Immune responses in cancer

www.elsevier.com/locate/pharmthera

Pharmacology & Therapeutics 99 (2003) 113–132

Immune responses in cancer

Jamila K. Adama, Bharti Odhavb, Kanti D. Bhoolac,*

aDepartment of Medical Science, Durban Institute of Technology (ML Sultan Campus), Durban, South AfricabDepartment of Biological Science, Durban Institute of Technology (ML Sultan Campus), Durban, South Africa

cAsthma and Allergy Research Institute, University of Western Australia, Ground Floor, E Block, Sir Charles Gairdner Hospital,

Hospital Avenue, Nedlands, WA 6009, Australia

Abstract

The complex of humoral factors and immune cells comprises two interleaved systems, innate and acquired. Immune cells scan the

occurrence of any molecule that it considers to be nonself. Transformed cells acquire antigenicity that is recognized as nonself. A specific

immune response is generated that results in the proliferation of antigen-specific lymphocytes. Immunity is acquired when antibodies and T-

cell receptors are expressed and up-regulated through the formation and release of lymphokines, chemokines, and cytokines. Both innate and

acquired immune systems interact to initiate antigenic responses against carcinomas. A new approach to the treatment of cancer has been

immunotherapy, which aims to up-regulate the immune system in order that it may better control carcinogenesis. Currently, several forms of

immunotherapy that use natural biological substances to activate the immune system are being explored therapeutically. The various forms of

immunotherapy fall into three main categories: monoclonal antibodies, immune response modifiers, and vaccines. While these modalities

have individually shown some promise, it is likely that the best strategy to combat cancer may require multiple immunotherapeutic strategies

in order to demonstrate benefit in different patient populations. It may be that the best results are obtained with vaccines in combination with

a variety of immunotherapy combinations. Another potent strategy may be in combining with more traditional cancer drugs as evidenced

from the benefit derived from enhancing the efficacy of chemotherapy with cytokines. Through such concerted efforts, a durable, therapeutic

antitumour immune response may be achieved and maintained over the course of a patient’s lifespan.

D 2003 Elsevier Science Inc. All rights reserved.

Keywords: Carcinogenesis; Immunity; Immunotherapy; Tumour antigens and surveillance; Vaccines

Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; APC, antigen-presenting cells; ATP, activated receptor pathway; Bcl-2, B-cell

lymphomal leukaemia-2 protein; CDK, cyclin-dependent kinases; CEA, carcinoembryonic antigen; CTL, cytotoxic T-lymphocytes; EGFR, epidermal growth

factor receptor; FGF, fibroblast growth factor; GM-CSF, granulocyte-monocyte colony stimulating factor; HER-2, human epidermal growth factor receptor;

HLA, human leucocyte antigen; HSP, heat shock protein; IFN, interferon; IKB, inhibitors of KB; IL, interleukin; IL-1R, interleukin-1 receptor; IL-1RA,

interleukin-1 receptor antagonist; LPS, lipopolysaccharide; MHC, major histocompatibility complex; NF-kB, nuclear factor-kB; NK, natural killer; PGE2,

prostaglandin E2; PMN, polymorphonuclear leucocytes; RAS, rat sarcoma gene product protein, p21ras; TGF-b, transforming growth factor-b; TNF, tumour

necrosis factor; VEGF, vascular endothelial growth factor; WT1, Wilms’ tumour gene.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

1.1. Immunity: historical preamble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

1.2. Innate immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

1.3. Acquired immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

1.4. Antigen recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

1.5. Cell populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

1.6. Immune cell regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

1.7. Immune cell modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

1.7.1. Interleukin-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

1.7.2. Tumour necrosis factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

0163-7258/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved.

doi:10.1016/S0163-7258(03)00056-1

* Corresponding author. Tel.: +61-8-9346-2954; fax: +61-8-9346-2816.

E-mail address: [email protected] (K.D. Bhoola).

J.K. Adam et al. / Pharmacology & Therapeutics 99 (2003) 113–132114

1.7.3. Interleukin-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

1.7.4. Interleukin-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

1.7.5. Interleukin-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

1.7.6. Granulocyte-macrophage colony stimulating factor. . . . . . . . . . . . . . . . 119

2. Carcinogenic cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

2.1. Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

2.2. Cell cycle progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

2.3. DNA replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

2.4. Evading apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

2.5. Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

2.6. Metastasis and invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

3. Tumour antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

4. Immune surveillance and detection of tumours . . . . . . . . . . . . . . . . . . . . . . . . . . 122

5. Immune-directed apoptosis of cancer cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

6. Tumour escape mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

7. Cancer immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

7.1. Molecular aspects of immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

8. Immunotherapeutic strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

8.1. Monoclonal antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

8.2. Immune response modifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

8.2.1. Interferon-a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

8.2.2. Interleukin-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

8.2.3. Interleukin-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

8.3. Colony stimulating factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

8.4. Vaccination against tumours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Table 1

Immune response modulators

Cytokines Lymphokines Chemokines Growth factors

IL-1a IL-2 IL-8 EGF

IL-1b IL-3 Gro-a TGF-bIL-6 IL-4 Gro-b FGF

IL-10 GM-CSF MIP-1a PDGF

IL-13 IF-g MIP-1b TGF-b family

TNF-a MCP-1, -2, and -3

1. Introduction

1.1. Immunity: historical preamble

The earliest record of treating a patient with cancer comes

from the Edwin Smith papyrus dated 1600 B.C. The papyrus

documents a surgical procedure that was established as the

primary method of treating solid tumours in the 19th century

and remains so today. Radiation therapy came into practice

around 1896, just 1 year after Roentgen first reported the use

of X-rays for diagnostic purposes in medicine. Chemother-

apy is an invention of the 19th century (Old, 1996). William

B. Coley, a New York City surgeon, is often credited with

first recognizing the potential role of the immune system in

cancer treatment (Coley, 1991). He observed that some of

his patients with sarcoma would undergo spontaneous

regression of their tumour. However, not until 1976 was it

possible to more fully understand how tumour recognition

and rejection was mediated at the cellular and molecular

level.

1.2. Innate immunity

The complex of cells and humoral factors that is known

collectively as the immune system may be divided into two

interrelated cascades. The first such functional system is

known as innate (natural) immunity. In general, the term

immunity implies acquired immunity with its antibodies and

T- and B-lymphocyte populations. Innate immunity involves

a large number of different cell populations such as epithe-

lial cells, monocytes, macrophages, dendritic cells, poly-

morphonuclear leucocytes (PMN), natural killer (NK) cells,

and various lymphocyte subpopulations (e.g., CD5-positive

B-lymphocytes and gd T-lymphocytes), which bridge the

divide between innate and acquired immunity. These cells

generally arise from precursor cell populations in the bone

marrow. Humoral systems are also important and include a

large number of cytokines (Table 1), certain enzymes (e.g.,

lysozyme), metal-binding proteins, integral membrane ion

transporters, complex carbohydrates, and complement path-

ways. Molecules that act as recognition ‘‘receptors’’ in

innate immunity comprise humoral proteins (c-reactive

protein, serum amyloid, mannose binding protein, and

CD14) and cellular receptors (scavenger and mannose

J.K. Adam et al. / Pharmacology & Therapeutics 99 (2003) 113–132 115

receptors, dendritic cell targets, CD14, CD35, CD21, and

CD11b [see Table 2]). Cytokine formation, release, and

target interactions form an important arm of the cellular

response in growth, repair, and cell proliferation. The

notable cytokines modulating the proliferation of tumour

cells are probably transforming growth factor-b (TGF-b)family, epithelial growth factor, and colony stimulating

factors.

1.3. Acquired immunity

The second, termed acquired or adaptive immunity,

evolved around 400 million years ago. Acquired immun-

ity compromises a unique mechanism whereby genetic

‘‘mutation’’ occurring in two specialised cell populations,

B- and T-lymphocytes (Fig. 1), produces numerous

molecular ‘‘shapes’’ that are expressed as antibodies and

T-cell receptors. If these molecules bind to structurally

related proteins called antigens, and provided costimula-

tory signals are present, proliferation of antigen-specific

lymphocytes occurs and a specific immune response is

generated. The specificity of immune response resides in

selective clonal proliferation of lymphocytes. Two further

families of molecules play major roles in acquired

immunity: major histocompatibility complex (MHC) gene

products and cytokines. They provide an essential link for

cell-to-cell communication and component of the acquired

(as well as the innate) immune response (Clark &

Ledbetter, 1994). The cell surface protein CD40 with its

receptor provides a costimulatory signal for the inter-

action between T-cells and antigen-presenting cells (APC)

(Chen et al., 2002).

Table 2

Immunological receptors

CD number Function

CD1a/1b/1c Peptide, lipid antigen presentation

CD2 T-cell adhesion to APC

CD3 T-cell activation

CD4 Th activation

CD5 T-cell activation

CD8 T-cell subpopulation marker

CD11a/CD18 Leukocyte adhesion protein

CD11b/CD18 Leukocyte adhesion protein

CD11c/CD18 Leukocyte adhesion protein

CD14 LPS binding protein

CD16 Phagocytosis and ADCC

CD19 B-cell activation/proliferation

CD23 B-cell activation/IgE regulation

CD28 Costimulatory molecule

CD54 Adhesion molecule

CD62E Vascular adhesion molecule

CD71 Receptor for transferrin



CD88 C5a receptor


1.4. Antigen recognition

Antibody synthesis is the molecular response to the

presence of nonself epitopes (Fig. 2). Antibodies are specific

immunoproteins formed by B-cells in response to nonself

molecules. Antibodies react directly against the antigen or

Fig. 1. Antigen recognition and antibody formation.

Fig. 2. B-lymphocytes.

Fig. 3. Dendritic cell interactions.


by activation of the complement system. One of the more

important complement effects includes opsonization and

phagocytosis. A product of the complement cascade

strongly activates phagocytosis by neutrophils and macro-

phages, which engulf nonself proteins. This type of cellular

event is called antibody-dependent cell-mediated cytotox-

icity (ADCC) and has the advantage of enhancing T-cell

activity. Antibodies have also been shown to kill cells by

blocking growth mechanisms, particularly in cancer cells.

Growth factor proteins such as human epidermal growth

factor receptor (HER-2)/neu can become overexpressed

after the gene that encodes the protein is amplified. Anti-

bodies specific for HER-2/neu bind to the molecule on the

surface of the cell and block the growth signal (Slamon et

al., 2001).

1.5. Cell populations

Cell populations (macrophages, dendritic cells, NK cells,

and neutrophils) play a major role in both innate and

acquired immunity. However, the two key cell populations

that essentially define acquired immunity are the B- and T-

lymphocytes (Gowans et al., 1962). Adaptive immunity,

therefore, involves a wide range of antigen receptors

expressed on the surface of T- and B-lymphocytes that

detect nonself molecules. T-cells instruct affected host cells

to either shut down protein synthesis or commit suicide. B-

cells respond to antigens by secreting their own antigen

receptors as antibodies. Antibodies also call on the innate

immune system for help. Stimulation of antigen through the

B-cell receptor followed by T-cell activation drives prolif-

eration and differentiation of antigen-specific naive B-lym-

phocytes into memory B-cells and plasma cells. Memory B-

cells mediate secondary immune responses and plasma cells

sustain antibody production for several months.

The different subpopulations of T-cells (Mason, 1987)

are recognized largely by their expression of surface pro-

teins (CD markers). All T-cells express CD3, a heterooligo-

meric protein that is part of the T-cell receptor complex, and

can be further subdivided into those cells that express CD4

and those that express CD8. The CD4 lymphocyte popu-

lation can be further functionally subdivided into two

subpopulations termed Th1 and Th2 lymphocytes (Mason,

1988). Th1 and Th2 cells are initially discriminated not on

the basis of cell surface markers but on the basis of the

patterns of cytokines they produce. In very recent years, it

has proved possible to identify these distinct CD4 subpo-

pulations on the basis of their expression of chemokine

receptors.

Dendritic cells constitute a family of APC defined by

their morphology and their capacity to initiate primary

immune response (Rafiq et al., 2002). Langerhans cells

are paradigmatic dendritic cells, described in 1868 by a

young medical student, Paul Langerhans, in Berlin. Langer-

hans cells are present with epithelial cells in the epidermis,

bronchi, and mucosae (Fig. 3). After antigenic challenge,

the dendritic cells migrate into the T-cell areas of proximal

lymph nodes where they act as professional APC. Langer-

hans cells originate in the bone marrow and CD34+ hae-

matopoietic progenitors are present in cord blood or

circulating blood. They are actively involved in skin lesions

of allergic contact dermatitis or atopic dermatitis in cancer

immunosurveillance (Schmitt, 2001).

Human neutrophil transcribes and secretes peptides

termed a-defensin-1, -2, and -3 in response to nonself

proteins. Defensins have the ability to signal activation of

cells involved in adaptive immunity, specifically CD8+ T.

a-defensin and b-chemokines (MIP-1a, MIP-1b, and

RANTES) may be cooperatively involved in cell defense.

b-defensins are small peptides of the innate immune system.

b-defensins2 may act directly on immature dendritic cells, as

an endogenous ligand for Toll-like receptor 4, to up-regulate

dendritic cell maturation, thereby triggering adaptive imma-

ture responses. It is suggested that b-defensins2 may provide

molecular immunosurveillance against tumour antigens.

The role of macrophages in tumour growth and devel-

opment is complex and multifaceted (Gough et al., 2001).

Whilst there is limited evidence that tumour-associated

macrophages can be directly tumouricidal and stimulate

the antitumour activity of T-cells, there is now contrasting

evidence that tumour cells are able to block or evade the

activity of tumour-associated macrophages at the tumour

site (Bingle et al., 2002).

1.6. Immune cell regulation

Important scientific discoveries in recent years have led

to an increase in our understanding of the role of helper T-

cells. T-cells are either cytotoxic (CD8+) or helpers

(CD4+). Unlike antibodies, which react with intact proteins

only, the CD8+ T-cells react with peptide antigens

expressed on the surface of a cell. Peptide antigens are

those that have been digested by the cell and presented as

peptides displayed in the MHC. MHC-encoded molecules

govern immune responses by presenting antigenic peptides

to T-cells. The peptide and the MHC together attract T-


helper cells. CD8+ T-cells are specific for class I MHC

molecules, while CD4+ T-cells are specific for class II

MHC molecules. After attaching to the MHC-peptide

complex expressed on a cell, the CD8+ T-cell destroys

the cell by perforating its membrane with enzymes or by

triggering an apoptotic or self-destructive pathway. The

CD8+ T-cell will then move to another cell expressing

the same MHC-peptide complex and destroy it as well. In

this manner, cytotoxic T-cells can kill many invasive cells.

Ideally, CD8+ T-cells could engender a very specific and

robust response against tumour cells. Cytotoxic T-lympho-

cytes (CTL) are considered to be essential effectors of the

cell-mediated immune response. The ability of CTL to

specifically recognize and lyse malignant cells expressing

the relevant surface antigens under optimal in vitro con-

ditions justifies attempts to boost their number and activity

through various forms of immunotherapy (Titu et al.,

2002).

NK cells also form a line of defense against host cells

that are stressed and/or cancerous. NK cells express surface

receptors that receive signals from the environment and

determine their response to foreign or malignant cells. NK

cells respond to these signals by producing effector mole-

cules, which can both directly suppress tumour growth and

convey important information to the rest of the immune

system (Smyth et al., 2002).

The helper T, or CD4+ T-cell, is the major regulator of

virtually all immune system activities. These cells form a

series of protein mediators called lymphokines that act on

other cells of the immune system and on bone marrow.

Some of the most important lymphokines secreted by the

helper T-cells include interleukin (IL)-2, IL-3, IL-4, IL-5,

IL-6, granulocyte-monocyte colony stimulating factor

(GM-CSF), and interferon (IFN)-g (see Table 1). Without

these lymphokines, the remainder of the immune system

does not function as effectively as it would with the

appropriate cytokine environment. Lymphokines produced

by helper T-cells also regulate macrophage response. The

lymphokines slow or stop the migration of macrophages

after they have been engaged, allowing macrophages to

accumulate at the site. These lymphokines also stimulate

more efficient phagocytosis, so they can destroy rapidly

increasing numbers of toxins. The T-helper cell amplifies

itself by secreting lymphokines, particularly IL-2. This

action enhances the helper cell response as well as the

entire immune system’s response to foreign antigens. Like

CD8+ T-cells, the CD4+ T-cells also recognize MHC-

peptide complexes in the context of class II MHC. CD4+

T-cells augment the immune response by secreting cyto-

kines that stimulate either a cytotoxic T-cell response (Th1helper T-cells) or an antibody response (Th2 helper T-cells).

These cytokines can initiate B-cells to produce antibodies

or enhance CD8+ T-cell production. The function of the

CD4+ T-cell depends upon the type of antigen it recognizes

and the type of immune response required. The immuno-

logical dysfunction associated with human cancer com-

prises changes within the immune network including

cytokine imbalance of Th1/Th2 origin (Lauerova et al.,

2002).

The immune response to carcinoma cells like allograft

rejection is initiated through activation of alloreactive T-

cells and APC (e.g., monocyte-macrophages, dendritic

cells, and B-cells). This process involves the activity of

antibodies, adhesion molecules, cytokines, and lympho-

kines. A characteristic feature is the infiltration of the

tumour or graft by host mononuclear cells (lymphocytes

and macrophages). Immunohistologically, these have been

characterized as T- and B-lymphocytes, macrophages, and

NK cells (Medawar, 1944; Mason & Morris, 1986). Stimu-

lated B-lymphocytes differentiate into antibody-producing

plasma cells, which secrete nonspecific and specific nonself

antibodies (Tilney et al., 1979). The immunological

response to the foreign tissue proteins comprises two limbs:

an afferent or sensitising limb and an efferent or effector

limb (Gowans et al., 1962). T-cell activation begins when

T-cells recognize intracellularly processed fragments of

foreign proteins embedded in the groove of the MHC

proteins, expressed on the surface of APC (Krensky et

al., 1990; Weiss & Littman, 1994).

CD4 and CD8 proteins, expressed by peripheral blood T-

cells, bind to human leucocyte antigen (HLA) class II and

class I molecules, respectively (Miceli & Parnes, 1991). The

complex of T-cell antigen receptor and CD3, CD4, and CD8

proteins physically associate with and activate several intra-

cellular protein tyrosine kinases, resulting in mobilization of

ionised calcium from bound intracellular stores by inositol

triphosphate. The increased intracellular free calcium and

sustained activation of protein kinase C promote expression

of genes central to T-cell growth.

Cytokines are a family of peptide molecules that are

responsible for direct cell-to-cell communication. They

mediate interactions between leucocyte populations and

between leucocytes and tissue cells (Table 3). Cytokines

of the IL family, derived from APC (namely, IL-1 and IL-

6), also provide costimulatory signals that result in T-cell

activation. T-cell-derived lymphokines (e.g., IL-2 and IL-

4), and contact between T- and B-cells through specific

pairs of receptors and coreceptors, provide signals essential

for B-cell stimulation (Clark & Ledbetter, 1994). T-cell

proliferation is the result of IL-2 expression that is depend-

ent on T-cell activation.

The net result of cytokine production is the emergence

of antigen specific, tissue infiltrating, and destructive T-

cells. Cytokines also activate macrophages and other

inflammatory cells and the production of antibodies by

stimulated T-cells. Cytokines can amplify the ongoing

immune response by up-regulating the expression of

HLA antigens and costimulatory molecules (such as B7)

on parenchymal cells and APC. The costimulators direct T-

cell differentiation, for example, into a CD4+ Th1 cell,

which secretes lymphokines, facilitating CTL killing of

cells, or differentiates into a CD4+ Th2 cell, which

Table 3

Cellular expression and actions of cytokines

Cytokine Source Actions

IL-1 Macrophages, fibroblasts,

synovial lining cells

. Stimulates production of

IL-6 and TNF-aT-and B-lymphocytes,

endothelial cells

. Augments T-cell proliferation

and B-cell activation

. Induces hepatic production

of acute phase proteins

. Activates neutrophils to

synthesize and release PG

. Increases binding of

lymphocytes and monocytes

to endothelial cells

. Induces endothelial cell

proliferation

TNF Macrophages, monocytes . Stimulates production of

IL-1, -6, and -8

. Increases PGE2 and

collagenase production

. Increases plasminogen

activity

. Increases release of FGF

. Modulates PMN function

such as the release of

oxygen metabolites,

phagocytosis, adhesion to

endothelium, and ability to

degrade cartilage

IL-6 Neutrophils, monocytes,

fibroblasts, T- and B-cells,

. Stimulates the release of

hepatic acute phase proteins

endothelial cells . Induces activated B-cells to

differentiate into plasma cell

IL-8 Neutrophils, fibroblasts,

hepatocytes, epithelial

and endothelial cells

. Stimulates and attracts

neutrophils

GM-CSF Macrophages, fibroblasts,

endothelial cells,

. Stimulates secretion

of IL-1, TNF-a, and PGE2

and activated lymphocytes . Activates chemotaxis,

phagocytosis, antibody

cytotoxicity, and oxidative

metabolism in granulocytes

. Induces HLA-DR expression

on monocytes

Abbreviations: IL, interleukin; TNF, tumour necrosis factor; PG, prosta-

glandin; FGF, fibroblast growth factor; GM-CSF, granulocyte-macrophage

colony stimulating factor.


stimulates antibody production by B-cells (Dallman, 1995).

Cell killing may occur via specific T-cell products, such as

granzyme B (a serine esterase protein) and perforin (a

pore-forming lytic protein), which have been reported to

correlate closely with acute rejection of grafts (Clement et

al., 1994). The type of organ grafted, HLA matching

between donor and host and the degree of presensitisation,

influence the acute rejection process. CD4+ T-helper cells

are the primary, initiating, and organizing component of

host immunoresponsiveness against grafts. CD8+ cells are

recruited secondarily to the site to complete the acute

rejection process (Mason & Morris, 1986; Mason, 1987).

It is considered that these cellular and molecular responses

observed during graft rejection will apply to transformed

cells as they become carcinogenic.

1.7. Immune cell modulation

1.7.1. Interleukin-1

The IL-1 family consists of IL-1a, IL-1b, and IL-1

receptor antagonist (IL-1RA), which are structurally related

to each other and have similar affinity for IL-1R on cells

(Dinarello, 1994). IL-1a and IL-1b are potent agonists that

elicit various biological responses, whereas IL-1RA blocks

the effects of the agonists by competing for binding sites on

the cell surface receptors (Arend, 1993). IL-1a, IL-1b, andIL-1RA are encoded by separate genes, designated as ILIA,

ILIB, and ILIRN, respectively. The three genes are clustered

on the long arm of human chromosome 2 in a region (q13–

q21) that spans more than 430 kb (Nicklin et al., 1994).

Three related cell surface proteins are involved in IL-1

binding and signalling: type 1 IL-1R, type 2 IL-1R, and

IL-1R-associated protein (Sims & Dower, 1994). The IL-1R

genes are members of the large immunoglobulin supergene

family and are located in the same region of human

chromosome 2 as their ligands.

IL-1 is produced during antigen presentation and is

secreted by macrophages, fibroblasts, endothelial cells, and

T- and B-lymphocytes (Lorenzo, 1991). It has a wide range of

biological actions and acts via modulating gene expression in

target cells. Both IL-1a and IL-1b possess comitogenic

properties, recruit cells to the cancer site, and stimulate the

production of proinflammatorymediators, including IL-6 and

tumour necrosis factor (TNF). IL-1 has been shown to

augment T-cell proliferation and B-cell activation in response

to antigenic challenge (Dinarello et al., 1986). The cytokine

activates neutrophils to synthesize and release prostaglandins

(Rossi et al., 1985), enhance binding of lymphocytes and

monocytes to endothelial cells, and induce neovasculariza-

tion, a process that may encompass tumour initiation of new

blood vessels.

1.7.2. Tumour necrosis factor

TNF is produced principally by macrophages and mono-

cytes. The production of TNF is stimulated by several factors,

including lipopolysaccharide (LPS), IL-1, and GM-CSF, and

mediates its effects by interaction with two related membrane

receptors: TNF-R1 and TNF-R2 (or type I and type II). TNF-

a is thought to be the controlling element in the ‘‘cytokines

network’’ and is responsible for the production of other

cytokines (e.g., IL-1, IL-6, and IL-8) (Brennan et al., 1992).


IL-6 is produced bymonocytes, T-lymphocytes, and fibro-

blasts. The synthesis of this cytokine is induced by IL-1 and

TNF-a (Wong & Clark, 1988). IL-6 stimulates activated B-

cells to differentiate into plasma cells, which produce immu-

noglobulins (Arend&Dayer, 1990).Circulating levels of IL-6

are elevated in prostate carcinoma (Adler et al., 1999).

y & Therapeutics 99 (2003) 113–132 119


IL-8 is a member of the chemokine supergene family.

The chemokines belong to two related polypeptide families,

C-X-C and CC chemokines, as defined by the location of

the two cysteine residues at the amino terminus. In the C-X-

C family, the cysteine residues are separated by a non-

conserved amino acid; in the CC family, the cysteine

residues are in juxtaposition (Baggiolini et al., 1994). The

C-X-C chemokines are clustered on human chromosome 4.

IL-8 is a kDa peptide of the C-X-C chemokine family. It is a

potent neutrophil attractant and stimulator and is produced

by neutrophils, fibroblasts, hepatocytes, and epithelial and

endothelial cells (Baggiolini et al., 1989). IL-1, TNF, and

LPS-stimulated neutrophils exhibit increased expression of

IL-8 mRNA and IL-8 production (Strieter et al., 1992).


The lymphokine IL-2 is essential in stimulating T- and B-

cell populations to divide and expand their clones. IL-2 is

produced by antigen-stimulated T-lymphocytes and must be

present in sufficient quantities in order to mount an effective

counterattack against cancer cells. Increased formation of

IL-2 is an important way of expanding Tc-lymphocyte

population. These killer cells can accomplish lysis of

tumour cells through cell-to-cell contact. Two categories

of this cell type are believed to exist, namely, (1) antigen-

specific, MHC-restricted (well-established) Tc lymphocytes

and (2) broad specificity, non-MHC-restricted Tc lympho-

cytes. Thus, antigen-specific killer cells would recognize

cells with specific tumour markers, whereas those with

broad specificity could lyse a variety of different targets

on the tumour cell.

1.7.6. Granulocyte-macrophage colony stimulating factor

GM-CSF is a growth factor that is synthesized by macro-

phages, fibroblasts, endothelial cells, and activated lympho-

cytes (see Table 3) (Groopamn et al., 1989). It was first

characterized based on promotion of growth and differenti-

ation of granulocytes and macrophages. GM-CSF stimulates

the secretion of IL-1 and enhances the secretion of TNF-aand prostaglandin E2 (PGE2) from macrophages (Fischer et

al., 1988; Heidenreich et al., 1989). In addition, GM-CSF

activates chemotaxis, phagocytosis, antibody-dependent

cytotoxicity, and oxidative metabolism in granulocytes

(Firestein, 1994) and induces HLA-DR expression on

monocytes (Xu et al., 1989).

J.K. Adam et al. / Pharmacolog

2. Carcinogenic cascade

2.1. Proliferation

A characteristic of cancer cells is its ability to undergo

extensive proliferation through overproducing growth fac-

tors (vascular endothelial growth factor [VEGF] and fibro-

blast growth factor [FGF]) and/or overexpressing receptors

for growth factors (HER-2, epidermal growth factor recep-

tors [EGFR], and platelet-derived growth factor receptor).

Cellular proliferation begins when cell surface receptors

recognize their appropriate growth factors. The function of

extracellular signal-regulated kinases is to control cell

division, namely, meiosis, mitosis, and postmitotic functions

in differentiated cells. They are members of a specific

molecular group that is activated by the protooncogene

Ras. The next event is a cascade of reactions mediated by

cytoplasmic protein kinases (rat sarcoma gene product

protein, p21ras [RAS], ras-associated factor, MEK, and

MAPK cascade) that culminates in transcriptional activa-

tion, cell cycle progression, and cell division. Inhibition of

such a growth factor-induced mitogenic signalling may be

achieved through (i) binding of the growth factor to its

receptor or receptor dimerization with a specific agent,

namely, a monoclonal antibody, (ii) preventing receptor

activation with small molecule inhibitors, which bind to

the activated receptor pathway (ATP)-binding site in the

intracellular domain of the receptor, and (iii) inhibition of

cytoplasmic proteins downstream of the ATP (namely,

preventing formation of membrane-bound ‘‘activatable’’

RAS). Mutations that convert Ras to an activated oncogene

are common oncogenic mutations in human tumours.

2.2. Cell cycle progression

The four phases of the cell cycle are the M phase (state of

active mitosis), the S phase (state of DNA synthesis), and

the two G or gap phases that separate the other phases.

Cyclins and cyclin-dependent kinases (CDK) govern the

progression of cells from one phase to another (Sender-

owicz, 2001). Inhibiting cells at any point in the cell cycle

inhibits their progression through the cell cycle, thereby

preventing mitosis and cell division. The tumour suppressor

gene (p53) (Levine, 1997) and CDK inhibitors (p15, p16,

p21, and p27) are negative regulators of cell cycle progres-

sion, causing growth arrest. Commonly used cytotoxins

block the cell cycle in the S and G2/M phases (Malumbres

& Barbacid, 2001). The question arises whether carcinoma

cells secrete cytotoxins that target specific components of

the cell cycle. Nucleostemin, a stem cell gene product, if

produced by the tumour cell would bind to p53 and arrest its

function. Mutations of p53 are implicated in the many

different cancers.

2.3. DNA replication

Tumour cells have infinite replicative potential. DNA

replication takes place in the S phase of the cell cycle. The

process of replication essentially duplicates the genetic

material with the help of the replication machinery, namely,

DNA polymerases, DNA ligases, and topoisomerases.

Inhibiting replication ensures that malignant cells do not

progress in the cell cycle. Telomerase is a key component in

immortalization of malignant cells by preserving the integ-

Fig. 4. Diapedesis of tumour cell into the circulation.


rity of telomeres. Consequently, immunotherapeutic inhibi-

tion of telomerase abolishes the potential of cancer cells to

become immortal. Specific, separate signalling pathways are

activated in normal, in contrast to tumour, cells in response

to DNA damage. In normal cells, DNA damage results in

p53-dependent transcription of the CDK inhibitor p21. This

causes activation of Rb through inhibition of the CDK that

inactivates Rb. In normal cells, DNA damage evokes

activation of the transcription factor p53, which in turn

up-regulates the expression of CDK inhibitor p21 and BH3-

domain-containing proapoptotic proteins, PUMA and

NOXA. As a consequence of p21 suppression of cyclin

CDK activity, the gene Rb is activated and prevents deam-

ination of Bcl-XL. The intact Bcl-XL inhibits the activity of

BH3-domain-containing proteins, PUMA and NOXA, to

activate Bak/Bax, release of cytochrome c, and caspase

proteolysis. In contrast in tumour cells that lack Rb, deam-

ination occurs leading via the signalling steps to apoptosis

of tumour cell (Li & Thompson, 2002).

2.4. Evading apoptosis

Cells undergo apoptosis through death sensors and

effectors (Evan & Vousden, 2001). Sensors that trigger the

death pathway are linked to apoptosis-inducing ligands.

Apoptosis-inducing factor, a mitochondrial oxireductase, is

released into the cytoplasm to induce cell death in response

to apoptotic signals. Many of the death signals converge on

the mitochondria where the release of cytochrome c cata-

lyses apoptosis (Griffiths et al., 1999). Caspases are death

effector molecules that ultimately transmit the death signal

(Cohen, 1997; Earnshaw et al., 1999).

Evading apoptosis requires mechanisms that inhibit the

transmission of the death signals. Proapoptotic effectors

include B-cell lymphomal leukaemia-2 protein (Bcl-2),

proteins that belongs to the antiapoptotic Bcl-2 family

(Bcl-XL, Mcl-1, and A1) (Attardi et al., 1993). Cytoplasmic

Bcl-2 is a key player in inhibiting signals that converge on

the mitochondria, leading to cell survival. The Bcl-2 family

regulates cell apoptosis in a biphasic manner; Bcl-2 and Bcl-

XL are antiapoptotic, whereas Bax and Bak are proapoptotic

(Evan & Vousden, 2001). Many human cancer cells show

resistance to apoptosis by increasing the ratio of anti to pro

(Evan & Vousden, 2001). Differential regulation of Bcl-XL

modulates sensitivity to apoptosis. Tumour-associated decri-

mination of Bcl-XL occurs because of mutations of two

tumour suppressor genes, p53 and Rb.

Nuclear factor-kB (NF-kB) and serine/threonine kinase

are also associated with cell survival. NF-kB induces the

expression of inhibitor of apoptosis proteins, which bind to

and inhibit certain caspases. NF-kB is a transcription factor

that cooperatively regulates the expression of genes, which

control both innate and adaptive immune responses (Ghosh

& Karin, 2002; Li & Verma, 2002). NF-kB regulates many

genes that control inter- and intracellular signalling, cellular

stress responses, cell growth, survival, and apoptosis (Pahl,

1999). In mammalian cells, NF-kB is regulated by three

isoforms of the inhibitors of KB (IKB) family. When the

NF-kB signalling pathway is activated by TNF, IKB kinases

cause degradation of IKB proteins, which permits accu-

mulation of NF-kB in the nucleus where it binds to DNA

and initiates expression of target genes.

2.5. Angiogenesis

Tumours need oxygen and nutrients, which are provided

by new blood vessels that permeate the tumour mass.

Cancer cells activate the angiogenic switch by secreting

VEGF (Ohm & Carbone, 2001) and acidic and basic FGF1/

2. These growth factors bind to their receptors (VEGFR) on

the endothelial cell and activate signalling pathways that

eventually lead to tumour vascularization. Growth factor

signalling via integrin receptors and transduction by cyto-

kine kinases contributes to angiogenesis. Inhibiting angio-

genesis stops blood vessel formation to the tumour and

starves the tumour of nutrients that are essential for its

survival.

2.6. Metastasis and invasion

Metastasis and invasion involve primary tumour cells

moving out of the tumour mass, invading adjacent tissue,

and travelling to distant sites (Fig. 4). Some mechanisms

through which this may occur involve a change in the

expression of adhesion molecules. The activation of metal-

loproteases, which break down the extracellular matrix

proteins (namely, laminin and collagen), also contributes

to the process of invasion and metastases. Moreover, cell

surface integrin receptors found on invasive and metastatic

cancer cells are able to bind better to the degraded matrix

proteins.

3. Tumour antigens

Antigens are foreign substances recognized by and

targeted for destruction by the cells of the immune system

(Fig. 5). Tumour antigens have been explored because they

Self antigen-presenting cell

Activated macrophage

Activated B cell

Self MHC class II + peptide

TCR CD4 T-cell

Tumour cell

TCR TCR

CD4 T-cell

CD8 T-cell

MHC class I

MHC class II

CD4 T cell cytokine production and clonal expansion

Perforin granzyme

Delayed type hypersensitivity

Antibody production

Cytotoxic T cell mediated cell death

Fig. 5. Cellular interactions that form the immune response against the tumour cell.


elicit an immune response in patients who have cancer but

not in volunteer blood donors or people who do not have

cancer. Tumour cells express specific antigens on the cell

surface, usually within the MHC molecules. The problem

with tumour cells is that they cannot stimulate a T-cell

response by naive T-cells partly because they lack neces-

sary costimulatory molecules. However, dendritic cells (a

type of APC, found in most parts of the body, in the

circulation and on the epidermis as Langerhans cells) can

provide the stimulus by attracting tumour antigens to its

surface by a variety of mechanisms (Chen et al., 2002).

The dendritic cell can then present the tumour antigens on

their surface, lodged within MHC molecules, in a ready

state to activate T-cells (see Fig. 3). Once the T-cells are

activated, they are capable of recognizing and destroying

antigen-expressing tumour cell. Antigen uptake receptors

on dendritic cells provide efficient imitation of antigen-

specific adaptive immunity. The recent recognition of

dendritic cells as powerful APC capable of inducing

primary T-cell responses in vitro and in vivo in combina-

tion with identification of tumour-specific antigens empha-

sizes the role of dendritic cells in antigen recognition

(Cannon et al., 2002).

Tumour-associated antigens are expressed on (i) tumours

caused by physical or chemical environment, (ii) DNA

virus-induced tumours, and (iii) RNA virus-induced

tumours. In the case of tumour-specific antigens induced

by physical or chemical carcinogens, each tumour expresses

unique cell surface antigens. Thus, chemically induced

tumours carry cell surface antigens unique to the specific

tumour but not unique to the inducing chemical. This fact

makes it difficult to develop antigen-specific immunother-

apy since resistance raised to one set of chemically induced

tumour antigens might not prevent the growth of a second

tumour induced by the same chemical.

Oncofoetal tumour antigens are expressed both as self-

antigens during normal foetal development and as cancer

(nonself) antigens later in some adult tumours. These anti-

gens are considered as nonself by the immune system

because they were originally expressed before the individual

became immunocompetent. Although these foetal antigens

are only weakly immunogenic, their presence can be used

diagnostically to detect early cancer growth. Two examples

of antigens normally expressed by foetal, but not adult,

tissue is a-foetoprotein and carcinoembryonic antigen

(CEA). Tumours of the liver, pancreas, and testes most

often express the a-foetoprotein antigen, which is actually

secreted by tumour cells and found in the circulation. CEA

is a membrane protein usually associated with colon, lung,

and bladder cancers. The presence of these two foetal

antigens suggests that undifferentiated tumour cells express

the two proteins as a result of gene activation. The main

value of oncofoetal proteins is diagnostic and in monitoring

the progress of cancer therapy.

Wild-type Wilms’ tumour gene (WT1) is expressed at

high levels not only in most of acute myelocytic, acute

lymphocytic, and chronic myelocytic leukaemia but also in

various types of solid tumours including lung cancer. TGF-bis suppressed by Wilms’ tumour suppressor WT1 gene

product (Dey et al., 1994). The WT1 protein has been

identified as a novel tumour antigen and recent investi-

gations provide a rationale for developing WT1-based T-cell

therapy and vaccination against various kinds of malignant

neoplasms (Oka et al., 2002).

Many tumour antigens have been defined in terms of

multiple solid tumours: MAGE 1, 2, and 3, defined by


immunity; MART-1/Melan-A, gp100, CEA, HER-2, mucins,

prostate-specific antigen, and prostatic acid phosphatase are

just a short list. Viral proteins—hepatitis B, Epstein-Barr,

and human papilloma—are important in the development of

hepatocellular carcinoma, lymphoma, and cervical cancer,

respectively.

Endogenous HER-2/neu-specific antibody is overex-

pressed in � 20% of human adenocarcinomas and is a

defined tumour antigen in breast cancer (Plunkett & Miles,

2002). HER-2/neu antibodies (titer� 1:100) are detected in

14% (8/57) of patients with colorectal cancer compared with

none of the control population (0/200). Detection of HER-2/

neu-specific antibodies in the patient population is signific-

antly associated with HER-2/neu protein overexpression in

the patients’ tumour (P < 0.01). Nearly half (46%) of the

patients with HER-2/neu-overexpressing tumours (6/13) and

5% of HER-2/neu-negative tumours (2/44) have detectable

HER-2/neu-specific antibodies (Kobayashi et al., 2000).

Tumours induced by oncogenic viruses display cell

surface antigens that are coded from the viral genome. In

case of DNA viruses, these antigens are encoded by virion

DNA sequences and expressed only on transformed cells.

There is extensive cross-reactivity between different onco-

genic DNA viral antigens of the same viral class. Thus,

antigens can be diagnostic of the specific DNA virus or

group and may respond to antigen-specific immunotherapy

(Wilson, 2002).

In contrast, cells transformed by RNA viruses, called

oncornaviruses, show tumour antigens that are also viral

protein products. Therefore, transformed and RNA virus-

infected cells produce the same antigens. Within the oncor-

navirus tumour antigens, one finds (1) group-specific deter-

minants or common antigens that are shared by all viruses in

that group, (2) type-specific products that are displayed only

by tumours induced by only a few closely related viruses

within a group, and (3) unique virus-specific determinants

for only one type of virus. Since it is possible for RNA-

induced tumours to have unique antigens expressed in some

cancers or common antigens in others, it may be possible to

develop separate immunotherapeutic reagents for each indi-

vidual tumour.

4. Immune surveillance and detection of tumours

The question to answer is why, if tumours are immuno-

genic, do cancers continue to proliferate and grow? What

prevents the immune response from destroying the tumour?

One explanation is that in cancer patients the immune

response may not be robust enough.

The immune system identifies tumour cells as ‘‘nonself’’

by several mechanisms, including recruitment of pro-

grammed cell death receptors that cause apoptosis of these

cells. However, tumour cells neutralize the immune system

by evading detection and thereby prevent an immune

response. Currently, there are two concepts regarding the

search for carcinoma cells by the immune system. Immune

surveillance is based on the specific identification of trans-

formed and normal cells through their different antigenic

determinants. The Erhlich-Thomas-Burnet theory of

immune surveillance considers that the immune system

protects against transformed cells carrying foreign, nonself

signals (Burnet, 1970), whereas the Grossman-Heberman

theory proposes an antitumour surveillance system that

regulates self-population of cells (Grossman & Herberman,

1986). In essence, the two concepts of immune surveillance

should theoretically assure apoptosis of cancer cells.

Although the precise nature of the immune system’s role

in cancer has not been fully elucidated, we know that

tumours are immunogenic and that cancer is caused by a

variety of genetic defects that occur in genes that encode for

proteins involved in cell growth. The components of the

immune system, antibodies and T-cells, do not recognize or

respond to defective genes but recognize and respond to the

abnormal proteins the cancer-causing genes encode. Thus,

the individual components of the immune system play a

major role in cancer.

Immune recognition of cancer falls into categories: (1)

the detection of tumour ‘‘markers’’ and (2) the evaluation of

the antitumour response of the host. Tumour products that

are secreted by cancer cells and find their way into the

circulation are the best tumour markers. These include the

oncofoetal antigens, activated fibrinolytic pathway (as

found in pancreatic cancer), and CEA (as it occurs in colon

cancer). If antibodies can be raised to specific tumour

antigens, then these can be subsequently radiolabeled or

configurated with probes and used to locate the tumour by

imaging techniques. Such markers may also be helpful in

determining if the primary tumour has spread into other

organ or tissue sites.

Determination of a tumour-specific immune response can

also be important as a means of demonstrating the presence

of a tumour in the host. Humoral responses can be assessed

by testing for the presence of certain antibodies in serum,

which are diagnostic for certain tumours (namely, melano-

mas and sarcomas). Cell-mediated responses can be meas-

ured after antigen stimulation of host T-cells. In one such

test, the isotope release assay, Tc-cell activity can be

measured. Lymphocytes from the cancer host are incubated

with radiolabeled tumour cells. If the lytic activity of Tc-

cells is high, the levels of radioactivity in the tissue culture

medium should also be high, since, as tumour cells die, they

lyse and release radioactivity into the medium.

Tumour markers and antitumour immune responses have

proved useful in evaluating the progression or regression of

the disease, in a patient’s responses to therapy, and in

determining the recurrence of the disease. For example, it

can be used to monitor activated fibrinolytic pathway and

CEA levels in patients with certain cancers following

primary tumour removal. A rise in the levels of the onco-

foetal antigens postsurgery usually indicates a relapse,

whereas low serum levels indicates continued remission.


The identification of neoplasm-associated markers rec-

ognized by cellular or humoral effectors of the immune

system has opened new perspectives for antigen-directed,

individualised antineoplastic immunotherapy (van den

Eynde, 2002). In preparation for this new era of targeted

immunotherapy, a number of neoplasm-associated antigen

families have been identified as targets for CD8+, cytolytic

T-lymphocytes in vitro and in vivo: (1) cytotoxic T-cell

antigens expressed in various neoplasms and in normal

testis, restricted to male germ cells, (2) melanocyte differ-

entiation antigens, (3) point mutations of normal genes, (4)

antigens overexpressed in neoplastic tissues, and (5) viral

antigens (Bodey, 2002).

5. Immune-directed apoptosis of cancer cells

As with foreign grafts (Krensky et al., 1990) and virus-

infected cells, the T-lymphocyte plays a major role in the

destruction of tumour cells in mammals (Scaffidi et al.,

1999). T-cell activation includes the generation of helper,

sensitised, and cytotoxic subset clones. The sensitised T-

cells can affect killing of tumour cells by means of the

lymphokines that they release. Lymphokines mobilize and

activate B-cells through B-cell growth factors and B-cell

differentiation factors. The expression of foreign tumour

antigens on the surface of a cancer cell can be provoked by

activation of immunocytes. This activation leads to the

formation of clones of plasma cells that produce antibodies

that are specific for determinants expressed on the surface of

cancer cells. In turn, these give rise to a range ADCC

reactions. At one level, complement-fixing antibodies can

directly bind to the tumour antigens and lyse the cells by

complement activation (Kulcsar, 1997a). On the other hand,

effector cells, such as cytotoxic killer cells and macro-

phages, carrying Fc receptors may be recruited for tumour

killing by Fc receptor binding to the cancer cell membrane

(Peipp & Valerius, 2002). The binding of antibody to the

tumour cell would trigger opsonization, which would facil-

itate phagocytosis of the cancer cell. In addition, some

lymphocytes (e.g., NK cells and lymphokine-activated killer

cells) can be similarly recruited. Moreover, complement

activation would lead to generation of C3a and C3b

complement fragments. C3a is chemotactic for neutrophils,

whereas C3b induces macrophages enzyme release. Thus,

complement-activated neutrophils and macrophages lead to

cytolysis of tumour cells. A second set of lymphokines may

activate phagocytes of the reticuloendothelial system. These

lympokines include migration-inhibiting and chemotactic

factors, and macrophages recruited by these lympokines

phagocytoze, digest, and kill the tumour cells.

In addition, the lymphokine IFN, an immunomodulator

produced by the T-cell immune response, regulates T-cell

functions and B-cell antibody production and enhances the

tumouricidal activity of NK cells and macrophages. A very

important regulatory role of IFN in tumour killing may be

achieved by amplifying the NK cell population, a hetero-

geneous group of granular lymphocytes that appear very

effective in lysing target cancer cells through cell-to-cell

contact without prior sensitisation. Tumour cells of various

types possess unique sets of tumour markers that can be

recognized by NK cells. In addition, the lytic process

involves the release of cytotoxic factors for which there

seem to be more receptors on the surface of target cancer

cells than on nonmalignant self cells. Thus, the efficiency of

tumour killing by NK cells is both high and specific for

tumour cell (Coleman et al., 1992).

Macrophages and NK cells exert their tumouricidal

effects through different mechanisms. From in vitro studies,

it appears that macrophages can eliminate tumour cells

through both cytolysis and phagocytosis (Gough et al.,

2001; Bingle et al., 2002). It has been shown that the

efficiency of cytolysis of tumour cells by macrophages is

increased by the presence of activated lymphocytes or their

product, the lymphokines (Hamilton & Adams, 1987; Lewis

& McGee, 1992; Paulnock, 1992). The events leading to

cytotoxic macrophage killing are still unclear. Lysis of

tumour cells by NK cells seem to be another important

immune defense against cancer. NK cells are null cells,

neither B-lymphocytes nor T-lymphocytes, comprising 5–

15% of the total lymphocyte population of blood that is

restricted from the spleen, lymph nodes, and bone marrow.

They have a broad specificity, are non-MHC restricted, and

seem to recognize cancer cells by an unidentified NK target

receptor. However, recent studies indicate that the degree of

response of NK cells to tumour cells is inversely related to

the expression of MHC class I antigens. In particular,

tumour cells that express little or no MHC class I antigens

are more readily attacked by NK cells than are those that

express greater amounts of MHC class I antigens. Never-

theless, although NK cell killing is not MHC restricted, it

appears to be influenced by MHC expression. The killing

process is mediated by cell-to-cell contact, leading to

cytolysis where the target cell is destroyed but not the NK

cell. In vitro studies have demonstrated that the NK cell is

responsible for cytolytic activity against a variety of tumour

cell lines. The role of immune modulators in regulating NK

activity is still unclear.

6. Tumour escape mechanisms

When cells become cancerous, they produce new, unfa-

miliar antigens. The immune system may recognize these

antigens as nonself and contain or even destroy them.

However, the immune responses elicited by tumour antigens

are not potent, since most are ‘‘self-proteins.’’ The immune

system tolerates self-proteins, thereby providing a mech-

anism by which cancer cells can evade immune recognition.

Decreased immune function in cancer patients is well

characterized, and tumour cells have developed a variety of

mechanisms to avoid antitumour immune responses.


Although not completely understood, immune escape strat-

egies include (1) ‘‘sneaking through,’’ (2) modulating

tumour antigens, (3) masking tumour antigens, (4) inducing

tolerance, (5) producing blocking antibodies, and (6) pro-

ducing or expressing immunosuppressants (Coleman et al.,

1992).

One way cancer cells can avoid immune cytolysis is

because some tumours are only weakly immunogenic, so

that the small number of cells do not elicit an immune

response. However, when their numbers increase enough to

provoke an immune response, the tumour load may be too

great for the host’s immune system to mount an effective

response. The immune system and tumour may have inter-

acted in such a manner so as to suppress the immune

system’s detection and subsequent cytotoxic response.

Some tumour cells modulate tumour cell surface antigens

and thereby avoid detection. Certain tumour cells may

transfer antigens from their surface into the cytoplasm,

making themselves immunologically invisible. Alternately,

tumour cells might stop expressing certain surface antigens.

For example, tumour cells that are H-2K + may be destroyed

by Tc-cells. However, tumour cells that are H-2K � do not

evoke this cytolytic response. This assures a loss of recog-

nition, making the tumour cells no longer foreign, thus

avoiding recognition and the resultant lysis. In addition,

tumour cells may alter expression of cell adhesion mole-

cules and thereby reduce the formation of stable contacts

with cytolytic cells. Antigen modulation can also be affected

by redistributing the antigen within the cell membrane in

such a way as to prevent immune reaction. Moreover,

tumour antigens can be removed from the surface of the

cancer cell by ‘‘shredding.’’ This loss of tumour antigens

desensitises the cancer cells and protects them against

cytolysis.

Another strategy used is to devise a cloaking mechanism

that renders the cell invisible by the formation of the

mucoprotein, sialomucin, which coats and masks surface

tumour antigens. Since sialomucin is a normal (self) com-

ponent, the immune system cannot ‘‘see’’ through the

surface microprotein layer and avoid detection by the host

immune system. Certain types of tumours can synthesize

various immunosuppressants, thereby be able to actively

suppress the immune response. This would result in a state

of immune tolerance to the cancer, which could then be free

to invade adjacent normal tissue.

The receptor of the Ig superfamily, PD-1, negatively

regulates T-cell antigen receptor signalling by interacting

with the specific ligands (PD-L). This receptor is suggested

to play a role in the maintenance of self-tolerance. The

expression of PD-L1 can serve as a potent mechanism for

potentially immunogenic tumours to escape from host

immune responses and that blockade of interaction between

PD-1 and PD-L may provide a promising strategy for

specific tumour immunotherapy (Iwai et al., 2002).

Cancer cells may stimulate the immune system to express

blocking antibodies, which cannot activate complement, so

lysis of the cell is not possible. This also means that no C3a

or C3b is formed. Blocking antibodies also cover the surface

of cancer cells, preventing Tc-cells from binding to the

hidden receptors. In this way, killing of tumour cells by

compliment and Tc-cells is prevented. Synthesis of the

blocking antibodies has been shown to enhance tumouri-

genesis. Furthermore, the associated decline in immunity

results in a progressive fall in immune responsiveness to all

foreign antigens as the disease progresses.

One mechanism for inhibition of immune cell function

by tumours is the production of soluble factors, such as IL-

10, TNF, TGF-b, and VEGF. The effects of these factors

appear to be 2-fold: to inhibit effector function and to impair

the development of immune cells by acting on earlier stages

of immunopoiesis. Immune suppression by tumours is

accomplished by a variety of cellular and molecular mech-

anisms, and virtually all branches of the immune system can

be affected. VEGF and its receptors have profound effects

on the early development and differentiation of both vas-

cular endothelial and haematopoetic progenitors. It induces

proliferation of mature endothelial cells and is an important

component in the formation of tumour neovasculature.

VEGF is abundantly expressed by a large percentage of

solid tumours and this overexpression is closely associated

with a poor prognosis. Some of the earliest haematopoietic

progenitors express receptors for VEGF (Ohm & Carbone,

2001).

7. Cancer immunotherapy

7.1. Molecular aspects of immunotherapy

Immune-directed cancer therapy essentially has to pre-

serve normal cells and kill tumour cells. The exploitation of

biological differences between normal and malignant cells is

a logical approach to novel treatments for cancer. A century

has passed since the first attempt was made to stimulate the

host immune system against cancer. Recognizing the

immune system’s remarkable ability to defend the body

against disease, a new approach to the treatment of cancer

has been immunotherapy. The aim of cancer immunother-

apy is to bolster the immune system so that it is better able

to combat cancer cells. Clinically effective cancer immuno-

therapy has been sought for more than 100 years. The

identification of tumour-associated antigens recognized by

cellular or humoral effectors of the immune system has

opened new perspectives for cancer immunotherapy. Differ-

ent categories of cancer-associated antigens have been

described as targets for CD8+ T-cells in vitro and in vivo:

(1) ‘‘cancer’’ antigens expressed in different tumours, (2)

melanocyte differentiation antigens, (3) point mutations of

normal genes, (4) antigens that are overexpressed in malig-

nant tissues, and (5) viral antigens (Jager et al., 2001a).

Immunotherapeutic protocols directed against the cytotoxic

T-cell antigens are being used to analyse the induction of


antigen-specific cellular and humoral immune responses in

vivo (Bodey, 2002).

Over the last decade, there has been a rapid expansion in

the field of tumour immunology. There is now convincing

evidence that both cellular and humoral arms of the immune

system are capable of interacting with tumour cells. As

understanding of how T-cells interact with the micro-

environment of the immune system and how antigens are

recognized has heightened, greater clarity has been attained

with regard to the immune response against cancer cells.

Understanding this antigen recognition pathway and the role

of helper T-cells in enhancing cytotoxic T-cells or antibodies

has encouraged new concepts for immunotherapy (Wang et

al., 2002). Therefore, the feasibility of stimulating specific

immune responses that would be therapeutically effective is

being pursued. In order to achieve this goal, the specific

arms that recognize particular antigens have to be identified.

The most significant advances have been in our under-

standing of cellular responses and the complex events that

lead to T-lymphocyte activation as well as in the identifica-

tion of tumour antigens recognized by T-lymphocytes. This

knowledge has led to the development of anticancer immu-

notherapies designed to produce tumour antigen-specific T-

cell responses, adding to the earlier antibody or whole-cell

vaccine approaches. In addition, new methods have been

developed to quantify antigen-specific T-cell responses, and

the emergent field of recombinant gene technology has led

to an increasing number of novel methods for vaccine

delivery (Smith et al., 2002).

Clinical studies with peptides and proteins derived from

these antigens have been initiated to study the efficacy of

inducing specific CTL responses in vivo. Immunological

and clinical parameters for the assessment of antigen-spe-

cific immune responses are defined-delayed type hypersens-

itivity, CTL, autoimmmune, and tumour regression

responses. Specific defined-delayed type hypersensitivity

and CTL responses and tumour regression have been

observed after the intradermal administration of tumour-

associated peptides alone. Peptide-specific immune reac-

tions are enhanced after using GM-CSF as the systemic

adjuvant and by increasing the frequency of dermal antigen

presented to Langerhans cells. Complete tumour regression

has been observed in the context of measurable peptide-

specific CTL. However, in single cases with disease pro-

gression after an initial tumour response, either a loss of

single antigen targeted by CTL or the loss of presenting

MHC class I allele points towards immunization induced

immune escape (Jager et al., 2001b).

Heat shock proteins (HSP) act as molecular chaperones

binding endogenous antigenic peptides and transporting

them to MHC. HSP chaperones a broad repertoire of

endogenous peptides including tumour antigens. For the

immunotherapy of tumours, a strategy using HSP may be

more advantageous than other procedures because the

identification of each tumour-specific antigen does not

become necessary (Sato et al., 2001).

8. Immunotherapeutic strategies

Currently, several forms of immunotherapy are being

explored. The majority if these approaches use natural

biological products obtained to activate the immune

system through genetic engineering and hybridoma tech-

niques. The various forms of immunotherapy fall into

three main categories: monoclonal antibodies, immune

response modifiers, and vaccines. The antibody-based

therapies are a form of passive immunotherapy (Kulcsar,

1997b). That is, the molecules or substances are intro-

duced into the body rather than the body creating its own

immune response. Vaccines, on the other hand, are

considered to constitute active immunotherapy because

they generate an intrinsic immune response. They are

also considered to be a form of specific immunotherapy

because they attempt to stimulate an immune response

that can directly target the tumour antigens in contrast to

nonspecific approaches such as cytokines that broadly

stimulate the immune system.

8.1. Monoclonal antibodies

In recent years, antibody therapy has become a new

treatment modality for tumour patients (Trauth et al., 1989).

Based on evidence that effector cell-mediated mechanisms

significantly contribute to antibody efficacy in vivo, several

approaches are currently pursued to improve the interaction

between Fc receptor-expressing effector cells and tumour

target antigens. These approaches include application of Fc

receptor-directed bispecific antibodies, which contain one

specificity for a tumour-related antigen and another for a

cytotoxic Fc receptor on immune effector cells. Thereby,

bispecific antibodies selectively engage cytotoxic trigger

molecules on killer cells, avoiding, for example, interaction

with inhibitory Fc receptors. In vitro, chemically linked

bispecific antibodies directed against the Fc g-receptors

FcgRIII (CD16) and FcgRI (CD64) and the Fc a-receptorFcaRI (CD89) are significantly more effective than con-

ventional IgG antibodies. However, results from clinical

trials have been less promising so far and have revealed

clear limitations of these molecules, such as short plasma

half-lives compared with conventional antibodies (Peipp &

Valerius, 2002).

The potential targets for such therapy include the

products of protooncogenes and oncogenes, the inhibition

of growth factor receptor signalling, and the immuno-

logical exploitation of antigenic differences between nor-

mal and malignant cells. Monoclonal antibody technology

was heralded as a potential ‘‘magic bullet’’ for cancer

therapy following its evaluation in the mid-1970s, but it

is only in the past few years that such technology has

entered mainstream clinical practice. Monoclonal antibod-

ies directed against tumour-specific agents have been

approved for the treatment of breast cancer (trastuzumab)

and non-Hodgkin’s lymphoma (rituximab) and for the


diagnosis of certain cancers (oncoscint) (Plunkett &

Miles, 2002).

Antibodies can be targeted to specific cancer antigens.

Monoclonals can then aid in the diagnosis and treatment of

antigens that distinguish cancer cells from normal cells.

Desmoplastic small round cell tumour is an aggressive and

often misdiagnosed neoplasm of children and young adults.

It is chemotherapy sensitive, yet patients often relapse after

therapy because of residual microscopic disease at distant

sites. Strategies directed at minimal residual disease may be

necessary for cure. Monoclonal antibodies selective for cell

surface tumour-associated antigens may have utility for

diagnosis and therapy of microscopic disease at distant, as

recently demonstrated in advanced-stage neuroblastoma

(Modak et al., 2002). Furthermore, monoclonals serve as

powerful tools to define, treat, and monitor recurrences of

previously treated cancers.

8.2. Immune response modifiers

These are molecules, either extrinsic or intrinsic to the

host, that affect the immune response. One group of

extrinsic modifiers is referred to as immune potentiators.

These include BCG, C. parvum and endotoxin, which are all

microbes or microbial products that have been shown to

modify the immune response and, under certain conditions,

to cause tumours to regress or grow more slowly than usual.

The intrinsic group, known as biological response modi-

fiers, includes IL-1, IL-2, IFN (a, b, and g), TNF, B-cell

growth factors, and haematopoietic growth factors (such as

colony stimulating factors). These agents exert their influ-

ence at different stages of the immune response.

Haematopoietic growth factors are often combined with

chemotherapy and radiotherapy to restore bone marrow

function. Thalidomide, which suppresses TNF-a produc-

tion and has antiangiogenic properties, is currently under

evaluation in several cancers. Currently, there are more

than 20 IL (IL-1 to IL-18) and at least 5 other proteins

have just been found which are likely to be termed IL. The

cellular sources and functions of these IL are provided in

Table 1. Cytokines, the messengers of the immune system,

are either proteins or glycoproteins, secreted by immune

cells. They have autocrine and paracrine functions, so that

they function locally or at a distance to enhance or

suppress immunity. Currently, in cancer therapy, cytokines

are used to enhance immunity. They also regulate the

adaptive immune system, the T- and B-cell immune

responses. In the immune system, cytokines function in

cascades. Thus, clinical trials of individual cytokines are

rarely useful, since cytokines tend not to work individu-

ally but probably cooperatively. Some of the individual

cytokines that have been tested and found ineffective for

cancer treatment include IL-1b, although it may be useful

still because it helps to mediate the severe toxicity of IL-

2. IL-2 is the most widely studied IL and is used for

immunostimulation in metastatic renal cell carcinoma and

malignant melanoma. Although the use of TNF certainly

sounded promising, but because it caused severe hypo-

tension when used systemically, its value is limited. IL-4

shows minimal anticancer activity and is toxic. IL-6 has

some activity against cancer cells but turns out to be a

growth factor for myeloma cells. GM-CSF, used prim-

arily in stem cell transplant to reconstitute the myeloid

series, has been studied for melanoma with controversial

results.

Which cytokines are important for cancer? IL-2 and IFN-

a-2b are two cytokines approved by the Federal Drug

Agent for treatment of cancer. IL-2 has demonstrated

activity against renal cell, melanoma, lymphoma, and leuk-

aemia. IFN has activity in the same histologies but also in

Kaposi’s sarcoma, chronic myelogenous leukaemia, and

hairy cell leukaemia (HCL). Overall, the major cytokines

IFN-a, IL-2, GM-CSF, and IL-12 are being currently

evaluated for cancer therapy, since they appear to have

application in the treatment of haematologic malignancies

or immunogenic tumours.

8.2.1. Interferon-�IFN was isolated in 1970 from white cells and called IFN

because it interfered with viral infection. IFN-a is actually a

family of molecules comprising at least two types. They are

encoded by closely related genes on chromosome 9, encod-

ing proteins that are variably glycosylated. These are com-

prised of about 150 amino acids and bind to certain

receptors on the surface of immune cells. They are known

to have profound and diverse effects on gene expression.

IFN-a has many roles. It up-regulates genes like MHC class

I, tumour antigens, and adhesion molecules. It is also an

antiangiogenic agent, which is very active in the immune

system, promoting B- and T-cell activity. IFN-a stimulates

macrophages and even dendritic cells and up-regulates Fc

receptors. The mode of action of IFN-g appears to be

through activation of the host immune system, which

depends on the intrinsic immunogenicity of the target

tumour cell (Gri et al., 2002).

IFN’s activity in cancer has been well documented

(Quesada et al., 1986; Dorval et al., 1987; Allan et al.,

1995; Kirkwood et al., 1996, 2000, 2001) and is indicated

for the treatment of certain leukaemias and Kaposi’s sar-

coma in order to inhibit tumour proliferation and angio-

genesis. The efficacy of IFN-a has been well established for

the treatment of advanced melanoma and renal cell carcin-

oma patients (Bukowski et al., 2002). Small but consistent

response rates have been observed in a number of other

studies. HCL is a rare lymphoproliferative disease. IFN-adramatically improves the survival of HCL with haemato-

logical values returning to normal and the disappearance of

circulating hair cells in most patients (Zinzani et al., 1997).

HCL is a B-cell malignancy that is highly sensitive to the

immune modulator, IFN-a, which acts by up-regulating

endogenous TNF-a and by restoring the efficacy of the T-

cell receptor family (Baker et al., 2002).

y & Therapeutics 99 (2003) 113–132 127


IL-2 is a T-cell growth factor that binds to a specific

tripartite receptor on T-cells. Current clinical trial data

suggest that combined IL-2 and IFN-a administered sub-

cutaneously in accordance with specified regimes achieves

long-term survival benefits in subsets of patients with

metastatic renal cell carcinoma (Atzpodien et al., 2002).

Combining immune modulators may be a way forward for

metastatic carcinomas. The immunobiological agents, IL-2

and IFN-a, when combined with meroxyprogesterone,

produces a good response rate and low toxicity (Naglieri

et al., 2002). However, it should be remembered that clinical

trials restricted to combinations of IL-2 and IFN-a alone

have given contradictory results in the treatment of meta-

static renal cell carcinoma (Ravaud et al., 2002).


IL-12 is a very exciting cytokine. It is a heterodimeric

protein that promotes NK and T-cell activity and is a growth

factor for B-cells. IL-12 plays a central role in T-cell-

mediated immune responses. Endogenously formed IL-12

confers T-cells with a tumour migratory capacity and at the

same time entices tumour cells to accept tumour-migrating

T-cells (Uekusa et al., 2002). It has demonstrated antitumour

activity in mouse models. Alone, IL-12 shows minimal

potential for therapeutic effect (Atkins et al., 1997). Essen-

tially, the efficacy of IL-12 is dependent on stimulating Th1cells to release IFN-g (Segal et al., 2002). Furthermore, IL-

12 may have value also as a vaccine adjuvant. When IL-12

was paired with peptide vaccines in patients with resected

stages 3 and 4 melanoma, IL-12 appeared to boost the

response to the vaccine (Lee et al., 2001).

8.3. Colony stimulating factor

Cancer vaccines composed of tumour cells engineered to

secrete the cytokine, GM-CSF, are currently being clinically

evaluated. Although immune recognition of tumours is

known to occur, the failure of the host to either suppress

or attenuate progression of the disease may reflect limited

immunogenicity arising from the absence of critical deter-

minants like the tumour augmenting family of cytokines

(Dranoff, 2002). To enhance the immunogenicity of GM-

CSF-secreting tumour cell vaccines, a novel approach

expressing GM-CSF as a membrane-bound form (mbGM-

CSF) on the tumour cell surface has been investigated. The

intent is to enhance antigen presentation by increasing

interactions between the tumour cell lines in the vaccine

and GM-CSF receptor-positive APC, notably the patient’s

Langerhans cells (dendritic cells) residing within the intra-

dermal injection site. Tumour cells have been engineered to

express either membrane-bound or secreted GM-CSF (Yei et

al., 2002). GM-CSF has been approved also for use in stem

cell and bone marrow transplant to reconstitute the myeloid

series. GM-CSF is also being evaluated as an adjuvant for

vaccine therapy.

J.K. Adam et al. / Pharmacolog

New advances in GM-CSF or cytokine-linked immuno-

therapy devised for pre-B acute lymphocytic leukaemia and

acute myeloid leukaemia attempt to modify acute leukaemia

cells into functional APC. These cells can then be used as

autologous vaccines at the time of minimal residual disease

after standard chemotherapy to stimulate host immune

responses against their own leukaemia cells. The various

approaches toward this aim include incubation of leukaemia

cells with cytokines or growth factors and gene manipula-

tion of these cells. In particular, ex vivo culture of acute

lymphocytic leukaemia cells with CD40 ligand, incubation

of acute myeloid leukaemia cells with GM-CSF and IL-4

(GM-CSF/IL-4), and lentiviral transduction of acute lym-

phocytic leukaemia and acute myeloid leukaemia cells for

expression of immunomodulators (CD80 and GM-CSF) are

current approaches for the development of autologous acute

leukaemia cell vaccines (Stripecke et al., 2002).

The serological prostate-specific antigen progression of

prostate cancer can be reduced significantly following

subcutaneous injection of GM-CSF (Rini et al., 2003).

Expression of plasmids encoding GM-CSF and IL-2 suc-

cessfully produce protective immune responses in animals

immunized with transfected malignant plasma cells (Galea

& Cogne, 2002). However, the optimum choice of antigen,

delivery vector and adjuvant, and administration regimen

for some of these biological response modifiers are still

being investigated (Gupta & Kanodia, 2002).

8.4. Vaccination against tumours

Efforts to treat cancer with vaccines date back to the

origins of immunology. Although cancer immunotherapy

was initiated by William Coley more than a century ago, the

field of cancer vaccines is in an early stage of development

(Espinoza-Delgado, 2002). Only recently, major advances in

cellular and molecular immunology have allowed a com-

prehensive understanding of the complex and high rate of

interactions between the immune system and tumour cells.

Tumour-immune system interactions result either in strong

immune antitumour response or in tolerance to tumour-

associated antigens.

It is now clear that many human tumour antigens can be

recognized by the immune system. These tumour antigens

can be classified into several groups including tissue-spe-

cific differentiated, overexpressed, and viral-associated anti-

gens. In many cases, there is a known molecular basis of

carcinogenesis, which provides the explanation for the

differentiated expression of antigens in tumours when

compared with normal cells. Improved understanding of

the biology of the immune response, particularly of immune

recognition and activation of T-cells, allows better design of

vaccines. Preclinical comparative studies permit evaluation

of optimal vaccine strategies, which can then be delivered to

the clinic. Currently, a range of cancer vaccines are being

tested including those using autologous and allogeneic

tumour cells, proteins, peptides, viral vectors, DNA, or

Table 4

Tumour cell vaccines

Type Vaccine Reference

Tumour cell

vaccines

Melanoma tranduced

with Ad-GM-CSF

Kusumoto et al., 2001

Allogeneic panc.,

tumour-secreting GM-CSF

Jaffee et al., 2001

CancerVax Hsueh et al., 1998

Autologous colon

CA+BCG

Hanna et al., 2001

Autologous GBM+

Newcastle virus

Schneider et al., 2001


dendritic cells (Dermime et al., 2002; Liu et al., 2002).

Patients have been injected with autologous and allogeneic

tumour and dendritic cell vaccines (Plunkett & Miles,

2002). However, measuring immune response has been

problematic. Now that several tumour antigens have been

identified and the immune response they provoke, progress

has been made in developing cancer vaccines. Vaccines

consisting of peptide or protein administered with an adjuv-

ant have been the most frequently used. These adjuvants

might be compounds such as tumour cell wall components

or cytokines such as IL-12 or GM-CSF that incite a local

response. Monocytes, neutrophils, eosinophils, and T-cells

are all recruited to the site where adjuvant is used, but it is

believed that in situ dendritic cells ultimately take up the

tumour proteins and peptides, process them if necessary, and

present them on the cell surface as peptides capable of

binding to the MHC molecules. Dendritic cells can then

stimulate T-cells that have the receptors to recognize those

particular peptides.

Substantial data from several preclinical models and

early human clinical trials have confirmed the ability of

cancer vaccines to induce immune responses that are tumour

specific and, in some cases, associated with clinical

responses. One future challenge will be to determine how

to appropriately stimulate the pathways leading to effective

interaction among APC, T-lymphocytes, and tumour cells. It

also is critical to develop monitoring strategies that may

allow the identification of patients who may benefit from

cancer vaccines (Espinoza-Delgado, 2002).

Tumour cell-based vaccines are another vital area of

research (Ward et al., 2002). Some studies have used

autologous tumour, in which tumour cells are extracted

from surgical resection or biopsy specimens. Allogeneic

cell lines have also been developed for tumours such as

melanoma that likely encompass many of the tumour-

associated antigens expressed by the melanomas of most

affected individuals (Lauerova et al., 2002). Tumour cells

can also be modified to make them more immunogenic. To

accomplish that, tumour cells may be infected with various

types of viruses so that viral proteins are expressed on the

surface and transduced with genes expressing cytokines

such as IL-2 and GM-CSF or genes for HLA molecules or

costimulatory molecules. The idea is to irradiate the cells so

they can no longer proliferate then inject the tumour cells

back into the patient. The expectation is activation of the

immune system by either the tumour cells or the inflam-

matory response that includes recruitment of dendritic cells.

As the injected tumour cells undergo apoptosis or are

destroyed by the inflammatory reaction, antigens are picked

up by the dendritic cells and represented to the T-cells

(Morse et al., 1999).

The results of some of the current research on tumour

vaccines are illustrated in Table 4. These studies employed

several strategies. One of the most popular is to transduce

with a vector containing GM-CSF so that the tumour

secretes GM-CSF and sets up an inflammatory response.

In animal studies, this strategy has been the most promising

in inducing a protective immune response. Newcastle dis-

ease virus has antineoplastic and pleitropic immune stim-

ulatory properties. It alters the immunogenicity of tumour

cells. The virus preferentially replicates in and kills tumour

cells. The attenuated Newcastle disease virus vaccine causes

regression of human neoplasms and has been used for the

treatment of malignant melanoma. The mechanism of its

oncolytic action appears to affect specific signalling path-

ways of the tumour cell (Termeer et al., 2000; Fabian et al.,

2001; Schirrmacher et al., 2001).

Use of Bacille Calmette-Guerin as an inflammation-

inducing adjuvant along with autologous colorectal cancer

cells showed increased defined-delayed type hypersensitiv-

ity but no survival benefit (Smith et al., 2002). In fact, most

of these tumour cell approaches show an immune response

but have again been of limited clinical value. Nonetheless,

Cancer Vax70 tested on nonrandomized studies has

recorded survival benefits in melanoma.

Despite the identification of tumour antigens and their

subsequent generation in subunit form for use as cancer

vaccines, whole tumour cells remain a potent vehicle for

generating antitumour immunity. This is because tumour

cells express an array of target antigens for the immune

system to react against, avoiding problems associated with

MHC-restricted epitope identification for individual pa-

tients. Furthermore, whole cells are relatively simple to

propagate and are potentially efficient at contributing to

the process of T-cell priming. However, whole cells can also

possess properties that allow for immune evasion, so the

question remains of how to enhance the immune response

against tumour cells so that they are rejected. Scenarios

where whole tumour cells may be utilised in immunother-

apy include autologous tumour cell vaccines generated from

resected primary tumour, allogeneic (MHC-disparate) cross-

reactive tumour cell line vaccines, and immunotherapy of

tumours in situ. Since tumour cells are considered poorly

immunogenic, mainly because they express self-antigens in

a nonstimulatory context, the environment of the tumour

cells may have to be modified to become stimulatory by

using immunological adjuvants. Recent studies have reeval-

uated the relative roles of direct and cross-priming in

generating antitumour immunity and have highlighted the

need to circumvent immune evasion (Ward et al., 2002).

Table 5

Viral vector and plasmid vaccines

Type Vaccine Reference

Viral vector and ALVAC-CEA B7.1 von Mehren et al., 2001

plasmid vaccines Vaccinia CEA/avipox-

CEA+GM-CSF

Marshall et al., 2000

Vaccinia CEA Conry et al., 1999

PSMA/CD86 plasmid Mincheff et al., 2000


The development of genetically modified tumour vac-

cines has been prompted by a better understanding of

antitumour immune responses and genetic engineering tech-

nologies as well as the identification of numerous tumour

antigens in several malignancies, which occasionally induce

spontaneous tumour regressions. Cellular vaccines are based

on autologous or allogeneic tumour cells genetically engi-

neered to secrete different cytokines, costimulatory mole-

cules, or allogeneic HLA molecules programmed to provide

a strong stimulatory signal. Another promising approach

that is targeted towards breaking immune tolerance to

tumour antigens exploits dendritic cells loaded with gen-

etically modified tumour antigens (Liu et al., 2002). Effect-

ive nonviral and viral gene delivery systems have been

constructed including a third generation of adenoviral,

lentiviral, and hybrid vectors (Nawrocki et al., 2001).

In active immunization, cancer prevention is attempted

by vaccinating with actual inactivated tumour cells or their

antigens. Tumour cells are emulsified in Freund’s adjuvant

and injected prior to tumour challenge in animals. One

oncogenic viral vaccine, feline leukaemia vaccine, has been

very effective in reducing the incidence of leukaemia in the

domestic cat population. However, immunization against

the tumour itself depends on both the presence of tumour

cell surface antigens that can be ‘‘seen’’ by the immune

system and the appropriate cytotoxic responses by the

immune system. To date, most anticancer vaccines have

failed—either because the tumour’s antigenicity is somehow

hidden or because the immune system responds by pro-

ducing blocking antibodies that enhance tumour growth.

Cancer is a multigenic disorder involving mutations of

both tumour suppressor genes and oncogenes. A large body

of preclinical data, however, has suggested that cancer

growth can be arrested or reversed by treatment with gene

transfer vectors that carry a single growth inhibitory or

proapoptotic gene or a gene that can recruit immune

responses against the tumour. Many of these gene transfer

vectors are modified viruses that retain the capability of the

virus for efficient gene delivery but are safer than the native

virus due to modifications that eliminate or alter one or more

essential viral functions. In the field of viral-based gene

transfer, vectors for the treatment of cancer, vectors and

indeed naked DNA in the form of plasmids encoding

tumour antigens, can be used to immunize people (Wilson,

2002). Initially, these vaccines were administered to muscle

cells, but it is likely that dendritic cells were ultimately the

targets infected by the virus or were picking up the antigen

released by apoptotic muscle cells. Poxviruses are another

popular way to apply this approach, and a considerable

amount of work has been done with vaccinia and avian and

fowl pox vectors. One of the most interesting strategies is

the prime boost approach. An example of this is when a

patient is first immunized with vaccinia virus encoding the

gene for CEA.

Vaccinia is very immunogenic, so it can only be used once

or twice—after the first injection patients develop high

neutralizing antibody titers. In subsequent immunizations,

the antibody immediately binds to the virus, making it

difficult to get true immunization against the encoded tumour

antigen. However, an avian vector expressing CEA is used

and better immunologic responses are observed (Marshall et

al., 2000). Prostate cancer is a disease that may be amenable

to immunotherapy approaches, as evidenced by the ability to

induce human cytotoxic immune responses against prostate

cancer cells. Recent interest in recombinant poxvirus vac-

cines coupled with the need for new prostate cancer therapies

has led to the development of several recombinant poxvirus

agents designed for prostate cancer treatment. Whether these

agents will be effective in treating prostate cancer is under

investigation in several ongoing and upcoming clinical trials

(see Table 5) (Hwang & Sanda, 1999).

Cancer vaccines are injected with appropriate boosters to

stimulate an immune response to a specific type of cancer.

Current development efforts are centred on vaccines for

melanoma, rectal cancer, and breast cancer as well as other

cancers. Vaccines may someday serve as cancer prophy-

lactic modules, which by creating a state of acquired

immunity stop specific types of cancer from recurring in

people previously afflicted by them.

Acknowledgments

Research grant support from the Medical Research

Council (MRC, SA) and National Research Foundation

(NRF, SA) is much appreciated. We thank Celia Snyman for

the figure drawings and Bonita Jeena for assisting with the

manuscript.

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