Lipid A in Cancer Therapy [1 ed.] 1441916024, 9781441916020

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Lipid A in Cancer Therapy

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY EditorialBoard: NATHAN BACK,State University ofNew Yorkat Buffalo IRUN R. COHEN, The Weizmann InstituteofScience ABEL LAJTHA, N.S. Kline Institutefor PsychiatricResearch JOHN D. LAMBRIS, University ofPennsylvania RODOLFOPAOLETTI, University ofMilan Recent Volumes in this Series Volume 660 PARAOXONASES IN INFLAMMATION, INFECTION, AND TOXICOLOGY Editedby Srinu Reddy Volume 661 MEMBRANE RECEPTORS, CHANNELS AND TRANSPORTERS IN PULMONARY CIRCULATION Editedby Jason X. -J. Yuan, and Jeremy P.T. Ward Volume 662 OXYGENTRANSPORT TO TISSUEXXXI Edited by Duane F. Bruley and Eiji Takahasi Volume 663 STRUCTURE AND FUNCTION OF THE NEURALCELLADHESION MOLECULE NCAM Edited by VladimirBerezin Volume 664 RETINAL DEGENERATIVE DISEASES Edited by Robert E. Anderson, Joe G. Hollyfield, and MatthewM. LaVail Volume 665 FORKHEAD TRANSCRIPTION FACTORS Edited by KennethMaiese Volume 666 PATHOGEN-DERIVED IMMUNOMODULATORY MOLECULES Editedby PadraicG. Fallon Volume 667 LIPIDA IN CANCERTHERAPY Editedby Jean-Francois Jcannin

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Lipid A in Cancer Therapy Editedby Jean-Francois Jeannin

Tumor Immunology and Immunotherapy Laboratory Ecole Practique des Hautes Etudes Inserm U866, University ofBurgundy, Dijon , Fran ce

Springer Science+Business Media, LLC Landes Bioscience

Springer Science+Business Media, LLC LandesBioscience Copyright©2009LandesBioscience and SpringerScience+Business Media,LLC All rights reserved. No part of thisbookmaybe reproduced or transmitted inany formor by anymeans, electronic or mechanical, includingphotocopy, recording, or any information storageandretrievalsystem,withoutpermission in writingfromthe publisher, with the exceptionof any materialsuppliedspecifically for the purposeof being enteredand executedon a computersystem; for exclusive use by the Purchaserof the work. Printedin the USA. SpringerScience+Business Media,LLC, 233 SpringStreet, New York, New York10013, USA http://www.springer.com Pleaseaddress all inquiriesto the publishers: LandesBioscience, 1002WestAvenue, Austin, Texas7870I, USA Phone: 5121637 6050; FAX: 512/637 6079 http://www.landesbioscience.com The chaptersin this book are availablein the MadameCurie Bioscience Database. http://www.landesbioscience.comlcurie

LipidA in Cancer Therapy, editedbyJean-Francois Jeannin. Landes Bioscience1Springer Science+Business Media, LLC dual imprint1 Springerseries: Advances in Experimental Medicineand Biology. ISBN: 978-1-4419-1602-0 Whilethe authors,editorsandpublisherbelievethatdrugselectionand dosageand thespecifications and usage of equipment and devices, as set forth in this book, are in accordwith current recommendations and practice at the time of publication, they make no warranty, expressedor implied, with respect to material describedin this book. In view of the ongoingresearch, equipmentdevelopment, changes in governmental regulationsand the rapidaccumulation of information relating to the biomedical sciences, the reader is urgedto carefullyreviewand evaluatethe information providedherein.

Library of Congress Cataloging-in-Publication Data LipidA in cancertherapy1 editedby Jean-Francois Jeannin. p, ; em. -- (Advances in experimental medicineand biology; 667) Includes bibliographical references and index. ISBN 978-1-4419-1602-0 1. Cancer--Immunotherapy. 2. Microbial lipids-Therapeutic use. 3. Endotoxins--Therapeutic use. r. Jeannin, Jean-Francois, 1948- II. Series: Advances in experimental medicine and biology, v. 667. 0065-2598 ; [DNLM: 1. Lipid A--pharmacology. 2. Lipid A--therapeutic use. 3. Neoplasms-drug therapy. WI AD559v.667 20091 QU 85 L7605 2009] RC27I.I45L572009 616.99'4061--dc22 2009035583

FOREWORD Cancer remains a major challenge for modem society. Not only does cancer rank among the first three causes of mortality in most population groups but also the therapeutic options available for most tumor types are limited. The existing ones have limited efficacy, lack specificity and their administration carry major side effects. Hence the urgent need for novel cancer therapies. One of the most promising avenues in research is the use of specific immunotherapy. The notion that the immune system may have important anti-tumor effects has been around for more than a century now. Every major progress in microbiology and immunology has been immediately followed by attempts to apply the new knowledge to the treatment of cancer. Progress has reached a point where it is well established that most cancer patients mount specific T cell responses against their tumors . The molecular identity of the antigens recognized by anti-tumor T cells has been elucidated and several hundreds oftumor-derived antigenic peptides have been discovered. Upon recognition of such peptides presented by self MHC molecules, both CD8 and CD4 T cells are activated, expand to high numbers and differentiate into effective anti-tumor agents. CD8 T cells directly destroy tumor cells and can cause even large tumors to completely regress in experimental mouse models . These observations have spurred intense research activity aimed at designing and testing cancer vaccines . Over 100 years ago Coley successfully used intratumoral injection of killed bacteria to treat sarcomas. The important anti-tumor effects observed in a fraction of these patients fueled major research efforts. These led to major discoveries in the 80s and the 90s. It turns out that bacterial lipopolysaccharides stimulate the production of massive amounts of a cytokine still known today as tumor necrosis factor (TNF-a). They do so by engagement of a rather complex set of interactions culminating in the ligation ofa Toll-like receptor, TLR-4. Ensuing signaling through this receptor initiates potent innate immune responses. Unfortunately the clinical use of both TNF-a and LPS can not be generalized due to their very narrow therapeutic margin. Importantly, synthetic Lipid A analogs have been identified that retain useful bioactivity and yet possess only mild toxicity. v

vi

Foreword

The relatively large body of information accumulated thus far on the molecular and cellular interactions set in motion by administration of LPS as well as by the synthetic Lipid A analogs allow to place this family ofbacterially-derived molecules at the crossroads between innate and adaptive immunity. By virtue of this key position, the therapeutic applications being pursued aim at using these compounds either as direct anti-tumor agents or as vaccine adjuvants. The clinical experience acquired so far on these two avenues is asymmetric. Few clinical trials using Lipid A analogs as single anti-cancer agents involving less than 100 patients with advanced cancer have been reported. In contrast, Lipid A has been tested in over 300,000 individuals in various vaccines trials, including therapeutic cancer vaccines. Clearly most of the work needed to develop Lipid A as effective anti-cancer agents and/or as vaccine adjuvant lies ahead in the near future . This book is a timely contribution and provides a much needed up-to-date overview of the chemical, biological and physiological aspects of Lipid A. It should be a beacon to all those involved in this field of research.

Jean-Charles Cerottini, MD UniversityofLausanne, FormerDirector, LudwigInstitutefor Cancer Research Lausanne Branch PedroRomero, MD University ofLausanne, Member, Ludwig Institutefor Cancer Research Lausanne Branch

ABOUT THE EDITOR...

JEAN-FRAN 60·C. Very interestingly. the triacyllipid A part structure OM-I?4 (structure Fig. 1B) with a normal bisphosphorylared disaccharide backbone exhibits a non-lamellar structure." This is. however, a direct micellar rather than an inverted micellar (HI) structure, connected with lower but not zero activity in the cytokine assay. which is in contrast to the tetraacyllipid A 406 exhibiting no agonistic activity (Fig. 3). Moreover. the latter is antagonistically effective by blocking the LPS-induced cytokine production when administered to the cells before LPS addirion." Also synthetic triacyl monophosphoryl lipid A part structures were investigated and showed a very specific dependence ofthe aggregate structure and the intramolecular conformation (see next section) on the binding sites to the sugar backbone." It will be shown later that these different structural preferences are ofimportance for the expression of biological activity. An overview ofaggregate structures and other physico-chemical parameters oflipid A ofdifferent origin and their relative biological activities is given in a previous review.26

Intramolecular Conformation

Byapplying FTIR spectroscopy using an attenuated total reflectance unit with polarized IR light. the orientation ofmolecular groups within lipid A could be determined." For enterobacterial lipid A. an inclination of the diglucosamine backbone (normal to the sugar plane) with respect to the

Conformation and SupramolecularStructure ofLipid A

HII

70

-.a ｾ

!

l!!

[ ｾ

E

29

coexistence region

x

Q212

60

50

coexistence region

Q224

Q229

40 UQ229

Q230

30 70

75

80

UQ212

85

Water content I %

90

95

Figure 2. Phasediagramof lipidAfrom LPS ofSalmonellaminnesota R595 inthe water concentration range 70 to 95% at low Mg2+ concentrations ([lipid A] : [M g2+] » 1). Llamellar phase, Q212, Q224, Q229, Q230 cubic phases of space groups 212, 224, 229 and 230, respectively.":" HII inverted hexagonal phase. X = unassigned cubic phase. Reproduced from: Brandenburg Ket al. Chem Phys Li pids 91 :53-69; ©1998 with permission from Elsevier." direction ofthe hydrocarbon chains ofmore than 45° was observed, lower acylated lipid A carrying a reduced number ofacyl chains (penta and tetra, Fig. IA,B) as well as some nonenterobacterial lipid A exhibit a significantly smaller angle (4 positive regional lymph nodes or in patients with locally advanced Stage III disease. The vaccine was well tolerated and seroconversion was demonstrated in a majority ofthe patients, the analysis of the T-cell response is ongoing. A Phase II trial ofthe recombinant Her2 protein (500 f-tg) with ASI 5 adjuvant is currently underway, with transient clinical response observed in 2/5 patients treated thus far.

Section 2: Vaccines Targeting Specific TAAs Expressed on Multiple Tumor Types Vaccines Targeting MAGE-3-Expressing Cancers

MAGE-3 is a cancer testis antigen that is normally expressed only on testicular germ cells. MAGE-3 is aberrantly expressed on a high percentage of a broad variety of tumors, making it a potential target for vaccine immun otherapy. The safety and efficacyofa cancer vaccine containing MAGE-3 protein fused to the N terminal portion ofa protein derived from H influenzae (protein D) and combined with AS02B adjuvant was recently tested in 57 patients with MAGE-3-positive tumors (51 with Stage III /IV melanoma, 3 with transitional carcinoma of the urinary bladder, 2 with nonsmall cell lung carcinoma, 2 with esophageal cancer and 1 with head and neck carcinoma, all Stage IV) .36.37As inclusion criteria. all patients were required to express at least 1 ofthe 3 HLA class I haplorypes known to present MAGE-3 peptides (l.e., HLA-Al, HLA-A2 and/or HLA-B44).The vaccination schedule comprised 4 injections at 3 week intervals, with 2 additional vaccinations administered at 6-week intervals to patients whose tumors stabilized or regressed. Escalating doses ofthe antigen combined with a fixed dose ofthe adjuvant were tested in the trial. The vaccine was well tolerated. A significant MAGE-3 specific IgG response was elicited in most vaccinated patients (96%) and all patients generated IgG antibodies specific for protein D. Using IFNy and IL-5 production as the readout, T-cell responses were detected in approximately 30% of evaluable patients. Five of thirty-three evaluable melanoma patients had partial responses or disease stabilization lasting 4 to 29 months, these five responder patients were all part of the 12 patients with lessadvanced melanoma (5/12 Stage III with non visceral disease). A partial response lasting 10 months was observed in one metastatic bladder cancer patients. A vaccine containing recombinant MAGE-3 protein (+/- AS02B adjuvant) was tested recently in the US by the Ludwig Institute for Cancer Research (LICR) in 17 patients with

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MAGE-3-expressing Stage I or II nonsmall cell lung carcinoma who had undergone surgical resection ofthe primary tumor." The patients had no evidence ofdisease at the onset ofthe trial. Three ofnine patients treated with the vaccine in the absence ofadjuvant developed modest, but significant, anti-MAGE-3 antibody responses and one patient in that cohort had a CD8+ T-cell response to the MAGE-3 peptide 243-258. In contrast, seven ofeight patients in the group receiving adjuvanted vaccine developed a marked increase in anti-MAGE-3 antibodies. The investigators used IFNy elispot and tetramer staining to show that inclusion of adjuvant in the vaccine was necessary to generate significant CD4+ and CD8+ T-cell responses to MAGE-3. No assessment of objective clinical response was conducted since the patients had no evidence ofdisease at the onset of the trial . Various tumor types have been targeted by MAGE-3-containing cancer vaccines produced by GSK Biologicals, with emphasis on non-small cell lung carcinoma (NSCLC) and cutaneous metastatic melanoma. 39,40 A Phase lIb, double-blind, placebo-controlled clinical trial was initiated in 2002 to test a MAGE-3 vaccine containing AS02B adjuvant in patients with MAGE-3-positive Stage IB and Stage II NSCLC (post surgical resection). Patients are receiving five intramuscular injections ofplacebo (n 60) or vaccine (300 !J.gMAGE-3; n 122) at three week intervals, followed by eight maintenance immunizations spaced three months apart. The primary endpoints are time to recurrence, disease-free and overall survival, recurrence rate at different timepoints, toxicity and tolerability. Humoral and cellular imm une responses are secondary endpoints. Although the safety aspect ofthe study is still blinded, the vaccine appears to be well tolerated, with mild grade 1 or 2 local or systemic reactions lastingless than 24 hours post-injection being the most common sequale. There have been only three grade 3 adverse events potentially related to treatment. Interim efficacy data was presented at the 2006 American Society of Clinical Oncology (ASCO) annual meeting showing a clear signal (although not significant) with a benefit of33% in recurrence rate. Core analysis ofthe results are expected by October 2006.

=

Vaccines Targeting MUCI-Expressing Cancers

=

Mucins (e.g., MUC-1) are high molecular weight (>500 kDa) glycoproteins expressed on the surface of normal and malignant cells.41•42 The core protein has an extracellular N-terminal domain, a transmembrane region and a C -terminal cytoplasmic domain. The oligosaccharides are attached to serines or threonines of the core peptide by O-glycosidic bonds. In normal cells, mucins are only expressed on the luminal surface and are therefore not exposed to the immune system. Mucins from cancer cells, on the other hand, are exposed uniformly on the cell surface, exposing them to immune surveillance." More importantly, mucins expressed by cancer cells are often underglycosylated, with shorter and simpler carbohydrate chains. Three cancer-specific carbohydrate epitopes have been identified: Thomsen-Friedenreich epitope, a precursor known as Tn and sialyl-Tn (STn). All three epitopes are commonly found on the cell surface ofepithelial cancers or as part ofthe mucins secreted by them, but not on normal epithelial cells, thereby maleing them potential TAAs.44 Most adenocarcinomas express MUC-1 on the cell surface and secrete underglycosylated MUC-1 mucin (expressing repeating STn epitopes) into the serum, resulting in exposure ofthe immune system to multiple tandem repeat core peptide epitopes. A vaccine consisting of a 100-amino acid peptide corresponding to five 20-amino acid long repeats ofMUC-1 and AS02B adjuvant was tested in a Phase I safety and immunogenicity study in 15 patients with resected and 1 patient with locally advanced pancreatic cancer." Patients (4 per dose) were vaccinated by intramuscular injection with one offour doses ofpeptide (100 !J.g, 300 /!g, 1000 !J.gor 3000 !J.g) admixed with the AS02B adjuvant every 3 weeks for a total ofthree doses. The vaccine was well tolerated, with toxicities limited to transient flu-like symptoms and tenderness/ erythema at the injections sites. Vaccination led to an increase in the percentage ofCD8+ cells in the peripheral blood and an increase in MUC-1-specific antibody was seen in some patients. Two ofthe 15 patients with resected tumors were alive and disease-free at 32 and 61 months. BLP25 liposome vaccine iStimuoax", Biomira, Inc.) is a cancer vaccine designed to elicit a cellular immune response to the exposed core peptide of MUCI. The vaccine is a lyophilized

Monophosphoryl Lipid A (MPL) asan Adjuvantfor Anti-CancerVaccines: ClinicalResults

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liposomal preparation containing BLP25 lipopeptide (25 amino acid peptide), MPL and the lipids cholesterol, dimyristoyl phosphatidylglycerol and dipalmitoyl phosphatidylcholine. MPL and BLP25 are present in the lipid bilayer of the liposome once the dry powder is rehydrated. Following a Phase I safety study ofBLP25 liposomal vaccine in Stage IIIB/IV nonsmall-cell lung cancer patients.t" clinical trials were conducted to test the safety and efficacy ofBLP25 vaccine in prostate" and nonsmall cell lung carcinoma patients (NSCLC).48 A single intravenous dose of cyclophosphamide (300 rng/rn' , maximum of 600 mg) was administered 3 days prior to vaccination. Such treatment has been demonstrated to augment delayed-type hypersensitivity responses, increase antibody responses, abrogate tolerance and potentiate anti-tumor immun ity in preclinical ｭｯ､･ｉｳＮ Ｔ ｾ Ｎ ｳ ｯ For primary treatment, the vaccine dose (1000 IJ.g ofBLP25 and 25 IJ.g MPL) was divided and administered subcutaneously at four sites weekly for eight weeks. In the prostate study, patients were reassessed following the primary treatment and those who did not require a change in therapy were eligible to continue with BLP25 vaccination every 6 weeks for up to 1 year. At the investigator's discretion, patients in the NSCLC trial continued to receive maintenance vaccinations every 6 weeks starting at week 13. In the study testing BLP25 vaccine in prostate cancer patients with biochemical failure (increasing prostate specific antigen; PSA) after radical prostatectomy, a total of sixteen individuals with a median age of60 were enrolled.? Fifteen ofsixteen completed the primary treatment and ten completed the maintenance period. After primary treatment, eight of sixteen patients had stable or decre ased PSA. Although only one patient maintained stable PSA by the last on-study measurement, six of sixteen patients had greater than 50% prolongation of PSA doubling time compared to prestudy measurements. To evaluate the effect of BLP25 vaccine on survival and toxicity in individuals with Stage IIIB or IV NSCLC, patients were randomly assigned to the BLP25 arm or to the best supportive care (BSC) arm. Overall, the 88 patients assigned to the BLP25 arm had a median survival time (MST) that was 4.4 months longer than the 83 patients in the BSC arm, though the difference was not statistically significant (p = 0.112). The greatest vaccine effect was observed in Stage IIIB locoregional disease, where the MST for 30 BSC patients was 13.3 months while the MST for 35 vaccinated patients was more than 30 months (p = 0.069, though MST had not been reached by end of study for vaccinated patients. A subsequent press release confirmed an MST of 30.6 months for the vaccine group). No significant toxicit y was associated with BLP25 treatment and quality oflife was maintained longer in vaccinated patients.

Vaccines Targeting STn-Expressing Carcinomas

Aberrant carbohydrate molecules, such as sialyl-Tn (STn) , are often expressed as part ofglycoproteins present on the surface ofcarcinomas.As discussed in the previous section, these molecules are not found on normal tissues and are considered TAAs. Antibodies specific for these molecules can be induced that mediate tumor cell lysis by complement or antibody-dependent cellular cytotoxiciry." When expressed on tumors, the mucin-associated STn epitope is a predictor of poor prognosis and is associated with increased metastatic potential.52·54STn is expressed on a significant proportion ofbreast cancer s (16-80%, depending on detection method and laboratory),55'58 with a tendency for higher expression in metastatic disease compared to primary tumors.l" For these reasons , STn is considered a promising target for a cancer vaccine. In the late 1980s, a vaccine consisting ofpartially desialylared ovine submaxillary gland mucin (modified OSM), which contained both Tn and STn epitopes, was tested in patients with metastatic colorectal cancer.ｓｾ Six patients were administered the vaccine alone, eight received the vaccine combined with Detox" adjuvant and six were treated with the vaccine plus BCG as adjuvant. Anti-STn antibody titers increased in 4 of8 patients in the Detox" group, 5 of6 in the BCG group and 0 of6 in the vaccine onl y group. Toxicity was limited to inflammatory skin reactions at the injection sites in patients receiving vaccine plus adjuvant. The result s demonstrated that STn-containingvaccines can induce specific humoral immune responses in cancer patients and that vaccines containing these molecules can be administered safely with immunological adjuvants.

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Lipid-Ain Cancer 'Iberapies

Tberatope" (Biomira, Inc.) is an investigational cancer vaccine consisting of synthetic STn conjugated to keyhole limpet hemocyanin (KLH) combined with Detox" adjuvant (early trials) or Enhanzyn adjuvant (later trials). Like Detox", Enhanzyn contains MPL and CWS, but is formulated as a stable emulsion with squalene oil instead ofsqualane oil. KLH is a carrier protein that promoted enhanced S'In -specific antibody responses in preclinical models. Because of the promiscuous expression ofSTn on adenocarcinomas derived from numerous tissues, Tbemtope" has been tested clinically as a treatment for a variery of tumor types (breast. colon, ovarian or pancreatic). The vast majority of patients (>1100 of 1500) have been treated for breast cancer. Preclinical testing in mice demonstrated that treatment with low-dose cyclophosphamide prior to immunization leads to enhanced antigen-specific antibody responses." presumably via the inhibition of suppressor cells. Some of the early clinical trials of the vaccine. therefore, also evaluated whether cyclophosphamide pretreatment would have the same effect in humans. In an initial dose-escalation study. 12 metastatic breast cancer patients were administered low-dose (300 mg/m 2) IV cy1cophosphamide on day -3 and four doses ofSTn-KLH (25 /-lg. 100 /-lg or 500 ug) combined with Detox" adjuvant at 2 week intervals, with four monthly injections thereafier." The 500 flg dose caused excessiveDTH reactions and was therefore eliminated from the protocol. At the lower doses. vaccine-associated toxicity was minimal. with side-effects limited to granulomas/ulcerations at the injections sites in 5 patients. All patients developed anti-STn IgG and IgM responses. Two patients had partial remissions lasting 6 months, while several others demonstrated disease stability for 3 -10 months. In a second trial. 100 /-lg ofthe STn-KLH vaccine combined with Detox"was administered to 23 metastatic breast cancer patients (with or without cyclophosphamide pretreatment) at weeks 0.2,5 and 9, with four additional monthly injections administered to patients with responding or stable disease after the first round of vaccinations.S Toxicity was again limited to granuloma formation at the injection sites. All patients developed anti-STn IgG and IgM responses and IgM responses were significantly higher in patients pretreated with cyclophophamide. Because 5 patients developed progressive disease during the initial 12 week period. only 18 patients received all four injections. ofwhich rwo had minor responses. Subsequent prospective trials evaluated the effect of different low-dose cyclophosphamide pretreatments on the response to STn-KLH combined with Detox" .63 Individuals receiving intravenous cyclophosphamide generated stronger anti-STn antibody responses and lived significantly longer than those administered the vaccin e with oral or no cyclophosphamide. An inverse correlation was observed between anti-STn antibody titers and tumor growth. and a lower percentage ofthe patients receiving intravenous cyclophosphamide pretreatment had progressive disease at 9 weeks. As a result of these trials, addition of cyclophosphamide pretreatment to the ｔｨ･ｲ｡ｴｯｰｾｶ｣ｩｮ protocol has become standard practice. In a randomized. double-blind Phase III trial, metastatic breast cancer patients whose tumors did not progress after front-line chemotherapy were treated with intravenous cyclophosphamide STn-KLH and Enhanzyn adjuvant) or intravenous cyclophosphamide folfollowed by ｔｨ･ｲ｡ｴｯｰｾＨ lowed by KLH alone combined with the Enhanzyn adjuvant (negative control).64.66An improved formulation of Ｑｨ･ｲ｡ｴｯｰ･ｾｴｨ｡ generated higher anti-STn antibody titers was used in this study, such that the STn/KLH ratio was increased compared to the preparation used in earlier studies. Concomitant endocrine hormone therapy was permitted and patients were stratified with regard to hormone therapy and response to initial therapy. The study was large (1028 patients) and subset analysis was conducted on patients that received hormone treatment. With respect to time to and control arms in the progression. there was no statistical difference berween Ｑｨ･ｲ｡ｴｯｰｾＭ､ study. regardless ofwhether selective estrogen -receptor modulators or arornatase inhibitors were used as adjunct hormonal therapy. Those patients who developed high titer antibody responses. however. had significantly longer survival times (41.1 months versus 25.4 months. p = 0.01). The failure of the Phase III trial may be related to the observations from early trials that induction of immune response to Theratope ｾ takes, on average, 17 weeks, while the time to disease progression in untreated patients is appoximately 12 weeks. Thus, the increased tumor burden after

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disease progression may suppress S'In -specific immune responses (via production ofimmunosuppressive molecules such as mucin) or may simply overwhelm the nascent immune response that is being generated to the vaccine. To test the vaccine in a situation where tumor burden was low during the vaccination regimen, Theratope-was evaluated in breast and ovarian cancer patients in conjunction with chemotherapy and autologous stem cell rescue. Trial designers reasoned that immune responses to the vaccine would have a chance to develop prior to disease progression in these patients. Since low tumor burden correlated with stronger immune responses and stronger immune responses correlated with longer survival times in initial trials, the strategy appeared sound. Beginning in 1995, 53 breast and 17 ovarian cancer patients were treated with Tberatope" vaccine beginning 30 to 151 days post-stem cell infusionY ·70Toxicity was limited to induration and erythema at injection sites, with some patients experiencing transient flu-like symptoms. Most patients developed elevated anti-STn IgG titers , usually after the third vaccination and approximately halfofthe patients developed S'In-specific T-cell proliferative and cytolytic responses. Induction of strong (versus weak) cytolytic T-cell responses by the vaccine was associated with longer remission times (p =0.047), suggesting that development ofanti-STn cytotoxic cells may also correlate with a clinical effect. Phase III trials to evaluate Theratope "in transplant patients have not been initiated at this time , though pilot studies are planned to determine whether addition ofIL-2 or GM-CSF to Theratope-treatment is beneficial in tran splant patients.

Vaccine Targeting Ras-Expressing Tumors The mutant oncogene ras is a well characterized TAA and is present in more than 20% of all

solid tumors, making it a prospective target for cancer immunotherapy. Taking advantage of the fact that a single point mutation (codon 12) in the rasgene accounts for 90% ofall ras mutations, investigators from the National Cancer Institute developed and tested vaccines containing one of the three B -mer mutant ras peptides expressed by tumor cells and Detox" adjuvant." Fifteen patients with a variety of cancers (colon, pancreas, nonsmall cell lung , duodenal, rectal and appendix) that carried defined ras mutations received three monthly subcutaneous vaccinations with the appropriate peptide at one of five doses (100 , 500 , 1000, 1500 or 5000 !!g) of peptide with a constant dose of Deiox: (25 !!g MPL and 250 !!g CWS). The vaccines were well tolerated, regardless ofdose, with local reaction (sterile granuloma) at the injection site in one patient after the third vaccination. T-cell proliferative responses directed against the vaccine peptide, but not against wild-type or other mutant ras peptides, developed in 3 of 10 evaluable patients as a result ofvaccination. HLA-A2/ras peptide-specific CD8+ cytotoxic T-cell responses also developed in two of three patients evaluated. These cellular responses were not dependent on dose. No major therapeutic responses were observed in any ofthe elevenpatients who received allthree vaccinations. One patient who showed stable disease after the three-dose regimen was administered three additional vaccinations and continued to show no evidence oftumor progression 10 months later.

Conclusion

A variety ofvaccines designed for cancer immunotherapy have been tested in clinical trials for more than two decades. Investigators realized early on that add ition ofadjuvants to cancer vaccines would be required to overcome the poor immune responses that are generally elicited to antigens contained within these vaccines. Although the effectiveness ofLPS as an immunomodulator has long been known, the pharmacologic use ofpurified LPS (or lipid A) asan adjuvant is precluded by its toxicity. In this regard , LPS is highly pyrogenic and promotes systemic inflammatory response syndrome." In an effort to uncouple the immunomodulatory effects oflipid A from its toxicity, Ribi er al developed 3-0-desacyl-4'-monophosphoryl lipid A (MPL), which comprises the lipid A portion ofLPS from which the (R)- 3-hydroxytetradecanoyl group and the l-phosphate have been removed? by successive acid and base hydrolysis. LPS and MPL induce similar cytokine profiles, but MPL is at least 100-fold less toxiC.9•1O MPL, as the active ingredient in MPI:' adjuvant or as one of the active ingredients in Detox"" adjuvant (with CWS and oil), AS02B adjuvant (with QS21 in an oil in water emulsion) or AS15 (a liposomal formulation with QS21 and CpG), has

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been administered to more than 300,000 hwnan subjects in studies ofnext-generation vaccines," including the patients enrolled in the clinical trials discussed in this chapter. At this time , MPL is the only lipid A derivative that has been clinically tested as an adjuvant for cancer vaccines. The reasons for the limited clinical success ofcancer vaccines in general are not clear, but may relate to the fact that most trials have been conducted in patients with late-stage disease. where bulky metastatic tumors may evade or suppress the immune system and prevent induction ofefficacious anti-tumor cellular and/or hwnoral adaptive immune responses. The very encouraging data obtained in an adjuvant setting in early stages NSCLC with the MAGE3 protein formulated in AS02B support that hypothesis. Another constraint is the weakly immunogenic nature ofmany TAAs, which are often "self" in nature and. therefore, not good targets for the induction ofeffector T-cell responses . Efforts to move beyond these limitations include adding potent adjuvants, such as MPL, to the vaccines, as well as combining immunotherapy with surgery. chemotherapy. radiation therapy and/or autologous stern-cell/dendritic cell therapy. The hope is that chemotherapyI radiation therapy will reduce tumor burden and deplete suppressive Tvcells, while optimized vaccination protocols will allow for enhanced induction, proliferation and activity oftumor-specific effector T-cells that can eliminate residual tumor, Such combination therapies are currently in the early stages of clinical testing and may lead to better options for the treatment ofcancer.

References

F, Schmid P, Mackensen A et al. Phase II trial of intr avenous endotoxin in patients with colorecral and nonsmall cell lung cancer. Eur] Cancer 1996; 32:1712-1718. 2. Engelhardt R. Mackensen A. Galanos C. Phase I trial of intravenously administered endotoxin (Salmonella abortus equi) in cancer patients. Cancer Res 1991; 51:2524-2530. 3. Mackensen A, Galanos C. Engelhardt R. Modulating activity of interferon-gamma on endotoxin-induced cyrokine production in cancer patients. Blood 1991; 78:3254-3258 . 4. Mackensen A. Galanos C. Wehr U et al. Endotoxin tolerance : regulation of cytokine production and cellular changes in response to endotox in application in cancer patients. Eur Cytokine Nerw 1992; 3:571-579. 5. Goto S. Sakai S. Kera] et al. Intradermal administration of lipopolysaccharide in treatment of human cancer. Cancer Immunol Imrnunother 1996; 42(4) :255-261. 6. Vosika G]. Barr C. Gilbertson D. Phase-I study of intravenous modified lipid A. Cancer Immunol Immunother 1984: 18(2):107-112. 7. Kiani A, Tschiersch A. Gaboriau E et al. Downregulation of the pro inflammatory cyrokine response to endotox in by pretreatment with the nontoxic lipid A analog SDZ MRL 953 in cancer patients . Blood 1997; 90(4) :1673-1683 . 8. de Bono ]S. Dalgleish AG, Carmichael] et al. Phase 1 study of ONO-4007. a synthetic analogue of the lipid A moiety of bacterial lipopolysaccharide. Clin Cancer Res 2000; 6(2) :397-405. 9. Myers KR. Truchot AT, Ward] et al. A critical determinant of lipid A endoroxic activity. In: Nowotny A. Spitzer ]]. Ziegler E], editors. Cellular and Molecular Aspects of Endotoxin Reactions. Amsterdam : Elsevier Sciences Publishers B V, 1990:145-156. 10. Ulrich ]T, Masihi KN . Lange W. Mechanisms of nonspecific resistance to microbial infections induced by trehalose dimycolate (TDM) and monophosphoryllipid A (MPL) . In: Masihi KN. Lange W; editors. Advances in the Bioscicnces, Great Britain : Pergamon Journals Ltd.• 1988:167-178. II. Evans]T. Cluff CW; Johnson DA et al, Enhancement of antigen-specificimmunity via the TLR4ligands MPL adjuvant and Ribi 529. Expert Review Vaccines 2003 ; 2(2) :219-229. 12. Woodlock T]. Sahasrabudhe DM . Marqu is DM er al. Active specific immunotherapy for metastatic colorectal carcinoma: Phase I study of an allogeneic cell vaccine plus low-dose interleukin-la.] Irnmunother 1999; 22(3) :251-259. 13. Sherye SF, Frodin ]-E. Christens son B. Immunohistochemical mon itoring of metastatic colorectal carcinoma in patients treated with monoclonal antibodies (MAb 17-01A). Cancer Immunol Imrnunorher 1988; 27:154 -162. 14. Neidhart]. Allen KO. Barlow DL er al. Immun ization of colorecral cancer patients with recombinant baculovirus-derived KSA (Ep-CAM) formulated with monophosphoryllipid A in liposomal emulsion, with and without granulocyte-macrophage colony-stimulating factor. Vaccine 2004; 22(5-6):773-780. 15. Papsidero LD. Kuriyama M. Wang MC er al. Prostate antigen : a marker for human prostate epithelial cells.] Nat! Cancer Insr 1981; 66(1) :37-42. 16. Oesterling ]E . Prostate specific antigen : a critical assessment of the most useful tumor marker for adenocarcinoma of the prostate.] Uro11991; 145(5):907-923. I.

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17. Harris DT, Matyas GR, Mastrangelo M] et al. Inducing immunity to prostate specific antigen (PSA) in prostate cancer patients. Proc ASCO 1999; 18:1693. 18. Meidenbauer N, Harris DT, Spitler LE et al. Generation of PSA-reactive effector cells after vaccination with a PSA-based vaccine in patients with prostate cancer. Prostate 2000; 43(2):88-100. 19. Mitchell MS, Kan-Mitchell ], Kempf RA et al. Active specific immunotherapy for melanoma: Phase I trial of allogeneic lysates and a novel adjuvant. Cancer Res 1988; 48(20):5883-5893. 20. Mitchell MS, Harel W; Kempf RA et al. Active-specific immunotherapy for melanoma. ] Clin Oncol 1990; 8(5):856-869. 21. Vose BM. Quantitarion of proliferative and cytotoxic precursor cells directed against human tumours: limiting dilution analysis in peripheral blood and at the tumour site. Inr ] Cancer 1982 ; 30(2):135-142. 22. Mitchell MS, Harel W; Groshen S. Association of HLA phenotype with response to active specific immunotherapy of melanoma.] Clin OncoI1992; 10(7):1158-1164. Sosman ]A, Sondak VK. Melacine: an allogeneic melanoma tumor cell lysate vaccine. Expert Rev Vaccines 2003 ; 2(3) :353-368. 23 . Elliott GT, Mcl.eod RA, Perez] er al. Interim results of a phase II multicenter clinical trial evaluating the activity of a therapeutic allogeneic melanoma vaccine (rheraccine) in the treatment of disseminated malignant melanoma. Semin Surg Onco11993; 9(3) :264-272. 24. Mitchell MS, Von Eschen KB. Phase III trial of Melacine melanoma theraccine versus combination chemotherapy in the treatment of stage IV melanoma. Proceedings of the American Society of Clinical Oncology 1997; 16,494a. 25 . Sosman ]A, Unger ]M, Liu PY et al. Adjuvant immunotherapy of resected, intermediate-thickness, node-negative melanoma with an allogeneic tumor vaccine : impact of HLA class I antigen expression on outcome, ] Clin Oncol 2002; 20(8):2067-2075. 26. Sondak VK, Sosman ]A . Results of clinical trials with an allogeneic melanoma tumor cell lysate vaccine: Melacine', Semin Cancer BioI 2003; 13:409-415. 27. Sosman ]A, Sondak VK. Melacine : an allogeneic melanoma tumor cell lysate vaccine. Expert Rev Vaccines 2003 ; 2(3) :353-368. 28. Mitchell MS, ]akowatz ], Harel W et al. Increased effectiveness of interferon alfa-2b following active specific immunotherapy for melanoma. ] Clin Onco11994; 12(2):402-411. 29. Vaishampayan U, Abrams ], Darrah D er al. Active immunotherapy of metastatic melanoma with allogeneic melanoma lysates and interferon alpha . Clin Cancer Res 2002; 8(12):3696-3701. 30. Mitchell MS. Immunotherapy as part of combinations for the treatment of cancer. Inc Immunopharmacol 2003; 3(8):1051-1059. 31. Mitchell MA, Abrams ], Kashani-Saber M et al. Interim analysis of a phase III stratified randomized trial of Melacine + low-dose Intron-A versus high-dose Intron-A for resected stage III melanoma. Am Soc Clin Oncol 2003; 22:709a . 32. Schultz N, Oratz R, Chen D et al. Effect of DETOX as an adjuvant for melanoma vaccine. Vaccine 1995 ; 13(5):503-508. 33. Eron 0 , Kharkevirch DD, Gianan MA er al. Active immunotherapy with ultraviolet B-irradiated autologous whole melanoma cells plus DETOX in patients with metastatic melanoma. Clin Cancer Res 1998; 4(3):619-627. 34. Wong R, Lau R, Chang] et al. Immune responses to a class II helper peptide epitope in patients with stage III /IV resected melanoma. Clin Cancer Res 2004; 10(15):5004-5013. 35. Lienard D, Rimoldi D, Marchand M er al. Ex vivo detectable activation of Melan-Avspecific T'cells correlating with inflammatory skin reactions in melanoma patients vaccinated with pepcldes in IFA. Cancer Immun 2004; 4:4 . 36. Marchand M, Punt C], Aamdal S et al. Immunisation of metastatic cancer patients with MAGE-3 protein combined with adjuvant SBAS-2: a clinical report. Eur] Cancer 2003; 39(1):70-77 . 37. Vantomrne V, Dantinne C, Amrani N. Immunologic analysis of a phase lIII study of vaccination with MAGE-3 protein combined with the AS02B adjuvant in patients with MAGE-3 -positive tumors. ] Immunorher 2004; 27:124-135. 38. Atanackovic D, Altorki NK , Stockert E et al. Vaccine-induced CD4+ T-cell responses to MAGE-3 protein in lung cancer patients.] Immunol 2004 ; 172(5) :3289-3296. 39. Brichard V. Development of cancer vaccines with the MAGE-3 protein. Cancer Immunity 2005; 5(1) :16. 40 . Brichard V. CVADD 2005; Portugal. 41. Zotter S, Hageman PC, Lossnitzer A et al. Tissue and tumor distribution of human polymorphic epithelial mucin . Cancer Rev 1988; 11-12:55-101. 42. Ho SB, Niehans GA, Lyfiogr C et al. Heterogeneity of mucin gene expression in normal and neoplastic tissues. Cancer Res 1993 ; 53(3) :641-651.

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43 Burchell J, Gendler S, Taylor-Papadimitriou J er al. Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin. Cancer Res 1987; 47(20) :5476-5482 . 44. Kjeldsen T, Clausen H, Hirohashi S et al. Preparation and characterization of monoclonal antibodies directed to the tumor-associated O-linked sialosyl-2-6alpha-N-acetylgalactosaminyl(sialosyl-Tn) epitope. Cancer Res 1988; 48(8) :2214-2220. 45. Ramanathan RK, Lee KM, McKolanis Jet al. Phase I study of a MUCI vaccine composed of different doses of MUCI peptide with SB-AS2 adjuvant in resected and locally advanced pancreatic cancer. Cancer Immunol Immunother 2005; 54(3) :254-264. 46. Palmer M, Parker J, Modi S er al. Phase I study of the BLP25 (MUCl peptide) liposomal vaccine for active specific immunotherapy in stage IIIB/IV nonsmall-cell lung cancer. Clin Lung Cancer 2001; 3(1):49-57. 47. North SA, Graham K, Bodnar D er al. A pilot study of the liposomal MUC 1 vaccine BLP25 in prostate specific antigen failures atter radical prostatectomy. J Urol 2006; 176(1) :91-95. 48. Butts C, Murray N, Maksymiuk A er al. Randomized phase lIB trial of BLP25 liposome vaccine in stage IIIB and IV nonsmall-celliung cancer. J Clin Onco12005; 23(27):6674-6681. 49. Machiels JP, Reilly RT, Emens LA et al. Cyclophosphamide, doxorubicin and paclitaxel enhance the antitumor immune response of granulocyte/macrophage-colony stimulating factor-secreting whole-cell vaccines in HER-2/neu tolerized mice. Cancer Res 2001; 61(9) :3689-3697. SO. Bass KK, Mastrangelo M]. Immunoprotentiation with low-dose cyclophosphamide in the active specific immunotherapy of cancer. Cancer Immunollmmunother 1998; 47:1-12. 51. Longenecker BM, MacLean G. Prospects for mucin epitopes in cancer vaccines. Immunologist 1993; 1:89-93. 52. Itzkowitz SH, Bloom EJ, Kokal WA et al. Sialosyl-Tn. A novel mucin antigen associated with prognosis in colorecral cancer patients . Cancer 1990; 66(9) :1960-1966. 53. Springer GF. T and Tn, general carcinoma autoantigens. Science 1984; 224(4654):1198-1206. 54. Kobayashi H , Terao T, Kawashima Y. Serum sialyl Tn as an independent predictor of poor prognos is in patients with epithelial ovarian cancer. J Clin Oncol 1992; 10(1):95-101. 55. Thor A, Ohuchi N, Szpak CA er al. Distribution of oncofetal antigen tumor-associated glycoprotein-72 defined by monoclonal antibody B72.3. Cancer Res 1986; 46(6) :3118-3124. 56. Nuti M, Teramoto YA, Mariani-Costantini Ret al. A monoclonal antibody (B72.3) defines patterns of distribution of a novel tumor-associated antigen in human mammary carcinoma cell populations. Inr J Cancer 1982; 29(5) :539-545. 57. Yoneawa S, Tachidawa T, Shin S. Sialosyl-Tn antigen : its distribution in normal human tissues and expression in adenocarcinomas. Am] Clin Parhol 1992; 98:167-174. 58. Longenecker BM, Reddish M, Miles D et al. Synthetic Tumor-Associated Sialyl-Tn Antigen as an Immunotherapeutic Cancer Vaccine. Vaccine Res 1993; 2(3) :151-162. 59. O'Boyle K, Zamore R, Adluri S et al. Immunization of colorectal cancer patients with modified ovine submaxillary gland mucin and adjuvants induces IgM and IgG antibodies to sialylated Tn. Cancer Res 1992; 52(20) :5663 -5667. 60. Berendt MJ, North R]. Tvcell-mediared suppression of anti-tumor immunity. An explanation for progressive growth of an immunogenic tumor. J Exp Med 1980; 151(1) :69-80. 61. MacLean GD, Reddish M, Koganty RR er al. Immunization of breast cancer patients using a synthetic sialyl-Tn glycoconjugate plus Derox adjuvant. Cancer Immunol Imrnunother 1993; 36(4) :215-222. 62. Miles DW; Towlson KE, Graham R et al. A randomised phase II study of sialyl-Tn and DETOX-B adjuvant with or without cyclophosphamide pretreatment for the active specific immunotherapy of breast cancer. Br J Cancer 1996; 74(8):1292-1296. 63. MacLean GD, Miles DW; Rubens RD er al. Enhancing the effect of THERATOPE Stn-KLN cancer vaccine in patients with metastatic breast cancer by pretreatment with low dose intravenous cyclophosphamide. J Immunorher 1996; 19(4):309-316. 64. Miles D, Ibrahim N, Roche H . An international randomized phase III clinical trial of STn-KLH (Therarope) therapeutic cancer vaccine in metastic breast cancer patients . Proceedings 27th Annual San Antonio Breast Cancer Symposium 2003;(36). 65. Ibrahim NK, Murray J, Parker]. Humoral immune-response to naturally occurring STn in metastatic breast cancer (MBC prs) treated with STn-KLH vaccine. Am Soc Clin Oncol 2004; 22:S174. 66. Majordomo J, Tres A, Miles D. Long-term follow-up of patients concomitantly treated with hormone therapy in a prospective controlled randomized multicenter clinical study comparing STn-KLH vaccine with KLH control in Stave IV breast cancer following front-line chemotherapy. Proc Am Soc Clin Oncol 2004 ; 22:1882S.

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67. Holmberg LA. Oparin DV. Gooley T er al. Clinical outcome of breast and ovarian cancer patients treated with high-dose chemotherapy. autologous stem cell rescue and THERATOPE STn-KLH cancer vaccine. Bone Marrow Transplant 2000: 25(12) :1233-1241. 68. Holmberg LA. Sandmaier BM. Theratope(R) vaccine (STn-KLH). Expert Opin BioI Ther 2001 : 1(5):881-891. 69. Holmberg LA. Oparin DV. Gooley T er al. The role of cancer vaccines following autologous stern cell rescue in breast and ovarian cancer patients: experience wirh the STn-KLH vaccine (Therarope). Clin Breast Cancer 2003: 3 SuppI4:S144-S151. 70. Holmberg LA. Sandmaier BM. Vaccination wirh Theratope (STn-KLH) as treatment for breast cancer. Expert Rev Vaccines 2004: 3(6) :655-663. 71. Khleif SN. Abrams SI. Hamilton JM et al. A phase 1 vaccine trial with pepridcs reflecting ras oncogene mutations of solid rumors. J Imrnunother 1999: 22(2):155-165. 72. Johnson AG. Molecular adjuvants and immunomodularors: new approaches to immunization. Clin Microbiol Rev 1994: 7(3):277-289.

CHAPTER

11

Antitumoral Effects ofLipids A, Clinical Studies Marc Bardon" and Daniele Reisser

Abstract

C

ancer remains the second leading causeofdeath. after cardiovasculardiseases.in industrialized countries. The first goal to achieve is to prevent cancer occurrence or to diagnose it at an early and curable stage. Some screening strategies have been developed, with controversies across countries. for several cancer type; colorectal, breasts or prostate cancer for example. Treatment ofcancer is generally based on surgery and radiotherapy for localized and attainable tumors. associated. in some cases, with adjuvant chemotherapy. Chemotherapy can also be used as first line treatment for disseminated diseases. The formulation of therapeutic strategies to enhance immune-mediated tumor destruction is a central goal ofcancer immunology. Substantive progress toward delineating the mechanisms involved in innate and adaptive tumor immunity has improved the prospects for crafting efficacious treatments LPS and their active component lipid A, have been used in tumor therapy since the 19th century. Studies in animal models have shown promising results on different models ofcancer but data from human trial are scarce. The published Phase-I cancer studies have shown that lipid A analogues are usually well tolerated. most ofthe side effects being likely related to immune response . i.e.•fever. chills and rigor. The administration ofseveral lipids A analogues was shown to result in a significant increase in circulating levels ofseveral cytokines but no objective antitumor responses were observed. Therefore clinical activity of such molecules deserves further experiments, likely in conjunction with chemotherapy.

Introduction

Even ifrecent data suggest a downward trend ofmortality rates for all cancers combined based on declining rates for many individual sites. including colon.' with only few exceptions affecting mainly females (e.g.• lung cancer)2.3 or specific sites such as liver," cancer remains the second cause of death. after cardiovascular diseases. in industrialized countries. together accounting for over half of all deaths.' Most cancer patients are treated by a combination of surgery. radiation and/or chemotherapy. Whereas the primary tumor can, in most cases, be efficiently treated by a combination ofthese standard therapies. preventing the metastatic spread ofthe disease through disseminated tumor cells is ofien not effective. The eradication ofdisseminated tumor cells present in the blood circulation and micro-metastases in distant organs therefore represents another promising approach in cancer immunotherapy. When the immune system is not destroyed by *Corresponding Author: Marc Bardou-Clinical Pharmacology Unit & Laboratory of Cardiovascular Experimental Physiology and Pharmacology, Faculty of Medicine, 7 Bd Jeanne d'Arc, BP87900, 21079 Dijon, France. Email: [email protected]

LipidA in Cancer Therapy, edited by jean-Francois jeannin. ©2009 Landes Bioscience and Springer Science+Business Media.

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chemotherapy, it is able to recognize tumor-specific antigens and eventually eliminate cancer cells. Furthermore a recent review highlights the fact that some cancers, such as colorectal cancer, cause direct inhibition ofthe host's immune response with a detrimental effect upon prognosis, suggesting that immunotherapy offers a therapeutic strategy to counteract these effects.6 Comprehensive analysis oftumor immunology and new immunization protocols suggest that immunotherapy can become an efficacious treatment in the near future. Combination with radiotherapy or chemotherapy should be investigated? One ofthe means to reverse this cancer-induced immune tolerance and to stimulate immune system might be the use oflipopolysaccharides (LPS). These are components ofthe outer membrane of Gram-negative bacteria, composed of a polysaccharide, an oligosaccharide core and a lipid A. These compounds have the property ofinducing the secretion ofvarious cytokines such as tumor necrosis factor (TNF), interferon (IFN)y, interleukin ＨｉｌＩＱｾ and IL6,8,9, as well as activation of immune cells including neutrophils," rnacrophages" and both CD4 and CD8 T-Iymphocytes, which infiltrate the tumors. I 2.13 Furthermore, these compounds have been shown to decrease supﾷｬＴ pressive cytokines like tumor growth factor ＨｔｇｆＩｾＮｉｏ The first assaysusing bacterial extracts containingLP S as a treatment for cancer were performed in 1898 as clinical trials." Half a century later, the antitumoral effect was attributed to LPS in mouse subcutaneous tumors" and finally it was demonstrated that this effect is due to the lipid A component ofLPS.1 7Since lipids A and their derivatives are less toxic than LPS, most anti-cancer treatments aimed at activating an immune response against tumors have been developed using natural lipids A or synthetic analogs, either alone or as adjuvant to enhance the efficacy of therapeutic anticancer vaccines. Animal models permit the investigation ofthe mechanisms ofthe antitumoral effect oflipids A (for review, see Reisser et al l8and the corresponding chapter ofthis book); therefore this chapter will summarize the results obtained in clinical trials.

Immunological Background Underlying the Clinical Potential Interest

Recent advances in molecular biology and immunology have contributed to a better understanding oftumor growth and tumor-host interactions and have shown that tumor progression is favored by the host tolerance to his tumor. In order to overcome the immunological unresponsiveness of patients to their growing tumors, endotoxins and their active component, lipid A , or synthetic analogs, may favor the settlement of, or increase the antitumoral immune response . Emphasis has been put on the role of dendritic cells on the onset of an efficient immune response.'? In the presence of a danger signal frequently delivered by bacterial produces," they release costimulatory factors that allow specific lymphocytes to differentiate and kill tumor cells they would have otherwise ignored. In vitro, LPS stimulate the production of cytokines by hu man dendritic cells" and monophosphoryllipid A (MPL) induces their maturation and their ability to activate T -cells, as does a lipid A analogue." However, no data document this question in cancer patients. Few studies dealt with the ex vivo activity ofimmune cells from patients treated with LPS or lipids A. Vosika et al23did not detected any clear effect ofa treatment with MPL on immune cell activity. On the contrary, monocytes from patients treated with LPS from Salmonella abortus equi(S.abortus equi) displayed an activated phenotype. 23 ln vitro , the lipid A OM-174 increases natural killer (NK) cytotoxicity ofperipheral blood cells from cancer patients," The number ofwhite blood cells (WBC) generally decreased, albeit transiently, after LPS or lipid A Injection," in particular, monocytes'? and lymphocytes.P'" The CD4:CD8 ratio was enhanced." Meanwhile the number ofgranulocytes always increased after the injections.23.26-28 .3o In spite ofthe decrease in the number ofWBC, in most ofthe studies published so far, treatments ofpatients with LPS or lipid A enhanced the levelofblood cytokines.Indeed the production ofcytokines is a biological marker ofthe activation ofimmune cells and LPS and lipid A have the property ofinducing the secretion ofvarious cytokines in humans."

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LPS from S. abortus equi increased the concentration ofTNF and the activity ofIL6, IL8, granulocyte colony-stimulating factor (G-CSF) and macrophage (M)_CSP6.27 while there was no change in ｉｌｾ and IL2 and IL7 was not detected. LPS from Pantoeaagglomerans increased the serum concentrations ofTNF, ILIa, IL6 and G-CSY SDZ MRL 953 increased, though not significantly, the serum concentrations ofTNF, ｉｌ ｾＬ IL8, G-CSF and IL6. 30 TNF and IL6 production was induced after a treatment by ON0-4007,28 while this lipid A analog had no effect on GM-CSF, IFN-y and neopterin. Mononuclear cells from patients treated with LPS were primed for cytokine production ex vivo." Lipids A induce the in vitro production ofcytokines by human peripheral blood cells and monocytes v'" and MPL that ofreactive oxygen species. 36 At the difference with LPS, ON0-4007 induced few, if at all, in vitro production of cytokines by monocytes or total peripheral blood cells unless they had been primed with GM-CSF.37The real impact of these cytokines on tumor evolution was not deciphered and one can regret no study was interested in the production of IFNy which is a marker oflymphocyte acrivation." In humans, LPS can induce coagulation via TNF involvemenr.'? but its importance for tumor irrigation and therefore its effect on tumor growth is difficult to evaluate in human. Treatment with S. abortus equP4 and ON0-4007 27led to no changes in coagulation parameters, disseminated intravascular coagulation and clotting parameters The hallmark ofa bacterial infection is fever due to LPS pyrogenicity. Ie may have some relevance in cancer as hyperthermia is tested as a cancer treatment on its own, as reviewed by Christophi et al.40 This effect is mediated by cyrokines (mainly ｉｌｾ｢ ),41 which production was inconsistently found after LPS or lipid A treatment. Indeed, treatments with LPS or lipid A induced fever in at least half the patients.22·24.26.29 Fever is generally opposed with anti-inflammatory drugs and Engelhardt? verified ibuprofen did not affect cytokine production. However the role of fever in LPS treatment efficacy was recently discussed by Hoption Cann et al43 who wondered whether fever inhibition might impede treatment efficiency.

Clinical Studies

Few Phase -I trials were performed with intravenous (i.v.) injections oflipids A, from S. typhimurium or S.minnesota,22 or the synthetic lipids A SDZ MRL 95Y9and ONO-4007. 27 Vosika et al22 have conducted a Phase -I trial with MPL prepared from S. typhimurium and S minnesota.

In this study patients received i.v.MPLA, twice weekly for a total of4 weeks, at the following dose levels of 10,25, 50, 100 and 250 micrograms/rn! body surface area. One additional patient was treated at the dose level of500 rnicrograrns/m', In this study, lipid A was generally well tolerated, the major clinical coxicity being fever, chills and rigor, which occurred in over 50% of the treatments at doses of 250 micrograms/m'. Two instances of bronchospasm occurred in one patient who received 250 rnicrograrns/rrr'. The patient who received 500 micrograrns/rn" became hypotensive. Sequential clinical data showed no evidence of renal or hepatic toxicity, Interestingly a transient decrease in the WBC and platelets was observed during the first 24 hours after therapy. Immune function testing suggested a shift in monocyte populations with activated cells moving into the tissue . Unfortunately no direct objective antitumor activity or necrosis was observed in this group ofpatients. From this trial, Vosika et al 22 recommended a dose of up co 100 micrograms/rn' co be used with acceptable toxiciry for further evaluation of its clinical activity as a single agent in combination with other immunomodulacors. More than ten years later Kiani et al 29 conducted another phase one trial on cancer patients using the synthetic lipid A analog SDZ MRL 953 which has been shown co be protective against endotoxic shock and bacterial infection in preclinical in vivo models. This authors, as part ofa trial ofunspecific imrnunostimulation in cancer patients, conducted a double-blind, randomized, vehicle-controlled Phase-I trial ofSDZ MRL 953 to investigate its

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biologic effects and safety ofadministration in humans and its influence on reactions to a subsequent challenge ofendotoxin (S. abortus equi). In this Phase-I trial, twenty patients were treated i.v, with escalating doses ofSDZ MRL 953 or vehicle control, followed by an i.v, application ofendotoxin (2 ng/kg ofbody weight [BW]). The first result of this study is that administration of the lipid A analogue, SDZ MRL 953, was safe and well-tolerated. On the contrary to the transient decrease in WBC count that was observed in the stu dy by Vosika et a122SDZ MRL 953 itselfincreased granulocyte counts. This increase was associated with and likely related to , an increase in serum levels of G-CSF and IL6. Surprisingly, the proinflarnmatory eytokines TNF, III ｾ and IL8 were not significantlyincreased by SDZ MRL 953 administration. In the second part ofthe trial , pretreatment with SDZ MRL 953 markedly reduced, compared with vehicle control, the release ofTNF, ｉｌｬｾＬ IL8, IL6 and G-CSF, but augmented the increase in granulocyte counts to endotoxin. Once again this study suggested an overall safety oflipid A analogue in cancer patients. More recently de Bono et aJ27 published the results of a Phase -I trial using the lipid A ON0-4007, that is a synthetic analogue ofthe lipid A moiety ofbacterial LPS, which, in animal models , exhibits antitumor activity by the induction of intratumoral TNF, the potentiation of tumor-infiltrating macrophages and the inhibition ofangiogenesis. ILla, IL6 and ILl2 induction by ONO-4007 activates NK cells to up-regulate IFNy and nitric oxide synthase activity. In this trial ON0-4007 was given to 24 patients (13 males and 11 females; median age, 53 years) as a 30-min i.v, infusion on day 1, followed on day 15 by a first treatment cycle consisting of three weekly infusions at the same dose, followed by a rest period of 1 week. Cohorts of six pat ients received up to a maximum offour treatment cycles at increasing dose levels (75, 100 and 125 mg). De Bono et a1 27 found a maximum tolerated dose of 125 mg, with grade 3 National Cancer Institute Common Toxicity Criteria toxicity (NCICTC; rigors with cyanosis) occurring in two ofsix patients at thi s dose level. An additional sixpatients were treated at 100 mg, the dose below the maximum tolerated dose. Other toxicities included grade 2 NCICTC myalgia, nausea and hypotension. In this study the pharmacokinetics of ON0-4007 appeared to be independent of dose and showed linearity with respect to time. ON0-4007 was described to have a low systemic clearance (approximately 1.3 ml/min) and a small volume ofdistribution (5-8 liters) with a long half-life of74-95 hours and the administration of this drug was shown to result in a significant increase in circulating levels ofTNF and IL6. As it was the case with the previously reported two phase one trials,22,29no objective antitumor responses were observed. Seven patients maintained stable disease for at least two cycles,whereas five patients maintained stable disease for the full four -cycle duration ofthe study. Limited toxicity was observed with Salmonella lipids A or ONO-4007 and the maximal tolerated dose (MTD) was not reached for SDZ MRL 953. One Phase-I study on cancer patients was conducted with 0 M-174 another analogue oflipid A, by Viens P and aI (unpublished results up to date) . This study was aimed to assessthe tolerance of and the biological response to, incr easing doses of OM-174 administered as single i.v, infusion. The first dose that was administered was half ofthe no adverse event level obtained in a phase one study conducted in healthy volunteers, i.e., 1.25 ug/kg or 50 !J.g/m2. Only one serious adverse event (SAE) was reported at the dose of800 ug/rn' with rigor hypotension , cyanosis and hypothermia. After i.v, acetaminophen this patient experienced full recovery within 24 hours and was discharged from hospital as initially scheduled. Three additional patients were created at the 800 ug/rn! and the study was continued up to the 1300 !J.g/m2dose without reaching the maximal tolerated dose. A biological response was assessed by cyrokine measurement. To summarize the findings that will be published as a full paper a TNF increase was noted, with very high levels (up to 4000 pg/

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ml) for some patients without clear correlation with the dose ofOM-174 that was administered. Furthermore even ifthe patient who experienced the SAE had a high levelofplasmatic TNF, some other patients had even higher levels ofTNF without experiencing SAE. Once again no antitumor activity was reported in this clinical trial . There is another ongoing (as of early 2007) Phase-I study with OM-174 in cancer patients aimed to assess safety, efficacy and biological response of OM-174 with an incremented doses (600,800 and 1000 flg/m 2) and injections (5,10 or 15) protocol. To summarize the results ofthese four clinical trials,with only three published as full paper.lipid A analogues administration in cancer patients was associated with a biological response, evidenced most consistently by a TNF and an IL6 production. But no clear dose-response was observed for cytokine production and no objective ant itumor activity was observed in any of thi s four trials.

Conclusion

From a general point ofview, treatments with lipids A alone have the advantage of bypassing the investigation of specific tumor antigens and are therefore easier to establish on a broader range ofcancers. However, despite the encouraging results obtained, most human trials use lipids A as adjuvant . This is mainly due to the need of minimal quantities oflipids A in vaccines, thus minimizing their eventual toxicity and the goal of generating a more specific immune response against particular tumors. Only hints were afforded concerning the mechanisms of action of treatments with LPS and lipids A in cancer therapy and further stu dies are needed to decipher how to take advantage of the different ways lipid A could oppose tumor growth. Treatments generally lead to endotoxin tolerance ; the beneficial or detrimental role is discussed in another chapter in the present book. None of the published studies with lipids A used as anticancer therapy has been able to show any beneficial effect in terms of tumor control. However, in a field which often lacks efficient therapy, the encouraging results obtained so far indicate that treatments with lipids A could represent a hope. In the situations where no effective therapy is available, it could be ofinterest to test immunotherapy and particularly lipid A, on small tumors. Immunotherapy with lipid A has already been used to activate or reactivate memory cells after chemotherapy. It may also be used after tumor burden reduction by surgery or radiotherapy as a complementary therap y to stimulate or recover the immune system. It is therefore worthwhile to expand further the stu dies and tr ials with these compounds to determine the best circumstances and conditions for the application of these treatments in view ofclarifying whether lipids A will become a clinical approach for the treatment of cancer.

References

1. Cress RD, Morris C, Ellison GL et al. Secular changes in colorectal cancer incidence by subsite, stage at diagnosis and racc/ethniciry, 1992-2001. Cancer 2006 ; 107(5 Suppl):1142-1152. 2. Becker N, Altenburg HP, Stegmaier C et al. Report on trends of incidence (1970-2002 ) of and mortalit y (1952-2002) from cancer in Germany. J Cancer Res Clin Oncol 2006. 3. Levi F, Lucchini F, La Vecchia C. Trends in cancer mortality in Switzerland, 1980-2001. Eur J Cancer Prev 2006; 15(1):1-9. 4. West J, Wood H. Logan RF er al. Trends in the incidence of primary liver and biliary tract cancers in England and Wales 1971-2001. Br J Cancer 2006 ; 94(11 ):1751-1758. 5. Ho yerr DL , Heron MP, Murph y SL er al. Deaths: final data for 2003 . Nat! Vital Stat Rep 2006 ; 54(13):1-120. 6. Evans C, Dalgleish AG, Kumar D. Review article: immune suppression and colorectal cancer. Aliment Pharmacol Ther 2006; 24(8):1163-1177. 7. Carpenti er AF, Meng Y. Recent advances in immunotherapy for human glioma. Curt Op in Onc ol 2006; 18(6):631-636. 8. van de Wiel PA, van der Pijl A. Bloksma N. Role of tumour necrosis factor in the tum our-necrot izing activity of agents with diverging toxicity. Cancer Immunol Irnrnunorher 1991; 33(2):115-120 . 9. Blondiau C, Lagadec P, Lejeune P er al. Correlation between the capacity to activate macrophages in vitro and the antitumor activity in vivo of lipopolysaccharides from different bacterial species. Immuno biology 1994; 190(3):243-254.

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10. Inagawa H . Nishizawa T. Takagi K et al. Antitumor mechanism of intradermal administration of lipopolysaccharide. Anticancer Res 1997; 17(3C):1961-1964. 11. Shimizu T, !ida K, Iwamoto Y et al. Biologicalactivities and antitumor effects of synthetic lipid A analog linked N-acylated serine. Int J Immunopharmacol1995; 17(5):425-431. 12. Onier N, Lejeune P, Martin M et al. Involvement of T-Iymphocytes in curative effect of a new imrnunornodulator OM 163 on rat colon cancer metastases. Eur J Cancer 1993; 29A(14) :2003-2009 . 13. On ier N, Hilpert S, Arnould L et al. Cure of colon cancer metastasis in rats with the new lipid A OM 174. Apoptosis of tumor cells and immunization of rats. Clin Exp Metastasis 1999; 17(4):299-306. 14. Lagadec P, Raynal S. Lieubeau B et al. Evidence for control of nitric oxide synthesis by intracellular transforming growth factor-beta1 in tumor cells. Implications for tumor development. Am J Pathol 1999; 154(6):1867-1876. 15. Coley WB. The treatment of inoperable sarcoma with the mixed toxins of Erysipelas and bacillus prodigiosus. j Am Med Assoc 1898; 31:589-595. 16. Shear MJ, Turner Fe. Chemical treatment of tumors. V. Isolation of the hemorrhage-producing fraction from Serratia rnarcescens (Bacillus prodigiosus) culture filtrate. J Nat! Cancer Inst 1943; 4(81-97). 17. Parr I, Wheeler E, Alexander P. Similarities of the anti-tumour actions of endotoxin , lipid A and double -stranded RNA. Br J Cancer 1973; 27(5) :370-389. 18. Reisser D, Pance A, Jeannin JF. Mechanisms of the antitumoral effect of lipid A. Bioessays 2002; 24:284-289. 19. Matzinger P. Tolerance, danger and the extended family. Annu Rev Immunol1994; 12:991-1045. 20. Verhasselc V. Buelens C, Willems F ec al. Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells: evidence for a soluble CDI4-dependent pathway.J Immuno11997; 158(6):2919-2925. 21. Ismaili J, Rennesson J, Aksoy E et al. Monophosphoryllipid A activates both human dendritic cells and T-cells. J Immunol 2002; 168(2):926-932. 22. Vosika GJ, Barr C, Gilbertson D. Phase-I study of intravenous modified lipid A. Cancer Immuno l Imrnunother 1984; 18(2):107-112. 23. Rcisser D, Arnould L, Maynadie M et al. Lipid A OM-174 increases the natural killer activity of peripheral blood cells from breast cancer patients. J Endotoxin Res 1999; 5(4) :189-195. 24. Engelhardt R. Mackensen A, Galanos C. Phase 1 trial of int ravenously administered endotox in (Salmonella abortus equi) in cancer patients. Cancer Res 1991; 51(10) :2524-2530 . 25. Mackensen A, Galanos C, Wehr U ec al. Endotoxin tolerance : regulation of cytokine production and cellular changes in response to endotoxin application in cancer patients . Eur Cytokine Netw 1992; 3(6) :571-579 . 26. Mackensen A, Galanos C, Engelhardt R. Modulating activity of interferon-gamma on endotoxin-induced cytokine production in cancer patients. Blood 1991; 78(12):3254-3258. 27. de Bono JS, Dalgleish AG, Carmichael Jet al. Phase I study of ON0-4007. a synthetic analogue of the lipid A moiety of bacterial lipopolysaccharide. Clin Cancer Res 2000; 6(2) :397-405. 28. Otto F, Schmid P, Mackensen A et al. Phase II trial of intravenous endotoxin in patients with colorectal and nonsmall cell lung cancer. Eur J Cancer 1996; 32A(1O):1712-1718 . 29. Kian i A, Tschiersch A, Gaboriau E er al. Downregulation of the proin£lammatory cytokine response to endotoxin by pretreatment with the nontoxic lipid A analog SDZ MRL 953 in cancer patients. Blood 1997; 90(4) :1673-1683. 30. van Devenrer SJ, Buller HR, ten Care JW et al. Experimental endotoxemia in humans: analysis of cyto kine release and coagulation, fibrinolytic and complement pathways. Blood 1990; 76(12) :2520-2526. 31. Goto S, Sakai S, Kera J er al. Intradermal administration of lipopolysaccharide in treatment of human cancer. Cancer Immunol Immunother 1996; 42(4) :255-261. 32. Engelhardt R, Otto F, Mackensen A ct al. Endotoxin (Salmonella abortus equi) in cancer patients . Clinical and immunological findings. Prog Clin Bioi Res 1995; 392:253-261. 33. Matsuura M, Kiso M, HasegawaA. Activity of monosaccharide lipid A analogues in human monocytic cells as agonises or antagonists of bacterial lipopolysaccharide. Infect Immun 1999; 67(12 ):6286-6292. 34. Tarnai R, Asai Y, Hashimoto M er al. Cell activation by monosaccharide lipid A analogues utilizing Toll-like receptor 4. Immunology 2003; 110(1):66 -72. 35. Martin M. Michalek SM, Katz J. Role of innate immune factors in the adjuvant activity of monophosphoryllipid A. Infect Immun 2003; 71(5) :2498-2507. 36. Saha DC, Barna RS. Astiz ME et al. Monophosphoryllipid A stimulated up-regulation of reactiveoxygen intermediates in human monocytes in vitro. J Leukoc Bioi 2001; 70(3):381-385 . 37. Matsumoto N, Aze Y, Akimoto A et al. ON0-4007. an antitumor lipid A analog, induces tumor necrosis factor-alpha production by human monocyres only under primed state : different effects of ON0-4007 and lipopolysaccharide on cytokine production. J Pharmacol Exp Ther 1998; 284(1) :189-195.

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38. Walzer T. Dalod M. Robbins SH et al. Natural-killer cells and dendritic cells: "l'union fait la force" Blood 2005 ; 106(7) :2252-2258. 39. Magder S. Neculcea J. Neculcea V et aL Lipopolysaccharide and TNF-alpha produce very similar changes in gene expression in human endothelial cells. J Vase Res 2006 ; 43(5):447-461. 40. Christophi C. Winkworth A. Muralihdaran V er al. The treatment of malignancy by hyperthermia . Surg Onco11998; 7(1-2):83-90. 4 I. Dinarello CA. Infection. fever and exogenous and endogenous pyrogens: some concepts have changed. J Endotoxin Res 2004; 10(4):201-222. 42. Engelhardt R. Mackensen A, Galanos C et al. Biological response to intravenously administered endotoxin in patients with advanced cancer. J Bioi Response Mod 1990; 9(5) :480-491. 43. Hoption Cann SA, van Nerren JP, van Nerren C. Dr Will iam Coley and tumour regression: a place in history or in the future . Postgrad Med J 2003; 79(938) :672-680.

CHAPTER 12

Conclusion ]ean-Franftois]eannin

O

ne of the major advances in the treatment of cancer with lipid A is the possibility of chemically synthesizing, lipid A analogs that are both biologically active in cancer patients and very well tolerated. Biotechnological production of different lipid A forms is also possible but the chemical route is preferable as it avoids LPS contamination. This significant step was somewhat unexpected as in the 1980s the debate had centered on the question "Is it possible to separate the toxic activity oflipid A from its anti-tumor activity?" and I will come back to this question here. We know very little about the anti -tumor mechanisms oflipid A analogs. Their effects are indirect, in the inflammatory pathway and probably mediated by cells ofthe innate immune system and the TLR4 receptor; however they induce a specific immune response and immunization without an auto-immune reaction. The action oflipid A analogs fits into the Danger model (see Introduction). Antigen-presenting cells (APC, mainly dendritic cells) have to receive (i) activating signals from injured tumor cells, but not from normal cells and (ii) activating signals to induce co-stimulation of helper T cells. It has been shown in this book that lipid A analogs activate APC, especially dendritic cells, to produce co-stimulatory molecules either directly via TLR4 or via cytokine produced by stroma cells. To get a specific immune response without an auto-immune response, only tumor cells should be killed while normal cells should be preserved, thus lipid A analogs must kill tumor cells specifically. Lipid A analogs are not cytotoxic by themselves, the inflammatory response they generate is not specific, so they cannot be involved in the cell injury. On the other hand, lipid A analogs are able to activate innate immune cells to become selectively toxic to tumor cells. NK cells and macrophages are able to discriminate between tumor and normal cells, killing tumor cells and leaving normal cells unharmed. Therefore the most likely hypothesis is that lipid A analogs activate innate immune cells (macrophages or NK cells) to kill tumor cells, then fragments oftumor cellswith antigens are phagocytosed. processed and presented to helper T cells by APC (dendritic cells) which at the same time are stimulated (directly or not) by lipid A analogs to co-stimulate helper T cells. Lipid A analogs induce active mechanisms and also inhibit suppression by inducing the switch from Tumor associated macrophages-2 (TAM-2, suppressor macrophages) to TAM-1 (anti-tumor macrophages) by inhibiting regulatory T cells. So coming back to the question "Is it possible to separate toxic activity from antitumor activiryr" we only have a partial answer. It is possible to induce anti -tumor activity without toxicity. Toxicity is due to huge inflammatory reactions of the innate immune system in which pro-inflammatory cascades are activated and the various pro-inflammatory mediators appear in an amplification loop. Anti-tumor activity comes with an inflammatory response that is local and moderate and probably supportS the anti-tumor response by creating a favorable micro-environment. Although toxicity and anti-tumor activity start by following the same inflammatory response mechanism, they differ in their intensity then diverge, toxicity going on to produce a huge and systemic inflammatory reaction , and the anti-tumor response to tumor cell-mediated toxicity, antigen presentation and a specific 'Corresponding Author: lean-Francois Jeannin-EPHE, Dijon, F-21 000, France; Inserm U866 , Dijon, F-21000, France. Email: [email protected]

LipidA in Cancer Therapy, edited by jean-Francoisjeannin. ©2009 Landes Bioscience and Springer Science+Business Media.

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immune response. Structure-function analysis of lipid A signalingindicates that the length and number ofacylsidechainsarecriticalforsignalingviaTLR4. Decreasingthe numberor lengthofthe attached fatty acidsor alteringthe overallchargeof lipidA can reducethe magnitude of the signal,' Preclinicalstudiesshowthat the most effective lipidA analogsin termsof antitumor activityin vivo do not havethe optimum structure in terms ofthe length and number of acylsidechainsto induce signaling. Is the signalingthe reasonfor differences between toxicityand the anti-tumor response? Do the lipid analogsbind TLR4 in a differentwaythan natural lipid A forms?Or is the difference upstream of the signaling, in the transport or in the transfer from LBP/CD14 to TLR4? The danger signal model is a good framework in which to assess non specific activeimmunotherapywith compounds isolatedfrom bacteriaor viruses. Until recentlythe mechanismsofaction ofthis class ofsubstanceremainedunknown.IncreasedunderstandingofTLR signalinghasallowed the molecularbasisof suchcompoundsto beelucidated. Themost effective treatment for superficial bladder cancer, and the only reallyeffective immunotherapyto date,ismediated byboth TLR2 and TLR4. 2The biologicalresponse modifierOK-342 isdependent on TLR-4 for itsanti-tumor effect.3 Double-strandedRNAI activates TLR3 on dendritic cells to release Type 1IFN that inducestumor cellapoptosisand NK-mediatedtumor cyroroxiciry," Unmethylateddeoxycytidyl-deoxyguanosine (CpG) dinucleotides are recognizedby the Toll-like receptor 9 (TLR9) and are effective in treating leukemia.' Bytargeting TLR these unrelated compounds may conform to the Danger model, inducing the innate immune systemthen activatingthe tumor adaptiveimmune response. Based on preclinical results, it is not possibleto define the type of cancer to treat. The lipid A analogs tested were effective in treating severaltypes of cancer (carcinoma, sarcoma, erc.) in different animal species (mice,rats, guinea pigs, rabbits, etc.). However there are very few results ofpreclinicalstudieswith hematological cancers. Does this type of cancernot respond to lipid A analogsor doesthe lackofresultssimplyreflectthe lackofexperiments? However, wecan conclude from preclinical results that it is necessary to repeat the injections of lipid A analogsand that the rhythm ofinjections is as important as the route, with the intra venous route being more effective than the intra peritoneal, intra muscullaror sub cutaneous routes. An important issueis addressedin this book-the tolerance induced bylipid A analogs. Is this tolerance necessary or not in the anti-tumor effectiveness of lipid A analogs? Toleranceis induced and maintained in vivowhen lipid A is administered with a specific rhythm ofinjecrions. In rats this rhythm is the best wayofproducing an anti-tumor effect,but it doesnot followthat tolerance is necessaryfor the anti-tumor effect.The question remainsopen. The rationale for usinglipid A analogsin clinicaltrials is to activateTLR4 on innate immune cells to induce adangersignal(tumor celldeath,antigenpresentationand APC activationto express co-stimulatorymolecules). Somecancers stillhaveno effective treatment,othersarecuredonlywhen they are treated earlyand in the absence of metastases, so lipid A analogs represent a significant sourceofhope for cancerpatients.

References

1. Miller SI, Ernst RK, and Bader MW. LPS, TLR4 and infectious disease diversity. Nat Rev Microbiol 2005; 3:36-46. 2. Tsuji S, Matsumoto M, Takeuchi 0 et al. Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmene-Guerin: involvement of toll-like receptors. Infect Immun 2000; 68:6883-6890. 3. Okamoto M, Oshikawa T, Tano T et al. Involvement of Toll-like receptor 4 signaling in interferon -gamma production and antitumor effect by streptococcal agent OK-432. j Narl Cancer Insr 2003; 95:316-326. 4. Whitmore MM, DeVeerM], Edling A et al. Synergistic activation of innate immunity by double-stranded RNA and CpG DNA promotes enhanced antitumor activity. Cancer Res 2004; 64: 5850-5860. 5. jahrsdorfer B. Muhlenhoff L, BlackwellSE et al. B-celilymphomas differ in their responsivenessto CpG oligodeoxynucleotides. Clin Cancer Res 2005 ; 11: 1490-1499 .

INDEX A Activeprincipleof endotoxin 17 Acylchain fluidity 33, 42 Adjuvant 2,3,71,83,85,92, 101, 103, 111-120,125,126,129 Aggregate structure 25,27-30,32,33, 42-45 Albumin 41,42,69,71 Animal model 3, 71,72, 76,84,86, 101, 105, 108, 125, 126, 128 Antagonist 12, 18,31,32,41,43,69,76, 83,91 Antitumoral activity 102, 104, 106

B Bactericidal/Permeability-Increasing Protein 40,42,45,70 B cell 2, 53, 56 Biosynthetic precursor to lipid a 6, 7 Blood cytokine 126

C C3H/He] 54, 59 Cancer 83-86,92,93,101-103,111,112, 113,115-120,125-129,133,134 Cancer immunotherapy 119, 125 Cancer vaccine 83,85,92,111-113, 115-120 Cathelicidin 40, 45, 46 CD14 2,34,39-41,43,45,46,53,55,59, 70,87,88,91,92, 134 Chemicalsynthesis 1,6,17,18,69 Clinical toxicity 127 Colorectal cancer 112, 113, 117, 126 Critical micellarconcentration 25, 26 Cytokine 28, 30, 32, 34,42, 43, 45, 46, 56, 59,60,62,63,70,71,73-76,81-85, 88-90,92, 101, 102, 105, 107, 111, 119, 125-129, 133

D

D-Glucosamine2-amino-2-deoxy-d-glucose (GleN) 5-11, 14-16,31 Dendritic cell 53,56,62,63, 71,84, 107, 134 111,120, 126, ＱＳｾ

E Endotoxicprinciple 5, 25, 33, 39 Endotoxin 1, 5, 9, 17,25,27,30,32-34, 36,39,41,42,46, 53, 56, 59, 82-85, 88-90,92, 126, 128, 129

F Freelipid A 8,9,27,28,30,41

G Gel to liquid crystallinephasetransition 44 GleN3N (3-dideoxy-d-glucose) 8, 11, 14-16 Glycolipid 5, 8, 17

H Heat shock protein 33, 54, 75 Hydrophilic backbone 5, 6, 8, 15, 16

K Kdo (3-deoxy-d-manno-octulosonicacid) 9, 14,18,27,41,44

L Lactoferrin 42, 44, 70 Leptospira interrogan 15, 54 LipidA 9,12-17,25-27,32,33,39,41,42, 44,46,53,54,59,65,69,70,72,73, 75,81-83,85,86,91,101,111,133 Lipid bilayer 5, 117 LipidX 7

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136

Lipopolysaccharide (LPS) 1,2, 5-9, 12, 14, 18, 19,25,27-34,39-46,53-56,59,60, 62-64,69,70,72-75,81 -92, 101, 102, 104, Ill, 119, 125-129, 133 Lipopolysaccharide-bindingprotein (LBP) 2,31,33,34,36,39-41,43-46,53,59, 70,91, 134 Lipoprotein 40, 42-44, 69

SDZ MRL 953 72, 83, 85, 86, 92, 103, 127, 128 Secondaryacylgroup 6, 8, 9, 11, 12, 16, 17 Sepsis 39,41,69,81-84,86 Supramolecular structure 3, 25, 28, 32, 43, 45

M

T

S

Pharmacokinetic 86, 128 Polar substituent 13-15, 19 Prirnaryacylgroup 6,8,9,11,17 Proteasome 63, 90-92

Taxol 54 Tcell 2,133 TlRAP 61-63 TLR2 54,62,87, 88,91,92, 134 TLR4 2,3,33,34,40,41,53,-56, 59-65, 69,70,72,87-89,91 ,92,111,113, 133,134 Tolerance 3,62,63,69,72-74,81-93, 105-107, 117, 126, 128, 129, 134 Toll-like receptor (TLR) 2, 33, 40, 53, 59-65,87,91, 134 Toll receptor 2, 54 TRAM 61-63 TRIF 60-64 Tumor 1,2,25,39,54,69-76,82-86,92, 101-108,111-120,125-129,133,134 Tumor associated antigens (TAA) 111-113, 115-117, 119, 120 Tumor growth 71,73-75,84, 102-107, 118, 126, 127, 129 Tumor inununity 117, 125

R

u

RP105 53,56

Unsaturatedacylgroup 12, 18

Massspectrometry(MS) 6, 16, 19 Molecularconformation 12,25,27,29, 31,32 Molecularmodelling 25, 31 Monophosphoryllipid A (MPL) 28,29, 32,69-72,82,83,85,91,92, 103, 111-114,117-120,126,127 MyD88 59-65,87,88,91,92

N NFKB 72,87 Nuclearmagneticresonance spectroscopy (NMR) 6, 16, 17, 19

p