**8. Loading of DCs with antigen**

There are several strategies for loading DCs with antigen ex vivo. A number of important trials have applied simple co-incubation of DCs with peptides, proteins or tumour lysates. Other interesting vaccine studies have made use of tumour-DC hybrids, i.e. tumour cells that are fused with DCs. Similar to DCs loaded with tumour lysates or tumour-RNA, these hybrids may combine the antigen repertoire of tumour cells with the stimulatory capacity of DCs. However, sufficient numbers of living tumour cells are required. In most patients, clinical scale vaccine production may therefore only be feasible from allogeneic cell lines, not from autolo‐ gous tumours.

DNA- or RNA-transfection represents other alternatives for DC-loading. It is accepted by the Radium group that mRNA has certain important advantages compared to DNA.

First, the use of mRNA bypasses the complex issues of transcriptional regulation.

Second, DNA requires entry into the nucleus, while mRNA has direct access to the translation machinery upon entry into cytoplasm.

In experiments with liposome-mediated loading of plasmid DNA, Saeboe-Larssen et al. found that only a minute fraction (10ˉ⁴) was detected in the nucleus.

Third, transfected DNA may persist in the cell and encode harmful proteins, while RNA will rapidly degrade.

The latter point is of particular relevance for the safety of using tumour-derived DNA/RNA, likely to encode proteins involved in tumour genesis.

The intrinsic instability of RNA, however, also carries a prominent obstacle to clinical use.

The tumour-mRNA may easily degrade if the tumour samples and RNA-preparations are not carefully handled right from the initial biopsy excision.

Contrary to tumour lysates or tumour/DC-hybrids, tumour–mRNA may be amplified from small tumour biopsies.

This may be of particular importance if the clinical trials are extended to patients with earlystage disease, where only small tumours will be available.

Moreover, mRNA-amplification may enable us to make vaccines from small biopsies of tumours located at difficult sites, e.g. in the brain or visceral organs.

The efficiency of RNA-transfection with conventional RNA/DC co-culture or liposomemediated loading is limited, probably reflecting degradation of RNA both outside of the cell and in endocytic DC compartments.

The limited efficiency results in low intracellular concentrations of the transfected mRNA.

Though immune responses have still been obtained, it is believed that a higher transfection efficiency is desirable for recruiting a wider spectrum of T-cell clones.

While some T-cell clones will respond even to low peptide concentrations on the DC surface, the low affinity clones will require higher concentrations.

Viral vectors represent an effective alternative for both, DNA- and RNA-transfection, but there are considerable safety concerns and regulatory obstacles regarding their clinical application.

The Radium group [2, 3, 4, 5, 6, 7, 8] has developed an efficient method for mRNA-transfection by square-wave electroporation, compatible with clinical use.

The electroporation procedure has been optimized for full-scale vaccine production, as worked out by the group, and applied both, in the melanoma and the prostate cancer trials in Radium. The measured transfection efficiency is substantially higher than was expected to be obtained with other methods like liposome-mediated delivery.

In addition to dendritic cells, the research group works also on Epstein-Barr-Virus-trans‐ formed cell lines, monocytes and several cancer cell lines, and the initial results indicate efficient transfection.

#### **9. Summary**

**8. Loading of DCs with antigen**

14 Immunopathology and Immunomodulation

machinery upon entry into cytoplasm.

that only a minute fraction (10ˉ⁴) was detected in the nucleus.

likely to encode proteins involved in tumour genesis.

carefully handled right from the initial biopsy excision.

stage disease, where only small tumours will be available.

tumours located at difficult sites, e.g. in the brain or visceral organs.

efficiency is desirable for recruiting a wider spectrum of T-cell clones.

gous tumours.

rapidly degrade.

small tumour biopsies.

and in endocytic DC compartments.

There are several strategies for loading DCs with antigen ex vivo. A number of important trials have applied simple co-incubation of DCs with peptides, proteins or tumour lysates. Other interesting vaccine studies have made use of tumour-DC hybrids, i.e. tumour cells that are fused with DCs. Similar to DCs loaded with tumour lysates or tumour-RNA, these hybrids may combine the antigen repertoire of tumour cells with the stimulatory capacity of DCs. However, sufficient numbers of living tumour cells are required. In most patients, clinical scale vaccine production may therefore only be feasible from allogeneic cell lines, not from autolo‐

DNA- or RNA-transfection represents other alternatives for DC-loading. It is accepted by the

Second, DNA requires entry into the nucleus, while mRNA has direct access to the translation

In experiments with liposome-mediated loading of plasmid DNA, Saeboe-Larssen et al. found

Third, transfected DNA may persist in the cell and encode harmful proteins, while RNA will

The latter point is of particular relevance for the safety of using tumour-derived DNA/RNA,

The intrinsic instability of RNA, however, also carries a prominent obstacle to clinical use.

The tumour-mRNA may easily degrade if the tumour samples and RNA-preparations are not

Contrary to tumour lysates or tumour/DC-hybrids, tumour–mRNA may be amplified from

This may be of particular importance if the clinical trials are extended to patients with early-

Moreover, mRNA-amplification may enable us to make vaccines from small biopsies of

The efficiency of RNA-transfection with conventional RNA/DC co-culture or liposomemediated loading is limited, probably reflecting degradation of RNA both outside of the cell

The limited efficiency results in low intracellular concentrations of the transfected mRNA.

Though immune responses have still been obtained, it is believed that a higher transfection

Radium group that mRNA has certain important advantages compared to DNA.

First, the use of mRNA bypasses the complex issues of transcriptional regulation.

The development and evaluation of immuno-gene therapy of cancer based on tumour-mRNA transfected dendritic cells, and focused on malignant melanoma and prostate cancer give certain optimism for future successful application of anti-cancer vaccines.
