**2. The first glance throws light on**

#### **2.1. Metastatic malignant melanoma – Prognosis and treatment**

Malignant melanoma (MM) is among the most common cancers in the developed countries and the incidence has increased substantially over the last decades. Surgery is frequently curative at an early stage, but the prognosis for patients with disseminated disease is generally bleak, with a medium survival of 6-10 months and a 5-year survival of about 5% only.

Decarbazin is extensively used for treatment of metastatic melanoma and has been reported to induce objective tumour responses in 5-29% of all patients. A number of studies have compared other single agents or multi-drug regimes to Decarbazin, without demonstrating a superior effect. To date, no randomized controlled trials have demonstrated improved survival after treatment with Decarbazin or any other drug.

Furthermore, there have been numerous reports of spontaneous immune responses [1] in melanoma patients, to some extent associated with a favourable clinical development. This has prompted the development of various vaccines [1] to target defined or undefined mela‐ noma antigens.

#### **Immunological responses against vaccine antigens have been demonstrated in a number of studies** [2, 3, 4, 5, 6, 7, 8]**, but there is limited evidence of clinical effect**

With regard to non-specific immunostimulation, high-dose interleukin-2 (IL-2) has been shown to induce complete remission in about 16% of melanoma patients, but is associated with considerable adverse effects. IL-2, Interferon-alpha (INF-alpha) and other cytokines are also investigated in combination with conventional chemotherapy. Adjuvant therapy with IFNalpha is believed to prolong the disease-free period, but most studies do not indicate improved survival. Taken together, there is an urgent need for improved therapy of metastatic malignant melanoma.

#### **2.2. The second glance throws light on: Metastatic prostate cancer – Prognosis and treatment**

Prostate cancer is the most commonly diagnosed cancer in the male population of the devel‐ oped countries world-wide. Though the majority of patients eventually die of other causes, prostate cancer is also among the most common cause of cancer death among males in Europe, North America and Japan.

Metastatic prostate cancer is usually treated with bilateral orchiectomy and/or androgen suppressive drugs. The resulting androgen deprivation frequently induces tumour regression and has a palliative effect. The treatment is also considered to give prolonged survival for subsets of patients. However, after a transient response period (median 12-24 months), virtually all patients develop progressive cancer refractory to hormone therapy.

In the RNA/DC-vaccine trial performed in Radium, all included patients had hormone refractory prostate cancer (HRPC).

At this advanced stage, the median survival is only 10-12 months. There has been no effective therapy for HRPC, and many physicians have thus recommended 'clinical observation'.

It is interesting that some separate trials, in other oncology centers, demonstrated prolonged survival after treatment with Doxetacel. This finding has been confirmed in subsequent studies, and Doxetacel is now considered as standard therapy for patients with HRPC.

It should be recalled, however, that the effect on mean survival is limited (2-3 months). Patients with HRPC may also to some extent benefit from different forms of palliative treatment, including certain cytotoxic drugs, bisphosphonates, second-line hormonal agents, glucocorti‐ coids and radiation therapy.

There is, however, an evident need for improved systemic treatment, and immunotherapy may represent an attractive option. Several small-scale studies [2, 3, 5] have demonstrated promising immune responses after vaccine therapy, but there is limited evidence of clinical affect. Interestingly, a recent placebo-controlled phase III trial on HRPC patients has suggested a possible survival benefit from therapy with dendritic cells (DCs) pulsed with a fusion protein of prostatic acid phosphatase and granulocyte-macrophage colony-stimulating factor (GM-CSF).

#### **3. General background for cancer immunotherapy**

#### **3.1. Immunosurveillance and immunoediting**

**1. Introduction**

4 Immunopathology and Immunomodulation

tumour cells.

on patients.

noma antigens.

melanoma.

**2. The first glance throws light on**

after treatment with Decarbazin or any other drug.

**2.1. Metastatic malignant melanoma – Prognosis and treatment**

The author analyzes the experience and the research projects worked out in the Dept. Cell Therapy of the University Hospital "Radium" (Inst. Cancer Research, Oslo, Norway) by the team of Prof. Gunnar Kvalheim, Prof. Jon Kyte, Prof. Jahn Nesland, the Bulgarian immunol‐

The colleagues from Radium have utilized gene-transfer technology for developing vaccine therapy with dendritic cells transfected with tumour-mRNA. These vaccines are designed to combine the immunostimulatory capacity of dendritic cells with the antigen repertoire of

There are two approaches to the project: preclinical/experimental evaluation and clinical trials

Malignant melanoma (MM) is among the most common cancers in the developed countries and the incidence has increased substantially over the last decades. Surgery is frequently curative at an early stage, but the prognosis for patients with disseminated disease is generally bleak, with a medium survival of 6-10 months and a 5-year survival of about 5% only.

Decarbazin is extensively used for treatment of metastatic melanoma and has been reported to induce objective tumour responses in 5-29% of all patients. A number of studies have compared other single agents or multi-drug regimes to Decarbazin, without demonstrating a superior effect. To date, no randomized controlled trials have demonstrated improved survival

Furthermore, there have been numerous reports of spontaneous immune responses [1] in melanoma patients, to some extent associated with a favourable clinical development. This has prompted the development of various vaccines [1] to target defined or undefined mela‐

**Immunological responses against vaccine antigens have been demonstrated in a number of**

With regard to non-specific immunostimulation, high-dose interleukin-2 (IL-2) has been shown to induce complete remission in about 16% of melanoma patients, but is associated with considerable adverse effects. IL-2, Interferon-alpha (INF-alpha) and other cytokines are also investigated in combination with conventional chemotherapy. Adjuvant therapy with IFNalpha is believed to prolong the disease-free period, but most studies do not indicate improved survival. Taken together, there is an urgent need for improved therapy of metastatic malignant

**studies** [2, 3, 4, 5, 6, 7, 8]**, but there is limited evidence of clinical effect**

ogist M.Sc. Biol. Paula Lazarova, and the author himself, for the last several years.

A tumour-specific immune response will depend on the ability of immune cells to discriminate the tumour from normal host tissues. In contrast to infectious microbes and allogeneic human cells, the tumour cells are largely similar to normal host cells. According to the theory of immunosurveillance, as suggested by Burnet in 1970, the immune system is still able to recognize and eliminate tumour cells. This concept was severely challenged in the following years. However, there is now convincing evidence that the immune system may recognize tumour cells due to their expression of altered antigens. The early concept of tumour surveil‐ lance has highlighted the importance of the immune system in protecting against cancer.

Recent modifications of this theory, now named immunoediting, provide increased insight into the role of the immune system in sculpting the tumour into an immunologically selected cell population. The immunoediting perspective points to a major challenge in cancer immu‐ notherapy: how to make the immune system destroy a tumour that has already escaped the immune attack.

A solution may be found in exploiting the difference between spontaneously occurring immune activation and optimally engineered immunization. This is the reason for the developing of immune-gene-therapy with tumour-mRNA transfected dendritic cells.

#### **3.2. Activation of T cells that recognized tumour-associated antigens**

T-lymphocytes express antigen-specific T-cell receptors (TCRs) that enable them to recognize target cells expressing a particular antigen. The antigens are presented as peptides on HLAmolecules on the target cell surface, and the recognition is mediated by binding of the T-cell receptor to the HLA/peptide complex. Proper T-cell stimulation, including TCR-binding, leads to activation and clonal expression of T cells with the relevant antigen specificity. During the development of a tumour, numerous mutations result in novel antigens and altered expression of normal antigens. The resulting tumour-associated antigens are presented as peptides on HLA-molecules.

Figure 1a shows how host T cells may recognize tumour cells by binding of the TCR to the HLA/peptide complex [7]. However, TCR-binding does not necessarily lead to T-cell activa‐ tion. In general, the activation of previously unstimulated T cells ('naïve' T cells) requires additional stimulation through co-stimulatory molecules like CD80 and CD86. If stimulated only through the TCR, the naïve T cells enter a stage of anergy and permanently lose their ability to be properly activated. The expression of co-stimulatory molecules is largely restricted to professional antigen presenting cells (APCs), including dendritic cells (DCs), macrophages and B cells [7].

Two major subsets of T cells exist. CD4+ T cells recognize peptides bound to HLA class II, while CD8+ T cells recognize peptides presented on HLA class I (Figure 1b).

Most human cells, except erythrocytes and testicular cells, express HLA class I. In contrast, HLA class II is expressed mainly by professional APCs, activated T cells and the cortical epithelium in the thymus. Thus, CD8+ T cells may be stimulated by most cells, while CD4+ T cells depend on stimulation from APCs. As it will be discussed a bit later, the activation of both T-cell subsets is probably important for an effective anti-tumour response.

Effector T cells and the Th1/Th2 delineation [7]:

Proper T-cell activation results in differentiation into effector T cells. CD8+ effector T cells are cytotoxic, i.e. capable of killing cells that express the relevant antigen (Figure 1c).

tumour cells due to their expression of altered antigens. The early concept of tumour surveil‐ lance has highlighted the importance of the immune system in protecting against cancer.

Recent modifications of this theory, now named immunoediting, provide increased insight into the role of the immune system in sculpting the tumour into an immunologically selected cell population. The immunoediting perspective points to a major challenge in cancer immu‐ notherapy: how to make the immune system destroy a tumour that has already escaped the

A solution may be found in exploiting the difference between spontaneously occurring immune activation and optimally engineered immunization. This is the reason for the

T-lymphocytes express antigen-specific T-cell receptors (TCRs) that enable them to recognize target cells expressing a particular antigen. The antigens are presented as peptides on HLAmolecules on the target cell surface, and the recognition is mediated by binding of the T-cell receptor to the HLA/peptide complex. Proper T-cell stimulation, including TCR-binding, leads to activation and clonal expression of T cells with the relevant antigen specificity. During the development of a tumour, numerous mutations result in novel antigens and altered expression of normal antigens. The resulting tumour-associated antigens are presented as peptides on

Figure 1a shows how host T cells may recognize tumour cells by binding of the TCR to the HLA/peptide complex [7]. However, TCR-binding does not necessarily lead to T-cell activa‐ tion. In general, the activation of previously unstimulated T cells ('naïve' T cells) requires additional stimulation through co-stimulatory molecules like CD80 and CD86. If stimulated only through the TCR, the naïve T cells enter a stage of anergy and permanently lose their ability to be properly activated. The expression of co-stimulatory molecules is largely restricted to professional antigen presenting cells (APCs), including dendritic cells (DCs), macrophages

Two major subsets of T cells exist. CD4+ T cells recognize peptides bound to HLA class II,

Most human cells, except erythrocytes and testicular cells, express HLA class I. In contrast, HLA class II is expressed mainly by professional APCs, activated T cells and the cortical epithelium in the thymus. Thus, CD8+ T cells may be stimulated by most cells, while CD4+ T cells depend on stimulation from APCs. As it will be discussed a bit later, the activation of

Proper T-cell activation results in differentiation into effector T cells. CD8+ effector T cells are

while CD8+ T cells recognize peptides presented on HLA class I (Figure 1b).

both T-cell subsets is probably important for an effective anti-tumour response.

cytotoxic, i.e. capable of killing cells that express the relevant antigen (Figure 1c).

Effector T cells and the Th1/Th2 delineation [7]:

developing of immune-gene-therapy with tumour-mRNA transfected dendritic cells.

**3.2. Activation of T cells that recognized tumour-associated antigens**

immune attack.

6 Immunopathology and Immunomodulation

HLA-molecules.

and B cells [7].

**Figure 1. a**) Mutations through the development of tumor lead to expression of mutated proteins. The mutated pro‐ teins are processed into peptides that are presented on HLA classI (HLA I) on the tumor cell surface. CD8+ T cells spe‐ cific for mutated peptides may therefore recognize tumor cells by binding of their T cell receptor (TCR) to the HLA/ peptide complex. **b**) Stimulation of tumor-specific T cells by dendritic cells (DCs). Tumor proteins are engulfed by DCs and processed into peptides. The tumor-associated peptides are presented by DCs on HLA class I and HLA class II, to CD8+ and CD4+ T cells respectively. For proper activation, previously unstimulated ("naive") T cells also requirestimu‐ lation from co-stimulatory molecules (e.g. CD80) and IL-2. Dendritic cells constitutively express HLA class II (and I) and co-stimulatory molecules, and the expression in up-regulated upon DC mutation. T cells start producing IL-2 when stimulated through TCR and co-stimulatory molecules. **c**) Activation of CD4+ and CD8+ T cells induce differen‐ tiation into effector T cells. CD8+ T cells differentiate into cytotoxic effector cells that specifically kill target cells ex‐ pressing the relevant antigen. CD4+ T cells differentiate into T-helper cells secreting high levels of cytokines. Based on their cytokine profiles, the CD4+ effector cells are conventionally divided into Th1- and Th2-cells.

CD4+ effector T cells are conventionally divided into T-helper 1 (Th1) or T-helper 2 (Th2) cells, based on their cytokine secretion profiles (Figure 1c).

Interferon-γ (IFNγ), tumour-necrosis factor-α (TNFα) and IL-2 are usually designated as

Th1-cytokines, while the Th2-cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13.

Th1 cells support cellular immunity, e.g. by secreting cytokines that induce up-regulation of HLA on target cells and stimulate macrophages and CD8+ T cells. Th2 cells promote antibody responses by interaction with B cells.

In cancer immunotherapy, Th1-type responses are generally believed to be desirable.

Antibody responses can only target surface antigens, whereas the CD8+ T cells are able to recognize intracellular antigens presented on HLA class I. Th1- and Th2-responses are mutually inhibitory. Th1-cytokines generally promote Th1-differentiation and inhibit Th2 differentiation, while Th2-cytokines have the opposite effects [5, 7].

Th2-cytokines may therefore suppress the development of cytotoxic anti-tumour responses.

There are considerable experimental data on the effects of individual Th1- or Th2-cytokines.

However, the validity of the Th1/Th2-delineation in humans may be questioned, which could be our next publication [8].
