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Breast Disease 20 (2004) 311 3IOS Press

Peptide-Based Vaccines in Breast Cancer

Mary L. Disis, Lupe G. Salazar and Keith L. KnutsonTumor Vaccine Group, Oncology, University of Washington, Seattle, WA, USA

Abstract. Human tumors are immunogenic and tumor-associated proteins that generate immunity in cancer patients have been

defined. Many of these proteins are involved in the malignant transformation and play a role in either initiating or maintaining

the malignant phenotype. Furthermore, due to technical advances in basic immunology over the last decade we have a betterunderstanding of the immune effector cell phenotypes that are potentially involved in tumor eradication and have developed

methods to quantitate and characterize these immune effectors. Breast cancer is an intriguing model tumor to target with ac-

tive immunization. Dozens of breast cancer antigens have been defined [1]. Although many patients with breast cancer can

be rendered free of disease with standard therapy such as surgery, radiation, and chemotherapy, some patients will have their

disease recur. However, relapse may not occur for many months to years after definitive treatment giving an extended period of

micrometastatic disease that may be amenable to immune eradication or modulation. Peptide based vaccines are one of the most

commonly studied vaccine strategies targeting breast cancer.

Abbreviations: APC: antigen presenting cell; CEA: carcinoembryonic antigen; CTL: cytotoxic T cell; DC: dendritic cell;

ECD: extracellular domain; GM-CSF: granulocyte macrophage colony stimulating factor; HLA: human leukocyte antigen; IFA:

incomplete Freunds adjuvant; IFN: interferon; IL: interleukin; KLH: keyhole limpit hemocyanigen; LC: Langerhans cell; MHC:

major histocompatibility antigen; PBMC: peripheral blood mononuclear cells; TCR: T cell receptor; Th: T helper cell

THE RATIONALE SUPPORTING PEPTIDE

BASED VACCINES

Vaccines have been one of the most successful clin-

ical interventions for the prevention of human dis-

ease. Prospective immunization against a variety ofpathogens has resulted in protection from the devel-opment of life-threatening infection. A recent reviewcited data derived from records kept by the Centers of

Disease Control since 1912 and outlined the decrease

in infectious disease since vaccines targeting specificpathogens have become widely available [2]. The inci-

dence of indigenous poliomyelitis has decreased 100%,diphtheria, measles and rubella have decreasedby 99%,

and whooping cough has had a 97% decrease in inci-dence associated with vaccination. Most cases of vac-

cine failure can be linked to insufficient immune re-sponses generated after active immunization [2]. The

Corresponding author: Mary L. Disis, Box 356527, Oncol-ogy, University of Washington, Seattle, WA 98195-6527, USA.Tel.: +1 206 616 1823; Fax: +1 206 685 3128; E-mail: [email protected].

purpose of a vaccine is to induce an antigen-specific

immune response that will result in disease prevention.

The identification of specific tumor antigens has sig-nificantly advanced the field of tumor immunology, in

particular, the development of breast cancer vaccines.

Improved understanding of the molecular basis of anti-gen recognition has resulted in the development of ra-

tionally designed breast cancer antigen-specific vac-cines based on motifs predicted to bind to human class I

or class II major histocompatibility molecules (MHC).

The use of synthetic peptides in breast cancer vaccinesoffers practical advantages such as relative ease of con-

struction and production, chemical stability, and a lack

of infectious or oncogenic potential. Peptides mayalso allow better manipulation of the immune response

through the use of epitopes designed for stimulatingparticular subsets of T cells (e.g. helper and cytotoxic

T cells). In animal models, peptide vaccines are par-

ticularly effective in generating immune responses toself-proteins [3]. Theoretically, immunization to for-

eign proteins normally elicits immunity to only a sub-set of potential epitopes, dominant epitopes, whereas

other potentially immunogenic epitopes, subdominant

0888-6008/04/$17.00 2004 IOS Press and the authors. All rights reserved

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4 M.L. Disis et al. / Peptide-Based Vaccines in Breast Cancer

epitopes, are ignored. There is evidence that T cells

are tolerant to the dominant epitopes of self-proteins,but may respond to the subdominant epitopes [4,5]. As

many newly defined tumor antigens are self-proteins,

peptide immunization may play a key role in the ability

to elicit an immune response to such antigens. Peptide

based vaccines can be designed to represent subdomi-

nant epitopes, thus, elicit immunity.

The development of a rational approach to breast

cancer vaccination must be based on how the immune

system is stimulated to respond to breast cancer asso-

ciated proteins. The goal of many infectious disease

vaccines is to stimulate a cellular immune response, i.e.

T cell response that would result in the development

of long-term memory T cells that would be capable of

responding to the pathogen when a patient is exposed.

Generation of T cell immunity would be the goal for a

cancer vaccine developed to prevent disease in patients

at high risk for relapse. T cells comprise the major-

ity of circulating peripheral blood lymphocytes. T cell

activation depends on recognition of MHC-antigenic

peptide complexes on the surface of antigen present-

ing cells (APC). Tumors themselves could present anti-

genic peptides in MHC molecules expressed on cell

surface. Vaccines composed of both class I and class II

peptides derived from breast cancer antigens have been

studied in human clinical trials [6,7].Peptide-based vaccines have advanced from pre-

clinical to human clinical studies over the last decade

and several important issues have been elucidated dur-

ing this clinical progression. First, investigators have

developed powerful paradigms to choose peptide(s) for

immunization and established the pre-clinical evalua-

tion needed to develop a peptide-based tumor vaccine.

Secondly, the importance of class II peptides in active

immunization is being increasingly defined. The gen-

eration of an endogenous CD4+ T cell response will

improve the magnitude and maintenance of CD8+ im-

munity generated with class I binding epitopes. Finally,

methods have been developed to modify peptides to

improve the immunogenicity of epitope-specific vac-

cines. Tumor antigen-specific peptide-based vaccines

have been an excellent model to test active immuniza-

tion in breast cancer patients over the last several years.

CD8+ cytotoxic T cells (CTL) can kill cells express-

ing an antigen. The action of CD8+ T cells requires

recognition of antigenic proteins that have been pro-

cessed and presented as peptide fragments in the con-

text of MHC class I. Epitopes recognized by CD8+ T

cells are, in general, peptides of 810 amino acids in

length anchored at each end within the major groove of

MHC class I molecules. As a consequence, MHC class

I molecules exhibit more amino acid sequence speci-ficity based on these anchoring residues, than MHC

class II molecules which may be more promiscuous in

the binding of peptides. A potential problem in the de-

velopment of CTL epitope-based vaccines is the large

degree of MHC polymorphism and the need for knowl-

edge of MHC restrictions in the population to be vacci-

nated. However, it is now known that that MHC class I

moleculescan be divided into several families or super-

types based on similar peptide-binding repertoires [8].

For example, the A2 supertype consists of at least eight

related molecules and of these, the most frequently ob-

served are HLA-A*0201, A*0202, A*0203, A*0206,

and A*6802. Many peptides that bind A*0201 also ex-

hibit degenerate binding, i.e. binding to multiple alle-

les, thus, an A2 supertype multi-epitope vaccine could

be designed to provide broad population coverage [8].

Initial studies of CTL epitopes were based on the

elution of naturally processed peptides from MHC

class I molecules on tumor cells. The identification

of sequence motifs from eluted peptides, as well as

crystallographic data of peptide binding to specific

MHC class I molecules, has allowed predicted bind-

ing of specific peptides. Recent investigations demon-

strate that computer algorithms designed for predict-

ing class I binding can be good predictors of pep-tides that are naturally-processed epitopes of tumor

antigens. Lu and colleagues used two computer-

based prediction algorithms that are readily available

on the Internet; syfpeithi.bmi-heidelberg.com and bi-

mas.dcrt.nih.gov/molbio/hla bind/hla motif search

info.html. The algorithms were able to identify three

candidate peptides of CEA specific for HLA-B7. Af-

ter constructing the peptides based on motif identifi-

cation, investigators demonstrated one of the peptides

generated CTL capable of lysing HLA-matched CEA

expressing tumors [9]. Similar strategies have been ex-

tremely useful for identifying class I peptide epitopes

from a variety of tumor antigens. For example, Rong-

cun and colleagues identified four HER-2/neu-specific

HLA-A2.1-restricted CTL epitopes which were able

to elicit CTL that specifically killed peptide-sensitized

target cells, and most, importantly, a HER-2/neu-

transfected cell line and autologous tumor cells. In ad-

dition, CTL clones specific for particular HER-2/neu

epitopes were isolated from tumor-specific CTL lines,

further demonstrating the immunogenicityof these epi-

topes as candidates for a potential breast cancer vac-

cine specifically designed to develop a tumor-specific

CTL response [10]. Thus, peptide based vaccines can

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M.L. Disis et al. / Peptide-Based Vaccines in Breast Cancer 5

be designed for eliciting CTL immunity and may even

be able to immunize across multiple class I haplotypes.CD4+ T helper cells (Th) augment the antigen-

specific immune response via cytokine secretion. Th1

subsets of CD4+ T cells secrete cytokines such as in-

terleukin (IL)-2 and interferon (IFN)-that will stimu-

late the proliferation and activity of CTL. Th2 subsets

of CD4+ T cells secrete cytokines such as IL-4, -5,

-6, and -10 that will result in production of high affin-

ity effective antibody response. The action of CD4+

T cells requires recognition of antigenic proteins that

have been processed and presented as peptide frag-

ments in thecontext of MHC class II. Unlike MHC class

I molecules, which typically bind peptides of eight to

ten amino acids in length, MHC class II molecules are

generally believed to be more permissive in length and

amino acid sequence of bound peptide [11]. For exam-

ple, in studies defining the T helper epitopes of tetanus

toxoid, panels of overlapping peptides were screened

for their ability to stimulate T cell proliferation in lym-

phocytes derived from tetanus-immune subjects. Pep-

tides of greater than 12 amino acids and less than 31

amino acids and typically in the range of 14 to 16amino

acids were found to be most efficient in defining rec-

ognized epitopes [12]. Moreover, T helper peptides

can be universal or promiscuous and bind to multiple

alleles. Promiscuous class II helper epitopes have beenidentified for the HER-2/neu [13], NY-ESO [14], and

human telomerase reverse transcriptase antigens [15],

three common immunogenic proteins associated with

breast cancer.

CD4+ T cells play an important role in the genera-

tion of an anti-tumor immune response. CD4+ helper

T cells initiate andmaintainCD8+T cell responses[16,

17]. Peptide-based cancer vaccines must consider the

important and potentially critical role of CD4+ T cells.

Similar to the identification of MHC class I binding

motifs, computer algorithms have played a major role

in the identification of MHC class II binding motifs.

MHC class II antigenic epitopes tend to share a com-

mon secondary structure, namely amphipathic he-

lices [18] suggesting antigenic epitopes recognized by

T cells can be predicted from the primary structure of

a protein based on predicted secondary structure [19,

20]. There are now several amphipathicity and hy-

drophobicity algorithms that have been demonstrated

to be useful in predicting T cell presented sequences

within potentially antigenic proteins [18,19,21]. These

algorithms have been used to identify potential MHC

class II-bindingepitopes from several tumor-associated

antigens. In vitro generation of peptide-specific T cells

from patients with cancer, similar to those used with

MHC class I peptides, is the endpoint of the identi-fication of epitopes appropriate for use in immuniza-

tion. This strategy was used to define putative class

II HER-2/neu binding epitopes for use in breast can-

cer vaccines [19]. Subsequently, when these peptides

were tested for binding to HLA-DR molecules in vitro,

the peptides which were the most immunogenic in vivo

were also those peptides that had the highest binding

affinity to HLA-DR [22].

BREAST CANCER VACCINES COMPOSED OF

CLASS I PEPTIDES AND DESIGNED TO

GENERATE A CD8+ CYTOTOXIC T CELL

(CTL) RESPONSE

In an initial clinical study, HLA-A2 positive patients

with metastatic HER-2/neu overexpressing breast,

ovarian, or colorectal carcinomas were immunized

with p369-377, a MHC class I peptide defined from

the HER-2/neu protein sequence, admixed in incom-

plete Freunds adjuvant (IFA) every three weeks [23].

Peptide-specific CTL were isolated and expanded from

the peripheral blood of patients after two or four immu-

nizations. The CTL could lyse MHC-matched peptide-

pulsed target cells but could not lyse MHC-matchedtumors expressing the HER-2/neu protein. Even when

tumors were treated with IFN- to upregulate MHC

class I, the CTLlines generatedfrom the patients would

not respond to the peptide presented endogenously on

tumor cells. A similar study performed with the same

MHC class I peptide in patients with metastatic breast

and ovarian cancer demonstrated peptide and tumor-

specific responses in some patients [24]. Another group

of investigators, immunizing patients with p369-377,

used GM-CSF as an adjuvant [6]. GM-CSF is a re-

cruitment and maturation factor for skin dendritic cells

(DC), Langerhans cells (LC) and theoretically may al-

low more efficient presentationof peptide epitopes than

standard adjuvants such as IFA. Six HLA-A2 patients

with HER-2/neu-overexpressing cancers received six

monthly vaccinations with HER-2/neu peptide, p369-

377, admixed with GM-CSF. The patients had either

stage III or IV breast or ovarian cancer. Immune re-

sponses to the p369-377 were examined using an IFN-

ELIspot assay. Following vaccination, HER-2/neu

peptide-specific precursors developed to p369-377 in

just two of four evaluable subjects. The responses were

short-lived and not detectable at five months after the

final vaccination. Immunocompetence was evident as

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patients had detectable T cell responses to tetanus tox-

oid and influenza.Another vaccine strategy commonly used in breast

cancer vaccination is the delivery of MHC class I pep-

tides loaded on DC and injected subcutaneously or in-

tradermally. Brossart and colleagues reported a vac-

cine trial immunizing breast and ovarian cancer patients

with autologous DC pulsed with MUC-1 or HER-2/neu

class I binding peptides [25]. In this trial, 10 patients

were immunized. Half the patients developed CD8+

T cell precursors to their immunizing peptides. More-

over, some patients developed new immunity to other

tumor antigens expressed in their cancers such as CEA

and MAGE-3, i.e. epitope spreading. Epitope, or de-

terminant spreading, is a phenomenonfirst described in

autoimmune disease [26] and represents the generation

of an immune response to a particular portion of an im-

munogenic protein and then the natural spread of that

immunity to other areas of the protein or even to other

antigens present in the environment. Epitope spreading

may representthe ability of the initial immuneresponse

to create a microenvironment at the site of the tumor

that enhances endogenous local immunity [27].

These phase I clinical trials demonstrate that patients

with even advanced stage breast cancer can be immu-

nized against antigen expressed by their tumors. Al-

though early trials have focused on only one or two de-fined breast cancer antigens, many class I peptide epi-

topesare beingdefined for novel immunogenic proteins

expressed in breast cancer. For example, investigators

have identified several HLA-A3 binding epitopes of

mammaglobin-A, a protein highly expressed on about

80% of breast tumors [28]. Similarly, MHC class I

binding epitopes have been defined for the near univer-

sal antigen, survivin [29]. The identification of an ar-

mamentarium of MHC class I binding peptides derived

from multiple immunogenic breast cancer proteins will

lay the foundation of multi-antigen approaches to pre-

vent breast cancer relapse in antigenically diverse tu-

mors across a broad patient population.


CLASS II PEPTIDES AND DESIGNED TO

GENERATE A CD4+ T HELPER CELL (TH)

RESPONSE

Potential pitfalls of the use of single MHC class I

binding peptides are highlighted in the trials described

above; immunity is short lived and the peptide-specific

T cells may not recognize endogenously presented

antigen. A successful vaccine strategy for generating

peptide-specific CTL capable of lysing tumor express-ing HER-2/neu and resulting in durable immunity in-

volved immunizing patients with putative T-helper epi-

topes of HER-2/neu which had, embedded in the nat-

ural sequence, HLA-A2 binding motifs of HER-2/neu.

Thus, both CD4+ T cell helper and CD8+ specific epi-

topes were encompassed in the same vaccine. HLA-

A2 patients with HER-2/neu-overexpressing cancers

received a vaccine preparation consisting of putative

HER-2/neuhelper peptides [6]. Contained within these

sequences were the HLA-A2 binding motifs. Patients

developed both HER-2/neu-specific CD4+ and CD8+

T cell responses. The level of HER-2/neu immunity

was similar to viral and tetanus immunity. In addi-

tion, the peptide-specific T cells were able to lyse tu-

mor, demonstrating generation of T cells that recog-

nized naturally processed antigen. The responses were

long-lived and detectable for greater than one year after

the final vaccination in some patients. These results

demonstrate that HER-2/neu MHC class II epitopes

containing encompassed MHC class I epitopes are

able to induce long-lasting HER-2/neu-specific IFN--

producing CD8 T cells.

Stimulating an effective T helper (Th) response, even

without concomitant CD8+ peptide vaccination, is a

way to boost antigen-specific immunity as CD4+ Tcells generate the cytokine environment required to

support an evolving immune response. In one recent

study, patients with advancedstage HER-2/neu overex-

pressing breast, ovarian, and non-small cell lung cancer

were enrolled on a trial of a multi-peptide HER-2/neu

Th vaccine [7]. Over 90% of patients developed T cell

immunity to HER-2/neu Th peptides and over 60% to a

HER-2/neu protein domain. Thus, immunization with

peptides resulted in the generation of T cells that could

respond to protein processed by APC. Furthermore, at

1-year follow-up, immunity to the HER-2/neu protein

persisted in over a third of patients. Immunity elicited

by active immunization with CD4+ T helper epitopes

was durable. An additional finding of this study was

that epitope spreading was observed in the majority of

patients and significantly correlatedwith the generation

of HER-2/neu protein-specific T cell immunity.

Vaccines targeting HER-2/neu are among the most

commonly tested immunotherapeutic strategies being

evaluated in breast cancer patients. However, vacci-

nation with longer peptides derived from the MUC-1

breast cancer tumor antigen has undergone extensive

clinical study. MUC-1 is a membrane-bound glyco-

protein and is a primary component of the ductal cell

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surface of most glandular cells. When a normal cell

becomes cancerous, MUC-1 is both overexpressed andaberrantly glycosolated. Overexpression of MUC-1 in

cancer cells interferes with cell adhesion and may fa-

cilitate the development of metastasis. Several peptide

based MUC-1 vaccines have been evaluated in clini-

cal trials using it as a model antigen for eliciting both

humoral and cellular immune responses. Early trials

using MUC-1 with BCG as adjuvant induced DTH re-

sponses, but only in a minority of patients [30]. For-

mulating MUC-1 as a mannan-MUC-1 fusion protein

and immunizing patients multiple times resulted in aug-

mentation of MUC-1 specific antibodies in about 50%

of patients, with titers higher than 1/20,000. However,

patient numbers were small [31]. A more recent study

describes vaccination of breast cancer patients with

MUC-1/KLH along with QS-21, an adjuvant specif-

ically developed to stimulate cellular immunity [32].

While antibody titers were elevated, and remained so

over 100 days after vaccination, no antigen-specific T

cell responses were detected. MUC-1 specific anti-

bodies generated after active immunization have, how-

ever, been demonstrated to bind to and lyse tumor in

vitro [33].

PEPTIDE-SPECIFIC T CELLS ELICITED BYACTIVE IMMUNIZATION AND DIRECTED

AGAINST A SINGLE BREAST CANCER

ANTIGENIC EPITOPE CAN BE REMARKABLY

DIVERSE IN PHENOTYPE AND FUNCTION

A theoretical drawback of the use of peptide epi-

topes as immunogens is the risk of generating very

restricted immune responses, e.g. T cells of a single

phenotype directed against the epitope. A strongly re-

stricted response may limit potential therapeutic effi-

cacy. Dissection of the peptide-specific immune re-

sponse after immunization has demonstrated a marked

phenotypic diversity of the vaccinated response [34].

T cell clones specific for HER-2/neu HLA-A2 pep-

tide p369-377 were isolated from a patient who had

been vaccinated with HER-2/neu helper epitopes that

contained HLA-A2-binding CTL epitopes within their

sequences. Throughout the course of immunization,

PBMC from this patient showed strong proliferative

responses to the HER-2/neu helper epitope p369-384.

Following vaccination, T cell clones specific for p369-

377, the HLA-A2 binding peptide were isolated by lim-

iting dilution and then characterized. The responding T

cell repertoire generated was both phenotypically and

functionally diverse. A total of 21 p369-377 T-cell

clones were isolated from this patient; peptide-specificCD8+, CD4+ and T cell clones. Of note, de-

spite being generated with an HLA-A2-binding pep-

tide, CD4+ T cells were generated. Although CD4+

T cells are predominantly associated with responding

to peptides associated with MHC class II, at lower fre-

quencies CD4 T cells play a role in regulating MHC

class I-restricted T cells, particularly T cells associ-

ated with cancers such as melanoma, colon, and pan-

creatic cancer [35,36]. The majority of the clones ex-

pressed the T cell receptor (TCR), however, two

clones expressed the TCR. Clones could lyse HLA-

A2-transfected HER-2/neu overexpressing tumor cells

as well as peptide loaded MHC-matched cells. In ad-

dition to their lytic capabilities these clones could be

induced to produce IFN- specifically in response to

p369-377 peptide stimulation. The TCR clones

expressed CD8 and lysed HLA-A2 HER-2/neu posi-

tive tumor cells, but not HLA-A2 negative HER-2/neu

overexpressing tumor cells. T cells are involved in

a wide range of immune responses to infectious and

non-infectious diseases, including malaria, mycobac-

terial infections, cancers, as well as autoimmune dis-

orders such as multiple sclerosis [37]. Often, T

cells clones are isolated from the tumor-infiltrating lym-

phocyte population of many tumors including dysger-minoma [38], seminoma [38], renal carcinoma [39],

lung [40], colorectal [41], and melanoma [42]. The

recruitment to and role of these unique cells in medi-

ating anti-tumor immunity is unknown. One hypoth-

esis is that, given their broad range of regulation by

multiple mechanisms of antigen presentation and nat-

ural localization to epithelial tissue, TCR T cells

are sentinels for the immune system and are capable of

alerting the immune system to the presence of danger

(e.g. infection, tumors, etc.). These results suggest

that a tumor antigen-specific T cell response can be

markedly polyclonal at multiple levels including T cell

subset and TCR even if peptide epitopes are used as the

immunizing antigens.


PEPTIDES AND DESIGNED TO GENERATE

BREAST CANCER SPECIFIC ANTIBODY

IMMUNITY

Another aspect of peptide epitope prediction would

be to identify peptide portions of a breast cancer

antigen, such as the HER-2/neu extracellular domain

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(ECD), appropriate to target with an antibody re-

sponse. Several monoclonal antibodies against theHER-2/neu ECD have been isolated and one such an-

tibody, trastuzumab, has demonstrated clinical efficacy

in the treatment of metastatic breast cancer [43] . Al-

though manyHER-2/neu-specific antibodiesinhibit the

growth of cancer cells, some antibodies have no ef-

fect on cell growth, while others even actively stimu-

late cancer growth [44]. This wide range of biologi-

cal effects is thought to be related to the epitope speci-

ficity of the antibodies and to consequent changes in

receptor signaling [44]. An alternative to the use of

passive antibody therapy would be active immuniza-

tion against the HER-2/neu ECD. However, inappro-

priately induced immune responses could have unto-

ward effects on cancer growth. Therefore, it is crucial

to identify epitopes on HER-2/neu that are targeted by

stimulatory and inhibitory antibodies in order to en-

sure the induction of a beneficial endogenous antibody

response.

In a recent study, investigators constructed HER-

2/neu gene fragment phage display libraries to epitope-

map a number of HER-2/neu-specific antibodies with

different biological effects on tumor cell growth [44].

Regions responsible for opposing effects of antibod-

ies were identified and then used to immunize mice.

Mice immunized with the peptides developed antibod-ies that recognized both the peptide and native HER-

2/neu. More importantly, HER-2/neu-specific antibod-

ies purified from mouse sera were able to inhibit up to

85% of tumor cell proliferation in vitro. Furthermore,

using peptide regions that contain multiple inhibitory B

cell epitopes is likely to be superior to the use of single

epitope immunogens [45].

THE NEXT GENERATION PEPTIDE BASED

VACCINES FOR USE IN BREAST CANCER

THERAPY

Initial clinical trials of peptide-based breast cancer

vaccines indicated that patients can be immunized, but

immune responses were generally not to the levels of a

vaccinated antigen such as tetanus toxoid. Therefore,

more recent investigations are focused on improving

the immunogenicity of peptide immunization.

The choice of adjuvant is an important considera-

tion in peptide vaccine trials, particularly as peptides

themselves are typically only weakly immunogenic.

Due to recent renewed enthusiasm for peptide-based

tumor vaccines coupled with the search for adjuvants

that promote cellular as well as antibody responses,

the list of adjuvants being studied continues to grow.The choice of adjuvant has largely been determined by

pre-clinical models. Similar to alum, oil-based incom-

plete Freunds-type adjuvants, such as Montanide ISA-

51 (Seppic, Paris, France) and TiterMax (CytRx Corp.,

Norcross, GA), have been used in human peptide vac-

cine studies [46,47], with their adjuvant effect likely

mediated by a depot effect,increasing the half lifeof the

peptide antigen at the site of immunization. Other pro-

inflammatory biologic adjuvants such as BCG, Detox

(mycobacterial cell-wall skeleton plus a lipid moiety)

(Corixa Corp., Seattle, WA), and influenza virosomes

have been used as adjuvants in peptide- and protein-

based human vaccine studies [4850]. One study com-

pared a variety of adjuvants in a rodent model targeting

human MUC-1 peptide antigens conjugated to keyhole

limpet hemocyanin (KLH), and found the saponin ad-

juvant QS-21 (Aquilla Biopharmaceuticals, Worcester,

MA) to be the most effective in promoting an antigen-

specific antibody responses [51]. Subsequently, QS-21

has been used as an adjuvant in human peptide cancer

vaccine trials [52,53], with the hope that the use of a

saponin may allow entry of peptides directly into MHC

complexes to stimulate a T cell response [54]. Another

type of adjuvant that has been studied in animal models

is polymer microspheres that encapsulate the peptideantigen, permitting slow release over variable lengths

of time [55]. Potential advantages to this type of ad-

juvant includes the ability to use an oral route of de-

livery [56], continuous release of antigen obviating the

need for booster immunizations, and the ability to in-

corporate other biologic adjuvants or cytokines within

the polymer [57]. Finally, the discovery of various

cytokines participating in the initiation of immune re-

sponses has suggested that cytokines themselves may

be useful immunologic adjuvants. As an example,

soluble GM-CSF is a vaccine adjuvant, and has the

ability to induce the differentiation of dendritic cells

and act as a chemoattractant for various immune cell

effectors [58,59].

The relationship between MHC class I affinity and

tumorantigen epitope immunogenicity is of importance

because tissue-specific and developmental tumor anti-

gens, such as those that are breast cancer antigens, are

expressed on normal tissues at some point in time at

some location within the body. T cells specific for these

self antigens could be functionally inactivated by T cell

tolerance. However, CTL responses to tumor epitopes

in both volunteer donors and breast cancer patients in-

dicate that tolerance to these tumor antigens, if it ex-

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ists at all, is incomplete [8,60]. It is unknown whether

T cells recognizing high affinity epitopes have beenselectively eliminated, leaving a repertoire capable of

recognizing only low affinity epitopes. One investiga-

tion evaluated several peptides derived from four dif-

ferent breast cancer tumor antigens, p53, HER-2/neu,

CEA, and MAGE proteins, for their capacity to in-

duce CTL in vitro capable of recognizing tumor target

lines [8]. In order to increase the likelihood of over-

coming tolerance, fixed anchor analogues that demon-

strate improved HLA-*0201 affinity and binding, were

used along with wild type peptides. A total of 20/20

wild-type and 9/9 single amino acid substitution ana-

logues were found to be immunogenic in primary invitro CTL induction assays, using normal PBMCs and

monocyte derived dendritic cells as APC. Cytotoxic T

cells generated by 13/20 of the wild type epitopes and

6/9 of single substitution analogues tested recognized

HLA-matched antigen bearing cancer cell lines. Fur-

ther analysis suggested that high binding affinity epi-

topes are frequently generated and can be recognized

as a result of natural antigen processing. Indeed, the

immunogenicity of CTL epitopes derived from human

tumor antigens such as CEA can be improved by alter-

ing the peptide motif to improve binding to MHC [61].

Studies such as these demonstrate that recognition of

self-tumor antigens is within the realm of the T cell

repertoire and that binding affinity may be an important

criterion for epitope selection.

CONCLUSION

The definition of multiple breast cancer antigen-

specific peptides, both class I and class II derived epi-

topes are rapidly being translated into human clinicalstudies. Investigations over the last decade have al-

lowed the development of technology such as com-

puter algorithms and high throughput peptide binding

assays to rapidly screen vaccine candidates. Clinical

trials of single peptides have demonstrated that cancer

patients can be immunized against self-tumor antigens

and some studies have shown early positive clinical re-

sults. Current effort in the field is focused on improving

the immunogenicity of individual MHC binding pep-

tides as well as developing multiple peptide vaccines

for the prevention and treatment of breast cancer.

ACKNOWLEDGEMENTS

This work is supported for MLD by grants from

the NIH, NCI (R01 CA75163 and CA85374, and K24CA85218) as well as funding from the Cancer Re-search Treatment Foundation, for LGS by NIH train-ing grant T32 (HL07093), and for KLK by a fellow-ship from the Department of Defense Breast CancerProgram (DAMD17-00-1-0492).

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