**6. Background for the present up-to-date tumour-RNA/DC vaccines**

#### **6.1. The use of dendritic cells in cancer immunotherapy**

Most cancer vaccines have been based on peptides/proteins or tumour lysates that are injected intradermally. These approaches depend on uptake of vaccine antigen by immature DCs in the skin, and subsequent DC maturation and migration to lymph nodes. Alternatively, DCs may be loaded with tumour antigens ex vivo, and then injected into the patient. This strategy appears attractive, as it may result in enhanced antigen presentation and more effective T-cell stimulation. Moreover, DC-based vaccines may offer the opportunity of directing the immune response, by manipulating the DC phenotype.

The first DC-vaccine trials in cancer patients were reported by Hsu et al. in 1996 and Nestle et al. in 1998 [7]. Promising T-cell responses were obtained, and in recent years, various ap‐ proaches to DC-vaccines have been explored.

Early studies applied immature DCs, but it is now generally believed that a mature phenotype is desirable.

Targeted loading of DCs in vivo represents another strategy.

Ralph Steinman's group has explored this option by use of antibodies targeting the receptor DEC-205 on the DC surface [7]. Their data from animal model indicate that potent T- and Bcell responses may be elicited.

Alternatively, vaccine antigens might be injected and subsequently transfected into tissue DCs by use of in vivo electropermeabilization.

#### **6.2. Large-scale generation of dendritic cells**

For clinical vaccine production, large quantities of autologous DCs are required. Most studies make use of monocyte-derived DCs (Mo-DCs).

On the other hand, DCs may be cultures from CD34+ cells obtained from bone marrow, umbilical cord blood or cytokine-mobilized peripheral blood progenitor cells.

DCs may also be purified from peripheral blood, e.g. after in vivo mobilization of DCs with Flt3-ligand.

At present, it is not clear which method results in the best DCs for cancer vaccination.

The only restriction of use of umbilical cord blood comes from the fact that the per cent of potentially active cells is rather limited and is not enough for bigger scales and clinical application.

In a study reported by Syme et al., Mo-DCs were compared to DCs generated from CD34+ cells 5, 6, 7). The results demonstrated higher expression of HLA class II and CD86 in the Mo-DCs, but no difference in the ability to elicit mixed lymphocyte reaction.

Immature DCs, with a phenotype resembling interstitial DCs, can be generated by stimulating monocytes from peripheral blood with IL-4 and GM-CSF.

The original methods made use of monocytes enriched by adherence.

Viral antigens represent attractive vaccine targets for virus-induced cancers and are included in the prophylactic vaccines mentioned above for cervical carcinoma (Human papilloma virus)

The differentiation antigens are tissue-specific, i.e. expressed only in normal and neoplastic cells from a particular lineage. These antigens may be utilized in cancer vaccines if an auto‐

For instance, prostatitis or vitiligo may represent acceptable adverse effects for patients with

Several differentiation antigens are extensively used in cancer vaccines, including prostatespecific antigen (PSA) and the melanoma antigens Melan a/Mart-1, gp100 and tyrosinase

Most cancer vaccines have been based on peptides/proteins or tumour lysates that are injected intradermally. These approaches depend on uptake of vaccine antigen by immature DCs in the skin, and subsequent DC maturation and migration to lymph nodes. Alternatively, DCs may be loaded with tumour antigens ex vivo, and then injected into the patient. This strategy appears attractive, as it may result in enhanced antigen presentation and more effective T-cell stimulation. Moreover, DC-based vaccines may offer the opportunity of directing the immune

The first DC-vaccine trials in cancer patients were reported by Hsu et al. in 1996 and Nestle et al. in 1998 [7]. Promising T-cell responses were obtained, and in recent years, various ap‐

Early studies applied immature DCs, but it is now generally believed that a mature phenotype

Ralph Steinman's group has explored this option by use of antibodies targeting the receptor DEC-205 on the DC surface [7]. Their data from animal model indicate that potent T- and B-

Alternatively, vaccine antigens might be injected and subsequently transfected into tissue DCs

For clinical vaccine production, large quantities of autologous DCs are required. Most studies

**6. Background for the present up-to-date tumour-RNA/DC vaccines**

and hepatocellular carcinoma (Hepatitis B virus).

10 Immunopathology and Immunomodulation

immune reaction to the relevant tissue is tolerable.

prostate cancer or malignant melanoma, respectively.

**6.1. The use of dendritic cells in cancer immunotherapy**

response, by manipulating the DC phenotype.

proaches to DC-vaccines have been explored.

Targeted loading of DCs in vivo represents another strategy.

[6, 7, 8].

is desirable.

cell responses may be elicited.

by use of in vivo electropermeabilization.

**6.2. Large-scale generation of dendritic cells**

make use of monocyte-derived DCs (Mo-DCs).

However, the handling of large numbers of adherent cells is time consuming.

**Figure 3.** Production of the present RNA/DC-vaccines against malignant melanoma or prostate cancer. Tumor-mRNA was extracted from autologous melanoma biopsies (melanoma vaccine) or from three prostate cancer cell lines (PC-3, LNCaP, DU-145; prostate cancer vaccine). Autologus DCs were generated from monocytes obtained from leukaphere‐ sis products. The monocytes were cultured 5 days with IL-4 and GM CSF fro differentiation into immature DCs. Tu‐ mor mRNA was then transfected into DCs by electroporation. After transfection, the DCs were cultured for 2 days with cytokines promoting maturation and frozen in vaccine batches.

The production procedure used in the trials performed in Radium Hospital (Inst. Cancer Research, Oslo, Norway) by the group of Gunnar Kvalheim, Jon Kyte and Paula Lazarova [2, 6, 7] is outlined in Figure 3.

The research group did not make use of adherence, but isolated monocytes from leukapheresis products by immunomagnetic depletion of lymphocytes.

The monocytes were transferred to gas permeable Teflon bags that allowed the intrinsically adherent cells to stay in the suspension.

After five days' culture with IL-4 and GM-CSF, the cells were transfected with tumour-mRNA (Figure 3).

Finally, the transfected DCs were matured for 2 days ex vivo with TNFα, IL-6, IL-1β and PGE2.

Contrary to most previous studies, a serum-free culture medium was used. Thereby, unwanted antigens from bovine or human serum were excluded from the vaccine product.

The procedure for DC generation was first established at Radium Hospital by experiments on healthy donors, as reported by Mu et al. [3, 4] and Lazarova et al. [2, 6, 8], later on patients as well.

Subsequently, the generation of clinical grade DCs from patients was evaluated as part of the full-scale preclinical evaluation of the research group in Radium, namely Paula Lazarova et al. [2, 6, 8].

## **7. Choice of vaccine antigens**

Defined tumour-associated antigens have been targeted in a number of interesting vaccine trials world-wide, resulting in antigen-specific immune responses. However, there is a limited evidence of clinical effect, and initial responses are probably vulnerable to tumour escape through loss of antigen expression. The spectrum of target antigens may be widened by use of peptide cocktails or allogeneic tumour cell lines. In the vaccine for prostate cancer, as proposed by the Radium group [2, 3, 4, 5, 6, 7, 8], the employed DCs are transfected with complete mRNA from allogeneic prostate cancer cell lines. To extend the number of antigens, three cell lines were combined.

The clinical trial was conducted on patients with hormone refractory cancer, and thus two hormone-insensitive tumour cell lines were selected (DU-145 and PC-3). A cell line expressing PSA (LN-CaP) was also included.

PSA is widely used for monitoring disease development and also represents an immunogenic tumour antigen.

It was considered that the allogeneic antigens included in the tumour cell lines may increase the risk of side effects, but may also be beneficial.

T-cell recognizing allogeneic antigens will be primed in the same lymph nodes as the tumourspecific T cells.

The production procedure used in the trials performed in Radium Hospital (Inst. Cancer Research, Oslo, Norway) by the group of Gunnar Kvalheim, Jon Kyte and Paula Lazarova [2,

The research group did not make use of adherence, but isolated monocytes from leukapheresis

The monocytes were transferred to gas permeable Teflon bags that allowed the intrinsically

After five days' culture with IL-4 and GM-CSF, the cells were transfected with tumour-mRNA

Finally, the transfected DCs were matured for 2 days ex vivo with TNFα, IL-6, IL-1β and PGE2.

Contrary to most previous studies, a serum-free culture medium was used. Thereby, unwanted

The procedure for DC generation was first established at Radium Hospital by experiments on healthy donors, as reported by Mu et al. [3, 4] and Lazarova et al. [2, 6, 8], later on patients as

Subsequently, the generation of clinical grade DCs from patients was evaluated as part of the full-scale preclinical evaluation of the research group in Radium, namely Paula Lazarova et

Defined tumour-associated antigens have been targeted in a number of interesting vaccine trials world-wide, resulting in antigen-specific immune responses. However, there is a limited evidence of clinical effect, and initial responses are probably vulnerable to tumour escape through loss of antigen expression. The spectrum of target antigens may be widened by use of peptide cocktails or allogeneic tumour cell lines. In the vaccine for prostate cancer, as proposed by the Radium group [2, 3, 4, 5, 6, 7, 8], the employed DCs are transfected with complete mRNA from allogeneic prostate cancer cell lines. To extend the number of antigens,

The clinical trial was conducted on patients with hormone refractory cancer, and thus two hormone-insensitive tumour cell lines were selected (DU-145 and PC-3). A cell line expressing

PSA is widely used for monitoring disease development and also represents an immunogenic

It was considered that the allogeneic antigens included in the tumour cell lines may increase

antigens from bovine or human serum were excluded from the vaccine product.

6, 7] is outlined in Figure 3.

12 Immunopathology and Immunomodulation

(Figure 3).

well.

al. [2, 6, 8].

adherent cells to stay in the suspension.

**7. Choice of vaccine antigens**

three cell lines were combined.

PSA (LN-CaP) was also included.

the risk of side effects, but may also be beneficial.

tumour antigen.

products by immunomagnetic depletion of lymphocytes.

The allo-reaction may therefore result in an inflammatory milieu promoting the development of effective anti-tumour responses.

It is argued that the majority of tumour antigens are probably specific to each patient and not even expressed in allogeneic cancer cell lines. The individual tumour antigens are believed to arise from numerous incidental mutations occurring during the development of tumour.

The melanoma RNA/DC-vaccine, worked out by the Radium group [2, 3, 4, 5, 6, 7, 8], represents individualized immune-gene therapy. The autologous tumour material as source of mRNA is used in the procedure (Figure 3) and thereby targets the entire spectrum of tumour antigens in each individual. Moreover, non-expressed tumour antigens are excluded.

In general, it is believed that personalized vaccines, targeting the unique spectrum of tumour antigens in each patient, may emerge as a major principle in cancer immunotherapy

The tumour-mRNA strategy is in principle applicable to any cancer form and may prove particularly useful in rarer cancer forms, where common tumour antigens have not yet been defined. Contrary to peptide vaccines, the use of cell line/tumour-mRNA bypasses require‐ ments for defined HLA alleles and for expression of identified antigens by tumours.

The mRNA can encode multiple epitopes and recruit a wide spectrum of T cell clones, including both CD4+helper and CD8+cytotoxic cells.

There is a number of cancer vaccine trials that have applied RNA-transfected DCs.

Certain parts of these studies include use of undefined antigens.

There are definite disadvantages related to the use of undefined antigens.

First, a wide array of possibly harmful autoantigens will be included, suggesting an increased risk of autoimmune side effects.

Second, the antigens recognized after vaccination will usually not be known.

If HLA-matching peptides can be obtained, T-cell responses to defined antigens may be demonstrated.

However, most antigens, including unique patient-specific targets, will remain unknown.

The T-cell responses can thus only be characterized to a limited extent.

Third, in a vaccine based on autologous tumour material, each individual will receive different vaccines.

This complicates the comparison of results from different patients. It should, however, be recalled, that in any trial on humans the inter-individual variability is immense, even though the vaccine itself may be fully standardized.
