**3. DNA vaccines**

DNA vaccines are focused to the TAA which is used in a specific approach to switch the patient's immune system. Most DNA vaccines have focused on tissue-specific (PAP, PSMA, PSCA and PSA) rather than tumor-specific antigens. The major advantages of DNA vaccines is, at least compared with DC-based vaccines, that they are easy and inexpensive to produce and that with some viral vectors used in this attempt there is a huge body of experience as they have been used in millions of persons for preventive vaccinations against infective disease (Fioretti et al., 2010).

### **3.1 Prostvac-VF approach**

298 Prostate Cancer – Diagnostic and Therapeutic Advances

subjects receiving the non-activated peripheral blood mononuclear cells. Such events included, but were not limited to, vomiting, fatigue, fever, chills, respiratory events (dyspnea, hypoxia, and bronchospasm), nausea, hypertension, and tachycardia. To minimize potential acute infusion reactions e.g. chills and/or fever, it is recommended to premedicate patients orally

The safety evaluation of sipuleucel-T, released by the FDA, is based on 601 PCa patients who received at least one dose of sipuleucel-T reported in four different clinical trials (Small et al., 2006; Harzstark et al., 2009; Higano et al., 2009a; Kanthoff et al., 2010a). Almost all (98.3 %) indivduals in the sipuleucel-T group and 96.0 % in the control group reported an adverse event. In 67.4 % of patients in the sipuleucel-T group, these adverse events were mild or moderate. Severe (grade 3) and life-threatening (grade 4) adverse events were reported in 23.6 % and 4.0 % of patients in the sipuleucel-T group compared with 25.1 % and 3.3 % of control group patients. Fatal (grade 5) adverse events were reported in 3.3 % of patients in the sipuleucel-T group compared with 3.6 % of patients in the control group. The FDA recommended the manufacturer to run a post-marketing study to assess the risk of cerebrovascular events in 1,500 PCa patients who receive sipuleucel-T and awaits

Each dose of sipuleucel-T requires a leukapheresis approximately three days prior to the infusion. AE´s that were reported within one day following the leukapheresis procedure included citrate toxicity (14.2 %), oral paresthesia (12.6 %), general paresthesia (11.4 %), and

Due to its novelty in the market as if the pricey production process and due to the high research and development costs sipuleucel-T as a pharmaceutical product is quite expensive – more than 90.000 USD. So there is a discussion whether or not these costs should be covered by the public. In comparison to other recently introduced drugs in other tumor entities (e.g. lung cancer or breast cancer) and based on the calculation of the achievable duration in life time the costs of sipuleucel-T has been calculate as about 10

A wide variety of other approaches using DCs have been studied, including evaluation of DCs pulsed with defined proteins like PSMA (Tjoa et al., 1999; Fishman, 2009), PSA (Barrou et al., 2004; Hildenbrand et al., 2007), xenogeneic PAP (Fong et al., 2001), PSCA (Thomas-Kaskel et al., 2006), and telomerase (Vonderheide et al., 2004; Su et al., 2005). Strategies based on peptides included pulsing DCs with multiple but defined peptides (prostate stem cell antigen (PSCA14–22), prostatic acid phosphatase (PAP299–307), prostate-specific membrane antigen (PSMA4–12), Prostein (31-39), Survivin (95–104), Trp-p8 (187–195), and prostate-specific antigen (PSA154–163)) (Fuessel et al., 2006; Waeckerle-Men et al., 2006). To expand antitumor reactivity and prevent tumor escape from the immune system, researchers have used DCs genetically engineered to express an enlarged range of antigens by the use of tumor cell lysates (Pandha et al., 2004), and allogeneic PCa cell lines (DU145,

DNA vaccines are focused to the TAA which is used in a specific approach to switch the patient's immune system. Most DNA vaccines have focused on tissue-specific (PAP, PSMA,

with an antihistamine prior to infusion of sipuleucel-T (Kanthoff et al., 2010b).

completion of this study till December 31, 2015 (FDA, 2010b).

fatigue (8.3 %) (FDA, 2010a).

times higher (Longo, 2010).

**3. DNA vaccines** 

**2.2 Other DC based vaccines** 

LNCaP and PC-3) messenger RNA (Mu et al., 2005).

This approach has been tested clinically in a number of phase I studies demonstrating safety of the vectors (Sanda et al., 1999; DiPaola et al., 2006; Arlen et al., 2007), and three phase II studies. Prostvac-VF consists of two genetically engineered viruses (recombinant Vaccinia (V) virus and Fowl pox (F) virus) administered in a sequential regimen. The virus strain used in Prostvac-V is a to some extent attenuated version of the virus used for smallpox immunization. Fowl pox viruses are unable to replicate in human cells but have been shown to be an effective way of boosting cellular immune responses primarily initiated using Vaccinia virus. The viral vectors are engineered to contain a gene encoding human PSA which contains an alteration in the HLA-A2 specific epitope that is planned to enhance the immunogenicity of the expressed antigen. In addition, these viruses both contain the genes encoding three co-stimulatory molecules, B7.1, ICAM-1 and LFA-3 (together named TRICOM). The academic work to establish this vaccine platform was done in cooperation with industrial sponsorship initially with Therion Biologics Cambridge, MA and subsequently with BN ImmunoTherapeutics, Garcia Ave, CA (Madan et al., 2009). The first phase II trial conducted by the Eastern Cooperative Oncology group enrolled 64 eligible patients and assigned them randomly to receive i) four vaccinations with fowl pox-PSA (rF-PSA), ii) three rF-PSA vaccines followed by one vaccinia-PSA (rV-PSA) vaccine, or iii) one rV-PSA vaccine followed by three rF-PSA vaccines. In this trial the TRICOM-component was not included. The prime/boost schedule was well tolerated with a small amount of adverse events. Of the eligible patients, 45.3% of men remained free of PSA progression at 19.1 months and 78.1 % demonstrated clinical progression free survival. There was a trend favouring the treatment group that received a priming dose of rV-PSA (Kaufman et al., 2004).

So in further trials using this approach rV-PSA priming was always followed by rF-PSA boostering. In the second phase II trial (n = 125 patients) it could be shown that at 3 years post study analysis, Prostvac-VF patients (n = 82) had a better OS with 25 (30 %) of 82 alive versus 7 (17 %) of 40 controls, longer median survival by 8.5 months (25.1 vs 16.6 months for controls), an estimated hazard ratio of 0.56 (95% CI, 0.37-0.85), and stratified log-rank p < 0.01. There was a minor imbalance in favour of the Prostvac-VF arm in mean and median of some laboratory values. But integration of these factors plus performance status in the Halabi nomogram revealed a 1-month mean and 2-month median difference in predicted survival (mean and median of 20.4 months for controls vs mean of 21.4 months and median of 22.5 months for Prostvac-VF). The observed survival difference of 8.5 months far exceeds that predicted by the Halabi nomogram. So these data are – despite of OS being not the primary end point – considered clinically meaningful and strongly suggests that Prostvac-VF immunotherapy may produce an OS benefit, but still regarded as hypothesis generating data as the authors state in the discussion of there recently published work (Kantoff et al., 2010b). The National Cancer Institute (NCI) has also recently completed a third phase II study in 32 PCa bearing men in whom immune and regulatory T-cell responses were

Entering a New Era – Prostate Cancer

treatment (McNeel et al., 2009).

adjuvant seems beneficial (Feyerabend et al., 2009).

alone (p < 0.05) (Noguchi et al., 2010).

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

**4.1 Prostate GVAX®** 

Immuno-Therapy After the FDA Approval for Sipuleucel-T 301

increased PSA-DT, 6.5 months pretreatment versus 9.3 months in the 1 year post

In a phase I/II trial a composition of 13 prostate-associated synthetic peptides (e.g. PSA, PSCA, PSMA, Survivin and Prostein) was used to counteract tumor escape mechanisms by genetic mutations or antigen loss. The TAA-epitopes were presented on HLA-A2 and on HLA-DR molecules, with the aim to activate a broad spectrum of CD8+ and CD4+ specific T-cells. The peptides were applied s.c. with or without immune stimulants (imiquimod, GM-CSF, MUC1-protamin complex). Four out of 19 men had a fivefold elevation of PSA-DT. Four individuals had minor PSA changes during vaccination and 11 patients showed progressive disease. In four men the vaccination was discontinued due to adverse events graded moderate. Although underpowered to draw definitive results imiquimod as an

In contrast to the later approach using a peptide cocktail it is also possible to induce immune responses (elevated DTH-reaction, increased IFNγ ELISPOT activity and decreased Treg-cell frequency) by only using a short active sequence existing of fourteen amino acids of the TAA HER2/neu. Such synthetic fragments (500 µg) were administered together with GM-CSF in a phase I trial using a schedule of six intradermal vaccinations (Perez et al., 2010). A slightly different but unique approach is used by a group at the university of Kurume, Japan. They tested an individualized method of peptide vaccination based on preexisting cytotoxic T-cell and immunoglobulin (IgG) reactivity. Each patient was tested for reactivity among 16 immunogenic peptides known to bind to HLA-A24. Peptides were derived from a number of targets, including PAP, PSMA, multidrug resistance protein, PSA, and a variety of other epithelial tumor antigens. Each patient was immunized with four peptides on the basis of his reactivity panel. This personalized medicine approach was tested as monotherapy and in combination with chemotherapy. As the peptides were injected s.c. vaccines were well tolerated and showed reactivity (DTH-reactions, CTL and IgG responses) and clinical activity (PSA declines up to 30 % in four out of 17 patients (Uemura et al., 2010). In a second trial the combination with estramustine showed a better PFS as estramustine

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

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

various tumor cell lines and are easier to set up (Risk & Corman, 2009).

evaluated. In that study, 13 of 28 evaluable patients had more than two-fold increases in PSA epitope specific immune responses, and four of five high responders (more than a sixfold increase) survived >40 months, while low or non-responders had a median OS of 20 months. The Halabi predicted survival of these metastatic CRPC patients was 17 months. Of interest is the Treg-cell course reported: Treg-cell suppressive function was shown to decrease following vaccine in patients surviving longer than predicted, and increase in patients surviving less than predicted (Gulley et al., 2010). Prostvac-VF immunotherapy is a promising approach, and a larger pivotal phase III trial is planned. If the data gained to date (OS benefit of 8.5 months) could be approved in a pivotal phase III trial – this platform is considered to be the next immunotherapy candidate for FDA approval.

Main components of this approach (rV-PSA, rF-PSA and rV-B7.1) have also been tested in a two armed phase II trial with or without combining the vaccine and docetaxel (plus dexamethasone) showing that docetaxel can be administered safely with immunotherapy without inhibiting vaccine specific T-cell responses. The authors state that patients previously vaccinated with an anti-cancer vaccine respond longer to docetaxel compared with a historical control of patients receiving docetaxel alone (Arlen et al., 2006).

#### **3.2 Other viral vector-based vaccines**

A group at the University of Iowa, USA, used an adenovirus genetically engineered to carry the genetic information for PSA and injected one single amount of recombinant virus. They included 32 patients with measurable mCRPC in a phase I trial. Patients were treated with a single s.c. vaccine injection at one of three dose levels, either suspended in a Gelfoam matrix or as an aqueous solution. The vaccine was judged safe in both administration forms at all doses. Anti-PSA antibodies and anti-PSA T-cell responses were detected in the majority of individuals. As PSA doubling time (PSA-DT) was increased and half of the patients survived longer than predicted by the Halabi nomogram this vaccine could be stated safe and potentially clinical effective and should proceed to phase II (Lubaroff et al., 2009).

A multicenter phase II trial in 40 patients with PSA progression used TG4010, a recombinant MVA vector expressing the tumor-associated antigen mucin 1 (MUC1) and Interleukin-2 (IL-2) as an adjuvant. Despite the primary endpoint of a 50 % decrease in PSA values from baseline was not observed, in 13 of 40 patients a more than two fold improvement in PSA-DT could be observed (p < 0.01 for all 40 patients) and ten patients had a PSA plateau for over 8 months demonstrating evidence of biologic activity (Dreicer et al., 2009).

#### **3.3 Other DNA-vaccines**

A phase I/II, dose escalation, DNA vaccination trial with plasmid DNA, coding for PSMA, fused to a domain (DOM1) of the C fragment of tetanus toxin was performed in patients with recurrent PCa. The DNA, was delivered either by i.m. injection without further manipulation or i.m. followed by electroporation. The PSMA epitope used in this study is a short stretch of 9 amino acids. Preliminary analysis of CD8+ T-cell reactivity against the PSMA target peptide indicated significant responses in three out of three patients and CD4+ T-cell responses against the DOM1. These data suggest electroporation as an effective method for stimulating the humoral system induced by DNA vaccination in humans (Low et al., 2009).

Results of a phase I/II trial, conducted with DNA vaccine encoding human PAP coadministered intradermally with GM-CSF, in PCa patients (stage D0) showed an

evaluated. In that study, 13 of 28 evaluable patients had more than two-fold increases in PSA epitope specific immune responses, and four of five high responders (more than a sixfold increase) survived >40 months, while low or non-responders had a median OS of 20 months. The Halabi predicted survival of these metastatic CRPC patients was 17 months. Of interest is the Treg-cell course reported: Treg-cell suppressive function was shown to decrease following vaccine in patients surviving longer than predicted, and increase in patients surviving less than predicted (Gulley et al., 2010). Prostvac-VF immunotherapy is a promising approach, and a larger pivotal phase III trial is planned. If the data gained to date (OS benefit of 8.5 months) could be approved in a pivotal phase III trial – this platform is

Main components of this approach (rV-PSA, rF-PSA and rV-B7.1) have also been tested in a two armed phase II trial with or without combining the vaccine and docetaxel (plus dexamethasone) showing that docetaxel can be administered safely with immunotherapy without inhibiting vaccine specific T-cell responses. The authors state that patients previously vaccinated with an anti-cancer vaccine respond longer to docetaxel compared

A group at the University of Iowa, USA, used an adenovirus genetically engineered to carry the genetic information for PSA and injected one single amount of recombinant virus. They included 32 patients with measurable mCRPC in a phase I trial. Patients were treated with a single s.c. vaccine injection at one of three dose levels, either suspended in a Gelfoam matrix or as an aqueous solution. The vaccine was judged safe in both administration forms at all doses. Anti-PSA antibodies and anti-PSA T-cell responses were detected in the majority of individuals. As PSA doubling time (PSA-DT) was increased and half of the patients survived longer than predicted by the Halabi nomogram this vaccine could be stated safe and potentially clinical effective and should proceed to phase II (Lubaroff et al., 2009). A multicenter phase II trial in 40 patients with PSA progression used TG4010, a recombinant MVA vector expressing the tumor-associated antigen mucin 1 (MUC1) and Interleukin-2 (IL-2) as an adjuvant. Despite the primary endpoint of a 50 % decrease in PSA values from baseline was not observed, in 13 of 40 patients a more than two fold improvement in PSA-DT could be observed (p < 0.01 for all 40 patients) and ten patients had a PSA plateau for

considered to be the next immunotherapy candidate for FDA approval.

**3.2 Other viral vector-based vaccines** 

**3.3 Other DNA-vaccines** 

in humans (Low et al., 2009).

with a historical control of patients receiving docetaxel alone (Arlen et al., 2006).

over 8 months demonstrating evidence of biologic activity (Dreicer et al., 2009).

A phase I/II, dose escalation, DNA vaccination trial with plasmid DNA, coding for PSMA, fused to a domain (DOM1) of the C fragment of tetanus toxin was performed in patients with recurrent PCa. The DNA, was delivered either by i.m. injection without further manipulation or i.m. followed by electroporation. The PSMA epitope used in this study is a short stretch of 9 amino acids. Preliminary analysis of CD8+ T-cell reactivity against the PSMA target peptide indicated significant responses in three out of three patients and CD4+ T-cell responses against the DOM1. These data suggest electroporation as an effective method for stimulating the humoral system induced by DNA vaccination

Results of a phase I/II trial, conducted with DNA vaccine encoding human PAP coadministered intradermally with GM-CSF, in PCa patients (stage D0) showed an increased PSA-DT, 6.5 months pretreatment versus 9.3 months in the 1 year post treatment (McNeel et al., 2009).

In a phase I/II trial a composition of 13 prostate-associated synthetic peptides (e.g. PSA, PSCA, PSMA, Survivin and Prostein) was used to counteract tumor escape mechanisms by genetic mutations or antigen loss. The TAA-epitopes were presented on HLA-A2 and on HLA-DR molecules, with the aim to activate a broad spectrum of CD8+ and CD4+ specific T-cells. The peptides were applied s.c. with or without immune stimulants (imiquimod, GM-CSF, MUC1-protamin complex). Four out of 19 men had a fivefold elevation of PSA-DT. Four individuals had minor PSA changes during vaccination and 11 patients showed progressive disease. In four men the vaccination was discontinued due to adverse events graded moderate. Although underpowered to draw definitive results imiquimod as an adjuvant seems beneficial (Feyerabend et al., 2009).

In contrast to the later approach using a peptide cocktail it is also possible to induce immune responses (elevated DTH-reaction, increased IFNγ ELISPOT activity and decreased Treg-cell frequency) by only using a short active sequence existing of fourteen amino acids of the TAA HER2/neu. Such synthetic fragments (500 µg) were administered together with GM-CSF in a phase I trial using a schedule of six intradermal vaccinations (Perez et al., 2010).

A slightly different but unique approach is used by a group at the university of Kurume, Japan. They tested an individualized method of peptide vaccination based on preexisting cytotoxic T-cell and immunoglobulin (IgG) reactivity. Each patient was tested for reactivity among 16 immunogenic peptides known to bind to HLA-A24. Peptides were derived from a number of targets, including PAP, PSMA, multidrug resistance protein, PSA, and a variety of other epithelial tumor antigens. Each patient was immunized with four peptides on the basis of his reactivity panel. This personalized medicine approach was tested as monotherapy and in combination with chemotherapy. As the peptides were injected s.c. vaccines were well tolerated and showed reactivity (DTH-reactions, CTL and IgG responses) and clinical activity (PSA declines up to 30 % in four out of 17 patients (Uemura et al., 2010). In a second trial the combination with estramustine showed a better PFS as estramustine alone (p < 0.05) (Noguchi et al., 2010).
