**2. Proof of the principle: A solid body of preclinical evidence**

The idea to actively prime cytotoxic T cells to specifically kill a tumor target cell has become a well established scientific fact. Several approaches all aiming to induce specifically activated, tumor-rejecting effector T cells have been investigated and found to be reproducible and reliable technologies. Genetically engineered tumor cells but especially autologous dendritic cells charged with tumor associated antigens have become the most

Immunotherapy for Malignant Gliomas: A Roadmap for the Future 415

now are equivalent to standard of care chemotherapy regimens, the final results of immunotherapy in these entities are still modest. Malignant gliomas, the most common primary brain tumors have always been considered to be not suitable for immunotherapy because of their location in the immune privileged central nervous system. To date however, we know that all the obstacles like the blood brain barrier, the lack of lymphatic vessels, the lack of residing antigen presenting cells in the brain, the low MHC expression on the tumor target cells are quite relative. They don't seem to hamper the documented immune responses in patients harboring such tumors(12). To the current understanding the real hurdles in glioma vaccination strategies are the multiple immunosuppressive pathways orchestrated by this type of cancer cells and the lack of universally expressed glioma associated antigens that are really crucial for tumor cell survival. Surprisingly, preliminary data might be pointing to an exploitable lack of spontaneous immunogenicity of malignant gliomas. As such, they develop in a micro-environment protecting them from too extensive immune editing: especially that characteristic could be of major help to try to reset the patients' immunity resulting in a much better immune rejection or control since the original immune-sensitive tumor clones have not yet been

Immune responses in the brain always elicit some fear for potentially disastrous consequences of an auto-immune attack by the patient's immunity. Unlike vitiligo or even destruction of normal prostate tissue, an auto-immune encephalomyelitis could result in devastating neurological symptoms and deficits. Until now however, no preclinical –other than in a heavily manipulated immune environment- and no clinical data have been published showing any suspicion of serious auto-immunity in the central nervous system, using tumor vaccination strategies. Moreover, numerous phase I and small phase II trials have been published showing the safety and the very attractive low toxicity profile of dendritic cell vaccines in brain cancer. As we're still dealing with a palliative treatment thus far, this perfect patient tolerance profile is highly valuable to build upon for upcoming vaccination strategies. Although today's technology to produce autologous dendritic cell vaccines is still very labour-intensive, it proved to be perfectly feasible to implement it in the daily clinical practice, both in the case of relapsed or newly diagnosed

The main criticism against tumor vaccine approaches, often raised till today, comprises the presumed low rate of objective tumor responses. Objective, radiological responses as defined according to RECIST(14) or McDonalds(15) criteria have indeed been developed for radio-and especially chemotherapy regimens during which one aims to reduce the measurable tumor load in the patient. Although they provide valuable information in terms of proof of the principle of the investigated chemotherapy, they should not be considered a synonym for clinically relevant efficacy: in only a few cases, one was really able to demonstrate a clear correlation between objective responses and overall survival benefits(16). More intriguing even is the existence of the so-called 'pseudo-progression' since radiochemotherapy with temozolomid became the standard of care(17). In up to 25% (or even 40%) of cases, one might see an initial increase in radiological tumor volume, rather early after the concomitant radiochemotherapy: strikingly, often especially these patients seem to have a better overall survival chance than the group not displaying these types of misleading radiological changes. Dendritic cell vaccination is known to cause similar radiological images in which transient contrast-enhancements on magnetic

eliminated by the natural immune surveillance mechanisms(13).

high grade gliomas.

potent and popular immunological adjuvants to install an active anti-tumoral immunity(3;4). An additional advantage of the latter is the activation of both the innate and adaptive immunity arms of the patient. The mechanisms underlying this antitumoral priming capacity of dendritic cells seem to be fully consistent with the established paradigms of antigen uptake, processing and presentation. The exclusive potential of DC to present and cross-present exogenous antigens in the same antigen presenting cell is the critical characteristic for a successful antigen presentation to cytotoxic and helper T cells. DC pick up tumor-associated antigens from diverse sources, process them and present them in both an Major Histocompatibility Complex (MHC) class I and II context to cytotoxic and helper T cells respectively. This has been clearly shown, also for glioma associated antigens from a whole-tumor-cell lysate(5). To date, efficient priming of the patient by dendritic cells implies four sequences of interaction between the dendritic cell and the T cell. Upon binding of the T cell receptor with the MHC complex on the antigen presenting cell displaying the appropriate epitope, a specific activation of the antitumor T cells with high enough avidity for the epitope takes place. Expansion of this T cell clones requires co-stimulatory molecules on the DC. A polarized immune response, preferentially with a Th-1 cytokine profile, should result from the previous interactions. Finally, some indication about the target location should be transferred to the T cells leading to the appropriate T cell homing properties(6-8).

Many rodent models have demonstrated that prophylactic vaccination of mice with tumor-antigen loaded DC can protect immune competent animals from tumor challenge and outgrow. The most relevant experiments are being performed with syngeneic micetumor models, to prove the efficacy of the vaccination in an autologous setting underscoring the possibility to break tolerance for self-derived antigens. Moreover, one can raise an anti-tumor immunological memory to make mice survive a re-challenge of cancer cells. More elaborated manipulations of the immune system like e.g. regulatory T cell depletion before vaccination even yield a much stronger protective effect in up to 100% of the exposed animals(9;10). The step towards therapeutic vaccination models, being more relevant for the clinical reality of all day can be made if the crucial timing of inoculation and vaccination is being respected. Probably also due to the aggressive nature of the investigated models, where all mice are dead within 3 weeks after tumor inoculation, it appears crucial to vaccinate the animals not later than one week after the tumor inoculations. After that period, the course of the disease can hardly be influenced, presumably because of the establishment of an efficient immune suppressive, pro-tumor micro-environment around the tumor inocula. This should be understood as the first evidence to use DC vaccinations as an adjuvant therapy in minimal residual disease settings, resembling most the prophylactic setting in which anti-tumor vaccines are so efficient.
