A paradigm shift in therapeutic vaccination of cancer patients: the need to apply therapeutic...

A paradigm shift in therapeuticvaccination of cancer patients:

the need to apply therapeutic

vaccination strategies in thepreventive setting

Summary: An extraordinary variety of potential therapeutic vaccinestrategies directed against a wide variety of tumor antigens has beenexplored in clinical trials. To date, none of these cancer immunotherapieshave been approved by the Food and Drug Administration for use inhumans. A significant problem is that the vast majority of such clinicaltrials are carried out in patients with advanced or metastatic cancer. Theimmune systems of these patients are considerably compromised as aresult of tumor- and treatment-mediated immunosuppression. Even incases where patients are immunized in the adjuvant setting, where there isminimal residual disease, vaccines directed against tumor-associatedantigens have failed to mediate eradication of tumors in the overwhelmingmajority of cases. Recently, we and others have experimented withadministering therapeutic cancer vaccines in the preventive setting. Thisis achieved by vaccinating at the earliest possible stage of carcinogenesis.These studies have demonstrated that early vaccination is extremelyeffective in eliciting an anti-tumor immune response that leads tounprecedented improvements in the survival of mice that spontaneouslydevelop cancer. Certain human cancers, notably prostate adenocarcinomaand cervical cancer, can currently be detected at very early stages ofcarcinogenesis. Therapeutic vaccines are available for these diseases,opening up the possibility of administering vaccinations early to patientsdiagnosed with pre-malignant lesions to halt disease progression. Inaddition, new technologies have become available in the past decade thatwill soon yield very sensitive and specific diagnostic tests for a plethora ofother cancers. Earlier detection of these cancers, combined with existingvaccines directed against them, will soon make them targets for therapeu-tic vaccination in the preventive setting. The ability to immunize patients atthe very earliest stages of carcinogenesis, when they have fully competentimmune systems, has the potential to cause a paradigm shift in howtherapeutic cancer vaccines are tested and used clinically.

Keywords: therapeutic cancer vaccines, immunotherapies, tumor immunity, vaccination,cancer, cancer prevention

Introduction

Immunotherapy has long been proposed as a method of

treating cancer. Hundreds of animal studies have shown that a

myriad of cancers can be treated specifically, safely, and

Andrew Gray

Adam B. Raff

Maurizio Chiriva-Internati

Si-Yi Chen

W. Martin Kast

Immunological Reviews 2008

Vol. 222: 316327

Printed in Singapore. All rights reserved

r 2008 The Authors

Journal compilationr 2008 Blackwell Munksgaard

Immunological Reviews0105-2896

Authors addresses

Andrew Gray1, Adam B. Raff1, Maurizio Chiriva-Internati2,3,

Si-Yi Chen1,4, W. Martin Kast1,4,5

1Norris Comprehensive Cancer Center, University of

Southern California, Los Angeles, CA, USA.2Department of Molecular Microbiology & Immunology,

Texas Tech University Health Sciences Center and

Southwest Cancer Treatment and Research Center,

Lubbock, TX, USA.3Division of Hematology & Oncology, Texas Tech

University Health Sciences Center and Southwest Cancer

Treatment and Research Center, Lubbock, TX, USA.4Department of Microbiology & Immunology, University

of Southern California, Los Angeles, CA, USA.5Department of Obstetrics & Gynecology, University of

Southern California, Los Angeles, CA, USA.

Correspondence to:

W. Martin Kast

University of Southern California

1450 Biggy Street, NRT 7507

Los Angeles, CA 90033, USA

Tel.:11 323 442 3870

Fax: 11 323 442 7760

e-mail: [email protected]

Acknowledgements

This review is partially based on studies that were sup-

ported by the Margaret E. Early Medical Research Trust and

NIH training grant T32 GM 067587 (Gray). W. Martin Kast

holds the Walter A. Richter Cancer Research Chair.

316

effectively using a wide variety of immunotherapeutic strate-

gies. The extraordinary successes in eliciting immune re-

sponses against tumor-associated antigens (TAAs) that are

capable of mediating eradication of tumors in animal models

of cancer have led to an explosion in clinical trials designed to

test the viability of translating similar strategies to treating

patients. However, no such immunotherapies have been ap-

proved by the Food and Drug Administration for human use.

A significant problem is that virtually all such vaccines have

been tested in terminally ill cancer patients who have failed

other therapeutic strategies. These patients generally have large

and/or metastatic tumors. Patients with advanced cancers are

not good candidates for immunotherapy, because they are

immunocompromised as a result of previous treatments and

the immune evasion mechanisms of their tumors. The field has

attempted to circumvent this problem by two main

approaches: (i) patients with minimal residual disease (e.g., in

patients that have undergone surgical, radiological, and/or

chemotherapeutic intervention) have been vaccinated in the

hopes that the immune suppression commanded by the tumor

will be diminished if tumor volume is reduced, and (ii) the

mechanisms of tumor immune evasion have been individually

targeted and eliminated or attenuated in an effort to allow the

anti-tumor immune response elicited by vaccination to

proceed more efficiently. These approaches have met with

limited success. As discussed herein, large tumors can and do

significantly alter the immune system, and these changes can

persist even after the tumor has been debulked. Furthermore,

the complex immunosuppressive networks developed by

advanced cancers have multiple, often redundant, mechanisms

of limiting the anti-tumor immune response. As a result,

targeting one or a few of these immune evasion mechanisms

within a tumor rarely elicits an improvement in the clinical

response to vaccination because other immunosuppressive

mechanisms remain that can blunt the anti-tumor immune

response induced by vaccination.

Given these considerations, it is time for a paradigm shift in

how therapeutic vaccination protocols are tested and applied in

the clinic. Advances in screening and detection of cancer are

arguably the greatest victories in the so-called war on cancer

to date. The improved cancer detection mechanisms that are

available and that will be coming online over the next decade

will undoubtedly allow the medical field to detect cancers at

their very earliest stages. We believe that this provides us a new

opportunity to apply therapeutic cancer vaccines preventatively

during the earliest possible stages of carcinogenesis. Here we

will propose that very early detection of cancer will allow us to

intervene with tumor immunotherapies before the presence of

the tumor and before the use of conventional anticancer

treatments have manifested changes to the immune system of

the patient. We summarize our own recent studies and those of

others that show that therapeutic vaccinations in the very early

stages of carcinogenesis can induce an extremely effective, long

lasting, and safe immune protection against cancer development.

Most therapeutic cancer vaccine clinical trials to datehave been carried out in end-stage patients and haveyielded limited clinical benefit

A plethora of therapeutic vaccination strategies have undergone

clinical trials in recent years. These have included vaccines

based on tumor cell lysates, peptides of TAAs, recombinant

proteins, dendritic cells (DCs) (mature and immature) loaded

with all of the above, DCs transfected with tumor RNA or with

DNA encoding TAAs, whole tumor cells, recombinant viral

vectors expressing TAAs, and direct immunization with plas-

mid DNA encoding TAAs. In addition, many of these strategies

have been combined with administration of other immuno-

modulatory molecules including but not limited to interleukin-

2 (IL-2), granulocyte macrophage colony-stimulating factor

(GM-CSF), and costimulatory molecules. Despite these many

and varied attempts at eliciting an anti-tumor immune re-

sponse, very few are capable of doing so, and so far none have

proven consistently capable of eradicating an existing tumor.

Virtually all therapeutic cancer vaccines that have undergone

clinical trials have been tested in patients with advanced or

metastatic cancer who have failed all conventional treatment

options. This philosophy of clinical trial design is exemplified

in the case of prostate cancer immunotherapy. Some recent

trials of therapeutic prostate cancer vaccines have been

summarized in Table 1 (115). The majority of these trials has

been conducted in patients with metastatic hormone-

refractory prostate cancer (HRPC), for whom only palliative

treatment is available. From an ethical standpoint, these

individuals make good candidates for experimental therapies.

However, these trials have produced very little positive data.

Measurable immunological responses to vaccination, often

measured by enzyme-linked immunospot assay (ELISPOT),

tetramer staining, cytotoxicity assay for cellular responses, and

enzyme-linked immunosorbent assay for the development of

humoral immunity, are usually limited within these studies.

Moreover, objective clinical responses are even rarer. Initially,

the disappointing results obtained led the field to conclude that

the immunogenicity of vaccines need to be enhanced by

targeting new TAAs, adopting completely new vaccination

strategies or by attempting to bolster the effectiveness of

existing ones, for example by the co-administration of

Immunological Reviews 222/2008 317

Gray et al Therapeutic vaccines in cancer prevention

Table1.

Summaryofrecentclinical

trialsoftherapeu

ticprostatecancervaccines

Aut

hor(

s)Y

ear

Vac

cine

Num

ber

of

pat

ient

sD

isea

sest

atus

Pre

vious

trea

tm

ent(

s)Pat

ient

resp

ons

eto

vacc

ine

Clin

ical

resp

ons

esR

efer

ence

Tjo

aet

al.

1998

Aut

olo

gous

DC

spul

sed

with

PSM

Apep

tides

.33

Met

asta

ticpro

stat

eca

ncer

RP,H

,pal

liativ

era

dio

ther

apy

DT

Hfo

rPSM

A9/3

3PR

,11/3

3SD

,13/3

3N

R(1

)

Mur

phy

etal

.1999

As

Tjo

aet

al.(

1)

37

Adva

nced

pro

stat

eca

ncer

(sta

geC

D

)R

P,X

RT

,B,H

DT

Hfo

rPSM

A1/3

7C

R,1

0/3

7PR

,8/3

7SD

,18/3

7N

R(2

)

Eder

etal

.2000

Vac

cini

a-bas

edva

ccin

ein

corp

ora

ting

PSA

33

Adva

nced

(up

toan

din

clud

ing

LNin

volv

e-m

ent)

but

nobony

met

sal

low

ed

XR

T,R

P,b

oth

14/2

5as

sess

able

pat

ient

sha

dD

TH

resp

ons

esto

vax

afte

ral

l3

dose

s.In

the

vax1

GM

-CSF

group

,7/1

0pat

ient

sha

dD

TH

resp

ons

esan

d5/7

had

cellu

lar

resp

ons

es(E

LISP

OT

)

SDin

6/3

3pat

ient

s(3

)

Smal

letal

.2000

As

Smal

letal

.(12)

31

Met

asta

ticH

PR

CR

P,X

RT

,B,C

RY

O10/2

6pat

ient

ste

sted

dev

eloped

T-c

ellr

espons

esto

PA

P,1

6/3

1te

sted

dev

eloped

anti-

PA

Pan

tibodie

s

Thr

eepat

ient

ssh

ow

ed4

50%

dec

reas

ein

PSA

leve

l,th

ree

more

show

eda

dec

reas

eof

2549

%.M

edia

nT

TP

for

pa-

tient

sw

hodev

eloped

anim

-m

une

resp

ons

ew

as34

wee

ksco

mpar

edw

ith13

wee

ksfo

rth

ose

that

did

not(Po

0.0

27)

(4)

Fong

etal

.2001

Aut

olo

gous

DC

slo

aded

with

xeno

geni

cm

urin

ePA

P

21

Met

asta

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stat

eca

ncer

,eith

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rmone

sens

itive

or

HPR

C

C,X

RT

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T-c

ellp

rolif

erat

ion

assa

yaf

ter

stim

ulat

ion

with

hPA

P/m

PA

P,

ELIS

Afo

rIL

-4,I

L-10,I

FN-g

and

TN

F-a,

ELIS

PO

Tfo

rIF

N-g

and

IL-4

.All

pat

ient

sdev

eloped

re-

spons

eto

mPA

P.1

1/2

1pat

ient

sdev

eloped

ace

llula

rre

spons

eto

hPA

P

8/2

1ha

ddisea

sest

abiliza

tion.

Pro

gres

sion-

free

surv

ival

very

sign

ifica

ntly

bet

ter

inth

egr

oup

that

dev

eloped

hPA

Pre

spons

esco

mpar

edw

ithth

ose

that

did

not(P

=0.0

087

)

(5)

Hei

seret

al.

2002

Aut

olo

gous

DC

str

ans-

fect

edw

ithPSA

RN

A13

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asta

tic(D

1to

D3)

m

ost

D3

XR

T,R

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one

,OA

llpat

ient

ssh

ow

edPSA

-spec

ific

ELIS

PO

Tre

spons

eD

ecre

ase

inse

rum

PSA

leve

lsfo

r1/7

eval

uable

pat

ient

s.D

ecre

ase

inlo

gPSA

leve

lfor

afu

rthe

r5/7

.6pat

ient

sex

clud

ed

[6]

Bur

chet

al.

2004

As

Smal

letal

.(1

2)

21

Met

asta

ticH

PR

CR

P,A

A,X

RT

,HPro

lifer

atio

nas

say

and

ELIS

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esto

PA

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M-C

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ndPA

2024.

Sign

ifica

ntce

llula

rre

-sp

ons

eat

wee

k4

(Po

0.0

001),

13/1

5pat

ient

sdev

eloped

anti-

body

resp

ons

esto

PA

202

4but

notPA

P

2pat

ient

ssh

ow

ed2550%

de-

crea

sein

PSA

(tra

nsie

nt).

One

pat

ient

show

eddec

line

ofP

SAto

undet

ecta

ble

leve

lsby

wee

k24

co

ntin

ued

to52

mont

hs

(7)

Kau

fman

etal

.2004

Vac

cini

a-an

dfo

wlp

ox-

bas

edva

ccin

esin

corp

ora

ting

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64

Org

an-c

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aine

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stat

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,RP,R

T,o

ther

46%

had

incr

ease

inPSA

-spec

ific

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ellr

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esN

oobje

ctiv

ere

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utaf

ter

2ye

ars

off

ollo

w-u

p,7

8.1

%ofm

enar

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om

clin

ical

disea

sepro

gres

sion

and

45.3

%ar

efr

eefr

om

PSA

pro

gres

sion

(8)

Suan

dD

annu

llet

al.

2005

DC

str

ansf

ecte

dw

ithhT

ERT

or

hTER

T-L

AM

Pm

RN

A

20

Met

asta

ticH

PR

CR

P,X

RT

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Incr

ease

sin

IFN

-gby

ELIS

PO

Ton

CD

41

and

CD

81

cells

No

obje

ctiv

ere

spons

es,b

utPSA

doub

ling

time

impro

ved

to100

mont

hsin

6-c

ycle

group

(P=

0.0

4).

No

impro

vem

ent

seen

in3-c

ycle

group

(P=

0.6

3).

(9)

318 Immunological Reviews 222/2008


Tra

nsie

ntcl

eara

nce

ofP

SA-

expre

ssin

gtu

mor

cells

obse

rved

inso

me

subje

cts

(by

real

-tim

ePC

Rfo

rPSA

mR

NA

)D

iPao

laet

al.

2006

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cini

a-an

dfo

wlp

ox-

bas

edva

ccin

esin

corp

or-

atin

gPSA

seque

nce

and

TR

ICO

M(B

7-1,I

CA

M-1

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dLF

A-3

).Pat

ient

str

eate

dw

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ccin

iavi

rus

then

boost

edw

ithfo

wlp

ox

10

HPR

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om

em

etas

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prior

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erap

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inic

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olo

gous

DC

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with

reco

mbin

antfu

sion

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tein

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-bin

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M-C

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nge)

127

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asta

ticH

PR

CN

otdiscu

ssed

indet

ail

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ysign

ifica

nt(Po

0.0

01)

incr

ease

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pons

eto

antig

enic

targ

et

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ifica

ntim

pro

vem

entin

ove

rall

surv

ival

(P=

0.0

1)

(12)

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mas

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kele

tal

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PSC

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dPSA

pep

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immunostimulatory cytokines or costimulatory molecules.

Despite these efforts, the results of prostate cancer

immunotherapy trials have been broadly similar to each other

regardless of the vaccination strategy used or TAA targeted.

Commonly, overall survival (OS) and/or time to progression is

significantly improved in patients that develop antigen-specific

cellular or humoral immune responses as a result of vaccination,

implying that the vaccines that have been developed are perfectly

capable of working in a subset of immunocompetent subjects.

Similar results have been observed in trials of therapeutic

vaccines for renal cell carcinoma (16, 17), melanoma (18, 19),

pancreatic adenocarcinoma (20), and breast cancer (21). It is

thus most likely that the failure to date of immunotherapeutic

agents to mediate tumor clearance is mostly due to the immuno-

compromised status of advanced cancer patients in which they

are tested. Several studies have attempted to assess the general

immune competence of test subjects, for example by measuring

their ability to mount recall responses to common antigens.

However, the ability of the immune system to respond to an

antigen to which it has already been exposed is not necessarily a

measure of its ability to mount a completely new immune

response upon vaccination. In recent years, the cellular

and molecular mechanisms underlying immune suppression

and their implications for therapeutic cancer vaccination have

been the subject of intense interest. These studies have revealed

multiple mechanisms of immune suppression in advanced

cancer patients that render them poor candidates for

immunotherapeutic intervention.

Large tumors fundamentally alter the immune systems ofpatients and limit their ability to mount an anti-tumorimmune response

Large tumors have been shown to have multiple, often redundant

pathways of immune escape. Tumor immune escape mechan-

isms have been the subject of excellent reviews (22, 23) and are

not covered in depth here. In summary, a tolerizing milieu

consisting of regulatory T cells (Tregs), suppressive/tolerogenic

DCs, and suppressive cytokines develops within the tumor

microenvironment. Antigen presentation and T-cell activation

are often compromised within tumors. In addition, corrupted

CD81 T-cell memory function results from chronic stimulation

and contributes to immune failure in cancer patients (24).

As mechanisms of tumor-mediated immune suppression

have been elucidated over recent years, a determined research

drive has been concentrated on eliminating or inhibiting them

to enhance the efficacy of therapeutic cancer vaccines.

Naturally occurring CD41CD251 Tregs have been the focus of

particularly intense study in this regard, although they do not

always seem to be involved in mediating the suppression

of immune responses elicited by cancer vaccines (25).

Nevertheless, natural Tregs have been accepted as central players

in tumor-mediated immune suppression in many cancers, and

several methods to deplete them or limit their suppressive

activity have been developed (26). For example, inhibition of

CD41CD251 Tregs by cyclophosphamide pretreatment before

immunization with a vaccine directed against HER-2/neu in

mice allowed the activation of latent antigen-specific CD81 T

cells that were capable of mounting an anti-tumor response

(27). Efforts to abrogate natural Treg activity to boost vaccine

efficacy have also included depletion via treatment with a

monoclonal anti-CD25 antibody (28) and administration of

denileukin diftitox (Ontak, Ligand Pharmaceuticals, San Diego,

CA, USA) (29, 30). Other approaches to increasing tumor

vaccine efficacy by limiting tumor-mediated immuno-

suppression have included attempts to block suppressive

pathways in tolerogenic DCs and efforts to limit the effects

of suppressive cytokines, for example by administration of

neutralizing antibodies. Our recent studies have shown the

emerging importance of hardwired negative regulators of

proinflammatory signaling in antigen-presenting cells in the

maintenance of self-tolerance. Suppressor of cytokine signaling

1 (SOCS1) is a key regulator of cytokine receptor-mediated Janus

kinase (JAK)/signal transducer and activator of transcription

(STAT) signaling. We found that SOCS1 has a central role in

regulating the duration and intensity of antigen presentation by

DCs. Inhibition or silencing of SOCS1 in DCs results in the over-

activation of antigen-presenting DCs that in turn leads to

enhanced antigen-specific anti-tumor immunity, providing a

new strategy to break self-tolerance and enhance the potency of

tumor vaccines (31, 32). However, despite these advances in

targeting and inhibiting individual mechanisms of tumor-

mediated immune suppression, abrogating their collective

effects may prove to be difficult (33).

Immunosuppressive tumor microenvironments inhibit the

local anti-tumor immune response, both natural and in response

to vaccination, but they can also produce suppressive cells of

multiple phenotypes that migrate from the tumor to lymphoid

organs where they can mediate systemic immunosuppression.

These cells can inhibit immune responses even after the original

suppressive network has been eliminated, for example by tumor

resection, as discussed below. Moreover, metastasis of tumor cells

to a sentinel lymph node (LN), the first LN away from the

primary tumor in the lymphatic drainage pathway, leads to local

immunosuppression within that LN (34). This is significant as the

sentinel node is the first lymphoid organ in which antigenic

stimulation occurs as the first stage in the development of a



systemic immune response. Although the local immuno-

suppression mediated by metastatic tumor cells within LNs does

not normally lead to complete systemic immunosuppression, this

process nevertheless represents a mechanism by which the

presence of tumors can compromise the immune response of

the patient.

Systemic immune dysfunction was recently demonstrated in

the patients enrolled in a failed Phase II clinical trial of a

melanoma vaccine based on the glycoprotein MPS160 (35).

Although many patients demonstrated an increase in vaccine-

specific cytotoxic T-lymphocyte (CTL) numbers, as measured by

tetramer analysis, these cells were broadly incapable of

producing interferon-g (IFN-g) when stimulated with therelevant MPS160 peptides in vitro. Repeated immunizations

simply caused an increase in the numbers of these antigen-

specific cells that fail to produce IFN-g. Furthermore, expansionsin this non-responsive CTL population were correlated with

tumor progression events. Analysis of the peripheral blood of

vaccinated patients and healthy control subjects indicated that

the profile of plasma cytokines was skewed toward

immunosuppressive molecules. The authors hypothesized that

these suppressive cytokines were being produced within the

tumor microenvironment and were spilling over into the

plasma, resulting in a failure of systemic immune competence.

Alterations in the plasma cytokine profiles of cancer patients

compared with healthy subjects and those with benign tumors

have been highlighted in several studies. We recently completed

a study profiling the serum levels of 14 cytokines in 187 ovarian

cancer patients and compared them with those of 45 patients

with benign ovarian tumors and 50 healthy control subjects

(36). New cytokine bead array technology allowed the

simultaneous analysis of multiple cytokines present in serum

samples. Univariate analyses demonstrated that serum IL-6, IL-

7, and macrophage chemotactic protein-1 (MCP-1) were

increased in ovarian cancer patients compared with healthy

controls and patients with benign ovarian tumors (Po 0.05 inall cases). Many other studies have highlighted differences in

serum levels of cytokines in prostate cancer patients compared

with controls (37). Altered serum levels of IL-4, IL-6, IL-10,

and transforming growth factor-b (TGF-b) have been noted inprostate cancer patients, with several studies demonstrating that

increased IL-6 levels are a negative predictor of disease outcome.

Current conventional anticancer therapies havesignificant effects on the immune system

Current conventional cancer therapies are well known to have

potent immunomodulatory effects that can either inhibit or

enhance the anti-tumor immune response elicited by vaccina-

tion. Radiation therapy and many chemotherapeutic agents are

known to be immunosuppressive, particularly at high dosages.

Lymphocytes responding to antigens, including those stimu-

lated by vaccination against TAAs, rapidly proliferate. Therefore

they are susceptible to anticancer therapies that preferentially

kill proliferating cells (38).

Despite the traditionally held view that radiotherapy and

chemotherapy are generally immunosuppressive, the effects

of conventional anticancer treatments on the immune system

are extremely complex. As a result, chemotherapy and

radiotherapy can be combined with vaccination strategies to

improve their effectiveness so long as due attention is paid

to dosages and the relative timing of each treatment. For

example, doxorubicin and melphalan were shown not to

impede the efficacy of two separate vaccination strategies in

micewhen they were administered shortly before immunization

(39). An excellent recent review has covered scenarios in

which cytotoxic chemotherapies have been combined

with immunotherapy to yield enhanced responses (40).

Radiotherapy has also been combined with immunotherapy.

For example, a course of radiotherapy was recently integrated

into a vaccination schedule in prostate cancer patients (41).

Thirty patients were either given radiotherapy alone or

radiotherapy plus vaccination with a recombinant vaccinia-

prostate-specific antigen (PSA) vaccine followed by monthly

boosting with recombinant fowlpox-PSA. In the combination

therapy arm, patients received radiotherapy between the third

and fifth boosts. Thirteen out of 17 patients in the combination

arm developed PSA-specific cellular responses, versus none in

the radiotherapy-only arm (Po 0.0005). Given that there wasno immunotherapy-only treatment arm in this study, it cannot

be concluded that radiotherapy enhanced the immune response

to vaccination. However, the study ably demonstrates that local

radiotherapy does not inhibit antigen-specific responses elicited

by vaccination. Finally, we demonstrated that a peptide vaccine

directed against human papilloma virus (HPV) was capable of

eliciting complete protection against challenge with HPV-16-

expressing tumors in mice, despite prior treatment with pelvic

radiation or cisplatin. This was true even though the radiation-

and cisplatin-pretreated mice had measurably lower peptide-

specific immune responses to vaccination than untreated

controls, as measured by IFN-g ELISPOT (42).Despite the success of combining conventional anticancer

treatments with immunotherapy in certain instances, this

approach may not always be feasible. In many cases, the

optimal chemotherapeutic agents and/or radiotherapeutic

schedules available to physicians are dictated by the nature of



the cancer they are treating. Not all of these treatment options

will be suitable for combination with immunotherapeutic

strategies, and therefore not all cancers will be viable targets

for developing combination treatment strategies. Furthermore,

cancer patients are most commonly enrolled in clinical trials

for therapeutic vaccines as a treatment of last resort. Most

clinical immunotherapy trials exclude patients that have

received prior treatments that may affect vaccine efficacy, but

this is usually limited to treatment received o 1 month beforethe start of the trial. As a result, patients enrolled in cancer

immunotherapy clinical trials have already received a variety of

prior treatments that may have had long-lasting effects on their

immune competence. The complexity of the interactions

between these various treatments and the immune system

makes accurate assessment of the results of clinical trials and

indeed comparisons between otherwise similar trials

exceedingly difficult.

Therapeutic cancer vaccines have limited efficacy inpatients with minimal residual disease

It is well accepted that tumor growth is frequently too rapid for

the immune system to contain, despite the induction of a

robust anti-tumor response (40). Given that immunotherapy

in many cases can be successfully combined with certain

conventional therapies, the cancer immunotherapy field has

actively investigated a paradigm in which vaccines are admi-

nistered in the adjuvant setting, where minimal residual disease

is present. Clinical trials in which patients have been vaccinated

after their tumors have been debulked by surgical, chemother-

apeutic, or radiological means have been conducted for several

cancers, most notably in melanoma (4355), and lymphoma

(56, 57). Other examples of therapeutic cancer vaccines being

used in the adjuvant setting include those directed against

ovarian cancer (58, 59), lung cancer (60, 61), pancreatic

carcinoma (20, 62), and breast cancer (63).

Most clinical trials of therapeutic cancer vaccines in the

adjuvant setting have had positive but limited results. While

disease-free survival (DFS) and/or OS are often improved

(sometimes very significantly so), therapeutic vaccination in

the adjuvant setting is rarely capable of eradicating the residual

tumor cells that are present after surgery and eliciting a complete

response. This inability is probably because debulking tumors

removes only one component of the systemic immune

suppression that they establish, namely the intratumoral

suppressive milieu. One mouse study has demonstrated that

immunocompetence of the host can be restored upon surgical

removal of the primary tumor, despite the continued presence of

metastases in a mouse model of mammary carcinoma (64).

However, the limited results of human clinical trials suggest that

this effect is abrogated in humans, indicating that the

suppressive T cells/DCs that have already escaped the tumor

may be active in the LN and the immunosuppressive cytokine

profile present in the peripheral blood of the patient are capable

of limiting vaccine efficacy even in the absence of the

immunosuppressive tumor microenvironment. Nevertheless,

the improvements in DFS and OS observed in many studies of

therapeutic vaccination in the adjuvant setting indicate that

tumor-mediated immunosuppression is a major factor in the

limited efficacy of therapeutic cancer vaccines and that by

partially disrupting that immunosuppression their effectiveness

can be increased. These findings have led us and others (65) to

hypothesize that immunization with a therapeutic cancer

vaccine at the earliest stages of carcinogenesis before local and

systemic immunosuppressive environments are established by

the tumor will yield the best immune responses to vaccination

and therefore confer excellent protection against the progression

of cancer development.

Vaccination at early stages of carcinogenesis is highlyeffective at inducing anti-tumor immune responses andimproving survival

Patients with advanced cancer display systemic and local

(intratumoral) immunosuppression that is only partially alle-

viated by debulking of the tumor. Furthermore, overcoming

the combined tumor immunosuppressive mechanisms once

they become established is proving to be difficult. With these

considerations in mind, our group and others have directed

their attention to using therapeutic cancer vaccines in the

preventive setting.

We have recently demonstrated that a therapeutic vaccination

strategy directed against two different prostate TAAs (66, 67,

and authors unpublished observations) at the earliest stage of

carcinogenesis can elicit superb long-term protection against

spontaneous prostate cancer development in transgenic

adenocarcinoma mouse prostate (TRAMP) mice. In both

studies, we used a heterologous vaccination scheme involving

priming mice with a plasmid containing cDNA encoding the

target antigen, followed by boosting with Venezuelan equine

encephalitis (VEE) virus replicon particles (VRP) expressing the

same antigen. VEE VRP have been shown previously to efficiently

induce anti-tumor immune responses (68), and heterologous

vaccination schemes have been shown to increase

immunogenicity (69). The TAAs targeted in these studies, six-

transmembrane epithelial antigen of the prostate (STEAP) and



prostate stem cell antigen (PSCA), are strongly overexpressed in

both human and murine prostate cancers (7072).

TRAMP mice vaccinated against PSCA at an early stage of

carcinogenesis, at 8 weeks of age when they have developed only

premalignant prostate intraepithelial neoplasia (PIN) lesions,

showed dramatically improved survival compared with

unvaccinated age-matched controls (67). The OS of the PSCA

vaccinated group was 90% at 360 days, while all of the

unvaccinated controls had reached the survival endpoint by

day 360 post-vaccination. In contrast, PSCA vaccination at

16 weeks of age, when TRAMP mice have developed prostate

cancer, conferred no significant protection of mice compared

with unvaccinated age-matched controls (unpublished

observations). Interestingly, TRAMP mice vaccinated against

PSCA early (i.e. at 8 weeks of age) did develop prostate tumors,

but the majority of the mice presented with small well-

differentiated focal adenocarcinomas with extensive hyperplasia

and multiple apoptotic zones. Greater numbers of immune

cells, including macrophages, DCs, and IFN-g-expressingCD41 and CD81 T cells, infiltrated the prostate tumors of

PSCA vaccinated TRAMP mice compared with unvaccinated

controls. Intratumoral IL-5, IL-4, and IFN-g cytokine levelswere significantly increased in response to early PSCA

vaccination. In addition, transcription of IL-2 was significantly

upregulated within the tumors of mice vaccinated early against

PSCA compared with unvaccinated controls. Overall, TRAMP

mice vaccinated early against PSCA appeared outwardly healthy

at 18 months of age and had small, low-grade prostate tumors

that were in stark contrast to the very large, high-grade prostate

tumors observed in control vaccinated mice that typically

resulted in death before 9 months of age.

In a parallel study, we investigated the effectiveness of

vaccination against STEAP using our immunization protocol in

protecting TRAMP mice from prostate cancer (66). Initial

studies were carried out to determine whether STEAP

vaccination could protect C57BL/6 mice against challenge

with TRAMP-C2 prostate cancer cells. Interestingly, CD41 T

cells expressing IFN-g, TNF-a, and IL-2 played a major role intumor rejection. Furthermore, the presence of high IL-12 levels

in the tumor environment was associated with tumor rejection.

The therapeutic efficacy of STEAP vaccination was more modest

than its prophylactic effectiveness, but nevertheless it induced a

small but significant delay in the progression of established

TRAMP-C2 tumors. To assess the efficacy of STEAP vaccination

in a more physiologically relevant setting, TRAMP mice were

vaccinated against STEAP. In a striking parallel with our PSCA

study, TRAMP mice vaccinated at 8 weeks of age displayed

vastly superior survival compared with age-matched controls,

while mice vaccinated at 16 weeks of age showed little

improvement in survival (authors unpublished observations).

The extraordinary survival differences between mice

vaccinated against PSCA or STEAP at 8 versus 16 weeks of age

are most likely attributable solely to the stage of carcinogenesis

at which immunization occurs. Identical vaccination protocols

and reagents were used in each group of mice, and there is still

significant thymic output at 16 weeks of age in mice (73). We

hypothesize that by 16 weeks of age a suppressive prostate

tumor microenvironment has become established that inhibits

the anti-tumor immune response induced by vaccination. At 8

weeks of age, there is most likely a microenvironment within

PIN lesions that is still favorable for the establishment of an

anti-tumor immune response. This idea is demonstrated by the

vaccine-induced infiltration by active immune cells into the

prostate tumor that subsequently develops from these PIN

lesions and by the immuno-activating cytokine profiles that

develop within those tumors. We are currently involved in

studies designed to determine precisely at which stage of

carcinogenesis the prostate tumor microenvironment switches

from immune favorable to immunosuppressive. These studies

will allow us to intervene with cancer immunotherapy at the

latest possible stage of tumorigenesis at which vaccination can be

applied and still elicit an effective anti-tumor immune response.

Prevention of the spontaneous development of tumors by

vaccination at an early stage of carcinogenesis has also been

demonstrated in BALB-neuT mice that spontaneously develop

mammary tumors. Nava-Parada et al. (74) demonstrated that a

single vaccination of BALB-neuT mice with a peptide vaccine

derived from the RNEU TAA with concomitant administration

of the Toll-like receptor agonist CpG can significantly delay

spontaneous tumor development (74). BALB-neuT mice

remained completely tumor free until approximately 23

weeks of age, when they were vaccinated at 8 weeks of age, at

which time they display diffuse atypical hyperplasia but not

overt carcinoma. Interestingly, the authors report that peptide

vaccination at later stages of carcinogenesis was less effective

in eliciting anti-tumor immune responses and controlling

tumor growth.

Several studies have demonstrated that vaccination of

women with premalignant cervical intraepithelial neoplastic

lesions (CIN) can cause their complete eradication or partial

regression to a lower-grade lesion. Muderspach et al. (75)

demonstrated three complete responses and six partial

responses in 12 patients with grade II/III CIN that were

vaccinated with a vaccine directed against HPV E7 peptides.

Immunization with a recombinant protein (SGN-00101)

consisting of bacterial heat shock protein (M. bovis Hsp65)



fused to the complete HPV-16 E7 sequence was recently shown

by Roman et al. (76) to confer clinical benefit to patients with

high-grade CIN lesions. Of the 20 women enrolled in the study,

seven showed complete regression of their CIN lesions, one

had a partial regression to a low-grade lesion, 11 had stable

disease, and one patient progressed (76). A phase III

randomized study of the same vaccine conducted by Einstein

et al. (77) yielded very similar results. Of the 58 patients that

had completed the full vaccination protocol, 13 had a complete

response, 32 had partial responses, 11 had stable disease, and

two progressed (77). A vaccine based on a vaccinia virus vector

expressing recombinant MVA E2 has also been demonstrated to

be effective in treating high-grade CIN lesions (78). Of the 34

women immunized in a recent phase II clinical trial of the

vaccine, 19 showed complete regression, and 15 showed partial

regression (79). Early trials of other therapeutic cervical cancer

vaccines have demonstrated the generation of excellent immune

responses in patients with CIN lesions, although no clinical

responses were assessed (80, 81). Generally, vaccination of

CIN patients elicits the development of very robust immuno-

logical responses. This indicates that these patients are generally

immunocompetent, as is expected at the early stages of carcino-

genesis (82). Given the availability of efficacious therapeutic

vaccines and the accessibility and reliability of current screening

methods, cervical cancer represents an excellent candidate for a

treatment modality in which premalignant lesions are treated by

therapeutic vaccination.

Although the circumvention of immunosuppression

mediated by tumors and their treatment is the primary

justification for vaccination against premalignant lesions, in

some cases administration of therapeutic cancer vaccines has

had unexpected beneficial effects on the outcomes of

subsequent conventional treatments. As has been recently

discussed (83), superior responses to standard therapies have

been observed in patients who have initially received a

therapeutic vaccine and have subsequently been given

conventional treatments upon disease progression. This

fascinating phenomenon has been observed in trials of

vaccines directed against small-cell lung cancer (84) and

prostate cancer (85, 86). These observations are particularly

significant in the case of prostate cancer. The trials in question

were conducted in patients with HRPC who were probably

immunocompromised, given that prostate cancer patients with

metastatic disease are less able to mount immune responses

than patients that have less advanced disease (87). Early

detection of prostate cancer is commonplace as a result of PSA

screening. The potential side effects of conventional prostate

cancer therapies are more severe than the consequences of

living with contained, low-grade prostate cancer. As a result,

men with rising PSA levels frequently undergo very long

periods of active surveillance and only elect to undergo

curative surgery when it becomes apparent that the disease

has begun to progress. It is conceivable that therapeutic

vaccination during this watchful waiting phase may clear

premalignant prostate intraepithelial lesions or delay their

progression to prostate adenocarcinoma. If and when this

occurs, prior vaccination may then help enhance the efficacy

of standard prostate cancer therapies that the patient receives.

In summary, early vaccination of premalignant prostate lesions

is an attractive proposition, because it may yield clinical benefit

to patients at multiple stages of disease progression.

Several cancers will be excellent candidates forvaccination at the early stages of carcinogenesis becausenovel early detection methods are being developed andtherapeutic vaccines are available

Advances in genomics and proteomics have led to the develop-

ment of novel methods of cancer detection. Analysis of genetic

and epigenetic markers associated with carcinogenesis can be

performed on DNA obtained from whole tumor cells in the

peripheral blood, sputum, tumor ascites, and urine, or on

naked DNA shed into these fluids by dying cancer cells.

Analysis of DNA markers is exquisitely sensitive thanks to

polymerase chain reaction-based amplification of the minute

amounts of tumor DNA that are normally obtained. The

development of DNA microarray technology and the ability to

analyze thousands of DNA molecules simultaneously that it

affords will also revolutionize cancer diagnostics. In addition,

new proteomics methodologies, including very high-through-

put mass spectrometry and protein chip analysis, allow scien-

tists to search for cancer-associated protein markers at an

unprecedented resolution (88). Very intense efforts to use

these technologies and others are currently underway to dis-

cover novel biomarkers that will allow the very early detection

of many cancers, including ovarian, lung, and breast cancer.

There are several examples of promising therapeutic cancer

vaccines that are apparently capable of eliciting substantial

immune responses but are not able to eradicate tumors in

clinical trials. The development of new diagnostic tools may

allow researchers and physicians to reassess the efficacy of these

vaccines in the preventive setting by administering them at the

earliest possible stages of carcinogenesis. For example, a

vaccine based on an anti-idiotypic antibody that mimics the

ganglioside GD3, Bec2/bacille Calmette Guerin (BCG), was

recently demonstrated to be ineffective in improving either OS

or progression-free survival in a phase III study involving 515



small-cell lung cancer patients (61). Despite the lack of success

in improving patient survival, this vaccine elicited humoral

responses in approximately one-third of patients. Furthermore,

there was a trend toward improved survival in patients with

humoral responses versus those without (P=0.085), although

this finding was weakened when the data were stratified for a

confounding factor, namely the administration of prophylactic

cranial irradiation being more prevalent in the group that

developed humoral responses. A requirement for enrollment

in this study was that patients had only limited disease as

defined by the Veterans Administration Lung Study Group

criteria. However, patients with limited disease are defined

by these standards as having primary tumor and nodal

involvement limited to one hemithorax (89). Given that even

this is a relatively advanced stage of disease, it remains possible

that the Bec2/BCG vaccine may yield improvements in patient

survival if it is administered at a much earlier stage of

carcinogenesis. Another example of a currently available

vaccine that may benefit from early administration resulting

from improved screening methodologies includes a breast

cancer vaccine consisting of a HER-2/neu peptide (E75)

mixed with GM-CSF that has been successful in preventing

disease recurrence in node-positive patients with minimal

disease burden (90). Similarly, oregovomab (mAb B43.13)

has been demonstrated to increase time to progression in

ovarian cancer, but only in the subgroup of patients that had

the smallest residual tumors (59). It is possible that one or both

of these vaccines would be even more successful if

administered earlier in the development of disease. Improved

screening and identification of biomarkers for lung, breast, and

ovarian cancers may soon allow the vaccines that are available

for them to be tested in the preventive setting, which may lead

to improved patient outcomes.

Conclusion

The results of therapeutic cancer immunotherapy trials con-

tinue to be disappointing, despite major advances in vaccine

technology over the past decade. It has become apparent that

while the vaccines themselves have the potential to elicit potent

anti-tumor immune responses, the cancer patients in which

they are tested are simply not capable of mounting robust

responses to the vaccines. The compromised immunity in

patients with advanced cancer is largely unavoidable and,

currently, untreatable. This is broadly true even in instances

where conventional anticancer treatments have been used to

generate a scenario of minimal residual disease in which to

administer immunotherapy. Tumor- and treatment-mediated

immune suppression in cancer patients can be avoided by

vaccinating them early in disease, before other treatments are

dispensed and before the establishment of systemic immune

failure by the tumor. Rapid advances in cancer screening have

been made, and improvements in early cancer detection will

continue to advance as genomic and proteomic technologies

become more integrated into cancer diagnostics. With todays

technology, early detection methods and efficacious therapeutic

vaccines exist for two extremely common human cancers,

prostate and cervical, that allow the vaccination of patients at the

very earliest stages of carcinogenesis. With these considerations

in mind, we propose a paradigm shift away from the use of

therapeutic cancer vaccines in patients with advanced disease and

toward their application in the earliest stages of tumorigenesis.

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