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olorectal Cancer Vaccines: Whate Know and What We Don’t Yet Know

argaret von Mehren

Humans have a sophisticated immune system that functions to clear invading organismsand abnormal cells. However, cancers are able to arise despite this immune system.Vaccines have the potential of benefiting cancer patients by stimulating an immuneresponse against tumor-associated antigens (TAA). Our enhanced understanding of howthe immune system processes and presents antigens has allowed an array of vaccinemodalities to be developed and tested. The TAA with the greatest number of vaccineplatforms tested in colorectal cancer is carcinoembyronic antigen (CEA). Trials to date havedemonstrated safety and evidence for the induction of an immune response against CEA.This article will review trials conducted with a variety of CEA vaccines. Most studiesconducted are phase I or II in the metastatic disease setting, limiting our understanding ofthe role of the immune response in controlling colon cancers. Phase III trials conducted todate have conflicting data with respect to improvements in disease-free and overall sur-vival. It is our challenge to determine if and which vaccines have sufficient benefit towarrant large-scale trials in the adjuvant and prevention settings.Semin Oncol 32:76-84 © 2005 Elsevier Inc. All rights reserved.

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ll patients have an immune system that theoretically canbe harnessed to recognize an antigen as foreign and de-

troy the antigen-bearing cell. Unfortunately, cancers do notypically elicit an immune response.1 This is because tumorsrise and grow without eliciting inflammation or local tissueestruction, tumor cells lack major histocompatibility com-lex (MHC) receptors and/or costimulatory molecules, andumor cells may secrete compounds that inhibit immune cellunction. In addition, many of the tumor-associated antigensre self-molecules and therefore the immune system has de-eloped tolerance to them.

accine Targetsost vaccines developed and studied have focused on stim-

lating an immune response against a tumor-associated an-igen (TAA). Table 1 lists some potential antigens. Antigenselected for vaccine development are those that have lowevels of normal antigen expression in adult tissues and that

edical Oncology, Fox Chase Cancer Center, Philadelphia, PA.upported in part by 1R21 CA92992-01. Dr von Mehren has served as an

advisor to Aventis-Pasteur and Novartis Oncology. She received less than$5,000 in honoraria from each company in 2003.

ddress reprint requests to Margaret von Mehren, MD, Fox Chase CancerCenter, 333 Cottman Ave, Philadelphia, PA 19111.E-mail: margaret.

avonmehren@fccc.edu

6 0093-7754/05/$-see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1053/j.seminoncol.2004.09.033

ppear to be overexpressed in malignant tissues. Mutatedroteins associated with cancer presumably avoid the risk ofutoimmunity. This class of antigens has not been extensivelyested in colorectal cancers. Recent preclinical work has alsoargeted components of the tumor microenvironment, vas-ular endothelial growth factor receptor 2 (VEGFR2) andbroblast growth factor receptor 1 (FGFR1). Xenogeneic ho-ologous proteins are used to develop an antibody immune

esponse and have shown some evidence of tumor regres-ions.2,3 Investigators have yet to determine which antigensr combinations of antigens will have the greatest efficacy foratients with colorectal cancer or patients at high risk foreveloping colorectal cancer.The majority of vaccine trials that have included colorectal

ancer patients have used carcinoembryonic antigen (CEA)s the TAA. CEA is an 180-kd glycoprotein self-antigenresent on endodermally derived neoplasms and in the gut ofhe human fetus.4 CEA is not abundant in adult colon, al-hough there are several CEA-like antigens in normal tis-ues.5 The majority of adenocarcinomas have CEA detectedy immunohistochemistry.6-9 Adenocarcinomas do not stim-late a significant immune response against CEA, probablyue to its expression during fetal development. Despite this,EA has been evaluated as an immunogen because there isinimal expression of CEA in adult tissues, limiting the pos-

ibility of a harmful autoimmune response. CEA vaccine tri-

ls to date have shown evidence of the induction of an im-

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Colorectal cancer vaccines 77

une response against CEA and minimal toxicity. Methodsf immune response measurements have not been consistent,aking comparisons of immunologic efficacy between stud-

es difficult. There is minimal evidence of tumor regressionsn the metastatic disease setting. An autologous tumor cellaccine given with bacillus Calmette-Guérin (BCG) has beenested in the adjuvant setting with varying findings in threeifferent phase II-III studies.

oals of Vaccinationhe goals of active vaccine strategies have been to break the

nherent immunologic tolerance and to elicit a cytotoxic T-ell (CTL) response against a TAA. The immune system haseveloped two different pathways for antigen presentationnd the induction of an immune response10 (Fig 1). Toresent an antigen, a foreign protein must be structurallyodified and move to the appropriate subcellular compart-ent. Processed antigens are presented to the immune sys-

em by MHC molecules. Intracellular antigens are processedia the class I pathway and stimulate a CTL response. Extra-ellular antigens are processed by antigen-presenting cellsAPC) using the class II pathway and stimulate an antibodyesponse. The class I pathway presents peptide fragments inhe context of MHC class I molecules.11 Soluble or processedntigens are presented primarily by MHC class II receptorsia the class II pathway.

When antigen is presented to naive T-cell receptor cells viaHC, the interaction can result in anergy or in the stimula-

ion and activation of the T cell. Activation of B lymphocyteso generate antibodies or CD8� T lymphocytes to derive CTLccurs only in the presence of costimulatory molecules onhe APC binding receptors on the naive T-cell; without this

able 1 Pros and Cons of Varying Vaccine Formats

Types of Vaccines Pros

hole cell vaccines Presents multiple antigenAllogeneic MHC molecul

immune activationNA transfection of viabletumor cells

Allows presentation of mconjunction with relevaGM-CSF

urified antigen/recombinantprotein

Allows processing of thepresentation of all poteepitopes

mmunogenic peptides Facilitates immunologic m

nti-idiotypic Propagates an ongoing aaked DNA/RNA Not restricted by HLA typ

Ease of usePotential for intracellular

endritic cell vaccines Utilizes the best antigenstimulate an immune re

Can present a multitudeirally encoded DNA Virus can allow the prese

and other important moAllows presentation of th

Cons

s on tumor celles may aid in

Immunologic testing difficult due to themultiplicity of antigens presented

ultiple antigens innt adjuvants, eg,

Requires ex vivo manipulation of tumorcells; number of viable tumor cells islimiting

entire antigen andntial antigenic

Requires isolation of protein orproduction of the protein

onitoring HLA restricted, limits eligible patientpopulation

ntibody response Does not clearly elicit a CTL responsee

antigen production

Requires isolation nucleic acid material

presenting cell tosponseof antigen types

Requires the collection and ex vivomanipulation of DC

ntation of antigenlecules

Requires engineeringRemote risk of insertional mutagenesis

nteraction, anergy develops. Examples of costimulatory t

igure 1 Antigen processing. An APC performing the two types ofntigen processing is shown. The upper portion of the APC is pro-essing intracellular proteins via the class I pathway, typified by airal antigen. The virus infects the cell and produces protein. Therotein is taken up by the proteosome and degraded into smallereptide fragments. These fragments are taken up by the transporterssociated with antigen processing (TAP) and moved to the endo-lasmic reticulum, where MHC class I (MHC I) receptors in thendoplasmic reticulum (ER) will bind the peptide. The MHC I–pep-ide complex is moved to the cell surface where it can interact withaive T cells. Class II antigen presentation involves proteins foundutside of the cell. APCs take up the protein into the cell by endo-ytosis or pinocytosis. This vesicle then fuses with a lysozome whereegradative enzymes break down the antigen into peptides. Anotherlasma membrane containing vesicles with MHC II receptor willuse with the lysozome. The MHC II–peptide complex is brought to

he cell surface to interact with naive T-cells.

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olecules are B7.1 (CD80) and B7.2 (CD86). Once stimu-ated, CD4 cells can stimulate antibody production (Th1) orCTL (Th2) response. The type of response is reflected in the

ype of cytokines produced at the time of antigen stimulation,ith production of interferon gamma (IFN-�), interleukin-2

IL-2), and transforming growth factor-beta (TGF-�) associ-ted with a CTL response and IL-4 and IL-10 with a humoral-cell response.Most clinical trials have focused on demonstrating the in-

uction of a CTL response. This is due to extensive evidencen animal models that the induction of a specific CTL re-ponse can lead to tumor rejection. The most common assayo detect a CTL response is the enzyme-linked immunospotELISPOT) assay that determines the number of peripheralononuclear cells that produce IFN-� after stimulation with

ntigen. The benefit of antibodies for protection against can-er progression is less well documented, although it is theainstay of protection from infectious disease agents and

here is increasing evidence of the role of antibodies in theherapy of some cancers.

ypes of Vaccinesaccines are presenting antigens in many formats (Table 2).hole cell vaccine approaches present multiple antigens, but

nless obtained from cell lines, are limited by the number ofiable tumor cells that can be collected from each individual.ome have taken autologous tumor cells and engineeredhem to secrete cytokines or costimulatory molecules. Thispproach may improve the immune response that is gener-ted; however, it requires ex vivo manipulation. Protein anducleic acid vaccines allow for processing of the antigen byach individual. This bypasses the difficulties of peptide vac-ines that bind to specific human leukocyte antigen (HLA)eceptors, limiting the patient population that can be treated.nti-idiotypic vaccines, based on the network theories of

erne12 and Lindenmann,13 do not primarily seek to elicit aTL response. With this approach, an antibody (Ab1) thatas specificity for a TAA is given. The binding site of thisntibody is seen as foreign by the immune system, and themmune response that follows produces a second antibodyAb2). Ab2 recognizes the binding site of Ab1 and also mim-cs the structure of the site where Ab1 binds to the TAA. Thisascade continues, providing continued regeneration of “vac-

able 2 Antigen Types in Colorectal Cancers

Antigen TypeExamples of

Antigens

eat shock protein Cellular proteinsarbohydrate antigens MUC-1, Lewis antigenlycoprotein antigen CEArowth factor receptors HER-2/neu, EGFRutant proteins RAS, P53icroenvironment antigens VEGFR2, FGFR1

ine” and “antigen.” v

hole Cell Vaccine Trialsaccines derived from whole cells are attractive as theyresent the entire spectrum of antigens on a patient’s cancer.n the other hand, the cancer was able to develop within theatient, suggesting that there has not been an effective im-une response. Whole cell vaccines have also used colon

ancer cell lines.14 Autologous vaccines require sufficientumbers of viable tumor cells and must be made for eachatient. In addition, determining whether the vaccine haslicited an immunologic response is difficult because of theultitude of antigens presented, many of which are un-

nown. When whole cell tumor vaccines have been used,hey have usually been combined with an immunologic ad-uvant.15,16

Whole cell tumor vaccines are the only approach that haseen tested in the adjuvant setting. Based on extensive pre-linical data, a phase III prospective randomized trial evalu-ted the benefits of an irradiated whole tumor cell vaccinedmixed with BCG compared with no vaccine.16 Patientsith Dukes B2-C3 colorectal cancer were included in the

rial. Rectal cancer patients received adjuvant radiation ther-py. No adjuvant chemotherapy was given. Three vaccinesere given 1 week apart. Delayed type hypersensitivity

DTH) testing revealed an increased response to autologousumor cells without an increase in other antigens tested, sug-esting a specific immune response. Analyzing the entiretudy population, there were no differences in overall sur-ival or disease-free survival. A subset analysis evaluatingolon and rectal cancer patients separately revealed an im-rovement in overall and disease-free survival in the colonancer patients who received vaccine compared to those whoeceived no adjuvant therapy. The Eastern Cooperative On-ology Group conducted a second trial in 412 stage II and IIIolon cancer patients. This study did not demonstrate anyurvival benefit, although up to 25% of the vaccines given didot meet manufacturing specifications.17 A subset analysis ofhe 307 patients who displayed a DTH reaction to their finalaccine again demonstrated an improvement in overall sur-ival, although not statistically significant. These results sug-ested that a longer course of vaccination might be required.subsequent study in the same patient population was done

sing three weekly vaccines followed by a booster vaccine atmonths.18 Eighty-seven percent of patients receiving vac-

ine demonstrated greater than 10-mm induration with theirhird vaccination that increased to 92% following the fourthaccination. There was a significantly longer recurrence-freeurvival in stage II patients but no statistically significantifference in disease-free survival in either stage II or stage IIIisease. These studies did not incorporate adjuvant chemo-herapy, now considered the standard of care.

rotein and Peptide Vaccinerials

accines using protein as the antigen source allow the indi-

idual receiving it to process the antigen and present the

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Colorectal cancer vaccines 79

pitopes that can bind their particular HLA receptors. Inontrast, peptide vaccines must be given to patients of apecific HLA type. Once a specific HLA epitope has beenefined, however, peptide can be modified to have enhancedinding to the HLA receptor and immunologic efficacy.19,20

eptides with strong binding affinity may activate differentenes than the natural peptide, thus accounting for theirnhanced immunologic efficacy.21

There is one trial reported that has used recombinant CEArotein.22 This study vaccinated 18 patients following their

nitial surgical resection for Duke’s A-C colorectal cancerssing CEA protein produced by baculovirus in insect cells.he goal of the study was to compare the immunologic effectsf giving the vaccine alone compared to giving the vaccineith granulocyte-macrophage colony-stimulating factor

GM-CSF). All nine patients receiving GM-CSF developed angG response and CTL response to the recombinant CEArotein, whereas only three of nine did so in the group re-eiving vaccine alone. When CTL response was measuredgainst CEA derived from liver metastases, only five of nineatients in the GM-CSF group developed a response; theon–GM-CSF group was not tested.The studies using CEA peptides have used autologous

endritic cells (DC) to present antigen.23 Preclinical dataemonstrated the ability of DC loaded with the CAP-1 CEAeptide to elicit a CTL response able to lyse CEA-expressingumor cells in an HLA-restricted manner in T cells from nor-al donors24 and from patients with metastatic colorectal

ancer.25 A phase I trial has shown the feasibility of generat-ng autologous DC from patients with metastatic colorectalancer and loading them with the CAP-1 peptide. Once gen-rated, vaccines were cryopreserved until ready for intrave-ous infusion. Patients received four weekly or biweekly in-usions of 1 � 107 to 1 � 108 DC per dose. In addition, aroup of patients also received intradermal injections of 1 �06 DC. One patient had a minor response.Hsp 96, a heat shock protein bound to its associated pro-

eins, elicits an immune response26 and has been tested as aaccine for patients with colorectal cancer. The normal func-ion of hsp 96 is to maintain homeostasis during cellulartress and also chaperone newly made and aberrant pro-eins.27 An initial report of five patients treated with autolo-ous hsp 96 vaccines following resection of liver metastasesesulted in increased CEA and epitheleal cell adhesion mol-cule (EpCAM)-specific CTL.28 A follow-up report of thistudy described 29 patients who underwent liver resectionor one to three (good prognosis) or four to five (poor prog-osis) metastases.29 Hsp96 was isolated from autologous

iver metastases and administered as four weekly injectionsollowed 2 months later by four bimonthly injections. Fifty-wo percent of patients were found to have an increase inEA-specific T cells following vaccination. In addition, sur-ival was correlated with disease prognosis and with the in-uction of an immune response. Sixty-seven percent of good-rognosis patients had post-vaccination increases in CEA-pecific CTL, in contrast to only 45% in the poor-prognosisroup. More intriguingly, there were statistically significant

ifferences in overall and disease-free survivals in patients s

ho achieved an immunologic response in comparison toatients who did not. The overall survival at 2 years was00% versus 50 % (P � .001) and the disease-free survivalas 51% versus 8% (P � .0001) for the immunologic re-

ponders versus the nonresponders.

nti-idiotypic Vaccine Trialss described earlier, the anti-idiotypic vaccine approach usesn antibody as the antigenic material. Two anti-idiotypic an-ibodies that mimic an epitope on CEA have been developed.he first, anti-Id MAb 708, has been reported in the literatureut has not been clinically tested.30 The second, 3H1, haseen studied in advanced colorectal cancer. A phase Ib trialnrolled 23 evaluable patients to study the safety and efficacyf 1, 2, or 4 mg of 3H1 given every other week for fournjections followed by monthly boost injections until theime of disease progression.31,32 3H1 has been shown to gen-rate both humoral and cellular immune responses.33 Thisaccine has been tested in patients status post resection ofrimary and metastatic disease in conjunction with 5-flu-rouracil.34 The addition of chemotherapy was not associatedith any loss of immune response. The addition of immuno-

timulatory oligonucleotides as an adjuvant to 3H1 leads toaster induction of an antitumor immune response comparedith Freund’s adjuvant in a murine model.35

ucleic Acid Vaccine Trialsucleic acids are not commonly thought of as antigenic;owever, they have been shown to elicit antibodies, T helperells, and CTL.36 Advantages of DNA vaccines include thease of purification of vaccine, stability of vaccine, ability ofhe antigen to be incorporated into MHC class I leading toTL induction, the possibility of intracellular antigen synthe-

is, and avoidance of viral vector complications such as im-une responses against the vector or risk of insertional mu-

agenesis. A DNA vaccine encoding CEA with hepatitis Burface antigen (HBsAg) has been developed.37 HBsAg wassed as a positive control for immune response. Patients werereated on a phase I trial with escalating doses of vaccineiven as an intramuscular injection, initially as a single injec-ion; subsequently patients received doses every 3 weeks.38

oxicity was minimal, with grade 1 fatigue, injection siteenderness, and elevations in creatinine kinase noted. Evi-ence of increased antibody levels against hepatitis B wereresent in six of eight patients who received repeated doses ofaccine, with four of these subjects achieving levels consid-red to be protective. No CEA-specific antibodies were de-ected. However, four of 17 patients demonstrated a lympho-roliferative response to CEA. The response was notorrelated with dose or schedule of vaccine.

An alternate approach that has been used is loading DCith RNA.25,39 Proof of concept for this approach was donesing healthy donor DC in cancer patients. In vitro, the DCransfected with CEA mRNA lead to the induction of CTLpecific for CEA. CEA mRNA modified with LAMP-1, a lyso-

omal targeting sequence, lead to an increase in helper T cells

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hat may be important for inducing and/or maintaining pro-uction of CTL. RNA has also been extracted from tumors forutologous tumor vaccines.40 This approach led to the induc-ion of cells able to cause the in vitro lysis of autologousumor cells.

irally Encoded Vaccinestudies

iruses are useful as a means to present a TAA antigen aloner with additional molecules. Genes of interest are insertednto the viral genome. The virus facilitates delivery of thentigen by infecting host cells and using their nucleotideeplication machinery to produce the proteins of interest.hese proteins will be presented and processed by the pa-

ient’s own MHC receptors. The virus can also function as andjuvant as many viruses can induce strong humoral andTL responses.The first viral vaccines used vaccinia.41,42 Vaccinia is aember of the pox virus genus. A recombinant vaccinia vac-

ine against CEA (rV-CEA) was associated with local reac-ions, including pruritus and erythema, as well as flu-likeymptoms, which were greatest following the initial injec-ion. Laboratory abnormalities attributable to vaccine were aeft shift in the white blood cell count without an increase inhe total count. Patients receiving fowl pox CEA (rF-CEA)ave demonstrated antibodies against the viral vector andgainst CEA.42,43 Importantly, the induction of CEA-specificcells against CEA has also been demonstrated.44 However,

he robust immune response against the vaccinia virus likelyimits the benefit of repeated vaccinations.

Avipox vectors such as Canary pox (also known asLVAC) and fowl pox also have been used. They have inher-nt characteristics that make them more attractive than vac-inia. Canary and fowl pox can only replicate productively invian species, unlike vaccinia, eliminating the rare risk of aaccinia infection.45-47 Although these viruses do not repli-ate in non-avian species, inoculations of recombinant avi-ox into non-avian cells result in the expression of the trans-ene(s).45,48 Recombinant pox virus vectors have andditional advantage in that they can accommodate a largeNA insert.49 ALVAC and fowl pox vectors expressing for-ign antigens can elicit protective immune responses to viralathogens in avian50,51 and non-avian species.48,52-56

ALVAC CEA was the first of these vectors to be tested.57

scalating doses of the vaccine were given as three monthlyntramuscular injections. Vaccine was well tolerated at allose levels tested and side effects were limited to local siteeactions. No tumor responses were noted in 20 patientsnrolled on the study, with one patient noted to have nor-alization of an elevated CEA. Limiting dilution assays weresed to demonstrate an increase in CEA-specific CTLs. Thereas no significant difference in the immune response with

scalation of the vaccine dose. CEA-specific T cells generatedrom seven HLA A2-positive patients were able to lyse allo-eneic colorectal cancer cells that expressed CEA and HLA

2, but not a cell line expressing CEA alone.58 A further study I

ested the combination of ALVAC-CEA with one dose of rV-EA. This approach was an attempt to enhance the immune

esponse against CEA by incorporating the known immuno-ogic benefits of the vaccinia CEA construct without limitingts benefits by repeating the rV-CEA injections (rV-CEA isighly immunogenic because of vaccinia; however, with re-eat immunizations, the benefit of stimulating the immuneesponse becomes lost because the rapid immune responsebrogates or limits any further benefit of repeat injections.)atients received four vaccinations, with half of the patientsiven rV-CEA as the initial vaccine and the other half receiv-ng it as the last vaccination. This approach, called prime andoost, found that the initial prime with vaccinia followed byoosting with ALVAC-CEA was superior to the reversechedule with respect to the induction of CEA-specific Tells.59

An alternate method to enhance immune response is giv-ng a vaccine that is capable of presenting antigen in conjunc-ion with costimulatory molecules. As described above, whenn antigen is presented by an APC, an immune response willnly develop if there is costimulation present. B7.1 and B7.2,lso known as CD80 and CD86, are important costimulatoryolecules. Binding of these costimulatory molecules to CD

8 results in production of multiple cytokines, includingL-2, and IFN-� in both CD4 and CD8 T cells. Production ofFN-� correlates with the stimulation of a CTL response.

ALVAC-CEA B7.1 (Aventis, Toronto, Canada) was the firstaccine to incorporate a costimulatory molecule.60-62 Vaccineite biopsies demonstrated evidence of leukocytic infiltrationnd CEA expression 48 hours following the first vaccination.iven the ubiquitous expression of B7.1, it was not feasible toetermine if the vaccine led to expression of B7.1. Two trialsvaluated the safety and immunologic efficacy of escalatingoses of the vaccine given intradermally (ID)60 or intramus-ularly (IM).62 Both trials demonstrated the induction ofEA-specific T cells using an ELISPOT assay. Thirty patients

eceiving the ID vaccine were evaluable for response havingompleted all four initial bimonthly vaccines; eight (27%)emonstrated stable disease that lasted an additional 1 to 7onths. Six of 31 patients with elevated serum CEA levelsad a decline in their levels that lasted 4 to 12 weeks. All sixf these patients were found to have stable disease after fouriweekly vaccinations. The trial with IM vaccines was ini-ially designed to administer three monthly vaccines. Threeatients with stable disease were re-treated 8 to 20 weeksfter their initial vaccinations. All three patients demon-trated an increase in their T-cell precursor frequencies thateturned to baseline off of vaccine; however, with furtheraccinations, there was again evidence of increased CEA-pecific T-cell precursors, a finding that supports continuedoost vaccinations.The most recent construct to be tested is rF-CEA-6D Tri-

om (Therion Biologics, Cambridge, MA). This agent uses theowl pox vector and contains the full-length gene for CEA,ith a sequence modification encoding for an HLA-A2

pitope that has enhanced immune stimulation. In addition,t contains the genes for three costimulatory molecules, B7.1,

CAM-1, and LFA-3. ICAM-1 is a member of the immuno-

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Colorectal cancer vaccines 81

lobulin superfamily that is upregulated in areas of inflam-ation. It binds to LFA-1, an integrin found on T cells.63

FA-3 is also a member of the immunoglobulin superfam-ly.64-66 LFA-3 binds to CD2 on T cells and enhances T-cellctivation and proliferation.67 Coexpression of all three co-timulatory molecules (Tricom) has been shown to be syner-istic for T-cell stimulation.68 T cells stimulated by Tricomave decreased spontaneous apoptosis compared with T cellstimulated by each individual costimulatory molecule. Ani-als vaccinated with rV-CEA-Tricom all survived challengeith a CEA-expressing tumor, whereas animals vaccinatedith rV-CEA or rV-CEA B7.1 all succumbed to tumor chal-

enge. The hypothesis underlying these Tricom vaccine trialss that T-cell stimulation, particularly a Th1-mediated cellu-ar immune response to CEA, will be enhanced by the simul-aneous engagement of three costimulatory molecules.

Two phase 1 trials have been completed. The first testedscalating doses of the vaccine alone and in conjunction withM-CSF as a recombinant protein or as a fowl pox vectorontaining the gene for GM-CSF. This study has demon-trated safety of the vaccine.69 The second incorporated GM-SF and the prime boost strategy using the same construct inaccinia.70 Fifty-eight patients were treated, with one patho-ogic complete response, five patients with decreasing serumEA levels, and 25 patients with stable disease for longer

han 4 months. Again, significant increases in CEA-specific-cell responses were reported.GM-CSF has been included as a vaccine adjuvant because

f its many effects on the immune system.71 GM-CSF haseen shown to enhance primary in vitro immune responsesue to enhanced APC efficiency.72 It stimulates the growth ofPCs such as DC and macrophages. Irradiated tumor cells

ransfected with GM-CSF have been used as vaccines in mu-ine models and have elicited enhanced immune responsesgainst tumors, even upon rechallenge with nontransfectedumor cells.73 Clinical trials using ALVAC-CEA,59 ALVAC-EA B7.1,61 and rF-CEA 6-D TRICOM70 in conjunction withM-CSF have suggested an improvement in stability of dis-ase and/or immune response. The one trial where there wasot an improvement in immune response with GM-CSF used50 �g GM-CSF given subcutaneously in the region to beaccinated beginning 2 days before vaccine injection andontinuing for a total of 5 days. This differed from the otherrials in that GM-CSF was begun prior to vaccination ratherhan with the vaccination. In addition, the dose used in thistudy was higher than had been used in many other studies.he recently completed study with rF-CEA6-D TRICOM isvaluating different doses (100 and 250 �g) and rF-GM-CSF,ll starting the day of vaccination, to further clarify the role ofM-CSF.

uture Approacheshis review has outlined the many vaccine approaches used.ost trials have revealed evidence of an immune response;

owever, not many of these had clinical responses. Few stud-

es have been designed to or had the statistical power to o

etermine if vaccines have an impact on the survival of pa-ients with colorectal cancer.

How might vaccines be improved to lead to clinical re-ponses and affect patient survival? Many vaccines have onlyargeted one antigen, as illustrated above by the studies usingEA. CEA is shed into the circulation, as are many cell sur-

ace antigens. One could argue that any immune responseenerated may not reach tumors because it is “soaked up” byhe peripheral circulating CEA and therefore may not be thedeal TAA. Combinations of antigens in a vaccine might gen-rate a greater number of immune response cells with varyingpecificity that might increase the likelihood of a tumor re-ponse. It is known that antigens have various epitopes formmune recognition, some of which are immunodominantnd generate a strong immune response, whereas others leado weaker immune responses. Epitopes vary depending onhe HLA receptor of the individual. The optimal vaccine maye one with multiple antigens. Peptide vaccines will requirearied peptides to bind specific HLA receptors and investiga-ors need to consider which peptides derive the best immuneesponse for specific HLA receptors.

Studies are also evaluating strategies to combine chemo-herapy with vaccines. Chemotherapy may impede the abilityf the immune response to be generated.60 However, there isn vitro data that 5-fluorouracil can upregulate the expressionf CEA.74,75 In addition, the breakdown of cells induced byhemotherapy may allow immune cells to take up additionalntigens, boosting and diversifying the response previouslynduced. A study has recently been completed in first-line

etastatic disease combining ALVAC-CEA-B7.1 with aeekly regimen of bolus 5-fluorouracil, leucovorin, and iri-otecan. This study will help assess the impact of chemother-py on the generation of an immune response.

The other method of eliciting an enhanced immune re-ponse is to manipulate the immune system. The use of ad-uvants such as GM-CSF or tetanus toxoid and the incorpo-ation of costimulatory molecules are methods alreadymployed to enhance the immune response. The latest suchpproach, not yet tested in colorectal cancer, uses an anti-TLA4 antibody, MDX-010. CTLA4 is found on activated Tells and binds B7 molecules.76 It functions to inactivate thectivated T cell, serving as a check on the immune re-ponse.77-79 By inhibiting the interaction of B7 with CTLA4,he stimulated immune response is not turned off and canontinue. A phase I study using MDX-010 with a peptideelanoma vaccine has been reported.80 MDX-010 treatmentas associated with diarrhea, which was easily managed and

eversible. Information on the immunologic benefits ofDX-010 in contrast to vaccine alone is not yet published.

ncreasing the numbers of T cells induced by a vaccinationay allow for a clinical response, but this has yet to be dem-

nstrated in human clinical trials.Vaccine trials to date have focused on patients with meta-

tatic colorectal cancer, many of whom have received multi-le therapies prior to vaccination. We have demonstratedhat the greater the amount of prior chemotherapy a patientas received, the less likely an immune response is to devel-

p.60 In addition, patients with metastatic disease have an

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82 M. von Mehren

mmune system that may not be able to effectively performytotoxic functions. Testing vaccines in patients who are noteavily pretreated and who do not have extensive metastaticisease will likely provide a better understanding of theirherapeutic potential.

onclusionstudies to date have looked at various vaccine platforms de-ivered by different routes and schedules either alone or inonjunction with adjuvants. Testing of varying prime boostchedules as well as the incorporation of costimulatory mol-cules has also been tested. The majority of these trials haveeen phase I studies and have demonstrated immunologicesponses. It is not clear that an increased “dose” of vaccineas led to a greater immune response. In addition, it is diffi-ult to assess which vaccine has led to the best immunologicenefit, as over time the methods used to test for immuneesponse have varied and cannot be compared.

Few clinical responses have been observed. The lack ofesponse in these early-phase studies has led some to suggesthat these agents do not have benefit. An important factoray be the impact of prior chemotherapy. Studies in earlier

tage disease or in patients with limited metastatic diseaseay allow a better understanding of what the immunologic

esponse generated means for colorectal cancer patients.ome data have suggested that in those patients who developn immune response, there may be a survival benefit thatoes not come at the expense of significant toxicity. How-ver, the studies conducted have had modest numbers ofatients, limiting the statistical power of these observations.What do we know? We know CEA-targeted vaccines have

ed to the induction of an immunologic response. We knowEA vaccines can be given safely without significant toxicity.nd what don’t we know? We do not know which of theseany vaccine platforms generates the best immunologic re-

ponse, nor do we know if that immunologic response haseaningful clinical efficacy and if so which patient popula-

ion will most benefit from this approach. To answer theseuestions may require large phase III trials in the adjuvantetting. The trials in the adjuvant setting to date do not clearlyocument a survival benefit. Vaccination in the resected met-static disease setting has suggested a survival advantagehen the vaccine elicits an immune response. Patients doenefit from modern chemotherapeutic agents and surgicalechniques, although with some toxicity. Our challenge is toelect the most promising vaccine and to design appropriatelinical trials to test the survival benefit of these nontoxicmmunologic agents.

cknowledgmenthe author thanks Veronica A. Levin of Special Services forer expert assistance in creating the figure.

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