the antitumor activity of cis-platinum. Also, oral rhLF increases IL-18 production in the intestinal tract, NK cell activity in the spleen and the CD8 + T-cell population in the blood. Abbreviations: b, bovine; BCA, bicinchoninic acid; GlcNAc, N-acetyl- glucosamine; LDH, lactate dehydrogenase; LF, lactoferrin; MACS, mag- netic cell sorting; NK, natural killer; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin; pI, isoelectric point; PMNC, polymorphonuclear cell; PVDF, polyvinylidene difluoride; rh, recombinant human; Th1, T- helper 1. *Correspondence to: Agennix, Inc., Eight Greenway Plaza Suite 910, Houston, TX 77046, USA. Fax: +713-552-0795. E-mail: avaradhachary@agennix.com Received 19 August 2003; Revised 2 January, 9 February 2004; Ac- cepted 17 February 2004 DOI 10.1002/ijc.20271 Published online 23 April 2004 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 111, 398 – 403 (2004) © 2004 Wiley-Liss, Inc. Publication of the International Union Against Cancer