**4. Whole-tumor-cell vaccines**

While autologous whole-tumor-cell vaccines are derived from the patient's own tumor cells in an often lengthy and pricey process, allogeneic whole-tumor-cell vaccines originate from various tumor cell lines and are easier to set up (Risk & Corman, 2009).

### **4.1 Prostate GVAX®**

The GM-CSF-secreting cancer cell immunotherapy platform (GVAX®) (managed by Cell Genesys, South San Francisco, CA) was set up to be used in diverse types of carcinomas. The prostate GVAX® form uses two different PCa cell lines (PC3 and LNCaP) which have been modified through adenoviral transfer to secrete GM-CSF (Ward & McNeel, 2007). Analyses of these two cell lines showed up many genes well-known in human PCa metastases, including previously described prostate TAAs. The PC-3 cell line was derived from a PCa bone metastasis, and LNCaP was derived from a PCa metastasis to a lymph node. LNCaP was shown to express PAP, PSMA, PSA, urokinase-type plasminogen activator and prostate stem cell antigen (PSCA) (Simons et al., 1999; Kiessling et al., 2002; Lu & Celis, 2002). PC-3

Entering a New Era – Prostate Cancer

I/II trials and released results.

**4.2 ONY-P1** 

**4.3 LNCaP-IL2-IFNγ**

due to this contradictory data the future of GVAX is unclear.

neural network analysis (Michael et al., 2005).

Immuno-Therapy After the FDA Approval for Sipuleucel-T 303

up. VITAL-1 involved 626 men with asymptomatic chemotherapy-naïve CRPC, and randomized them to GVAX or docetaxel/prednisone, with OS as the primary endpoint. The trial was terminated in October 2008 based on the results of a previously unplanned futility analysis conducted by the study´s Independent Data Monitoring Committee (IDMC), which indicated that the trial had a less than 30 % chance of showing OS (predefined primary endpoint) (Higano et al., 2009b). VITAL-2 was designed initially to enrol 600 men with symptomatic mCRPC, randomizing them to docetaxel/prednisone or docetaxel/GVAX. It was haltered after having enrolled patients for two years (n = 408) in August 2008 as mortality appeared to be higher in men on the investigational arm receiving docetaxel/GVAX (67 vs. 47 respectively). Preliminary analysis revealed no significant difference in the patients baseline characteristics of toxic effect that could explain the unexpected discrepancy in death rate. A survival advantage (14.1 vs 12.2 moths; HR 1.7, 95 % CI 1.15-2.53) was seen in the control arm (doxetacel-prednisone) over the experimental (GVAX-doxetacel) arm (Small et al., 2009). Further evaluation has not yet been released and

Two other whole-tumor-cell vaccines (ONY-P1 and LNCaP-IL2-IFNγ) have finished phase

ONY-P1 (managed by Onyvax, Ltd, London, UK) consists of three irradiated PCa cell lines given to 26 patients with nonmetastatic CRPC intradermally (2.4 × 107 cells per injection), once a month for up to 12 month. In total 11 of the 26 patients demonstrated a prolonged decrease in their PSA-velocity (PSAV). None of the treated patients experienced any significant toxicity. Median time to disease progression was 58 weeks. PSAV-responding patients showed a titratable TH1 cytokine release profile in reply to restimulation with a vaccine lysate, while non-responders showed a mixed TH1 and TH2 response. Furthermore, immunologic profile correlated with PSAV response by artificial

This approach uses only LNCaP cells retrovirally transduced with a N2/huIL2/huIFNγvector, resulting in IL-2 and IFNγ secretion. The two cytokines chosen for this approach have been used for immunostimulation solely and in combination in a variety of tumors (Brill et al., 2007; Dieli et al., 2007) and both substances are FDA approved single agents. Expression of tumor-associated antigens is upregulated after treatment with IFNγ via IFNγinducible genes, thereby increasing the susceptibility of tumors to MHC restricted CD8+CTL-mediated killing (Gansbacher et al., 1990a; Shankanan et al., 2001; Propper et al., 2003; Dunn et al., 2005). IL-2 is a well-known T cell growth factor, which is traditionally implicated in the agonistic stimulation of immune responses (Gansbacher et al., 1990b; Rosenthal et al., 1994) and FDA approved for systemic application against metastatic renal cell cancer. IL-2 is the only cytokine to date not been detected to be produced by any cancer. LNCaP cells, as mentioned above (see GVAX) express some relevant TAAs but in contrast to other PCa cell lines used as cancer vaccines, do not express transforming growth factor (TGF)-β. After detailed investigation of the safety profile with special attention to induction of autoimmunity (n = 3 patients at a dose level of 7,5 × 106 cells) (Brill et al., 2007), further patients (n = 27) were scheduled to receive four intradermal vaccine injections (dose level of

was shown to express glutathione S-transferase, mutant p53, CEA, and urokinase-type plasminogen activator (Warren & Weiner, 2000). GM-CSF – the cytokine the GVAX inventors have chosen as to further augment their vaccine – has shown some impact in PCa patients (PSA modulations) if administered as a solely therapeutic agent (Small et al., 1999; Dreicer et al., 2001; Schwaab et al., 2006). However, the use of GM-CSF might be challenged by counterregulatory immune responses that aim to reduce the expansion of cytotoxic T cells, thereby limiting antitumor activity. The use of GM-CSF for anti-cancer immunostimulation has caused some concerns as GM-CSF is associated with the presence of CD34+myeloid suppressor cells. Besides that it has been shown that GM-CSF is secreted by some carcinomas (Bronte et al., 1999) with a clinically relevant worse outcome (e. g. higher rate of recurrence) as in tumors with lesser CD34+cells which release Transforming Growth Factor (TGF) β inhibiting T-cell functions (Young, et al., 1997). This knowledge is important to determine the best use of GM-CSF and generally, low doses of GM-CSF are associated with greater stimulation of the immune response than higher doses which might create a counterproductive immune response via inducible nitric oxide synthase (iNOS) in well designed mouse data. This immunosupression could be abandoned by the specific iNOS inhibitor, L-NMMA, resulting in restored antigenspecific T-cell responsiveness in vitro (Serafini et al., 2004). Therefore, it is critical to optimize the use of GM-CSF, in order to improve, rather than hamper, the immune response (Harzstark et al., 2009).

In a first phase I/II trial (coded G9802) a fixed total cell dose of 1.2 × 108 cells (6 × 107 per cell line) was used in hormone therapy-naïve patients with PSA recurrence following radical prostatectomy and absence of radiologic metastases. GM-CSF secretion from the clinical lots used in this trial was 150 ng/106 cells/24 h (LNCaP) and 450 ng/106 cells/24 h (PC-3). Patients were vaccinated weekly via intradermal injections for 8 weeks and resulted in one patient having a partial PSA response of 7 month duration. The injection sites were found to have invasion of inflammatory cells and APCs on histopathology. At 20 weeks after the first treatment, 16 of 21 treated patients showed a statistically significant decrease in PSA velocity (slope) compared with prevaccination PSA course (Simons et al., 2006) (Urba et al., 2008). Two further uncontrolled single-arm phase II studies included asymptomatic CRPC patients with (n = 34) or without (n = 21) metastases (G9803 trial) and only men with metastases (n = 80) (G0010 trial). The two trials have shown anti-tumor effects of prostate GVAX®, the first one (G9803 trial) demonstrating an overall survival benefit of 34.9 versus 26.2 months in the mCRPC subgroup (n = 34) (Small et al., 2007) and the other (G0010 trial), a study in which the vaccine was re-engineered to secrete a higher dose of GM-CSF, showing an OS ranging from 20.0 to 29.1 months (n = 80) depending on dosing regimen. Dose levels ranged from 1 × 108 cells q28d × 6 to as many as 5 × 108 cells prime/3 × 108 cells boost q14d × 11. Besides the differences in OS also the proportion of men who generated an antibody response to one or both cell lines increased with dose and included 10 of 23 in the low-dose up to 16 of 18 in the highest dose group (p < 0.01; Cochran-Armitage trend test) (Higano et al., 2008). A combined expanded retrospectively analyses of antibody response using the data from the three above mentioned trials indicated a significant (p < 0.05) association of alternative reading frame protein (TARP) antibody induction and median survival time (Nguyen et al., 2010). No dose-limiting or autoimmune toxicities were seen. The most common adverse events in both studies were injection-site erythema, myalgias, fatigue, malaise, and arthralgias. Based on these promising findings, two powerful sized randomized phase III studies of GVAX immunotherapy (VITAL-1 and VITAL-2) were set up. VITAL-1 involved 626 men with asymptomatic chemotherapy-naïve CRPC, and randomized them to GVAX or docetaxel/prednisone, with OS as the primary endpoint. The trial was terminated in October 2008 based on the results of a previously unplanned futility analysis conducted by the study´s Independent Data Monitoring Committee (IDMC), which indicated that the trial had a less than 30 % chance of showing OS (predefined primary endpoint) (Higano et al., 2009b). VITAL-2 was designed initially to enrol 600 men with symptomatic mCRPC, randomizing them to docetaxel/prednisone or docetaxel/GVAX. It was haltered after having enrolled patients for two years (n = 408) in August 2008 as mortality appeared to be higher in men on the investigational arm receiving docetaxel/GVAX (67 vs. 47 respectively). Preliminary analysis revealed no significant difference in the patients baseline characteristics of toxic effect that could explain the unexpected discrepancy in death rate. A survival advantage (14.1 vs 12.2 moths; HR 1.7, 95 % CI 1.15-2.53) was seen in the control arm (doxetacel-prednisone) over the experimental (GVAX-doxetacel) arm (Small et al., 2009). Further evaluation has not yet been released and due to this contradictory data the future of GVAX is unclear.

Two other whole-tumor-cell vaccines (ONY-P1 and LNCaP-IL2-IFNγ) have finished phase I/II trials and released results.
