CHAPTER 10

Monophosphoryl LipidA (MPL) asan Adjuvant forAnti..CancerVaccines:Clinical ResultsChristopherW. Cluff·

Abstract

A s technological advancesallow for the identification oftumor-associated antigens (TAAs)against which adaptive immune responses can be raised , efforts to develop vaccines for thetreatment ofcancer cont inue to gain momentum. Some ofthese vaccines target differen-

tiation antigens that are expressed by tumors derived from one particular tissue (e.g., Melan -A/MART-I , tyrosinase , gp 100). Some target antigensare specifically expressed in tumors ofdifferenttypes but not in normal tissues (e.g., MAGE-3), while other possible targets are antigens that areexpressed at low level in normal tissues and are over-expressed in tumors of different types (e.g.,HER2, Muc 1). Oncogenes (H ER2/ neu, Ras, E7 HPV 16), tumor supp ressor genes (pS3 ) ortumor-specific post-translational modified proteins (underglycosylated Muc 1) can also be usedas cancer vaccine candidates. In either case, th ese antigens tend to be poorly inmmunogenic bythemselve sand vaccines containing them generally require the inclusion ofpotent immunologicaladjuvants in order to generate robust anti-tumor immune responses in humans. Many adjuvantscurrently under evaluation for use in cancer vaccines activate relevant antigen presenting cells,suchas dendritic cells and macrophages, via toll-like receptors (TLRs) and promote effective uptake,processing and presentation ofantigen to T-cells in draining lymph nodes.

Lipid A, the biologically active portion of the gram-negative bacterial cell wall consti tu entlipopolysaccharide (LPS) , is known to possess strong immunostimulatory properti es and hasbeen evaluated for more than two decades as an adjuvant for promoting immune respon ses tominimally immunogenic antigens, including TAAs. The relatively recent discovery ofTLRs andthe identification ofTLR4 as the signaling receptor for lipid A have allowed for a better under-standingofhow this immunostimulant functionswith regard to induction ofinnate and adaptiveimmune response s.

Although several lipidA species, includingLPS and synthetic analogs, have been developed andtested asmonotherapeutics for the treatment ofcancer,}·8only 3-0-desacyl-4'-monophosphoryllipidA (MPL) has been evaluated as a cancer vaccine adjuvant in published human clinical trials.MPLcomprises the lipid A portion ofSalmonella minnesota LPS from which the (R)-3-hydroxytetradecanoyl group and the l -phosphate havebeen removed by successive acid and base hydrolysis," LPSand MPL induce similar cytokine profiles, but MPL is at least 1OO-fold less toxiC.9,IOMPL has beenadministered to more than 300 ,000 human subjects in studies ofnext-generation vaccines.I I

In this chapter, published clinical trials conducted to evaluate the safety and/or efficacy ofvarious cancer vaccines containingMPL, eith er alone or combined with other immunostirnulants,

*ChristopherW. C1uff-GlaxoSmithKline Biologicals, 553 Old CorvallisRoad, Hamilton,MT 59840, USA. Email: christopher.w.c1 uff@gskbio.com

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

112 Lipid-A in Cancer Therapies

such as cell wall skeleton (CWS) ofMycobacterium pble; (in the adjuvantDetox"; Biomira, Inc.),the saponin QS-21 (in the adjuvants ASOIB and AS02B; GSK Biologicals) or with QS -21 andCpG oligonucleotides (in the adjuvant AS 15; GSKBiologicals)will be summarized. CombiningMPL with other immunostimulants has been demonstrated to be advantageous in many casesand may be required to elicit the full complement of activities necessary to achieve an effectiveimmune response and overcome the ability of tumors to evade attack by the immune system . Inthis chapter, information relating to vaccines targeting specific cancerswill be presented in the firstsection, while information relating to vaccines targeting multiple tumor types by the induction ofimmune responses to shared TAAs is presented in the second section.

Section 1: Vaccines Targeting Specific Cancer Types

Vaccines Targeting Colorectal CarcinomaColorectal carcinomas have been demonstrated to express TAAs that are shared among

tumor-bearing individuals. In an effort to induce immune responses to common TAAs, a vaccineconsistingoffour irradiated allogeneic colon carcinoma cell lines combinedwith Detox" adjuvant(25 flgMPL,250 flg CWS, squalane oil andTween-80 as emulsifier) was administered three timesat three week intervals by intradermal injection to eight patients with low-volume metastatic col-orectal carcinomaY The hypothetical basis for this strategy derived from the fact that commonTAAs are present in a vast majority ofcolorectal carcinomas, suggesting that immunization withallogeneic carcinoma cell lines would immunize against autologous tumors. Seventeen morepatients were treated similarly, but also received subcutaneous interleukin-Ia (IL-Ia), 0.3-0.5 flg/m 2 per day for 8 days after each vaccination. In the patients receiving vaccine only, toxicity waslimited to transient erythema, induration andpruritis at the injection site. Addition ofIL-Ia causedfevers, chills and rigors that began within 4 hours ofadministration and lasted 1-2 hours. IL-I atreatment also caused fatigue by day 8 ofthe treatment regimen. Six patients were DTH-posiriveto one or more ofthe colon cancer cell lines before vaccination. Nineteen oftwenty-two assessedpatients had positive skin reactions to one ofthe vaccine cell lines (HCTII6) at the completionoftreatment, indicating that immune responses were generated to the vaccine antigens.

KSA (also known as Ep-CAM, GA733, COI7-IA, EGP and KSI-4) is a 40 kDa proteinexpressed on the cell surface ofmultiple human cancers, including colorectal carcinoma. KSA isconsidered an excellent target for cancer vaccines designed to treat colorectal cancer, since it isexpressed by more than 85% ofmetastatic lesions and more than 80% ofcells within this type ofrumor.'? A Phase I safety and immunogenicity trial of a liposomal vaccine containing recombi-nant baculovirus-derived KSA and MPL (OncoVax-CLb;Jenner Biotherapies, Inc.) formulatedin an oil-in-water emulsion was conducted in patients with histologically documented Stage IVKSA-positive colorectal cancer," The vaccine was administered via the subcutaneous route everyfour weeks for a total of four immunizations. In addition, some of the vaccinated patients wererandomly assigned to receive recombinant granulocyte-macrophage colony stimulating factor(rGM-CSF; Sargramostim, Imrnunex Corp.). Eleven patients were treated and vaccinations withor without addition of rGM-CSF treatment were tolerated equally well, with no injection sitereactions or regional lymphadenopathy noted. Seven of eleven patients developed significantKSA-specific cellular immune responses (as measured by IFNy ELISPOT and lymphoprolifera-tion assays), nine ofnine patients tested developed DTH responses to KSA and eight ofelevenpatients developed KSA-specific antibody responses. There were no significant difference in im-mune responses between vaccine only patients and those receiving vaccine plus rGM-CSF. Nosignificant clinical responses to the vaccination regimen were observed.

Vaccines TargetingProstate CancerProstate-specific antigen (PSA) is normally secreted into the seminal fluid by prostate cells

and is present at low levels in the serum of disease-free individuals. I I Because PSA is expressedexclusively on prostate tissue and is over-expressed in nearly all advanced prostate cancers.P:" it isa logical choice as a vaccine target.A liposomal vaccine consisting ofrecombinant PSA and MPL

MonophosphorylLipid A (MPL) asanAdjuvantfir Anti-CancerVaccines: ClinicalResults 113

(JBT 1001 or OncoVax-P;Jenner Biotherapies, San Ramon, CA) was tested in pilot studies ofprostate cancer patients. 17 Patients with histologically confirmed prostate cancer received subcu-taneous injections of the vaccine three times (days 0,30 and 60) , combined with subcutaneousadministration ofrGM-CSF (Sargmostim, Irnmunex Corp., Seattle ,WA) on days 0-4 , 30-34 and60-64. A second set ofpatients received intramuscular injections ofthe vaccine in an oil-in-wateremulsion according to the same schedule. The vaccine induced high titer anti-PSA IgM and IgGin a majority of patients evaluated, while there was considerable heterogeneity in terms of themagnitude and timing of cellular responses (measured by ELISPOT assay).18The small numberofpatients in the trial precluded evaluation ofthe vaccine for clinical efficacy.

Prostein is a prostate tissue-specificprotein that is uniquely and abundantly expressed in normaland cancerous prostate tissues. Due to this expression profile , prostein is considered a vaccinecandidate for prostate cancer. GlaxoSmithKline (GSK) Biologicals is currently testing a vaccineconsistingofa recombinant prostein protein and a GSK proprietary adjuvant AS15 in a EuropeanPhase 1111 safety and efficacystudy in prostate cancer patients.The Zl-patient study isfully enrolled,but no results are available at this time. The AS15 adjuvant is a liposome formulation containingQS-21 and ligands for both TLR4 (i,e.,MPL) and TLR9 (i.e., CpG oligonucleotide).

Vaccines TargetingMelanomaIn 1985, Dr. Malcolm Mitchell began clinical testing of a melanoma vaccine containing

homogenates oftwo melanoma cell lines combinedwithDetox"adjuvant .19.20Aswith the colorectalcancer vaccines made from allogeneic carcinoma cell lines, the melanoma vaccine strategy arosefrom the hope that common TAAs present on allogeneic melanomas would immunize againstautologous tumors. In the initial Phase I and II trials, the vaccine was found to be tolerable andapproximately 20% oftreated patients had partial (15%) or complete (5%) remission. The stron-gest correlate ofclinical response in each of the trials was an increase in cytolytic Tdymphocyreprecursors (pCTL), which were measured by a mixed lymphocyte-tumor cell assayadapted fromVose." Retrospective statistical analysis ofpatients treated with the vaccines revealed an associa-tion between the expression of three HLA Class I alleles (HLA-A2, HLA-BI2 and HLA-C3)and clinical response."

Positive results from seven open-label Phase II trials ofMelacine" (a total of 139 Stage III/VImelanoma patients) confirmed Mitchell's initial findings" and prompted Ribi ImmunoChem toinitiate a multicenter Phase III clinical trial ofMelacine~versusthe "Dartmouth" combinationchemotherapy regimen (dacarbazine, cisplatin, carmustine (BNCU) and tamoxifen) in 1991,24Although objective responses to the vaccine were not significantly different from chemotherapyand survival times were similar for the two treatments, quality-of-lifeduring treatment significantlyfavoredMelacine-' The results led to submission and approval ofMelacine~in Canada for treatmentofmetastatic melanoma.At this time,Melacine"remains the only cancer vaccine to pass regulatoryreview (Canada) and be marketed commercially (note: the vaccine was recently removed from themarket for business reasons) .

In 1992, the Southwest Oncology Group (SWOG) initiated a Phase III clinical trial to evalu-ate Meladne'tu: patients with intermediate thickness melanoma and tumor-free regional lymphnodes (i.e., Stage II disease) in a postsurgical resection setting.25•27 The rationale for targetingStage II melanoma derived from the theory that cancer vaccines often fail because higher grade,disseminated tumors (Stage III and IV) actively evade immune surveillance and/or suppress im-mune responses in general (hence, the high rate of infection in advanced cancer patients). If thevaccine was administered early in the disease process when the tumor had not metastasized andthe immune system was still functional, induction ofadaptive immune responses that can destroyresidual tumor should have a better chance of occurring. In add ition, since surgical resectionalone is the standard ofcare for Stage II melanoma, inclusion of relatively nontoxic vaccinationwith Melacine~waseasily justified, whereas treatment with chemotherapy or high-dose IFNa -2btreatment, both ofwhich are usually associated with significant side-effects, was not. TreatmentwithMelacine~appearedto provide little benefit versus surgery alone (p = 0.34 corrected for all

114 Lipid-A in CancerTherapies

prognostic factors) , although retrospective analysis of the data revealed a statistically significantbenefit for patients expressing HLA-A2 and/or HLA-C3. There have been no further trials toprospectively testMelacine"in patients expressing these MHC haplotypes.

At approximately the same time as the Ribi ImmunoChem-sponsored clinical trials were takingplace, Mitchell tested the efficacyofMelacine~combinedwith an approved immunotherapy forStage III/IVmelanoma, namely interferon-alpha-2b (Intron A"', Schering Plough Corp.).28.29Thelarge proportion ofpatients whose disease stabilized upon treatment in these trials led Mitchellto initiate a Phase III trial ofMelacine~plusIntronA~versus IntronA~alone in 253 patients withStage IV melanoma." While overall survival and objective response rates were not different forthe two arms, the duration of response favored the combination therapy (p =0.038) . A secondPhase III trial (604 patients) was subsequently initiated to compareMeladne"plus Intron A~ tohigh-dose Intron A~alone . Accrual onto the trial was completed in 2003 and preliminary resultspublished in abstract form showed no significant difference in relapse-free or overall survivalbetween the two arms."

In 1995, Schultz et al reported the results ofa Phase 1 clinical trial conducted to evaluate theimmunogenicity and efficacy of a polyvalent melanoma antigen vaccine admixed with Detox"adjuvant at two doses (20 I-tg MPL/200 I-tg CWS or 10 I-tg MPLIlOO I-tg CWS).32The antigenswere prepared from material shed into serum-free culture medium by four melanoma cell lines .Nineteen patients with resected Stage III melanoma were immunized with the mixtures (7 withlow-dose and 12with high-doseDetox~) every 3 weeks for a total of4 vaccinations .A nonrandom-ized control group (35 patients) was administered the vaccine with alum as the adjuvant.WhileDetox"markedly potentiated antibody responses to the antigens, DTH responses were minimaland the vaccine did not appear to improve disease-free survival when compared to the prepara-tion containing alum .

The clinical activity, toxicity and immunological effects of immunotherapy with ultravioletB-irradiated autologous tumor cellsplusDetox"adjuvant was tested in melanoma patients in themid-1990s.J3 Non-anergic patients (i.e., positive for DTH reaction to foreign antigens) who hadundergone surgical resection ofmetastatic melanomawere administered 107 irradiated tumor cellsplus Detox" by intradermal injection every 2 weeks for six weeks, then monthly until progressionofdisease or exhaustion ofvaccine supply.Toxicity was limited to irritation at the vaccine injectionsite and, in 10% ofthe patients, malaise lasting 24-48 hours. Small granulomas (<2 ern diameter)occurred at some injections sites and became lessapparent over weeks to months. The induction ofcell-mediated cytotoxicity against autologous tumor in 10 of35 assessablepatients indicated thatthe intradermal treatments resulted in tumor-specific systemic immune responses and detection ofthis immune reactivity correlated significantly with survival (p =0.009). Among 24 patients withindicator lesions, the vaccination regimen resulted in two major responses in soft-tissue sites andin a bone lesion. The results indicated that there was a small, but clinically significant, therapeuticeffect with this vaccination strategy (<20% response, with 95% confidence).

Beginning in 2001, the immunogenicity of a vaccine consisting of aT-helper epitope ofMART-l and AS02B adjuvant (100 I-tg MPL and 100 I-tg QS21 in an oil/water emulsion, GSKBiologicals, Rixensarr, Belgium) was tested in three Stage III /IVmetastatic melanoma panenrs."Patients were immunized once per month for 6 months, then once every 3 months, for a total of8immunizations in one year.Treatment-associated toxicitywasminimal and included injection sitediscomfort, aswell as transient flu-likesymptoms and fatigue. Immune reactivity to the peptidewasdemonstrated using DR4-peptide tetramer stainingand enzyme-linked immunospot assayoffreshand restimulated CD4+ T-cells obtained from the patients throughout the course of treatment.MART-I-specific CD4+T-cells obtained postvaccination generally exhibited a mixed Th I/Th 2phenotype. The T-cells proliferated when cultured with peptide bound to syngeneic antigen-pre-senting cells. CD4+ T-cells from one patient secreted granzyme B and exhibited MHC-restrictedcytolysis when incubated with HLA-matched antigen-expressing tumor cells. At the time ofthereport (August, 2004), two of the three patients were alive and disease-free at 25 and 34 monthsafter entering the trial.The third patient relapsed at 17 months and underwent additional therapy.

MonopbosphorylLipidA (MPL)asanAdjuvantfor Anti-Cancer Vaccines: ClinicalResults 115

The results demonstrated that a class II-restricred epitope ofa melanoma-associated antigen is im-munogenic in humans and suggests that inclusion ofsuch epitopes in future melanoma vaccinesmay be worthwhile.

A study published in mid-2004 was conducted to evaluate the safety, immunogenicity and ef-ficacyofMelan-A-peptide-basedvaccines containingeitherAS02B adjuvant or incomplete Freund'sadjuvant (IFA) in HLA-A2-positive patients with Melan-A-positive Stage III/IVmelanoma.v Ihevaccines were well tolerated and in vivo expansion ofMelan-A-specific CD8+ Tcells, measuredusing HLA-A2IMelan-A peptide tetrarners and IFNy ELISPOT assays,was observed in 13 ofthe49 patients (1112 in the AS02B group and 12117 in the IFA group). Four patients had stable diseasefor a period oftime after treatment and the two patients with the strongest Melan-A-specific T-cellresponses (1 from the AS02B group and 1from the IFAgroup) experienced regressionofmetastases.Both patients, however, relapsed and died at 14 and 21 months after entering the study.

Vaccines TargetingHER2-Positive Breast CarcinomaThe HER2 protein is a cell-surface glycoprotein receptor that is expressed in many types of

epithelial cellsand isover-expressed in many types ofcancer, includingapproximately25% ofbreastcancers. The high level ofexpression on such cancers makes HER2 a good target for antibody andvaccine therapies.

GSK Biologicals conducted a Phase I trial with a recombinant HER2 protein -containingvac-cine (plus AS15 adjuvant) in breast cancer patients.Multiple doses (20,100,500 !!g) ofantigen andtwo immunization schedules (6 doses versus 3 doses +/- boost) were tested in Stage II patientswith >4 positive regional lymph nodes or in patients with locally advanced Stage III disease. Thevaccine was well tolerated and seroconversion was demonstrated in a majority ofthe patients, theanalysis of the T-cell response is ongoing. A Phase II trial ofthe recombinant Her2 protein (500f-tg) with ASI 5 adjuvant is currently underway, with transient clinical response observed in 2/5patients treated thus far.

Section 2: Vaccines Targeting Specific TAAs Expressedon Multiple Tumor Types

Vaccines TargetingMAGE-3-Expressing CancersMAGE-3 is a cancer testis antigen that is normally expressed only on testicular germ cells.

MAGE-3 is aberrantly expressed on a high percentage ofa broad variety of tumors, making it apotential target for vaccine immunotherapy.The safety and efficacyofa cancervaccine containingMAGE-3 protein fused to the N terminal portion ofa protein derived from H influenzae (proteinD) and combinedwith AS02B adjuvant was recently tested in 57 patients withMAGE-3-positivetumors (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 carci-noma, all Stage IV) .36.37As inclusion criteria. all patients were required to express at least 1 ofthe3 HLA class I haplorypes known to present MAGE-3 peptides (l.e., HLA-Al, HLA-A2 and/orHLA-B44).The vaccination schedule comprised 4 injections at 3 week intervals, with 2 additionalvaccinations 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 mostvaccinated patients (96%) and all patients generated IgG antibodies specific for protein D. UsingIFNy 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 ordisease stabilization lasting 4 to 29 months, these five responder patients were all part of the 12patientswith lessadvanced melanoma (5/12 Stage III with non visceral disease).A partial responselasting 10 months was observed in one metastatic bladder cancer patients.

A vaccine containing recombinant MAGE-3 protein (+/- AS02B adjuvant) was testedrecently in the US by the Ludwig Institute for Cancer Research (LICR) in 17 patients with

116 Lipid-A in Cancer Therapies

MAGE-3-expressing Stage I or II nonsmall cell lung carcinoma who had undergone surgicalresection 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, butsignificant, anti-MAGE-3 antibody responses and one patient in that cohort had a CD8+ T-cellresponse to the MAGE-3 peptide 243-258. In contrast, seven ofeight patients in the group receiv-ing adjuvantedvaccine developed amarked increase in anti-MAGE-3 antibodies. The investigatorsused IFNy elispot and tetramer staining to show that inclusion of adjuvant in the vaccine wasnecessary to generate significant CD4+ and CD8+ T-cell responses to MAGE-3. No assessmentofobjective clinical response was conducted since the patients had no evidence ofdisease at theonset of the trial .

Various tumor types have been targeted by MAGE-3-containing cancer vaccines producedby GSK Biologicals, with emphasis on non-small cell lung carcinoma (NSCLC) and cutaneousmetastatic melanoma.39,40A Phase lIb, double-blind, placebo-controlled clinical trial was initiatedin 2002 to test aMAGE-3 vaccine containingAS02B adjuvant in patients withMAGE-3-positiveStage IB and Stage II NSCLC (post surgical resection). Patients are receiving five intramuscularinjections ofplacebo (n =60) or vaccine (300 !J.gMAGE-3; n =122) at three week intervals, fol-lowed by eight maintenance immunizations spaced three months apart.The primary endpoints aretime to recurrence, disease-free and overall survival, recurrence rate at different timepoints, toxicityand tolerability. Humoral and cellular immune responses are secondary endpoints. Although thesafety aspect ofthe study is still blinded, the vaccine appears to be well tolerated,with mild grade1or 2 local or systemic reactions lastingless than 24 hours post-injection being the most commonsequale. There have been only three grade 3 adverse events potentially related to treatment. Interimefficacy data was presented at the 2006 American Society ofClinical Oncology (ASCO) annualmeeting showing a clear signal (although not significant) with a benefit of33% in recurrence rate.Core analysis ofthe results are expected by October 2006.

Vaccines TargetingMUCI-Expressing CancersMucins (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-terminaldomain, a transmembrane region and a C -terminal cytoplasmic domain. The oligosaccharidesare 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 immunesystem. 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 areoften underglycosylated, with shorter and simpler carbohydrate chains. Three cancer-specificcarbohydrate epitopes have been identified: Thomsen-Friedenreich epitope, a precursor knownas Tn and sialyl-Tn (STn). All three epitopes are commonly found on the cell surface ofepithelialcancers or as part ofthe mucins secreted by them, but not on normal epithelial cells, therebymale-ing them potential TAAs.44Most adenocarcinomas expressMUC-1 on the cell surface and secreteunderglycosylatedMUC-1 mucin (expressing repeatingSTn epitopes) into the serum, resulting inexposure 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 longrepeats ofMUC-1 and AS02B adjuvant was tested in a Phase I safety and immunogenicity studyin 15 patients with resected and 1patientwith locally advanced pancreatic cancer." Patients (4 perdose) were vaccinated by intramuscular injection with one offour dosesofpeptide (100 !J.g, 300 /!g,1000 !J.gor 3000 !J.g) admixed with the AS02B adjuvant every 3 weeks for a total ofthree doses.Thevaccine 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 inthe peripheral blood and an increase in MUC-1-specific antibodywas seen in some patients. Twoofthe 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 acellular immune response to the exposed core peptide ofMUCI. The vaccine is a lyophilized

Monophosphoryl LipidA (MPL) asanAdjuvantfor Anti-CancerVaccines: ClinicalResults 117

liposomal preparation containing BLP25 lipopeptide (25 amino acid peptide), MPL and thelipids cholesterol, dimyristoyl phosphatidylglycerol and dipalmitoyl phosphatidylcholine. MPLand 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 lungcancer patients.t" clinical trials were conducted to test the safety and efficacy ofBLP25 vaccinein prostate" and nonsmall cell lung carcinoma patients (NSCLC).48 A single intravenous dose ofcyclophosphamide (300 rng/rn' ,maximum of600 mg) was administered 3 days prior to vaccina-tion. Such treatment has been demonstrated to augment delayed-type hypersensitivity responses,increase antibody responses, abrogate tolerance and potentiate anti-tumor immun ity in preclini-cal modeIs.4~.so For primary treatment, the vaccine dose (1000 IJ.g ofBLP25 and 25 IJ.g MPL) wasdivided and administered subcutaneously at four sites weekly for eight weeks. In the prostatestudy, patients were reassessed following the primary treatment and those who did not require achange in therapywere eligible to continuewith BLP25 vaccination every 6 weeks for up to 1year.At the investigator's discretion, patients in the NSCLC trial continued to receive maintenancevaccinations every 6 weeks starting at week 13.

In the study testing BLP25 vaccine in prostate cancer patients with biochemical failure (in-creasing prostate specific antigen; PSA) after radical prostatectomy, a total ofsixteen individualswith a median age of60 were enrolled.? Fifteen ofsixteen completed the primary treatment andten completed the maintenance period. After primary treatment, eight of sixteen patients hadstable or decreased PSA. Although only one patient maintained stable PSA by the last on-studymeasurement, six of sixteen patients had greater than 50% prolongation of PSA doubling timecompared to prestudy measurements.

To evaluate the effect of BLP25 vaccine on survival and toxicity in individuals with StageIIIB or IV NSCLC, patients were randomly assigned to the BLP25 arm or to the best supportivecare (BSC) arm. Overall, the 88 patients assigned to the BLP25 arm had a median survival time(MST) that was 4.4months longer than the 83 patients in the BSC arm, though the differencewas not statistically significant (p =0.112). The greatest vaccine effect was observed in Stage IIIBlocoregional disease, where the MST for 30 BSC patients was 13.3 months while the MST for35 vaccinated patients was more than 30 months (p =0.069, though MST had not been reachedby end of study for vaccinated patients. A subsequent press release confirmed an MST of 30.6months for the vaccine group). No significant toxicit y was associated with BLP25 treatment andquality oflife was maintained longer in vaccinated patients.

Vaccines Targeting STn-Expressing CarcinomasAberrant carbohydrate molecules, such as sialyl-Tn (STn) , are often expressed as part ofglyco-

proteins present on the surface ofcarcinomas.Asdiscussed in the previous section, these moleculesare not found on normal tissues and are considered TAAs. Antibodies specific for these moleculescan be induced that mediate tumor cell lysis by complement or antibody-dependent cellular cy-totoxiciry." When expressed on tumors, the mucin-associated STn epitope is a predictor ofpoorprognosis and is associated with increasedmetastatic potential.52·54STn isexpressed on a significantproportion ofbreast cancers (16-80%, depending on detection method and laboratory) ,55 '58 witha tendency for higher expression in metastatic disease compared to primary tumors.l" For thesereasons , 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 bothTn and STn epitopes,was tested in patients with metastaticcolorectal cancer.S~ Sixpatients were administered the vaccine alone, eight received thevaccine com-binedwithDetox" adjuvant and sixwere treated with the vaccine plus BCG as adjuvant.Anti-STnantibody titers increased in 4of8 patients in the Detox"group, 5 of6 in the BCG group and 0 of6in the vaccine onlygroup. Toxicitywas limited to inflammatory skin reactions at the injection sitesin patients receiving vaccine plus adjuvant.The result s demonstrated that STn-containingvaccinescan induce specific humoral immune responses in cancer patients and that vaccines containingthese molecules can be administered safelywith immunological adjuvants.

118 Lipid-Ain Cancer 'Iberapies

Tberatope" (Biomira, Inc.) is an investigational cancer vaccine consisting of synthetic STnconjugated to keyhole limpet hemocyanin (KLH) combined with Detox" adjuvant (early tri-als) or Enhanzyn adjuvant (later trials). Like Detox", Enhanzyn contains MPL and CWS, but isformulated as a stable emulsion with squalene oil instead ofsqualane oil. KLH is a carrier proteinthat promoted enhanced S'In -specific antibody responses in preclinical models. Because of thepromiscuous 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 orpancreatic). 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 toimmunization leads to enhanced antigen-specific antibody responses." presumablyvia the inhibi-tion of suppressor cells. Some of the early clinical trials of the vaccine. therefore, also evaluatedwhether cyclophosphamide pretreatment would have the same effect in humans.

In an initial dose-escalation study. 12 metastatic breast cancer patients were administeredlow-dose (300 mg/m2) IV cy1cophosphamide on day -3 and four doses ofSTn-KLH (25 /-lg. 100/-lg or 500 ug) combined withDetox"adjuvant at 2 week intervals, with four monthly injectionsthereafier." The 500 flg dose caused excessiveDTH reactions and was therefore eliminated fromthe protocol. At the lower doses.vaccine-associated toxicitywasminimal. with side-effects limitedto granulomas/ulcerations at the injections sites in 5 patients. All patients developed anti-STnIgG and IgM responses. Two patients had partial remissions lasting 6months, while several othersdemonstrated disease stability for 3 -10 months.

In a second trial. 100 /-lg ofthe STn-KLH vaccine combined withDetox"was administered to23 metastatic breast cancer patients (with or without cyclophosphamide pretreatment) at weeks0.2,5 and 9, with four additional monthly injections administered to patients with respondingor stable disease after the first round ofvaccinations.S Toxicity was again limited to granulomaformation at the injection sites. All patients developed anti-STn IgG and IgM responses and IgMresponseswere significantly higher in patients pretreatedwith cyclophophamide. Because5patientsdeveloped progressive disease during the initial 12week period. only 18patients received all fourinjections. ofwhich rwo had minor responses.

Subsequent prospective trials evaluated the effect of different low-dose cyclophosphamidepretreatments on the response to STn-KLH combined with Detox" .63 Individuals receivingintravenous cyclophosphamide generated stronger anti-STn antibody responses and lived sig-nificantly longer than those administered the vaccine with oral or no cyclophosphamide. Aninverse correlation was observed between anti-STn antibody titers and tumor growth. and a lowerpercentage ofthe patients receiving intravenous cyclophosphamide pretreatment had progressivedisease at 9 weeks. As a result of these trials, addition ofcyclophosphamide pretreatment to theTheratope~vaccination protocol has become standard practice.

In a randomized. double-blind Phase III trial, metastatic breast cancer patients whose tumorsdid not progress after front-line chemotherapywere treated with intravenous cyclophosphamidefollowed by Theratope~(STn-KLH andEnhanzyn adjuvant) or intravenous cyclophosphamide fol-lowed byKLH alone combined with the Enhanzyn adjuvant (negative control).64.66An improvedformulation of 1heratope~thatgenerated 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 therapywas permitted and patients were stratified with regardto hormone therapy and response to initial therapy. The studywas large (1028 patients) and subsetanalysis was conducted on patients that received hormone treatment. With respect to time toprogression. there was no statistical difference berween 1heratope~-treated and control arms in thestudy. regardless ofwhether selective estrogen -receptor modulators or arornatase inhibitors wereused 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 in-duction of immune response to Theratope~ takes, on average, 17 weeks, while the time to diseaseprogression in untreated patients is appoximately12weeks.Thus, the increased tumor burden after

Monopbospboryl LipidA (MPL)asanAdjuvantfor Anti-CancerVaccines: ClinicalResults 119

disease progression may suppress S'In -specific immune responses (via production ofimmunosup-pressive molecules such as mucin) or may simply overwhelm the nascent immune response thatis being generated to the vaccine. To test the vaccine in a situation where tumor burden was lowduring the vaccination regimen, Theratope-was evaluated in breast and ovarian cancer patientsin conjunction with chemotherapy and autologous stem cell rescue. Trial designers reasoned thatimmune responses to the vaccine would have a chance to develop prior to disease progression inthese patients. Since low tumor burden correlated with stronger immune responses and strongerimmune responses correlated with longer survival times in initial trials, the strategy appearedsound. 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 indurationand 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 ap-proximately halfofthe patients developed S'In-specific T-cell proliferative and cytolytic responses.Induction ofstrong (versus weak) cytolytic T-cell responses by the vaccine was associated withlonger remission times (p =0.047), suggesting that development ofanti-STn cytotoxic cellsmayalso correlatewith a clinical effect. Phase III trials to evaluate Theratope"in transplant patients havenot been initiated at this time , though pilot studies are planned to determine whether additionofIL-2 or GM-CSF to Theratope-treatment is beneficial in tran splant patients.

Vaccine TargetingRas-Expressing TumorsThe mutant oncogene ras is a well characterized TAA and is present in more than 20% ofall

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

ConclusionA variety ofvaccines designed for cancer immunotherapy have been tested in clinical trials for

more than two decades. Investigators realized earlyon that addition ofadjuvants to cancer vaccineswould be required to overcome the poor immune responses that are generally elicited to antigenscontained within these vaccines. Although the effectiveness ofLPS as an immunomodulator haslong been known, the pharmacologic use ofpurified LPS (or lipid A) asan adjuvant isprecluded byits toxicity. In this regard , LPS is highly pyrogenic and promotes systemic inflammatory responsesyndrome." In an effort to uncouple the immunomodulatory effects oflipid A from its toxicity,Ribi er al developed 3-0-desacyl-4'-monophosphoryl lipidA (MPL), which comprises the lipid Aportion ofLPS from which the (R)-3-hydroxytetradecanoyl group and the l-phosphate have beenremoved? by successiveacid 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 asone of the active ingredients in Detox"" adjuvant (with CWS and oil), AS02B adjuvant (withQS21 in an oil in water emulsion) or AS15 (a liposomal formulation with QS21 and CpG), has

120 Lipid-Ain Cancer Therapies

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 isthe 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 mayrelate to the fact that most trials have been conducted in patients with late-stage disease. wherebulky metastatic tumors may evade or suppress the immune system and prevent induction ofef-ficacious anti-tumor cellular and/or hwnoral adaptive immune responses. The very encouragingdata obtained in an adjuvant setting in early stages NSCLC with the MAGE3 protein formulatedin AS02B support that hypothesis.Another constraint is the weakly immunogenic nature ofmanyTAAs, which are often "self" in nature and. therefore, not good targets for the induction ofeffectorT-cell responses .Efforts to move beyond these limitations include addingpotent adjuvants, such asMPL, to the vaccines, aswell as combining immunotherapywith surgery. chemotherapy. radiationtherapy and/or autologous stern-cell/dendritic cell therapy. The hope is that chemotherapyI radia-tion therapywill reduce tumor burden and deplete suppressive Tvcells, while optimizedvaccinationprotocols will allow for enhanced induction, proliferation and activity oftumor-specific effectorT-cells that can eliminate residual tumor, Such combination therapies are currently in the earlystages ofclinical testing and may lead to better options for the treatment ofcancer.

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